UNIVERSITY OF ILORIN

THE SIXTY-FOURTH INAUGURAL LECTURE

THE SICLE CELL MEMBRANE:

TIP OF THE ICEBERG

BY

PROFESSOR CLEMENT OLATUBOSUN BEWAJI

B.Sc.; M.Phil.; Ph.D. (Ibadan)
Professor of Biochemistry and Head,
Department of Physiology & Biochemistry

Thursday, 27th February, 2003

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This 64th Inaugural Lecture was delivered under the Chairmanship of

The Vice-Chancellor
Professor S. O. O. Amali
B. A. (Ibadan); Ph.D. (Wisconsin)

Published by
Library and Publications Committee
University of Ilorin, Ilorin, Nigeria

February, 2003

Printed at
Unilorin Press
University of Ilorin, Nigeria

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PROFESSOR CLEMENT OLATUBOSUN BEWAJI
B.Sc.; M.Phil.; Ph.D. (Ibadan)
Professor of Biochemistry and Head,
Department of Physiology & Biochemistry

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The Sickle Cell Membrane: Tip of the Iceberg

The Vice-Chancellor, Professor S. O. O. Amali,

Chairman, Committee of Deans, Professor A. O. Soladoye,
Deans of Faculties, Postgraduate School and Student Affairs,
Registrar, and other Principal Officers of the University here present,
Heads of Department and other members of Teaching and Non-Teaching Staff,
Our Invited Guests and Dignitaries,
Distinguished Ladies and Gentlemen of the Press,
Greatest Unilorin Students !!

Introduction

Nature has bestowed on tropical Africa two deadly diseases: Malaria and Sickle
Cell. These two diseases are regarded in research circles as two sides of the same coin. It
is believed in research circles that the sickle cell gene arose as a result of nature’s
compensation for the prevalence of malaria in the tropical sub-region (Fig. 1). This
theory has stood the test of time. But the question which many of us have asked, and will
continue to ask, is this: which is more devastating, malaria or sickle cell?
What we now refer to as sickle cell was first described by Dr. J. B. Herrick in a
paper published in the Archives of Internal Medicine in 1910. Dr. Herrick was, at that
time, a physician in Chicago. Six years earlier (i.e. in 1904) he had examined a twenty-
year old black medical student who had been admitted to the hospital because of cough
and fever. The patient felt dizzy and had severe headache. For about a year he had been
experiencing palpitation and shortness of breath. The patient appeared well developed
physically. However, there was a tinge of yellow in the white of his eyes, and the visible
mucous membranes were pale. His heart was distinctly enlarged. Examination of the
blood showed that he was markedly anaemic; the red blood cell count was about half the
normal value. Examination of the blood smear revealed the presence of unusual red cells
which were described by Herrick in the following words:

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Fig. 1: Malaria/Sickle-cell endemic areas of the world.

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 The shape of the red cells was very irregular, but what especially attracted attention was
the large number of thin, elongated, sickle-shaped and crescent-shaped forms.

Dr. Herrick prescribed rest and nourishing food. After four weeks in the hospital
the patient felt much better and was less anaemic. However, his blood still exhibited “a
tendency to the peculiar crescent-shape in the red blood cells though this was by no
means as noticeable as before.”
Dr. Herrick was puzzled by the clinical picture and laboratory findings. He waited
six years before publishing the case report and, even then, he asserted that “not even a
definite diagnosis can be made.” He highlighted the chronic nature of the disease and the
diversity of abnormal physical and laboratory findings: cardiac enlargement, a
generalised swelling of lymph nodes, jaundice, anaemia and evidence of kidney damage.
He concluded that the disease could not be explained on the basis of an organic lesion in
any particular organ. He published his report in a paper titled: “Peculiar elongated sickle-
shaped red blood corpuscles in a case of severe anaemia.”

My adventure into membrane studies

When I started my research activities in Professor Bababunmi’s laboratory at the

Department of Biochemistry, University of Ibadan in 1980, I was fortunate that the
research focus in that laboratory at the time was on the red cell membrane, particularly
the cation pumping enzymes embedded in that membrane. The membrane is generally
regarded as the Customs and Excise Department of the living cell. It regulates what
comes into, and what goes out of, the cell. In a way, it was fortuitous that the research
focus of this laboratory led me to take a closer look at the membrane which houses the
sickle cell haemoglobin. What I eventually found out was that the membrane was just the
tip of the iceberg.
There is an analogy which can be drawn between an integral protein floating in
the lipid bilayer of a biological membrane and the iceberg floating on an ocean (Figs. 2 &
3).

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Fig. 2: A schematic three-dimensional and cross-sectional representation of the fluid
mosaic model of biological membranes, showing peripheral and integral membrane
proteins. [Singer and Nicholson, 1972].

Fig. 3: The lipid-globular protein mosaic model of biological membranes. The protruding
proteins have on their surfaces ionic residues while the non-polar residues are largely
embedded in the lipid layer. [Singer and Nicholson, 1972].

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Those who have watched the tragic film “The Titanic” will remember that the great ship
was sunk when it collided with an iceberg. The Titanic disaster was one of the worst
maritime disasters in history. The British luxury liner weighing 46,000 gross tons was on
its maiden voyage from Southampton to New York when it struck an iceberg about 153
km south of the grand banks of Newfoundland just before midnight on April 14, 1912.
The ship had been proclaimed unsinkable because of its watertight compartments, but the
iceberg punctured five of them and the Titanic sank in less than three hours.

