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Validamycin and Its Derivatives
Validamycin
and Its Derivatives
Discovery, Chemical Synthesis,
and Biological Activity

Xiaolong Chen, Yuele Lu,

Yongxian Fan and Yinchu Shen
Institute of Fermentation Engineering,
Zhejiang University of Technology, Hangzhou, PR China
Elsevier
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Chapter 1

An Introduction to Validamycins
and Their Derivatives
1.1 IMPORTANCE OF ANTIBIOTICS
The term “antibiotics” was first coined by the American microbiologist
Selman Waksman and his colleagues to describe chemical substances pro-
duced by microorganisms and having antagonistic effects on the growth of
other microorganisms. It excluded synthetic antimicrobials (sulfur drugs) and
biological products of nonmicrobial origin having antagonistic effects on bac-
teria. Though antibiotics were introduced into clinical practice only in the
middle of the 20th century, the use of microorganisms for the management of
microbial infections in ancient Egypt, Greece, China, and some other places
in the world is well documented. The modern era of antibiotics started with
the serendipitous discovery of penicillin from the culture filtrate of a fungus,
Penicillium notatum, by Alexander Fleming in 1928 (Fleming, 1929).
In the present scenario, antibiotics available in the market are either
produced by microbial fermentation or are derived via semisynthetic route
using the existing antibiotic backbone structure. They are classified into
different chemically defined groups. Antibiotics target bacterial physiology
and biochemistry, causing microbial cell death or the cessation of growth.
A significant number of these antibiotics affect cell walls or membranes
(e.g., β-lactam and glycopeptides), while several others exert their anti-
bacterial activity by targeting protein synthetic machinery via interaction
with ribosomal subunits, and these include antibiotics such as macrolides,
chloramphenicol, tetracycline, linezolid, and aminoglycosides. Other “mech-
anistic” groups include molecules that interfere with the nucleic acid
synthesis [e.g., fluoroquinolones (FQ) and rifampin], while some others exert
their effects by interfering with the metabolic pathways (e.g., sulfonamides
and folic acid analog) or by disruption of the bacterial membrane structure
(e.g., polymyxins, daptomycin, and others).
The successful use of antibiotics against bacterial diseases of human
beings has led to a large-scale screening of antibiotics’ effect for plant
disease control in the world.

Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00001-0

© 2017 Elsevier Ltd. All rights reserved. 1
2 Validamycin and Its Derivatives

1.2 AGRICULTURAL ANTIBIOTICS FOR PLANT PATHOGENS

Many antibiotics developed for medical purposes were investigated for activity
against plant pathogens. Furthermore, screening of soil organisms for production
of antibiotic substances was started with the prime purpose of plant disease con-
trol. However, the results obtained with antibiotics and antibiotic-containing cul-
ture broth did not fulfill the high expectations. Many of them were too
unstable under field conditions or showed toxic side effects on plants. Most anti-
biotics were rather expensive, even when used as a crude product. In the 1950s,
soon after the introduction of antibiotics in human medicine, the potential of
these "miracle drugs" to work wonders in controlling plant diseases was
explored (Mcmanus and Stockwell, 2000). Nearly 40 antibiotics were screened
for plant disease control. To be a viable candidate for disease control, the antibi-
otic needed to (1) be active on or inside of the plant; (2) tolerate oxidation, UV
irradiation, rainfall, and high temperatures; (3) be safe to plants; and (4) select
for resistant pathogens only at a low or nondetectable rate. Of the screened com-
pounds, fewer than 10 were used commercially and only streptomycin, tetracy-
cline, cycloheximide, and griseofulvin saw significant use worldwide (Kumar
et al., 2005). Streptomycin, the first antibiotic introduced in agriculture, was
used in the United States for the control of pear fire blight. This antibiotic and a
mixture of streptomycin and tetracycline have been used for the control of bac-
terial plant diseases, while cycloheximide and griseofulvin have been used for
the control of fungal plant diseases. Cycloheximide is a very powerful fungicide,
but unfortunately, is highly toxic to plants, which restricts its use against plant
diseases. Griseofulvin is a much less phytotoxic systemic fungicide, but its use
is also restricted, because the relation of its manufacturing cost to its perfor-
mance under field conditions is not quite satisfactory.
Later, in Japan (Misato, 1976), blasticidin S and kasugamycin have been
in practical use for rice blast control instead of mercuric fungicides, and
polyoxins and validamycin have been used to control the sheath blight of the
rice plant instead of arsenic fungicides. Since then, more and more antibio-
tics were found to be applied in plant diseases.

1.3 VALIDAMYCINS: MAGIC AGRICULTURAL ANTIBIOTICS

Validamycin (Asano et al., 1990; Horii et al., 1972; Iwasa et al., 1970; Kameda
et al., 1986), also called as jinggangmycin in China (Agricultural Antibiotic
Group, 1975a, 1975b), is a magic agricultural antibiotic produced by fermenta-
tion with Streptomyces hygroscopicus var. limoneus or S. hygroscopicus var.
jinggangensis, and has been widely used in Asia as the rice and wheat protectant
against Rhizoctonia solani since the 1970s. Sheath blight caused by R. solani is
a major disease of rice and wheat that greatly reduces yield and grain quality.
And validamycin is a nonsystemic antibiotic with fungistatic action and exhibits
remarkable therapeutic effects on the disease by inhibiting the extension of
R. solani without its growth inhibition (Shibata et al., 1982, 1989; Trinci, 1984).
 An Introduction to Validamycins and Their Derivatives Chapter | 1 3

Validamycin can also be used for the control of R. solani in potatoes, vegetables,
strawberries, tobacco, ginger, and other crops, and damping-off diseases of
cotton, rice and sugar beet, etc. Besides its excellent control effect, low price,
low drug-resistance, and low toxicity are the other outstanding merits of valida-
mycin, and it is now one of the most important agricultural antibiotics with the
biggest production in China (Shen, 1996).
Validamycin is a mixture of aminoglycoside compounds. There have
been eight members termed A to H (Fig. 1.1) (Asano et al., 1990; Horii

R5OH2C

R6O
OH HO
HO
HN

R3

OH
R1

R2OH2C
R4O

Compound R1 R2 R3 R4 R5 R6
Validamycin A H H H β-D-Glc H H

Validamycin B H H OH β-D-Glc H H

α-D-
Validamycin C H H H β-D-Glc H
Glc

Validamycin D H α-D-Glca H H H H

α-D-Glc(1-4)- β-
Validamycin E H H H H H
D-Glc

Validamycin F H H H β-D-Glc H α-D-Glc

Validamycin G OH H H β-D-Glc H H

β-D-Glc(1-6)- β-
Validamycin H H H H H H
D-Glc
aGlc=Glucopyranosyl.

FIGURE 1.1 Chemical structures of validamycins.

