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Effect of Parasite Diversity and Age on Antibody Responses

This study was aimed at investigating the effect of parasite diversity and age on the levels of antibody responses to P. falciparum in individuals living in an area of seasonal malaria transmission. Two blood stage antigens (MSP1(19), AMA1) and two liver stage antigens (CSP and celTOS), making a total of four antigens were selected to be used for the study. These antigens were tested in archived plasma samples with ages ranging from 1year to 70 years. The samples were collected close to the end of the rainy season in the months of November and December (wet season), and April the following year at the end of the dry season (dry season).
Of the samples collected in the wet season, 34.1% had parasites whiles those collected in the dry season was 15.5% (table 1). Appawu (2004) reported the seasonality of malaria transmission in a neighbouring district (Kasena Nankana District), being high in the wet season and low in the dry season. The high transmission in the wet season could be due to favourable breeding grounds for mosquitoes as a result of several collections of water when it rains and in turn increase the number of vectors and hence the high number of parasites individuals are exposed to (Ahmed, 1989). This data is in agreement with an earlier finding in northern Ghana by Apawu and his group where they found high parasite carriage, and high multiplicity of infection (MOI) in the wet season compared to low carriage and MOI in the dry season. Parasite carriage here means the proportion of participants with P. falciparum at enrolment. There was however no statistically significant difference in the parasite densities when compared between the seasons (p=0529) table 1. There was no difference in the overall ages between the wet and the dry season. (p=0.937). This was expected as most of the samples collected were from the same individuals in the two seasons.
To explore age related pattern of P. falciparum infection, participants were categorized into three (3) age groups: under 5 years, between 5 and 15 years, and over 15 years. The data shows the 5-15 year olds having higher infected proportion in the two seasons (table 2). Parasitaemia was also determined using a more sensitive method, the polymerase chain reaction (PCR). The PCR data confirmed the microscopy data because the 6-15 year group also had higher proportion of individuals carrying parasites at enrolment. This could be due to the fact that they are the more exposed group with incompletely developed immune systems. In contrast, under-fives who are much younger are likely to receive better care from parents thus preventing them from being infected with the parasites. Bed net usage could be one way of protecting the children. Also because of their younger ages they may not be allowed roam around freely. Adults on the other hand despite having possible similar exposure as 5-15 year olds, seem to have less parasites because of their developed anti-malarial immunity. This partial immunity is gained with age and repeated exposure thus making the younger individuals more susceptible to infection (Pratt-Riccio et al., 2005; Dodoo et al., 2008). Thus the over 15 year group who are older, had the least proportion of infection (table 2).
The study also sought to determine the relationship between IgG levels and age. Figures fig 7 and 8 shows the correlation plots between age and IgG levels in the wet and dry season respectively. There was a significant positive correlation between and IgG levels raised against AMA1-FVO, MSP1, CSP and CelTOS in the wet season. And for the dry season, the significant correlation was between age and IgG levels to AMA1-FVO, MSP1, CSP, and Celtos. The correlation coefficient (r2) and the p values are indicated on each plot. There was however no correlation between age and AMA1-3D7 (r2 0.0142, p 0.0699) for the wet season, and AMA1-FVO (r2=0.0114, p= 0.0594) for the dry season. The significant correlations were however very weak. This indicates that though IgG levels increased with age, the association between the antibody levels and age was not so strong and that the influence of age on the amount of antibodies produce against most of the antigens in the population studied was not that much. However the age dependent increase in IgG response to the antigens tested may be due to the mature immune system in adults, and could also be due to cumulative exposure to infection over time (Nebie 2008). It is however not known what might be the reason for the lack of correlation with age to AMA1-3D7, and AMA1-FVO, in the wet and dry season respectively.
IgG responses to the two AMA1 alleles (3D7, and FVO) was plotted against each other to determine which is more predominant in the study population. There was a strong correlation between the antibody levels of AMA1 alleles of 3D7 and FVO (r2=0.8382, p<0.0001) implying that both strains are circulating in the study site during the wet season. A similar trend was observed in the dry season but with a lower correlation coefficient compared to the wet season (r2= 0.5302, p<0.0001).