The nature and prevalence of sickle cell disease

It is now well established that sickle-cell disease is a genetically transmitted

disorder which affects several million black people in tropical Africa, the Middle East,
the West Indies and the United States of America. The disease is inherited as an
autosomal recessive trait and is associated with the presence, in the red blood cell, of
sickle-cell haemoglobin (HbSS), one amino acid residue of which is different from
normal adult haemoglobin (HbAA). This difference arose as a result of a mutation which
caused the substitution of valine for glutamic acid at the 6th position of the beta-globin
chain of the haemoglobin molecule (Fig. 4).
Patients who are homozygous for the gene which produces the abnormal
haemoglobin suffer from sickle-cell anaemia while those who receive the abnormal gene
from one parent but its normal allele from the other parent have the sickle-cell trait and
are usually not symptomatic.
The earliest documented report of sickle-cell disease, as I have said earlier, was
the observation by Herrick (1910) who examined a twenty-year-old black medical
student who had been admitted to the hospital for cough and fever, but was discovered to
also suffer from severe anaemia. It was from this observation that the disease acquired its
original name of sickle-cell anaemia. It was subsequently discovered that patients suffer
not only from anaemia but from a group of ailments, hence the more appropriate term,
sickle-cell disease or sickle-cell disorder, is now preferred.

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Fig. 4: Mutation and amino acid substitution in the β-chain of human haemoglobin.

The recognition by Pauling et al. (1949) that sickle-cell anaemia is a molecular

disease was a major landmark in medicine. This was quickly followed by the suggestion
by Haldane (1949) that the genetic polymorphism observed in man might have been
established largely as a result of adaptive changes to some disease conditions. This
suggestion was supported by the demonstration by Allison (1954) that the heterozygous
state of the sickle-cell gene confers resistance to malaria caused by Plasmodium
falciparum. Subsequently, several other erythrocyte traits such as C and E
haemoglobinopathies, the thalassaemias and primaquine sensitivity (resulting from
glucose-6-phosphate dehydrogenase, [G-6-PD] deficiency) have been associated with
resistance to falciparum malaria (Motulsky, 1960).
The haemoglobin molecule has become one of the best known proteins as a result
of the pioneering studies of the Italian Biochemist, Max Perutz. Although it is not an
enzyme, the haemoglobin molecule has provided the basis of our understanding of
enzyme mechanisms and regulation. It is composed of four polypeptide chains: two

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alpha chains, each with 141 amino acid residues, and two other chains designated beta,
gamma or delta, each with 146 residues. The delta chains differ from the beta chain in 10
amino acid residues while the gamma chains differ from the beta chains in 39 residues.
Each chain surrounds an iron porphyrin (heme) molecule which contains a central
iron atom to which oxygen is reversibly bound. The ability of the heme group to
transport oxygen is conferred on it by the presence of the globin chains attached to the
molecule.
It has been proposed that the high incidence of the sickle-cell gene in Africa is
due to the protection conferred against malaria. In some parts of Africa the gene
frequency could be as high as 40%. This means that Nigeria, with a population of one
hundred million, could have as many as forty million people carrying the sickle-cell gene,
either in the homozygous or heterozygous state (i.e. HbAS or HbSS). In this part of the
world, malaria is endemic and it has been found that those carrying the sickle-cell trait
have a much better chance of surviving to adulthood than those with the normal gene
who may die of malaria before reaching maturity. Deaths from malaria among children
in certain parts of Africa could be as high as 50%.

Sickle Cell and Malaria

The mechanism by which sickle cell trait protects against malaria infection has
not been completely resolved. However, it has been shown (Friedman, 1978) that the
high intracellular concentration of K+ is crucial to the survival of the malaria parasite.
The K+ loss observed during the sickling of erythrocytes containing HbSS could,
therefore, provide an environment which is not conducive to the growth of the parasite
(Figs. 5 & 6). For this experiment, a strain of Plasmodium falciparum was used. Red
cells containing HbAA, HbAS or HbSS were artificially infected with the parasite. It was
found that only when the oxygen level was lowered sufficiently to cause sickling was the
parasite affected. Further tests showed that it was the sickling, not only the depletion of
the oxygen in the environment, which caused the malaria parasite to die.
It was further shown that when red cells containing HbAS or HbSS are made to
sickle, they begin to leak potassium. Red cells containing HbAS were then incubated in a
medium containing 3 percent oxygen but an elevated intracellular potassium content.

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The cells sickled as usual due to the low oxygen tension, but the cellular potassium
remained high and the parasites survived. When the potassium was allowed to leak the
parasites died.
What attracted me to do research on sickle red cells was the report by Eaton et al.
(1973) that the concentration of calcium in sickle cells was eight times that in normal red
cells. These workers also went on to show that loading of normal erythrocytes
(containing HbAA) with Ca2+ using the specific Ca2+-ionophore A23187 induced sickling
in the erythrocytes (Eaton et al., 1978). These observations strongly suggest that elevated
Ca2+ level may play a role in the pathology of sickle cell anaemia.
Most of my colleagues are familiar with my work on adenosine triphosphatases
(ATPases), particularly the calcium-pumping ATPases of erythrocyte membranes. What
they may not know is that my interest in ATPases was motivated by the desire to
understand the nature of the pump responsible for removing calcium from the cytoplasm.
I proposed the hypothesis that this pump may be defective in sickle cells, hence
accumulation of calcium by these cells (Bewaji, 1983). My research work thereafter was
aimed at validating or invalidating the working hypothesis. Unfortunately, the work had
to be done at the molecular level, involving the isolation of membranes from red cells and
purifying the enzyme (Ca2+-ATPase or calcium pump) from these membranes.
Sickle cell patients usually have insufficient blood in their system, hence they
require frequent blood transfusion to keep them going. Therefore, to get them to donate
blood for my research work on sickle cell membranes was like asking for the impossible.
On one occasion, I achieved what seemed impossible. I was able to collect 10 ml of
blood from each of six sickle cell patients at the University College Hospital (UCH),
Ibadan. I set out to perform the technically difficult task of preparing membranes from
the blood collected. In normal circumstances, I would have needed at least 500 ml of
blood to prepare membranes conveniently. Then NEPA struck. That is the expression
for the now familiar power outage which was to last for two weeks during that period.
My blood samples got trapped in the refrigerated centrifuge for 2 weeks, and that was the
end of the experiment.