4 Validamycin and Its Derivatives

CH2OH CH2OH
CH2 OH
HO
OH OH OH NH2
NH2 OH NH2 OH
OH
OH OH OH
Valienamine Validamine Hydroxyvalidamine

CH 2OH CH2 OH
OH
OH OH
OH OH N
NH2 OH
OH H
OH OH OH
Valiolamine Voglibose
FIGURE 1.2 Chemical structures of valienamine and its related compounds.

et al., 1972; Iwasa et al., 1970; Kameda et al., 1986) isolated from the broth
of S. hygroscopicus var. limoneus, with validamycin A [1L-(1,3,4/2,6)-2,3-
dihydroxy-6-hydroxymethyl-4-[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-hydroxymethyl-
cyclohex-2-enylamino] cyclohexyl β-D-glucopyranoside] as the main member
and the most active. So the content of validamycin A is the most important
quality parameter of the commercial products of validamycin.
Many researchers also focused on the chemical synthesis, biosynthesis
genes, and derivatives of validamycins. Among them, the most interesting
work is that validamycins were lysed to produce valienamine or validamine
(Fig. 1.2) (Asano et al., 1984; Chen et al., 2005a, 2005b; Kameda et al.,
1980, 1981), which could be applied to synthesize valiolamine (Horii et al.,
1985; Kameda et al., 1984), and then voglibose (Babu et al., 2005; Chen
et al., 2006; Floss et al., 1999; Fukase, 1997; Kameda and Horii, 1986; Lu
et al., 2005; Yuan et al., 2005). Voglibose (code number: AO-128, trade
name: Basen), is an N-substituted derivative of valiolamine, which is a
branched-chain aminocyclitol, or pseudoamino sugar. Voglibose has attracted
considerable interest due to its wide range of therapeutic and pharmacologi-
cal properties, which include its excellent inhibitory activity against
α-glucosidases and its action against hyperglycemia and various disorders
caused by hyperglycemia. It has shown strong antiobesity and antidiabetic
activities, as it is a new, potent glucosidase inhibitor and is a drug used for
NIDDM (noninsulin-dependent diabetes mellitus) in Japan, China, and
Korea. Therefore, the industry of validamycin has converted from the agri-
cultural antibiotic to pharmaceutical.
Valienamine could also be used to synthesize N-octyl-4-epi-β-valienamine
(NOEV) (Iwasaki et al., 2006; Ogawa et al., 2004; Suzuki, 2006, 2008,
2013; Suzuki et al., 2007) and N-octyl-β-valienamine (NOV) (Ogawa et al.,
1996, 1998; Suzuki, 2013) (Fig. 1.3), two highly potent drug candidates
 An Introduction to Validamycins and Their Derivatives Chapter | 1 5

OH OH
HO

H HO
H
HO N HO N
(CH2 )7 CH3 (CH2 )7 CH3
HO HO
NEOV NOV
FIGURE 1.3 Chemical structures of NEOV and NOV.

for chemical chaperone therapy. NOEV and NOV are promising thera-
peutic agents for human β-galactosidase deficiency disorders (GM1-
gangliosidosis and Morquio B disease) and β-glucosidase deficiency
disorders (phenotypic variations of Gaucher disease), respectively (Suzuki,
2013). Originally NOEV and NOV had been discovered as competitive
inhibitors, and then their paradoxical bioactivities as chaperones were
confirmed in cultured fibroblasts from patients with these disorders.
Subsequently, GM1-gangliosidosis model mice have been used for con-
firmation of clinical effectiveness, adverse effects, and pharmacokinetic
studies. Orally administered NOEV entered the brain through the blood
brain barrier enhanced β-galactosidase activity, reduced substrate storage,
and improved neurological deterioration clinically. Computational analysis
revealed pH-dependent enzyme chaperone interactions. The recent study
(Suzuki, 2013) indicated chaperone activity of a new 1-deoxygalactonojirimycin
derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also
showed the chaperone effect toward several β-glucosidase gene mutants in
Gaucher disease. Furthermore a commercial expectorant drug, ambroxol, was
found to be a chaperone for β-glucosidase. A few Gaucher patients
responded to this drug with remarkable improvement of oculomotor dys-
function and myoclonus. It is hoped that chaperone therapy will become
available for some patients with Fabry disease, GM1-gangliosidosis,
Gaucher disease, and other lysosomal storage diseases particularly with
central nervous system involvement.

1.4 AIMS OF THE WORK

Validamycins have nice characteristics, such as high and long efficiency,
low toxicity and safe to the environment, and low production cost. Most
important of all, there is no report about discovery of validamycin-
resistant strains of R. solani. Thus, since the production of validamycins in
large scale, the yield was more than 30,000 40,000 ton per year (in 5%
preparations) for 1 1.3 3 107 hm2 rice field against R. solani and the loss
of rice yield was decreased 5000, 000 ton per year at least in China
6 Validamycin and Its Derivatives

(Shen, 1996). In recent years, the production of validamycins in other

Southeast Asian countries, such as India, Vietnam, Laos, has been boom-
ing. In addition, three drugs, voglibose, NEOV, and NOV, were derived
from validamycins. These drugs will give a second rise to validamycin
production and make products from the agricultural industry to the medical
industry.
The aims of the work are to review the magic agricultural antibiotics,
validamycins, including their discovery, biosynthesis, production, biological
activities, chemical synthesis, important derivatives, etc., and to provide
references in the research of antibiotics.

REFERENCES
Agricultural Antibiotic Group SIOAPS, 1975a. Classification and identification of jinggangmy-
cin producing strains. Weishengwu Xuebao 15 (2), 110 113.
Agricultural Antibiotic Group SIOAPS, 1975b. Isolation and identification of jinggangmycins.
Weishengwu Xuebao 15 (3), 223 226.
Asano, N., Kameda, Y., Matsui, K., Horii, S., Fukase, H., 1990. Validamycin H, a new pseudo-
tetrasaccharide antibiotic. J. Antibiot. 43 (8), 1039 1041.
Asano, N., Takeuchi, M., Ninomiya, K., Kameda, Y., Matsui, K., 1984. Microbial degradation
of validamycin A by Flavobacterium saccharophilum. Enzymatic cleavage of C N linkage
in validoxylamine A. J. Antibiot. (Tokyo) 37 (8), 859 867.
Babu J.S., Chavan G.J., Khanduri C.H., Kumar Y., Ray P.C., (Ranbaxy Laboratories Limited,
India). assignee. 2005 20050324. Processes for the purification of voglibose and the
preparation and purification of its intermediates. Application: WO patent 2005-IB777,
2005092834.
Chen, X., Zheng, Y., Shen, Y., 2005a. A new method for production of valienamine with micro-
bial degradation of acarbose. Biotechnol. Prog. 21 (3), 1002 1003.
Chen, X., Zheng, Y., Shen, Y., 2005b. Quantitative analysis of valienamine in the microbial deg-
radation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase
high-performance liquid chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life
Sci. 824 (1 2), 341 347.
Chen, X., Zheng, Y., Shen, Y., 2006. Voglibose (Basen, AO-128), one of the most important
alpha-glucosidase inhibitors. Curr. Med. Chem. 13 (1), 109 116.
Fleming, A., 1929. Classics in infectious diseases: on the antibacterial action of cultures of a
penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp.
Pathol. 10, 226 236.
Floss H.G., Lee S., Tornus I., (Bayer A.-G., Germany). assignee. 1999 19990329. Preparation of
valiolone as synthon for acarbose and voglibose. Application: WO patent 1999-EP2141,
9950217.
Fukase, H., 1997. Development of voglibose (Basen), an antidiabetic agent. Yuki Gosei Kagaku
Kyokaishi 55 (10), 920 925.
Horii, S., Fukase, H., Kameda, Y., 1985. Stereoselective conversion of valienamine and valida-
mine into valiolamine. Carbohydr. Res. 140 (2), 185 200.
Horii, S., Kameda, Y., Kawahara, K., 1972. Validamycins, new antibiotics. VIII. Isolation and
characterization of validamycins C, D, E, and F. J. Antibiot 25 (1), 48 53.
 An Introduction to Validamycins and Their Derivatives Chapter | 1 7