Antibody titres against recombinant antigens have often been linked to protection from clinical disease (Polley 2004, Nebie 2008, Dodoo 2008, Dodoo 1999, Cavanagh 2004). Where high anti-malaria antibodies is interpreted to mean protection from clinical disease. The important role of antibodies was demonstrated by the passive transfer of purified IgG from immune donors to individuals with P. falciparum infection, which reduced parasitaemia (Cohen 1961, sabchareon 1991). Apical membrane antigen-1 AMA1 and MSP119 have been associated with reduced risk of clinical malaria (Branch 1998, Osier et al 2008). Anti-CSP antibodies have also been found to be partially protective where in Kenya high anti-CSP, anti-LSA, and anti-TRAP ( pre-erythrocytic ) antibodies were demonstrated to be associated with relative protection from reinfection (John CC 2005, 2008). The most advance malaria vaccine, RTS’S which is a subunit of CSP, has been found to protect to about 35-55% in children 5-17 months for about 8 months (Alonso 2004,) . Also anti-CelTOS antibodies have been shown to inhibit invasion of herpatocytes by sporozoites in mice (Bergmann-Leitner 2010). In this study, antibody responses to the antigens, AMA1-3D7, AMA1-FVO, MSP119, CSP, and CelTOS were determined using indirect ELISA. The optical densities (OD) of the antigens were converted to arbitrary units (AU) where the highest OD for each antigen was awarded an arbitrary unit of 4, to allow for comparison between antigens the antigens tested. Antibodies to AMA1 in individuals living in malaria endemic regions have been reported to be high, (Thomas,1994, chelimo, 2005), and this study thus reports high antibodies to AMA1-3D7, and AMA1-FVO compared to MSP1(19), CSP, with anti-CelTOS antibodies being the least. (Fig: 3) in both wet and dry seasons. The data also shows total IgG responses in the wet season was higher than the in the dry season for all the antigens (p<0.0001) except MSP119 where no statistical differences was found between the two seasons (p=0.85) (Figure 3). The seasonal changes in antibody response could be as a result of higher number of vector (mosquitoes) in the community in the wet season which could in turn increase the exposure of the population to parasites and also introduce new parasite clones/strains in the population and hence the high anti-malaria antibodies found in the wet season. In contrast, the dry season cause a reduction in the number of breeding sites for the vectors reducing their population resulting in minimal exposure and hence the low antibody levels.
In malaria endemic regions, the number of different clones of Plasmodium falciparum parasites infecting a person could be a transmission indicator, an indicator of the hosts immune status, and a useful parameter in evaluating malaria control interventions (Babiker 1999, Arnot 1998, mayengue 2009). It has also been reported that parasite diversity in high malaria transmission areas are high and that individuals could carry multiple genotypes (clones) but the opposite pertains in low endemic areas with most infections being monoclonal (Peyerl-Hoffmann 2001, Bakiker 1997, Haddad 1999). Genotyping was done using block 3 region of the MSP 2 gene (Smythe et al. 1990) because of its high polymorphic nature (Felger et al.,1994 1999; Robert et al., 1996). Also MSP2 was selected to be used for this study because of the high allelic diversity observed in Ghana and other countries bordering Ghana with as many as 154 alleles in Ghana and about 50 genotypes in Côte d’Ivoire. ( Silue 2006, falk 2006). The results from the two seasons using the 3D7 allelic family primers showed high mean multiplicity of infection in the wet season compared to the dry season (1.76, and 1.46, p=0.001) table 3. In both seasons, the number of infections per person ranged from 0 to 4. A similar degree of multiple infections has been reported in other African settings (Ntoumi et al., 1995,Beck 1997, Engelbrecht 1999). The samples used for this study was collected in asymptomatic individuals and the high multiplicity of infection found in the wet season could probably be due to high rate of exposure as a result of favourable breeding conditions compared to the dry season. This study did not find any significant correlation between MOI and age in both seasons (p=0.5768 for wet season, and p=0.4158 for the dry season). The samples were then grouped into two based on whether positive or negative using PCR to detect Plasmodium parasites. No differences were detected in the IgG levels between the antigens tested in both seasons when compared based on the PCR data. This result is similar to that found when microscopy was used except that anti-CelTOS IgG in the parasitaemic group was higher than the non-parasitaemic group when microscopy was used. The disparity in the CelTOS report could be due to the lower sample size analysed using PCR compared to the microscopy.
Apical membrane antigen AMA1 has been found to be a promising blood stage vaccine candidate antigen but this potential has been dampen due to extensive polymorphism (Remaque 2008).

Concepts of Evolution and DNA in Biology

Because the fossil record did not exhibit Darwin’s predicted slow and gradual evolution with transitional forms, some paleontologists sought to find a theory of evolution where, “changes in populations might occur too rapidly to leave many transitional fossils” (see Figure from Gould and Eldredge 1977 .
In 1972, Gould and Eldredge proposed the theory of “punctuated equilibrium” where most evolution takes place in small populations over relatively rapid geological time periods. By reducing the numerical size of the transitional population and the number of years for which it exists, punctuated equilibrium greatly limits the number of organisms bearing transitional characteristics. Since many organisms are not fossilized, this increases the likelihood that transitional forms would not be fossilized. One strength of this theory is that Gould and Eldredge claim it is predicted by population genetics. But what are the implications of punctuated equilibrium?