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Fig. 5: Transmembrane cation gradients induced by the activities of membrane-bound
ATPases.

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Fig. 6: A chemiosmotic model for the mechanism of Ca2+ translocation across the
erythrocyte membrane, showing (A) a Fluid-Mosaic representation of Ca2+ fluxes and,
(B) the balance of charges during the operation of the Ca2+ pump. [From Bababunmi,
Olorunsogo & Bewaji, 1994; act = activator of the pump).

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 This incident made me to switch my attention to a comparative study of the
calcium-pumping ATPases in mammalian red cells (Figs 7 & 8). This was quickly
rewarded with the discovery that the activity of the Ca2+-ATPase in pig erythrocyte
membranes was 8 – 10 times that found in the membranes of other mammalian species
studied, such as goat, sheep, cow and man. This finding was published in Comparative
Biochemistry and Physiology (Bewaji, Olorunsogo and Bababunmi, 1985b) followed by
a review article in the World Review of Nutrition and Dietetics (Bababunmi, Olorunsogo
and Bewaji, 1990). We thought, at that time, that the high activity of the enzyme in pig
erythrocytes was due to the lipid environment which served as an activator. It was not
until I moved into Professor Ernesto Carafoli’s laboratory in Zurich, Switzerland, that I
was able to purify the enzyme in pig erythrocytes and demonstrate that the high activity
was actually due to an increase in the amount of pump protein in pig erythrocytes. This
was reported and published in Biochemical Journal (U.K.) (Bewaji and Bababunmi,
1987a), followed by another publication on the full characterization of the enzyme
(Bewaji and Bababunmi, 1987b).
These publications came at a time when scientists in many laboratories around the
world were trying to determine the amino acid sequence of the human erythrocyte Ca2+-
ATPase but were having difficulties in obtaining sufficient quantities of the enzyme. I
wrote another paper providing indirect evidence for sequence similarities between human
and porcine erythrocyte Ca2+-ATPases and sent it to a Nigerian journal for publication.
The journal sat on it for one year before taking a decision on the manuscript. Science is
moving at a terrific speed; if publication of a manuscript is unduly delayed, its scientific
content stands the risk of deteriorating. It’s much like manufactured food items that must
carry expiry dates. Scientists are also competing with one another in determining the first
person to report an important finding. The information which I tried to pass across to the
scientific community in 1987 was subsequently published in 1991 (Bewaji, 1991a,
1991b).

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Figs. 7: Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of ghost
membranes from (A) goat (B) sheep (C) pig (D) cow and (E) man. [From Bewaji et al.
(1985b)].

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Fig. 8: Desitometric scan of membrane proteins from (A) goat (B) sheep (C) pig (D) cow
and (E) man. [From Bewaji et al. (1985b)].

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 This incident propelled me, along with some colleagues in the Department of
Biochemistry, to look into ways of enhancing the dissemination of scientific information
in Nigeria. Our efforts led to the birth of the Nigerian Society for Experimental Biology
(NISEB) and two journals: Bioscience Research Communications and Biokemistri.
My studies on the properties of the calcium pump also led me to examine the
intracellular Ca2+-ATPases that are found in the endoplasmic reticulum as well as the
sarcoplasmic reticulum. Some chemical inhibitors such as, vanadium, thapsigargin and
cyclopiazonic acid were used as tools in these studies (Bewaji, 1993, 1995a, 1995b,
1996a, 1996b; Bababunmi, Olorunsogo and Bewaji, 1994).

Anti-sickling agents

Despite the fact that the molecular biology of sickle cell disease is well
understood, there are at present no specific drugs that can permanently cure the disease.
Previous attempts to inhibit the sickling of human erythrocytes containing HbSS have
been directed towards inhibition of gelation (polymerisation) through the modification of
the haemoglobin molecule either covalently with chemicals such as sodium cyanate or
non-covalently with the alkylureas. The possibility of inhibiting sickling independent of
Hb gelation has also been considered. The use of zinc and procaine hydrochloride are
examples of this approach. These compounds are thought to exert their effects on the red
cell membrane.
Other anti-sickling agents that have been employed include urea at high
concentrations, carbamyl phosphate, the bifunctional cross-linking reagent dimethyl
adipimidate, cetiedil, bepridil and potassium tellurite. Some of these anti-sickling agents
exhibit varying degrees of toxicity and are therefore unsuitable for clinical use. For
example, it has been shown that cyanate (and isocyanate) generally carbamylate the
amino acid terminals of proteins. When haemoglobin S is carbamylated, it is maintained
in a conformation which is not susceptible to sickling. However, it has been reported that
long term oral cyanate therapy produces neuropathy in patients.
The hope of finding an ideal, non-toxic anti-sickling agent was raised when it was
reported by Rumen (1975) that three amino acids, L-homoserine, L-asparagine and L-

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glutamine decreased the CO-binding affinity of haemoglobin S, while no effect of these
or any other amino acids could be demonstrated with haemoglobin A. This observation
suggested the possibility that these amino acids might inhibit the sickling of erythrocytes
and might be useful in the treatment of sickle cell disease by dietary means. Ekong et al.
(1975) also reported that a butanoic acid derivative, 3,4-dihydro-2,2-dimethyl-2H-1-
benzopyran-6-butanoic acid (DBA) efficiently prevents and reverses sickling with no
acute toxicity in mice.
Mr. Vice-Chancellor, Sir, I used to think that it is only people in medical and legal
professions that are in love with technical jargons. Organic chemists and biochemists
have now caught the infection. Instead of saying enlarged liver, our medical colleagues
would say hepatomegaly. Enlarged spleen is splenomegaly. I remember the story of a
medical student who was suffering from severe headache and had to go to the University
Clinic to see a doctor. On entering the doctor’s consulting room he was asked to describe
how he was feeling. The boy wanted to impress the doctor with the use of the jargons he
had learnt in the medical school. He said, “Sir, a corrosive exigency is spasmodically
emanating from and producing a prolific source of irritability in my cranium”. He was
promptly referred to see a psychiatrist.