Iwasa, T., Yamamoto, H., Shibata, M., 1970. Validamycins, new antibiotics. I. Streptomyces
hygroscopicus var. limoneus nov. var., validamycin-producing organism. J. Antibiot.
(Tokyo) 23 (12), 595 602.
Iwasaki, H., Watanabe, H., Iida, M., Ogawa, S., Tabe, M., Higaki, K., et al., 2006. Fibroblast
screening for chaperone therapy in beta-galactosidosis. Brain Dev. 28 (8), 482 486.
Kameda, Y., Asano, N., Teranishi, M., Matsui, K., 1980. New cyclitols, degradation of valida-
mycin A by Flavobacterium saccharophilum. J. Antibiot. 33 (12), 1573 1574.
Kameda, Y., Asano, N., Teranishi, M., Yoshikawa, M., Matsui, K., 1981. New intermediates,
degradation of validamycin A by Flavobacterium saccharophilum. J. Antibiot. 34 (9),
1237 1240.
Kameda, Y., Asano, N., Yamaguchi, T., Matsui, K., Horii, S., Fukase, H., 1986. Validamycin G
and validoxylamine G, new members of the validamycins. J. Antibiot. 39 (10), 1491 1494.
Kameda, Y., Asano, N., Yoshikawa, M., Takeuchi, M., Yamaguchi, T., Matsui, K., et al., 1984.
Valiolamine, a new alpha-glucosidase inhibiting aminocyclitol produced by Streptomyces
hygroscopicus. J. Antibiot. (Tokyo) 37 (11), 1301 1307.
Kameda Y., Horii S.; (Takeda Chemical Industries, Ltd., Japan). assignee. 1986 19860423.
Valiolamine derivatives. Application: EP patent 1986-303048, 199591.
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impact on the terrestrial environment. Adv. Agron. 87 (05), 1 54.
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Zhongguo Yiyao Gongye Zazhi 36 (9), 525.
Mcmanus, P., Stockwell, V., 2000. Antibiotics for plant diseases control: silver bullets or rusty
sabers. Core Discussion Papers Rp (May), 27 51.
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48 55.
Ogawa, S., Ashiura, M., Uchida, C., Watanabe, S., Yamazaki, C., Yamagishi, K., et al., 1996.
Synthesis of potent β-D-glucocerebrosidase inhibitors: N-alkyl-β-valienamines. Bioorg. Med.
Chem. Lett 6 (8), 929 932.
Ogawa, S., Kobayashi, Y., Kabayama, K., Jimbo, M., Inokuchi, J.-I., 1998. Chemical modifica-
tion of β-glucocerebrosidase inhibitor N-octyl-β-valienamine: synthesis and biological evalu-
ation of N-alkanoyl and N-alkyl derivatives. Bioorg. Med. Chem. 6 (10), 1955 1962.
Ogawa, S., Sakata, Y., Ito, N., Watanabe, M., Kabayama, K., Itoh, M., et al., 2004. Convenient
synthesis and evaluation of glycosidase inhibitory activity of α- and β-galactose-type valie-
namines, and some N-alkyl derivatives. Bioorg. Med. Chem. 12 (5), 995 1002.
Shen, Y.C., 1996. Research and development on jinggangmycins for 25 years. Zhiwu Baohu
(Plant Protect.) 22 (4), 44 45.
Shibata, M., Kido, Y., Honda, Y., Shimizu, K., 1989. Hyphal extension inhibitors I and II with
similar inhibitory spectra to validamycin, isolated from hyphae of Rhizoctonia solani. Agric.
Biol. Chem. 53 (3), 869 873.
Shibata, M., Mori, K., Hamashima, M., 1982. Inhibition of hyphal extension factor formation by
validamycin in Rhizoctonia solani. J. Antibiot. 35 (10), 1422 1423.
Suzuki, Y., 2006. β-Galactosidase deficiency: an approach to chaperone therapy. J. Inherit.
Metab. Dis. 29 (2/3), 471 476.
Suzuki, Y., 2008. Chemical chaperone therapy for GM1-gangliosidosis. Cell. Mol. Life Sci.
65 (3), 351 353.
Suzuki, Y., 2013. Chaperone therapy update: Fabry disease, GM1-gangliosidosis and Gaucher
disease. Brain Dev. 35 (6), 515 523.
8 Validamycin and Its Derivatives

Suzuki, Y., Ichinomiya, S., Kurosawa, M., Ohkubo, M., Watanabe, H., Iwasaki, H., et al., 2007.
Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis. Ann. Neurol.
62 (6), 671 675.
Trinci, A.P.J., 1984. Antifungal agents which affect hyphal extension and hyphal branching.
Symp. Br. Mycol. Soc. 9 (Mode Action Antifungal Agents), 113 134.
Yuan J., Shao C., Chen D., Ye W.; (Shanghai Laiyi Biopharmaceutical Research and
Development Center Co., Ltd., Peop. Rep. China; Xinchang Pharmaceutical Factory,
Zhejiang Medicine Co., Ltd.). assignee. 2005 20050303. Preparation of valiolamine as
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Chapter 2

Production of Validamycins
2.1 DISCOVERY OF VALIDAMYCINS
In the course of screening for new antibiotics effective in the control of
sheath blight, a destructive disease of rice plants caused by Pellicularia sasa-
kii (Shirai) S. Ito, validamycins were first discovered in the broth of
Streptomyces hygroscopicus var. limoneus T-7545, which was isolated from
a soil sample collected in Akashi City, Hyogo Prefecture, Japan in 1970
(Iwasa et al., 1970). Five years later, they were also discovered in the broth
of S. hygroscopicus var. jinggangensis Yen. TH82, which was isolated from
a soil sample of Jinggang Mountain in Jiangxi, China (Agricultural
Antibiotic Group, 1975). Since then, they have been widely used in Asia as
the rice and wheat protectant against P. sasakii. Now, eight validamycins
have been purified and identified, including A, B, C, D, E, F, G, and H.

2.2 MICROBES FOR PRODUCING VALIDAMYCINS

2.2.1 General Characteristics for Microbes
Microbes for producing validamycins belong to S. hygroscopicus, including
S. hygroscopicus var. limoneus (Iwasa et al., 1970) and var. jinggangensis
(Agricultural Antibiotic Group, 1975). The strains were similar except in
some respects, such as the morphology and cultural characteristics. The mor-
phological characteristics of the strains are shown in Fig. 2.1. The aerial
mycelium of strain T-7545 was simply branched and terminated in coils of
three to five volutions. The spores were oval or cylindrical and measure
1.0
1.3 3 1.0
1.5 μm. Their surfaces were smooth. On the other hand, the
aerial mycelium of strain TH82 was simply branched and terminated in coils.
The spores were oval and not of a uniform size. Their surfaces were smooth.
The cultural characteristics of S. hygroscopicus var. limoneus T-7545 and
var. jinggangensis Yen. TH82 are listed in Table 2.1. The colors of aerial
mycelium are grey to grey and yellow, and black moist areas form in the
aerial mycelium on glucose asparagine agar and starch agar. The colors of
the vegetative mycelium on most of the media are bright yellow to ochre
with, in some cases, a slight greenish tinge. A light yellow to faint yellowish
brown diffusible pigment was noted in various media, but because dark

Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00002-2

© 2017 Elsevier Ltd. All rights reserved. 9
10 Validamycin and Its Derivatives

(A)

(×950) (×10,000×1/1.5)
(B)

(×7500) (×7500)
FIGURE 2.1 Morphology of S. hygroscopicus var. limoneus T-7545 (A) and S. hygroscopicus
var. jinggangensis Yen. TH82 B. (A) Reprinted with permission courtesy of Japan Antibiotics
Research Association (JARA).

brown soluble pigment on proteinaceous media was not observed, T-7545

and TH82 are considered to be nonchromogenic.
Physiological characteristics of T-7545 and TH82 are shown in
Table 2.2. As show in the table, a wide temperature range and rather high
optimum temperature for growth are characteristic for these two organisms.
Starch hydrolysis and milk peptonization tests are positive, whereas tyrosi-
nase, nitrate reduction, cellulose decomposition, and serum liquefaction are
negative for T-7545. On the other hand, for TH82, starch hydrolysis and
milk peptonization tests are positive, whereas cellulose decomposition and
serum liquefaction are negative. Furthermore, nitrate reduction for TH82 is
positive in Czapek’s solution and negative in peptone solution. Gelatin is
slowly liquefied for two strains. Products of T-7545 are validamycins A.
However, products of TH82 are validamycins and other antibiotics.
For T-7545 and TH82, a variety of carbon sources, such as D-xylose,
L-arabinose, L-galactose, D-glucose, D-fructose, L-rhamnose, melibiose, malt-
ose, sucrose, lactose, raffinose, inositol, D-mannitol, mannose, and glycerol,
 Production of Validamycins Chapter | 2 11

TABLE 2.1 Cultural Characteristics of S. hygroscopicus var. limoneus

T-7545 and var. jinggangensis Yen. TH82

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
Czapek’s agar Growth (G): Moderate, R: Yellowish Brown to
colorless, folded Maroon
Reverse (R): Raw Sienna AM: Pink White to Jasmine
(Rda.a, III 17-i) to Sudan Buff to Mouse Grey with
Brown (Rdg., III 15-k) black moist areas
Aerial mycelium (AM): SP: Faint Brownish tinge
Tilleul-Buff (Rdg., XL
17’’’-f) to Light Buff
(Rdg., XV 17’-f), partially
Mouse Grey (Rdg.,
LI 15’’’’) along the
periphery of the colony
Soluble pigment (SP):
Yellow with a faint
Brownish tinge
Glucose Czapek’s agar G: Moderate, colorless to G: Banana-Buff to Maroon
Sulphin Yellow (Rdg., IV
21-i), folded
R: Raw Sienna AM: Faint Yellow to Dust
Grey
AM: Tilleul-Buff to SP: Faint Brownish tinge
Massicot Yellow (Rdg.,
XVI 21’-f), partially Light
Olive-Grey (Rdg., LI
23’’’’’-d) along the
periphery of the colony
SP: Yellow with a faint
Brownish tinge
Glycerol Czapek’s agar G: Moderate, colorless to G: Sulphin Yellow to
Orange-Citrine (Rdg., IV Brown
19-k), folded
R: Raw Sienna AM: Faint Grey to Almond
Yellow to Mouse Grey
AM: Tilleul-Buff to SP: Faint Brownish tinge
Massicot Yellow,
partially Light Olive-grey
SP: Yellow with a faint
brownish tinge
(Continued )
12 Validamycin and Its Derivatives

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
Glucose asparagine agar G: Colorless G: Grey Green to Brown
R: Old Gold (Rdg., XVI AM: Mouse Grey with
19’-i) to Antimony yellow areas to black
Yellow (Rdg., XV 17’-b) moist areas
to Cinnamon-Brown
(Rdg., XV15’-k)
AM: Light Olive-Grey to SP: Faint Yellow
Mouse Grey, with yellow
patches and black moist
areas
SP: Light brown
Calcium malate agar G: Primuline Yellow G: Colorless
(Rdg., XVI 19’)
R: Primuline Yellow. AM: Faint Yellow
AM: Sparse at first, but
later Tilleul-Buff to Light
Olive-Grey
SP: Pale Yellow SP: Faint Yellow
b
Starch agar G: Colorless to Barium G: Pheasant Brown to Tea
Yellow (Rdg., XVI 23’-d) Brown
R: Deep Colonial Buff AM: Grey White to Mouse
(Rdg., XXX 21’’-b) to Grey with black moist
Snuff Brown (Rdg., XXIX areas
15’’-k)
AM: Cartridge Buff (Rdg., SP: Faint Yellow
XXX 19’’-f) to Mouse
Grey, with black moist
areas
SP: Light Brown.
Hydrolysis of starch was
observed
Gelatin (25 C) G: Very poor

AM: None
SP: None. Liquefaction,
slow
Nutrient gelatin (25 C) Same results as with

gelatin
(Continued )
 Production of Validamycins Chapter | 2 13

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
Whole egg (37 C) G: Colorless

AM: None
SP: None
Tyrosine agar G: Colorless to Strontium R: Bamboo Shoot Brown
Yellow (Rdg., XVI 23’) to Maroon
AM: Ic43’ to Id64’
R: Pale Ochraceous-Buff SP: Faint Brown
(Rdg., XV 15’-f) to Light
Ochraceous-Buff (Rdg.,
15’-d)
AM: None
SP: None
Yeast extract agar G: Colorless, folded R: Colorless
R: Cream Color (Rdg., AM: Grey White
XVI 19’-f)
AM: White SP: Colorless
SP: Light brown

Nutrient agar (37 C) G: Colorless R: Colorless
R: Colorless AM: None
AM: None SP: Colorless
SP: None
Glucose nutrient agar G: Colorless wrinkled R: Pheasant Brown
(37 C)
R: Cartridge Buff to Pale AM: Ic 11’ to Id 42’ with
Ochraceous-Buff wrinkles
AM: None SP: Pale brown
SP: None
Peptone glucose agar G: Chartreuse Yellow R: Yellowish Brown to
Brown
R: Honey Yellow (Rdg., AM: Grey White to
XXX 19’’) Jasmine Yellow
AM: Thin, Cream Buff SP: Pale brown
SP: Yellow with a
Brownish tinge
(Continued )
14 Validamycin and Its Derivatives

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
Nutrient broth (37 C) G: Surface growth G: Surface growth
colorless and colorless colorless and colorless
flocculent growth at flocculent growth at
bottom of tubes bottom of tubes
AM: None AM: None
SP: None SP: None
Glucose nutrient broth G: Surface growth