The discovery of DBA

Investigations which led to the discovery of DBA arose from studies on the
properties of Fagara xanthoxyloides, a small tree with shining aromatic leaves which is
common in coastal areas of West Africa. The roots, which have a strong spicy taste, are
used in Nigeria as chewing sticks (locally referred to as orin ata) which are chewed to
make a brush and used to clean the teeth. We subsequently showed that DBA activates
the (Ca2+ + Mg2+)-ATPase in the membranes of normal erythrocytes and those of
erythrocytes with sickle-cell haemoglobin (HbSS) (Bewaji, Olorunsogo and Bababunmi,
1985a; Bewaji and Odutuga, 1987; Ogunleye and Bewaji, 1990). This suggests a
possible mechanism for the anti-sickling effect of DBA.

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 My current research interests include investigations on the antisickling properties
of hydroxyurea, thiocyanate and NIPRISAN (an antisickling drug developed at the
Nigerian Institute for Pharmaceutical Research and Development in Abuja).
Mr. Vice-Chancellor, Sir, it is necessary for me to explain here that I am not a
Clinician, so that I don’t wake up tomorrow morning to find a long line of patients in
front of my office. All I have been trying to do is to study the permeability characteristics
of the sickle cell membrane and the mechanism of action of antisickling agents.

The other side of sickle-cell research

Mr. Vice-Chancellor, Sir, let us now take a look at what lies beneath the iceberg,
what do we see? The ultimate solution to the sickle-cell problem lies in gene
replacement therapy (Fig. 9). However, I wish to assert here that we may not be able to
completely solve this problem until we have addressed the problem of malaria: the two
problems are intertwined. That is why I have one leg in sickle cell research and the other
in malaria research, using bioinformatics as a tool. I am pleased to announce that a
breakthrough is about to be made on both fronts as a result of the recent completion of
some genome sequencing projects. The first phase of the Human Genome Project was
completed two years ago (Lander et al., 2001; Venter et al., 2001); the final draft of the
complete human genome (with all the annotations) is expected to be published later this
year. We have already entered the phase of examining Single Nucleotide Polymorphisms
(SNPs), the classical example of which is the sickle cell mutation. These studies will
enable us to better understand the genetic variability in human populations and,
ultimately, the mechanism of gene expression. We already know that in order to account
for the synthesis of different types of haemoglobins, there must be at least four different
genetic loci which code for the different chains α, β, δ, and γ. The α chain locus has
been located on the long arm of chromosome 16, while the β gene is on the long arm of
chromosome 4. The δ and γ genes are thought to be closely linked and both of them are
on chromosome 11 (Deisseroth et al., 1978). We also know that the genetic information
for most (if not all) eukaryotic proteins is stored in the exon segments of their genes; the
introns are spliced off during messenger RNA processing.

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Fig. 9: Proposed scheme for the treatment of genetically transmitted diseases by gene
replacement therapy.

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 The human β-globin gene has 3 exons (coding for amino acids 1 – 30, 31 – 104
and 105 – 146) separated by 2 introns. The whole gene for β-chain of adult haemoglobin
is about 1,500 base pairs long, but the 146 amino acids in the chain would require only
438 base pairs. From the genetic code (Table 1) we know that three bases code for one
amino acid in a protein or polypeptide molecule.

The Politics of Human Cloning

Human cloning became prominent recently. The Italian gynaecologist, Professor

Severino Antinori, informed the world last November that the first human clone would be
born in January 2003. However, it appears another group has beaten him to it: A group
called Clonaid announced, in December 2002, the birth of the world’s first human clone,
a baby girl which they named Eve. The human clone was produced from an egg
fertilized by the skin cell of a 31-year old woman.
Clonaid is the medical wing of the Raelian sect, a religious group founded by
Claude Vorilhon commonly referred to by his nickname Rael. The recent cloning
announcement was made by the Secretary of the group, Brigitte Bosselier, a 46-year old
mother of three who works as a Senior Scientist with a company in France. She also
claimed that three other cloned babies would soon be born, including those from two
couples in Asia who decided to clone their dead children, the third by a lesbian couple in
Europe. The controversy already generated by this announcement will go on for quite a
long time. Unfortunately, Mr. Vice-Chancellor, Sir, the technology that will enable us to
gain a better understanding of genetic manipulation and gene expression is the same that
leads to human cloning. That is why the United States government has allowed the use
of public funds for stem cell research but not for wholesale human cloning.

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Table 1: The Genetic Code*

UUU Phe (F) UCU Ser (S) UAU Tyr (Y) UGU Cys (C)
UUC Phe (F) UCC Ser (S) UAC Tyr (Y) UGC Cys (C)
UUA Leu (L) UCA Ser (S) UAA STOP UGA STOP
UUG Leu (L) UCG Ser (S) UAG STOP UGG Trp (W)
CUU Leu (L) CCU Pro (P) CAU His (H) CGU Arg (R)
CUC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R)
CUA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R)
CUG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R)
AUU Ile (I) ACU Thr (T) AAU Asn (N) AGU Ser (S)
AUC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S)
AUA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R)
AUG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R)
GUU Val (V) GCU Ala (A) GAU Asp (D) GGU Gly (G)
GUC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G)
GUA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G)
GUG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

*Given the position of the bases in a codon, it is possible to find the corresponding amino acid. For example, the codon
AUG on mRNA specifies methinone, whereas CAU specifies histidine. UAA, UAG and UGA are termination signals.
AUG is the initiation codon and it codes for internal methionine as well (Nirenberg, 1968).