Cartridge Buff, and
colorless flocculent
growth at bottom of
tubes
AM: None
SP: None
Potato plug G: Colorless to Pale G: Colorless to White
Ochraceous-Buff
AM: Tilleul-Buff to AM: Grey White to Green
Mouse Grey. Color of Grey to Pale Brown
the plug turned to Sayal
SP: Faint Yellow
Brown (Rdg., XXIX 15’’-i)
Carrot plug G: Colorless R: Colorless
AM: White to Mouse AM: Grey White to Mouse
Grey. Color of the plug Grey to Faint Brown
turned to Cinnamon-
SP: Pale Yellow
Rufous (Rdg., XIV 11’-i)
to Cinnamon-Brown
Cellulose G: Poor growth, R: Colorless
Chartreuse Yellow (Rdg.,
AM: Mouse Grey and
XXXI 25’’-d) to Reed
grown poorly
Yellow (Rdg., XXX
23’’-d)
AM: Mouse Grey SP: Pale Yellow

Litmus milk (37 C) G: Surface growth Cream

Color to Seashell Pink
(Rdg., XIV 11’-f)
AM: None
SP: Army Brown (Rdg.,
XL 13’’’-i). Peptonization
(Continued )
 Production of Validamycins Chapter | 2 15

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
with or without weak
coagulation. Reaction of
the medium, weakly
acidic
Löeffler’s medium (37 C) G: Naples Yellow (Rdg.,

XVI 19’-d) becoming
Light Buff
AM: None
SP: None. No
liquefaction
a
Rdg.: Ridgway.
b
Soluble starch 1%, potassium monohydrogen phosphate 0.3%, calcium carbonate 0.3%,
magnesium sulfate 0.1%, ammonium sulfate 0.2%, sodium chloride 0.05%, agar 2%.

TABLE 2.2 Physiological Properties of Strains T-7545 and TH82

Properties

T-7545 TH82

Temperature and Growth occurs at 15
45 C, Growth occurs at 15
45.
pH rangesa better growth at 37
45 C, no Growth occurs at pH 5
10,
growth at 10 C and 50 C. better growth at pH 6
7
Growth occurs at pH 5
10,
no or poor growth at pH 4,
optimum range pH 6
7
Gelatin Slow liquefaction Slow liquefaction
Starch Hydrolysis. Diameter of Hydrolysis
hydrolyzed area/diameter of
colony 5 33 mm/8 mm
Tyrosinase Negative Nb
reaction
Litmus milk Peptonization. Coagulation, Peptonization. Coagulation,
doubtful. Reaction, weakly doubtful. Reaction, weakly
acidic acidic
Reduction of Negative (in peptone solution Positive in Czapek’s
nitrate to nitrite and Czapek’s solution) solution. Negative in
peptone solution
(Continued )
16 Validamycin and Its Derivatives

TABLE 2.2 (Continued)

Properties

T-7545 TH82
Cellulose Negative Negative
decomposition
Chromogenicity Negative Negative
Liquefaction of Negative N
serum
Products Validamycins Validamycins and other
antibiotics
a
On glucose asparagine agar.
b
Not tested.

are well utilized for growth. In view of the relatively high optimum tempera-
ture for growth of T-7545 and TH82, the cultural characteristics at 45 C
were observed and compared with those at 28 C. The characteristics at the
higher temperature were found to be almost the same as those at 28 C.
A few properties, such as sparse formation of white aerial mycelium on
Czapek’s agar, Czapek’s glucose agar, Czapek’s glycerol agar, and filter
paper, and a slightly deeper color of the vegetative mycelium on calcium
malate agar, were noted (Table 2.3).
The characteristics of T-7545 and TH82 are summarized as follows: good
growth and abundant aerial mycelia and coiled chains of spores form at
25
45 C; on a variety of media, grey to grey and yellow aerial mycelium,
bright yellow to ocher vegetative mycelia and faint, brownish yellow diffus-
ible pigment form; on certain media, black moist spots form in the aerial
mycelium. Dark brown diffusible pigment is not produced on proteinaceous
media, i.e., the strain is nonchromogenic.

2.2.2 Complete Genomes for the Two Microbes

Recently, complete genomes of the two strains, S. hygroscopicus var. jing-
gangensis 5008 (Wu et al., 2012) and var. limoneus KCTC 1717 (Leea et al.,
2016), were sequenced.

2.2.2.1 General Features of the S. hygroscopicus

var. jinggangensis 5008 Genome
Except for a linear chromosome, the strain 5008 also harbors a linear plas-
mid pSHJG1 and a 73,282-bp large circular plasmid. With a total length of
10,383,684 bp, the genome of strain 5008 is larger than most published
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Title: Married or single?, Vol. 1 (of 3)

Author: B. M. Croker

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Language: English

Original publication: London: Chatto & Windus, 1895

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*** START OF THE PROJECT GUTENBERG EBOOK MARRIED

OR SINGLE?, VOL. 1 (OF 3) ***
 MARRIED OR SINGLE?
BY
B. M. CROKER
AUTHOR OF “DIANA BARRINGTON,” “A FAMILY LIKENESS,” ETC.

IN THREE VOLUMES
VOL. I.

LONDON
CHATTO & WINDUS, PICCADILLY
1895
 CONTENTS OF VOL. I.
CHAPTER PAGE
I. The Pupil-teacher 1
II. No News 23
III. The Breaking-up Dance 41
IV. The Last Train 67
V. Expelled 89
VI. “Poverty comes in at the Door” 102
VII. A Telegram for Miss West 117
VIII. Not Married after All 135
IX. Bargaining 157
X. Mrs. Kane becomes Affectionate 167
XI. Change of Air and Scene 190
XII. “She will do!” 205
XIII. Mr. West’s Wishes 224
MARRIED OR SINGLE?
 CHAPTER I.
THE PUPIL-TEACHER.

MRS. AND THE MISSES HARPER.

SELECT ESTABLISHMENT
FOR
YOUNG LADIES.
The above, engraved in bold characters on a highly-polished brass
plate, may be read on the gate of an imposing mansion situated in
the far-spreading suburbs of Riverside, one of the principal
mercantile towns in England. “Harperton” is a solid and secluded
residence, standing in its own grounds (of two acres, one perch). It is
planned to resemble a country house of some pretensions, but the
symmetry of its proportions is spoiled by a long, low building jutting
out at the side, that may be taken for anything from a stable to a
billiard-room, but is, in fact, the scene of Mrs. Harper’s scholastic
labours, erected at her own cost—in other words, the schoolroom.
This apartment is illuminated by six windows, the lower halves of
which are, of course, of muffled glass. The floor is carpeted here and
there, as it were, in squares or plots, and in the midst of each square
there is a desk and a comfortable cushioned chair. These indicate
the localities of the various classes. The schoolroom walls are
covered with maps, book-cases, lists of rules, and practising hours,
and lined with narrow desks and benches. A worn piano, a prim,
white-faced clock, and a high wire fender comprise most of the
furniture—ornamental and otherwise; unless we include the two
young ladies who are sitting at one of the far desks, making the most
of their time whilst the boarders are out for their usual walk. One of
these damsels has mendaciously pleaded ear-ache in order to
escape the hateful daily promenade. The other—that nondescript
character, a pupil-teacher—is fulfilling a part of her duties, and
diligently darning the “little ones’” stockings, whilst her companion,
with both elbows on the desk, and both hands in her ruffled hair,
watches her and talks.
“This must be perfectly awful for you, Maddie dear,” she was
saying. “Don’t you loathe it all, and wish you could run away? I
should, if I were in your shoes.”
“Run away! What nonsense, Flo! Where could I run to, even
supposing such an insane idea had entered my head, which it never
has done? You forget that I have no friends in England; and, after all,
I am not such an object of pity as you seem to imagine,” darning
steadily all the time.
“If you are not, I should like to know who is!” demanded her
schoolfellow, emphatically. “You are one day at the top of the tree,
the head of the first class, the best pupil Herr Kroot ever had, adored
by the Harpies”—here Miss Blewitt alluded to her respected
instructress and daughters—“always exquisitely dressed, with heaps
of pocket-money, sleeping in the best room, allowed a fire in winter,
every extra—claret and coffee—and I don’t know what! After years
and years of this style of thing, and when you are seventeen, and
almost finished, your father suddenly stops supplies, you are not
paid for for three whole terms, and the hateful Harpies make you into
a regular drudge—a pupil-teacher, a nursery governess, a servant!
You sleep in the attic with those odious little Smiths—wash, dress,
and teach them; you go messages to the shops, and even into
Riverside—you, who were never allowed to stir one yard alone; you
mend and darn and teach.”
She paused, not from lack of words, but from want of breath.
“And a very good thing that I can do something to pay for my
living,” remarked the other, with composure. “If I could not sew and
mend and teach, what would become of me, I should be glad to
know? I could scarcely expect the Harpers to go on keeping me at
their own expense; and now I take the fifth class, the little ones’
music, and I save a servant for those Indian children, I work for my
bread—and I am worth it.”
 “I should rather think you were,” rejoined her listener, sarcastically.
“You are worth a hundred a year to them as teacher, besides being
dressmaker and nursery-maid. It makes me wild—I feel quite crazy
—when I see all that they get out of you, early and late, and the
shameful way they treat you! Once upon a time you were ‘darling
Madeline’—their ‘dear, bright-faced girl,’ their ‘model pupil,’ now you
are ‘Madeline West,’ or ‘Miss West,’ and you are ‘slow,’ ‘awkward,’
‘lazy,’ and ‘impertinent.’ Oh dear me! dear me! sometimes I feel as if
I should like to fly at Miss Selina and bite a piece out of her, I am so
savage.”
“I hope to goodness you will restrain your feelings,” said Madeline,
with a smile, as she threaded a long needleful of black wool, and
commenced on a gaping heel. “The Harpers are only human, after
all! It was very hard on them, my father having failed; and all my
music-lessons, and painting, and singing, and German, for two
terms, had to be paid for out of their own pockets. Signor Squaletti
charges half a guinea an hour. Then there were my clothes. I feel hot
all over when I remember the quantity of money I laid out, believing
that it would be all settled, as usual, by father’s cheque at Christmas.
There was that white dress for the breaking-up party——”
“In which you made such an impression on the Wolfertons’ friend,
young Mr. Wynne,” interrupted Florence, with a meaning nudge. “Oh
yes, I remember the white dress!”
“Don’t, Flo! Your elbow is like a knife,” expostulated her friend, with
some discernible increase of colour. “As to Mr. Wynne, what you say
is nonsense, and you know Mrs. Harper forbids us to speak of—of—
such things.”
“I know that Mrs. Harper was most uneasy in her mind when she
saw him dancing four times with you running—yes, dance after
dance—and she came up and introduced him to Julia Flowers’ two
red-haired sisters, and said that gentlemen were so scarce, and her
girls were not out, and all that sort of rubbish; and she sent him down
to supper with old Mrs. Browne, and she sent you to bed because
you looked pale! Oh yes, I saw it all—all. I saw that Mr. Wynne never
danced again, but stood with his back to the wall for the rest of the
evening, looking as cross as two sticks. Very likely he would never
have given you a thought, if you had not been so plainly and openly
banished: absence makes the heart grow fonder! Mrs. Harper put
the idea into his head by making such a stupid fuss—and she has
only herself to thank. He sent you those flowers, he came to our
church, and Miss Selina took it all to herself—the ridiculous old cat!
As if he would look at her! She closed on the flowers: much good
may they do her!”
“Now, Flo, how do you know that they were not for her?” asked her
companion with a smile. “But, don’t let us talk about them. It is an old
story.”
“But I will talk about them,” persisted Flo, angrily. “I’ll talk about
your nice green tailor-made, and your winter coat trimmed with fur,
and your opera cloak, and your white dress—the white dress, which
they took away from you!”
“Well, they had paid for them, you see,” rejoined Madeline quietly.
“I am glad they did take them—I owe them the less.”
“Thank goodness your gloves and boots were too small,”
continued Flo, in a tone of fervent congratulation, “otherwise they
would have gone also. They are rather different from the Harpers’
chaussure, which is of the canal-boat type and size. Now I know
what pedestrians mean when they talk of ‘covering’ miles of ground.”
“Well, my dear excited Flo, they did not make their own feet,” said
the other coolly.
“How philosophical you are becoming! Quite an old head on young
shoulders! Who made their tempers, I should be glad to know?—or
their tongues? Thank goodness, this is my last half! Good-bye to
early rising, lectures, scoldings, resurrection pies, milk and water,
and rice puddings. Good-bye to Harperton—penitentiary and prison.
Good-bye to Harpies, and hurrah for home!”—throwing, as she
spoke, a dictionary up to the ceiling; failing to catch which, it fell
open, face downwards, with a bang.
“That is May’s dictionary, Flo,” remonstrated the other. “You will not
improve its poor back.”
 “If you stay here long, Madeline, you will certainly become just as
preaching and particular as one of the Harpies themselves. You are
tremendously sobered as it is. Who would think, to look at you
darning away so industriously, that this time last year you were the
queen and moving spirit of the school; always getting up charades,
dances, and concerts, and carrying your point on every question,
and figuratively snapping your fingers at the Harpies if they interfered
with your schemes—which, to do them justice, was very seldom! Ah!
my poor Maddie, since then what a change has come o’er the spirit
of your dream! It is terrible. If you had always been a pupil-teacher it
would be another matter, or if you had gone to another school, where
no one knew that you had fallen from your high estate; but here, the
scene of your triumphs, to make the descent to the very foot of the
ladder, is—is frightful. I often wonder how you can bear it so well.”
“I often wonder too,” said Madeline shortly, winking her tears back
with a great effort. “You are not going the best way to work to help
me to endure my lot, Flo, raking up all these things. Bad or good, I
must submit. I have no alternative—nowhere to go, until my father
comes home. The best thing I can do is to be patient, and try and
repay the Harpers for some of the money they have expended on
me.”
“Repay them!” echoed Miss Blewitt, scornfully. “They made a very
good thing out of you for nine years—large profits and quick returns.
Now, although your father has not sent his usual remittance—is not
that the word?—and they have heard that he is in business
difficulties, yet I think they might have given you a little more law—a
longer day. They might have exercised some patience. You have not
heard of your father for more than a year, have you?” she added
bluntly.
“No, not for sixteen months,” answered the pupil-teacher.
“But even if he were dead,” proceeded Flo, with a fine disregard of
her friend’s feelings, and an open defiance of the laws of good
breeding, such as is occasionally to be found in girls of her age, “you
could not honestly pretend to be very much cut up! You have not
seen him since you were a small child. You left Australia when you
were seven years old. He is a stranger to you.”
“A stranger, certainly, in one way; but still he is my father, and I
have a presentiment that we shall meet again, and before long,”
rolling up a pair of stockings as she spoke, and averting her eyes
from her outspoken schoolfellow.
“Pooh! I don’t believe in presentiments. I had a presentiment that
father was going to give me a cart and cob last holidays, and it
ended in smoke. If your father had been in the land of the living,
surely you would have heard. I know I am saying this very baldly and
plainly, but there is no use in beating about the bush—is there? You
must face the position sooner or later.”
“You mean the position of being an orphan?” said Madeline,
tremulously. “But I refuse to accept that until I have not one grain of
hope left. It is easy for you, who have your father and mother and
five brothers at home, to talk in this way. Remember, I have only one
relation in the world, and when I lose him I lose all.”
“Well, all I can say is, that I hope your presentiment will turn out
better than mine! Oh, here are the girls coming back!” she exclaimed
peevishly, as a long file of figures appeared, passing the windows
two and two. “What a bore they are! They seem to have only been
out a quarter of an hour, and here they come marching in, disturbing
our nice comfortable little talk.”
Florence Blewitt, who so successfully practised the art of plain
speaking and trampling on other people’s susceptibilities—people
were welcome to trample on hers, she declared; she had none—was
a short, squarely-built girl of sixteen, with a sharp nose, thick brown
hair, intelligent grey eyes, and a very dark skin—a skin that betrayed
no soupçon of foreign blood, but was, nevertheless, more brown
than white. She was brusque, eccentric, clever, and indolent.
Florence could—if she would—but she so seldom would. She
preferred the ease of an undisturbed seat at the very bottom of the
class to ambitious battlings and feverish strivings for the first place.
She was the spoiled only daughter of a wealthy merchant and
shipowner, and, being deferred to and made much of at home, was
disposed to be both arbitrary and independent at school. Moreover,
she was selfish, which is not a taking trait in a young woman’s
character, and was anything but a popular idol. She would borrow
readily, but hated to lend; and the only thing with which she was
generous was her advice; the sole present she was ever known to
make was her opinion—gratis. Few were honoured by her liking, and
if she had a friend at Harperton, it was the girl who sat beside her,
conscientiously mending a basketful of most hopeless-looking
stockings.
“I wonder what your fate will be, Maddie?” said Flo, staring at her
meditatively, and studying her delicate profile, her pencilled
eyebrows, her shining hair.
“I wonder too,” echoed Madeline, with a profound sigh.
Madeline West had been born in Melbourne, and sent home at the
age of seven to Mrs. Harper’s establishment, where she had
remained for ten years. From a skinny, elf-like, wildly excitable child,
she had grown up into an extremely pretty girl, with what the
drawing-master termed “wonderful colouring.” Her hair, eyebrows,
and lashes were dark, her eyes two shades lighter, but it was in her
complexion and the exquisite modelling of her head and features
that her chief beauty lay. Her head was small, and beautifully set
upon her shoulders; her skin was of creamy fairness, with a faint
shade of carmine in her cheeks—a colour so delicate that it went
and came at a look or word. She was tall, slight, and wonderfully
graceful; full of vivacity, activity, versatility and resource, ready to
throw herself warmly into any scheme for amusement or mischief—
that was to say, twelve months previously. She was by far the most
striking-looking and admired of Mrs. Harper’s forty boarders, and,
notwithstanding this drawback to feminine goodwill, was a great
favourite with pupils, teachers, and servants. Her popularity had
even survived that terrible test of altered circumstances—that dire
fall from the wealthy Australian heiress to the unpaid slavey of the
establishment. She changed, of course, her ringing laugh and her
happy air; her merry repartee and snatches of songs had
disappeared with the pretty frocks and hats and shoes which she
had loved so well. She was developing a staid, grown-up manner,
according to her fellow-pupils; she held back from their advances—
abdicated of her own accord, and her place as queen of the school
was filled, after a decent interregnum, by a rich Cockney, who was
as lavish of her shillings, as she was frugal in the matter of h’s, and
who, according to Flo Blewitt, was “a harmless, good-natured,
vulgar, poor creature.”
It must not be supposed that Madeline West did not keenly feel
her altered position. Many a bitter tear she shed in secret; many a
sleepless hour she lay awake, when all her companions—with only
to-morrow’s lessons on their minds—were slumbering peacefully in
the arms of Morpheus. Every small indignity, every slighting speech
and sharp glance entered as an iron into her soul, but she made no
remonstrance or reply; her swiftly changing colour was the sole
index to her feelings, and what were a school-girl’s—a pauper
school-girl’s—feelings to Mrs. Harper? To tell the truth, Madeline had
never asserted herself even in her days of sunshine. She never
could face an unpleasant situation; she put aside a crisis with a
laugh or a gay word; her sensitive, luxurious nature shrank
instinctively from all unpleasant things. She was a moral coward,
though no one suspected it.
The present clouds on her sky had brought out, in an unexpected
manner, unexpected depths in her character. Madeline, the humble
semi-nursemaid, was an industrious, prudent, self-possessed
person, who laboured gravely, doggedly from morning to night, a
totally different girl to the extravagant, generous, easy-going
Madeline, the butterfly who had fluttered the happy hours away for
nine whole years. She was now at another seminary. Adversity is
said to be an excellent school, and offers a fine test of character.
Anomalous as it sounds, Madeline West had risen to the state of life
into which she had fallen.
 CHAPTER II.
NO NEWS.