In a way, some people regard me as a biochemical prophet because I have been

teaching human cloning (at the theoretical level) in this University for some eighteen
years. We have not started experimenting with human cloning because our hands are tied
legally, morally and financially. Some of my students in the Department of
Biochemistry, Faculty of Science, would remember my first lecture on human cloning in
1985. I emphasized in that lecture that the first human clone was actually born in 1973 –
a fact which was documented in a book titled “In His Image: The Cloning of a Man”
written by David Rorvik and published in 1978 by Hamish Hamilton Publishers. I
moved into the Faculty of Health Sciences in 1990 but continued with my lectures on
Genetic Engineering and manipulations, including cloning. These lectures generated
considerable interest which became heightened with the announcement of the first cloned
sheep named “Dolly” in 1997.

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 Before the birth of recombinant DNA technology in 1972, geneticists and
molecular biologists were contended with the cloning of bacteria and some other
microorganisms as well as cells in tissue culture. Plant breeders also cloned higher plants
by propagating them asexually through simple techniques such as cutting and grafting.
Higher animals do not reproduce asexually. Therefore, in order to clone an
animal, it is necessary to microsurgically remove the nucleus from a fertilized egg or
inactivate it with ultraviolet radiation, then replace it with a nucleus taken from a somatic
cell. The first of such experiments was carried out with frogs in 1968 by Dr. J.B. Gurdon
at Oxford University. Unlike mammalian eggs that are very small and can only be seen
with the aid of a microscope, the eggs of frogs are quite large and easy to manipulate.
Thus, the announcement of the successful cloning of higher mammals made big headlines
around the world. A group of scientists at the Roslin Institute in Edinburgh, headed by
Dr. Ian Wilmut, successfully cloned a sheep which they named “Dolly”. Another group
of scientists at the Oregon Regional Primate Research centre in the United States
simultaneously announced the cloning of two rhesus monkeys. It is generally accepted
by scientists that the monkey is the “next-of-kin” to man in the phylogenetic scale of
biological evolution. The question was then asked: Can man be cloned? If the answer is
yes, then the next question is: “Should man be cloned?” This is actually not a scientific
question; it is an ethical question. As far as scientists are concerned, if it can be done, it
will be done – it’s only a matter of time. There is a strong suspicion in scientific circles
that the human immunodeficiency virus (HIV) which causes AIDS is the result of a
genetic engineering experiment that went wrong.
The procedure for cloning an animal or a human being requires the implantation
of a body cell (or somatic cell) nucleus, containing a full set of 46 chromosomes, in an
ovum, the nucleus of which had been destroyed. The resulting embryo would then be
implanted into the uterus of a woman who will then carry the pregnancy for nine months.
Therefore, animal cloning is an advancement of the technique of in vitro fertilization
(IVF) developed in 1973. Babies born by this procedure are still referred to as “test-tube
babies”. The IVF technology made it possible to remove eggs from the ovaries of
women who were healthy in every sense, except that they were infertile as a result of

23
irreparably damaged fallopian tubes. Their eggs would be fertilized in the test tube with
the sperms of their husbands and then implanted in their own wombs.
As it always the case, every technique developed to solve a medical problem is
also subject to abuse. It soon became normal practice to remove eggs from one woman,
fertilize it in a test-tube and then transplant it into the womb of a second woman who
could not produce her own eggs. Furthermore, women who simply did not want to
undergo the difficulties of carrying a pregnancy or going through labour pains began to
hire other women to carry the pregnancy for them – at a reasonable fee. This practice is
now common place in the United States where surrogate mothers provide wombs for rent
at a fee of $150,000 only, for a period of nine months. It is much cheaper in India where
you would only require the equivalent of £20,000 (sterling) to rent a womb for nine
months. There already speculations about by-passing surrogate mothers by developing
artificial wombs (incubators) in which the growing embryo could be developed and the
baby collected after nine months! (Ron-El et al., 2000; Rojansky, 2000).
The development of the IVF technique was not without some unpleasant
incidents. This technique is credited to Dr. L.B. Shettles, a Professor at the Columbia-
Presbyterian Medical centre in New York. He set out to help a Florida couple (who had
been childless for years) to produce babies of their own. In collaboration with Dr.
Williams Sweetney, a Clinical Professor of Obstetrics and Gynaecology at Cornell
University, he was able to achieve a test-tube conception and the resulting embryo was
incubating in his laboratory, waiting to be implanted into the uterus of the woman whose
infertility was caused by damaged fallopian tubes (Shettles, 1970). Then disaster struck.
His superior at Columbia, Dr. Raymond Vande Wiede, abruptly terminated the
experiment by violating the sterile conditions under which the embryo was growing,
accused Dr. Shettles of breaking the law by attempting to create a monster and sent him
packing from the hospital. The Florida couple promptly filed a $1.5 million lawsuit
against Dr. Vande Wiede, claiming that the termination of the experiment without their
consent denied them the only chance open to them to have a baby (Rorvik, 1978).
The take-off of the IVF technology was thus delayed by about a year. In 1974,
Dr. Douglas Bevis of Leeds University in England, who also did his experiments
surreptitiously, announced in a press conference that three human embryos, conceived in

24
test-tubes, had been successfully implanted in the wombs of three women who later gave
birth to healthy babies. All the three women were regarded as hopelessly infertile due to
damaged or absent fallopian tubes.