Three months had passed, and still no sign or token from Mr.
Robert West. How anxiously his daughter’s eyes followed Miss
Selina’s skinny fingers, as they dealt out the letters every morning
during breakfast time—these letters having previously been
thoroughly turned over, examined, felt, and even smelt, by that lady
and her relatives. It was always the same in answer to Madeline’s
unspoken appeal. “No, nothing for you, Madeline,” or, “No letter yet,
Miss West,” according to the frame of mind in which Miss Selina
found herself. And then Mrs. Harper, who was seated behind an
immense copper tea apparatus, would peer round it, with her keen
little eyes and bobbing grey curls, and shake her head at the pupil-
teacher, in a manner which signified that she did not approve of her
at all! As if poor Madeline was not sick with hope deferred, and wild
with a frenzied desire to get away and never pass another night
under that lady’s roof-tree; only there was one big but, one immense
drawback to her own most eager wishes, she had nowhere else to
go.
The Miss Harpers, who were fully alive to Madeline’s value, were
by no means equally anxious for her departure. She corrected
exercises, ruled copybooks, relieved them of several distasteful
duties, and took the little ones’ music—an agonizing ordeal. She
really did as much as any two paid teachers, and—an ecstatic fact—
for nothing! Moreover, they had the delicious sensation that they
were performing a charitable action all the time, and looked primly
self-conscious and benevolent when their friends exclaimed: “How
good of you, you dear, kind, Christian people, to keep that
unfortunate Australian girl!”
Miss Selina, who was forty, with a complexion like that of a wax
doll who has been left lying in the sun, would sigh softly and murmur
the word “duty,” when perhaps at that very moment the unfortunate
Australian was fulfilling the least agreeable of hers—putting those
fretful, ungovernable, sickly little Anglo-Indians to bed—and to sleep.
They were too young for school routine; spoiled, fractious,
disobedient, and mischievous, they were Madeline’s almost entire
charge. Happy Madeline!
It is winter when we once more enter the schoolroom at Harperton,
a bitterly cold day, and the small fire behind the wire screen does not
half heat that great bare apartment, with its numerous doors and
windows. Those at a distance are “out in the cold” indeed, for a
double file of girls is gathered closely round the fender, talking four at
a time, and making noise enough for a rookery. This is the half-hour
after tea, and exclusively their own; they are indemnifying
themselves for many hours of silence and French—which almost
amounts to the same thing. Their speech is vigorous and unpolished,
for no teacher is present except Madeline—if teacher she can be
called. She is standing at a remote desk, mounting a drawing by the
light of a cheap little hand-lamp. The gas is never turned on in the
schoolroom until half-past six, because the twilight is so delightful (so
economical they meant), quoth the thrifty Miss Harpers.
The coals, which have been angrily stirred up, throw a good blaze,
and reveal the faces and figures of the fire-worshippers assembled
round the screen, especially the face and figure of Isabella Jones,
the present reigning potentate. She has hitched herself up on the
edge of the fire-guard, holding on there by the mantelpiece, and from
this elevated position is dispensing law, wit, snubs, and patronage.
She is very tall and thin, stoops a good deal, and is the proprietor of
a tip-tilted nose, a pair of quick little brown eyes, and millions of
freckles. She is also the proprietor of a quantity of pretty dresses, of
unlimited pocket-money, a vast amount of self-esteem, and the
largest and reddest hands in the room.
Mrs. Harper’s seminary is only intended for the offspring of
wealthy folk. Izzie’s father has made his pile in margarine, and has
desired that his daughter may have the best of everything—every
accomplishment, every extra, just like a duchess. Izzie has,
accordingly, a separate bedroom, and lessons from the most
expensive masters; nevertheless, she is far—oh! very far—from
being like a duchess. Her education was begun too late; she is
naturally dull.
“I say, girls,” she is screaming sociably, “isn’t it grand to think that
in ten days more we shall all be at ’ome?

“‘This day fortnight, where shall I be?

Not in this academee,
Eating scrape and drinking tea.
This day fortnight, where shall I be?’”

She chanted in a sing-song voice, more or less through her nose.

“And there is the breaking-up dance,” put in one of her satellites; “I
don’t want to go home till that is over.”
“Gracious! I should hope not. What fun it will be,” exclaimed Miss
Jones. “I hope there will be lots of men this time. I ’inted as much to
Miss Selina. What is the use of going to the expense of supper, and
us all getting new dresses, just for the day boarders? That’s what I
say.”
“What good, indeed!” put in Flo, sarcastically, as she elbowed her
way to the very middle of the fire. “But pray do not make yourselves
unhappy about the expense of the supper, my dear young friends. It
will not concern us. I heard Mrs. Harper telling mademoiselle that
they did not intend to have the girls in on this occasion, gobbling up
the ices and confectionery, like so many locusts.”
“I did not know that locusts went in for confectionery,” remarked
Isabella, with a sniff of scorn.
“This marvellous discovery in natural history was Mrs. Harper’s,
not mine,” said Flo, with swelling dignity. “However, the meaning is
plain. We are not to sup. We are to ’ave”—mimicking her
schoolfellow—“buns and egg-sandwiches ’anded round in the
schoolroom, whilst the company are carousing downstairs.”
 The “take-off” was entirely lost on Isabella, who was far too much
impressed with the intelligence to be alive to Flo’s impertinence. A
dead silence followed this disagreeable announcement, which was
at length broken by Miss Jones, who, sliding from the top of the
screen in the excitement of the moment, shrilly exclaimed—
“Well, I declare! I won’t stand it! I shall tell Mrs. H. so to her face.
Why, our parents pay for the supper! Locusts, indeed! My father
pays handsomely for extras and everything, breaking-up party and
all; and to be put off with a bun! I think I see myself—I just do!
Why”—warming with her theme—“supper is ’alf the fun! There are
the crackers and mottoes and jokes, and every one taken down by a
gentleman, arm-in-arm. I’ll go to supper for one, and stay up to the
last. I did not get my new pink dress just to dance with girls, and eat
an egg sandwich and go to bed. Rather not. Leave it to me, girls”—
looking round on her companions with an air of friendly
encouragement—“I shall have a word with Miss Selina. We shall all
go to supper, or Isabella Jones will know the reason why.”
“Oh, you dear, good Izzy!” cried two voices simultaneously. And
one continued, “You know you can do anything with Snappy, and if
you ask, it will be all right. But about partners, I am afraid they will be
few and far between; Snappy and Miss Harper keep the best for
themselves and their friends. Anything is good enough for the girls.
Last time I was thankful to dance all night with a little boy in a jacket;
however, it was a shade better than sitting-out.”
“There are the Wolfertons,” observed Flo, “and they generally
bring two or three men. Last year there was Mr. Wynne, who was
tremendously struck with Madeline.” Then raising her voice, “Maddie,
do you remember Mr. Wynne? Come over here, and let us see if you
are blushing.”
“Mr. Wynne, Fred Wolferton’s friend!” cried Isabella, with great
animation. “He is a barrister, and, of course, without a penny to jingle
on a milestone—poor as Job. My father don’t approve of my getting
to know these paupers. You know I’m an heiress”—giggling—“and
father says——”
 “Oh never mind your father!” broke in Flo, rudely. “You need not be
alarmed; Mr. Wynne won’t look at you as long as Madeline is in the
room—and perhaps he may not come. Who else are invited—the
Sangsters, the Wallers, the Rays?”
“All common sort of people,” remarked the grand-daughter of a
baron. “Very worthy in their way, and well enough for a girls’ school
breaking-up; but I should not dream of knowing them at home, or of
bowing if I met them anywhere;” and she threw up her chin, and
looked about her superciliously.
No one combated this dire announcement; they were all a little in
awe of Miss De Ville and her ancestors—especially of the one who
had fought in Palestine—and they were silent and impressed, being
young. At length a word was whispered, which quickly set every
tongue wagging. That magic word was “dress.” What were they all
going to wear? One lacked new shoes, another gloves; a fan was
lent—in prospect—in return for good offices in the hair-dressing line.
Amidst this gabble, Isabella’s piercing voice was heard high and
shrill above all, describing the body of her new pink dress. Madeline
had joined the crowd, looking white and cold—and no wonder.
“Keep away your fingers, my dear, if they are sticky,” said Flo;
“and, by the way, what are you going to adorn yourself in? Your white
dress was taken by the Harpies, as most unsuitable to you now.”
“I have nothing but my black cashmere,” she returned, “and this”—
holding out a shabby serge sleeve.
“They really must give you something!” cried Isabella,
impressively, “if only for the look of the thing. For the credit of the
establishment, they can’t have you appear like an old rag-picker.”
Madeline coloured vividly. “I don’t mind giving you a dress myself,
if you will take it.”
“Now, I call that a French compliment, Isabella Jones,” remarked
Flo, with her usual candour, “and you know it. If Madeline has to
wear the old black, so much the worse; but, whatever she wears,
she will always look a—lady,” accompanying the remark with a
glance at Miss Jones that gave it point and significance, and made