Sickle-Cell, Malaria and Evolution

Mr. Vice-Chancellor, Sir, what we are now witnessing is the attempt by man to
directly influence the course of evolution rather than leave it to chance or natural
selection. Some scientists have already coined the term “participatory evolution” to
describe this human intervention. The controversies surrounding recent reports on animal
and human cloning are very much like the test-tube babies controversies we witnessed
some thirty years ago. Most of the cloning experiments are being done surreptitiously
and they will only be made public when they are successful. We may soon be witnessing
legal tussles in the law courts over the rights of the babies or their parent or over some
ethical issues. My prediction is that just like the furor of protests over test-tube babies
faded out, the current indignation over whether or not to clone will also subside and the
technique will become perfected as respectable scientists move into this hitherto
controversial area. The decision to clone will then be no more controversial than the
decision to terminate an unwanted pregnancy.
A hundred and fifty years ago, the British Naturalist, Charles Darwin proposed
what is now known as the “Theory of Evolution by Means of Natural Selection”. This
theory simply states that all living things on earth have developed as a result of descent,
with modifications, from a common ancestor. It implies that all species have not been
placed on earth in their present form, but have, through a process of gradual change,
evolved from pre-existing, different species (Darwin, 1859).
Darwin’s theory immediately evoked intensive, social, scientific and ideological
controversies, as well as personal attacks. He was (and probably still is) more famous
and controversial than any of the founding fathers of biology. However, it is unfortunate
that his followers misrepresented his views more than his adversaries.
At first even Darwin was reluctant to suggest the possibility of such a complex
creation as man without a Creator. He sat on his theory of evolution for 20 years before

25
he finally agreed to publish it in 1859. This theory led him to an inevitable conclusion.
Man was not a unique creation; he was rather the most capable, most efficient and most
intelligent creature to have evolved through the process of natural selection. The human
race was the magnificent end product of millions of years of evolution in a world in
which only the fittest survived, as Darwin explained it.
This assertion has put people like me on a spot. I find myself caught between the
devil and the deep blue sea. I believe in the Creator of the physical universe and all life
in it, including man. I am also a scientist, a biochemist in particular, so I am compelled
to examine facts and figures and draw conclusions. In a way, this could be similar to the
situation in which Galileo must have found himself when confronted with the scientific
facts that the earth was not the centre of the universe but rather a planet revolving around
the sun. This was contrary to the widely held belief in religious circles in his days.
Darwin’s theory was first announced in 1858 in a paper jointly presented with
Alfred Russel Wallace, a young naturalist who had come independently to the theory of
natural selection. The complete theory was published in 1859, in a book titled On the
Origin of Species. This book, often referred to as the “book that shook the world”, sold
out on the first day of publication and subsequently went through six editions.
The question of the origin of life (and species) has plagued scientists for several
centuries. The authoritative answer to this question may eventually come from the
various genomic projects embarked upon by scientists around the world. We now have
empirical data to answer some, if not most, of the questions raised by the speculative
ideas put forward by Darwin about a hundred and fifty years ago. The question scientists
like me may have to resolve is this: Is evolution the mechanism by which the Creator
sustains the biological universe, just as he sustains the physical universe by gravity?

Bioinformatics: An Emerging Academic Discipline

Large-scale gene sequencing became popular following the development of

techniques for sequencing DNA. Many laboratories around the world are now engaged
in projects aimed at sequencing the entire genomes of various organisms, the most widely
publicized is the Human Genome Project (HGP) which began in 1990. Prior to this, in

26
the mid-1980s, the UNDP/World Bank/WHO-TDR Malaria Sequence Project was
initiated. By 1990, several genes from various Plasmodium species had been sequenced.
The project of building up a database for all sequenced genes and proteins in malaria
parasites was coordinated by Professor Ross Coppel at the Walter and Eliza Hall Institute
of Medical Research, Australia and the information contained in the database was freely
distributed on floppy discs to interested scientists around the world.
In the past few years there has been a dramatic increase in the number of
sequencing projects as a result of the interest shown in these projects by public funding
agencies and the pharmaceutical industry. The vast amount of data being generated by
various genome sequencing projects and functional genomic projects with the aid of
microarrays and proteomics simply cannot be assimilated with conventional methods.
The use of new computer-assisted methods has resulted in the birth of a new discipline
known as computational molecular biology or bioinformatics. It has become a powerful
tool to collect, store and analyze vast amounts of data which, in turn, will lead to new
discoveries in the area of diagnosis and treatment of disease. A method based on direct
sequence-to-structure-to-function paradigm was also introduced recently.
Bioinformatics is still primarily concerned with access to and analysis of the
databases of published gene and protein sequences. Increasingly, this is being augmented
by similar methods applied to databases of macromolecular structures and genetic maps.
At present, over one billion DNA and protein sequences have been determined and
deposited in computerized databases. These sequences contain a wealth of information
hidden within them, including protein structure, disease mechanisms and drug target
sites. The challenges facing researchers in biomedical and pharmaceutical disciplines is
how to extract biologically useful information from millions of sequences. This is what
bioinformatics is suppose to address, using a multidisciplinary approach which combines
computer science, information theory and molecular biology.
The Plasmodium falciparum genome sequencing project was officially launched
in 1996. An international consortium comprising of three sequencing centres was
established. The sequencing centres include: The Institute for Genomic Research
(TIGR) in Rockville, Maryland, USA; the Sanger Centre at Hinxton, near Cambridge,
U.K. and the Stanford University, California, USA. It was envisaged that the consortium

27
would completely sequence the genome and annotate it by 2003. There are 14
chromosomes in P. falciparum and these were divided among the three participating
groups when sequencing began.
The Human Genome Project was initiated in 1990 as an international cooperative
research effort with the aim of gaining a basic understanding of the entire genetic
blueprint of a human being. This genetic information is found in each cell of the body,
encoded in the chemical deoxyribonucleic acid (DNA). A genome is the complete
collection of an organism’s genetic material. The human genome is composed of some
40,000 genes located on 23 pairs of chromosomes. A single human chromosome may
contain more than 250 million DNA base pairs, and it has been estimated that the entire
human genome consists of about 3 billion base pairs (Albert and Klug, 2000; Hadden, S.
2000).
The project was intended to identify all the genes in the nucleus of a human cell;
to establish, by a process known as mapping, where those genes are located on the
chromosomes in the nucleus, and to determine, by a process known as sequencing, the
genetic information encoded by the order or arrangement of the DNA molecules. The
ultimate goal of genomic mapping and sequencing is to associate specific human traits
and inherited diseases with particular genes at precise locations on the chromosomes. It
is expected that the outcome of the project will be an unparalleled understanding of the
fundamental organization of human genes and chromosomes. This will have a great
impact on both therapeutic and preventive medicine by providing a better understanding
of disease mechanisms. This is based on the fact that all diseases have a genetic
component, whether inherited or resulting from the body’s responses to environmental
stresses such as parasitic and viral infections or exposure to toxins. For example, the
genes associated with diseases such as sickle cell anaemia, cystic fibrosis, muscular
dystrophy and Huntington’s disease have already been identified. Further research will
also help us to understand why some people are more susceptible to diabetes, heart
disease, stroke or cancer.
Mr. Vice-Chancellor, Sir, the pace of scientific activities on the African continent
is still very slow. This is because the pursuit of science is inextricably tied to the
economic well-being of the various countries within the continent. Research is heavily

28
dependent on equipment and reagents that must be imported from countries outside
Africa, necessitating the use of hard currency which is often difficult to procure. The
cost of an ultracentrifuge, for example, is about $30,000 or N4 million. Add to this cost
of an electron microscope, HPLC (high performance liquid chromatography) equipment,
mass spectrophotometer, then you would understand why most Universities and Research
Institutes in Africa cannot compete favourably in research with their counterparts in
Europe, North America or Japan.
Various governments are also faced with the problems of setting priorities in the
face of competing demands for scarce resources. The result is that research may be rated
very low when put side by side with the provision of food, shelter, adequate healthcare,
good roads, etc.
The advent of Bioinformatics has suddenly brought research at the cutting edge to
a level playing field. Scientists in the developed countries no longer have a competitive
advantage over those in the developing countries. The first draft of the complete human
genome was published in February 2001, and the publication of the final draft (with all
the annotations) is expected later this year (Rogers et al., 2001). The first draft of the
Plasmodium falciparum genome was also published in October 2002 (Gardner et al.,
2002 a, b); other genomic projects are in various stages of completion. With the
completion of the human genome project, experimental biology, as we know it, may be
getting near the end of its rope. It is gradually giving way to theoretical biology,
biocomputing and bioinformatics. Biomedical research in the 21st century will be carried
out mainly in silico (i.e. with computer chips or silicon chips) and in the laboratories of
the mind. The new tools will be ‘state-of-the-art’ computers and the information
superhighway (the Internet) where you can access various genomic and crystallographic
databases. That may be the lead African Scientists have been waiting for to remove the
handicap of depending on sophisticated equipment which are usually unaffordable. You
will then be able to move your entire ‘laboratory’ into a small room equipped with a
desktop or laptop linked to the Internet!
I am one of the early converts to the new field of Bioinformatics because it is less
capital and equipment intensive than the conventional research on molecular biology or
biotechnology. My fascination with Bioinformatics started during my Sabbatical leave in

29
the United Kingdom in 1992. Before then my research cycle was based on spending six
years in Nigeria, one year abroad mainly because the equipment that will enable me do
world-class research are not available here. Suddenly bioinformatics made it possible for
me to continue to do high level research in my laboratory in Nigeria without travelling
anywhere (Bewaji, 1999; Bewaji, 2000 a,b,c,d; Bewaji and Bababunmi, 2003).
That is why I have been persuading the University of Ilorin authorities to take the lead
in this new field by ensuring that the Bioinformatics Centre that will serve the West
African sub-region is established in this University. I have already made some moves in
this direction by getting the National Biotecnhology Development Agency (NABDA),
Abuja, and UNESCO involved in a project on Capacity Building in Bioinformatics in
Nigeria.
There is only one Bioinformatics Centre of International standard in Africa at the
moment, i.e. the South African National Bioinformatics Institute, located on the Campus
of the University of Western Cape, South Africa. The University of Ilorin should
immediately set in motion the establishment of the West African Bioinformatics
Research Institute (WABRI). The set up does not require more than what several
cybercafes in Ilorin already have. The only difference is that the Institute will be engaged
in high level research and not just checking e-mails. That is my only recommendation in
this Inaugural Lecture.

Concluding Remarks

It is now some ninety-three years since the first reported case of sickle-cell
anaemia but we are yet to find a permanent cure for this disease. Some people have
suggested genetic counselling of couples intending to get married to prevent the
continued propagation of the sickle-cell gene, but this has produced very limited success
mainly because love is blind – as they say. In the meantime, sickle-cell disease continues
to have its devastating and debilitating effect on the African society. The miseries that
people who suffer from this disease, or their parents and other relations, go through is
better imagined than described. The search for a permanent solution continues.

30
 A lot has been written on the possibility of curing sickle-cell disease (and other
genetic diseases) by gene replacement therapy. However, the only attempt so far to cure
two patients suffering from beta-thalassaemia through this technique were unsuccessful.
Successful replacement of defective genes may have to wait until we have a deeper
understanding of the regulation of gene expression.
Another way of curing diseases like sickle-cell anaemia and thalassaemia is to
induce the production of foetal haemoglobin (HbF) which do not have beta chains. The
gene which controls the synthesis of gamma-globin in the foetus is normally switched off
after 30 weeks of gestation. The switching off of this gene has been associated with an
increase in DNA methylation. Experiments with baboons have shown that the drug 5-
azacytidine, which inhibits DNA methylation, can induce a transient increase in foetal
haemoglobin. This drug has also been tested in some patients with severe beta-
thalassaemia. The risk here is that 5-azacytidine may also switch on other genes,
including lethal genes such as oncogenes. Nevertheless, it is my belief that we may
gradually be approaching the ultimate solution to sickle-cell disease and other
haemoglobinopathies.
I wish to conclude this lecture with an admonition to scientists on the continent
of Africa to embrace the new and emerging field of Bioinformatics. This will involve
active collaboration between hitherto strange bedfellows: mathematicians, statisticians,
physicists, computer scientists, molecular biologists, information scientists (if there is any
such discipline). This is because bioinformatics will continue to influence the way we
think and the way we do science. Research findings that make big headlines are usually
the results of cooperative efforts that are multidisciplinary in nature. The foundation for
what is now known as genetic engineering was laid in 1953 by the classical finding of
James Watson and Francis Crick. Both of them are still alive. Watson is a biochemist,
but many people may not know that Crick is a physicist! He also proposed the original
Central Dogma of Molecular Biology which states that information flows from DNA to
RNA to protein.
As we move further into the 21st century, students of Biochemistry, genetics or
Molecular Biology will need to know more about the new discipline if they intend to get
employment in the pharmaceutical or biotechnology industries. Acquiring the needed

31
skills in bioinformatics will immediately put you at a competitive advantage in a global
market place which cuts across the Atlantic.
This is a very important point because after the compulsory National Youth
Service Corps (NYSC) year most people are confronted with the problem of find a job. I
always like to tell the story of an Anatomy graduate from a Nigerian university who
looked for a job for two years after graduation but could not find any. She was advised to
set up a private mortuary business which would be first of its kind or become a funeral
“undertaker” but all these did not work. She then decided to travel to the United
Kingdom.
A week after her arrival in the UK she saw an advertisement, in the papers, for the
position of a Museum Curator in a London Zoo and she promptly applied for the job.
Unfortunately, someone had beaten her to it and the position was already filled before she
got there. She was very dejected and it showed on her face. The Zoo Manager was moved
with compassion, so he decided to offer her an alternative job.
“I can see you are very disappointed, but if you don’t mind there is another job I
can offer you. You see, the only chimpanzee we have in this zoo has just died and we
have not been able to find a replacement. It was a crowd puller and money spinner for the
zoo.
“What we have decided to do is to build a costume and find someone who could
act like a chimpanzee to wear it. We figure that this will make up for the loss of revenue
we have suffered since the death of the chimpanzee. This is the only job available if you
don’t mind taking it”.
She agreed to take up the job. Seeing her wearing the costume one would not be
able to distinguish between her and a real chimpanzee. The usual crowd was back in less
than a week after she took the job. The crowd was fond of throwing bananas and peanuts
at her which she gladly accepted and ate as she climbed up and down in the cage. She
was at this for two weeks when an unfortunate incident occurred.
One morning the caretaker who fed the animals in the zoo brought the daily ration
into her cage but forgot to lock the door between her cage and the adjoining cage. She
was jumping about frantically in the cage to the cheer of the crowd when her foot kicked
the door open and found herself landing in the adjacent cage which belonged to a lion!

32
 The lion surged forward and the “chimpanzee” cried, “Please help! I’m dead!!!”
She was surprised when the lion replied, “Relax! I’m also a Nigerian!!”

ACKNOWLEDGEMENTS
I wish to thank the Almighty God who has made today possible. I also wish to
thank the following for their contributions towards my achievements in life:

• My parents who brought me into this world and gave me the education and
moral upbringing that have sustained me up till now.

• My brothers, sisters and other members of the extended family, particularly

my uncle, Pastor Titus Oluwole Agbeniyi who was my guardian throughout
my stay at the Government College, Ibadan.

• I thank my teachers at all levels, particularly, Mr. Victor Olufokunbi who

ensured that I passed the entrance examination into six secondary schools in
1962; my mathematics teachers at the secondary school, Mr. M. A. Badmus,
Professor Olufunso Olorunsogo at the Biomembrane Research Laboratories,
University of Ibadan and Professor A. A. Odutuga, whom we regard as the
father of Biochemistry at the University of Ilorin.

• My appreciation also goes to the best of the teachers, my mentor and

supervisor at the Postgraduate level, Professor Enitan Abisogun Bababunmi,
former Vice-Chancellor, Lagos State University. He literally took me through
a sound biochemical education, the best money can buy. He is also still very
much interested in my career development up till today.

• I thank the University of Ilorin for the Staff Development Award that enabled
me pursue a Ph.D degree and for a conducive atmosphere for my research
activities over the years.

• Members of the tea group in the Department of Biochemistry, University of

Ilorin, notably Professor Musbau Akanji, Professor O.B. Oloyede, Mr.
Rapheal Kolade and the part-time member, Dr. (Mrs.) A.T. Oladiji. Although
the tea group became moribund when some of us took up political
appointments, I recall with nostalgia that may great ideas emanated from our
discussions over tea, in line with the tradition in many parts of the world.
Most people would not know that the establishment of journals like:
Bioscience Research Communications and Biokemistri, as well as what has
now become the Nigerian Society for Experimental Biology started with
discussions over tea.

33
 • I thank my children who had to bear the brunt of my perennial search for new
knowledge around the world, it is now time for them to receive the baton.

• Last, but not the least, the bone of my bones, the flesh of my flesh, my only
confidant, my twin sister who has also been my wife for almost 25 years. I
would like to invite everyone here to our 25th wedding anniversary in August
this year.

I thank you all for your attention!!

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