Under punctuated equilibrium, species usually change little as, “gradual change is not the normal state of a species.” Large populations may experience, “minor adaptive modifications of fluctuating effect through time” but will “rarely transform in toto to something fundamentally new.” This is called “stasis.” But small “peripheral” populations may allow for more change at a quicker rate. Gould argued that most macroevolutionary change takes place in such populations during “speciation” such that there is insufficient time for the transitional forms to be fossilized:
“Speciation, the process of macroevolution, is a process of branching. And this branching €¦ is so rapid in geological translation (thousands of years at most compared with millions for the duration of most fossil species) that its results should generally lie on a bedding plane, not through the thick sedimentary sequence of a long hillslope.”
What is meant by phylogeny? Give an account on phylogeny of humans.
Ans- The context of evolutionary biology is phylogeny, the connections between all groups of organisms as understood by ancestor/descendant relationships. Not only is phylogeny important for understanding paleontology, but paleontology in turn contributes to phylogeny. Many groups of organisms are now extinct, and without their fossils we would not have as clear a picture of how modern life is interrelated. We express the relationships among groups of organisms through diagrams called cladograms, which are like genealogies of species.
Phylogenetics, the science of phylogeny, is one part of the larger field of systematics, which also includes taxonomy. Taxonomy is the science of naming and classifying the diversity of organisms.
In humans- it is used to the transfer of genes.
In general, organisms can inherit genes in two ways: vertical gene transfer and horizontal gene transfer. Vertical gene transfer is the passage of genes from parent to offspring, and horizontal gene transfer or lateral gene transfer occurs when genes jump between unrelated organisms, a common phenomenon in prokaryotes.
Horizontal gene transfer has complicated the determination of phylogenies of organisms, and inconsistencies in phylogeny have been reported among specific groups of organisms depending on the genes used to construct evolutionary trees.
Carl Woese came up with the three-domain theory of life (eubacteria, archaea and eukaryotes) based on his discovery that the genes encoding ribosomal RNA are ancient and distributed over all lineages of life with little or no horizontal gene transfer. Therefore, rRNAs are commonly recommended as molecular clocks for reconstructing phylogenies.
This has been particularly useful for the phylogeny of microorganisms, to which the species concept does not apply and which are too morphologically simple to be classified based on phenotypic traits.
DNA is genetic material. Describe two classical experiments to support this statement.
Ans- Clarification came during the First World War. During the war, hundreds of thousands of servicemen died from pneumonia, a lung infection caused by the baceterium Streptococcus pneumoniae. In the early 1920s, a young British army medical officer named Frederick Griffith began studying Streptococcus pneumoniae in his laboratory in the hopes of developing a vaccine against it. As so often happens in scientific research, Griffith never found what he was looking for (there is still no vaccine for pneumonia), but instead, he made one of the most important discoveries in the field of biology: a phenomenon he called “transformation.”
Dr. Griffith had isolated two strains of S. pneumoniae, one of which was pathogenic (meaning it causes sickness or death, in this case, pneumonia), and one which was innocuous or harmless. The pathogenic strain looked smooth under a microscope due to a protective coat surrounding the bacteria and so he named this strain S, for smooth. The harmless strain of S. pneumoniae lacked the protective coat and appeared rough under a microscope, so he named it R, for rough .
Dr. Griffith observed that if he injected some of the S strain of S. pneumoniae into mice, they would get sick with the symptoms of pneumonia and die, while mice injected with the R strain did not become sick. Next, Griffith noticed that if he applied to the S strain of bacteria, then injected them into mice, the mice would no longer get sick and die. He thus hypothesized that excessive heat kills the bacteria, something that other scientists, including Louis Pasteur, had already shown with other types of bacteria.
However, Dr. Griffith didn’t stop there – he decided to try something: he mixed living R bacteria (which are not pathogenic) with heat-killed S bacteria, then he injected the mixture into mice. Surprisingly, the mice got pneumonia infections and eventually died (Figure 3).
Dr. Griffith examined samples from these sick mice and saw living S bacteria. This meant that either the S bacteria came back to life, an unlikely scenario, or the live R strain was somehow “transformed” into the S strain. Thus, after repeating this experiment many times, Dr. Griffith named this phenomenon “transformation.” This discovery was significant because it showed that organisms can somehow be genetically “re-programmed” into a slightly different version of themselves. One strain of bacteria, in this case the R strain of S. pneumoniae, can be changed into something else, presumably because of the transfer of genetic material from a donor, in this case the heat-killed S strain.
Scientists around the world began repeating this experiment, but in slightly different ways, trying to discover exactly what was happening. It became clear that, when the S bacteria are killed by heat, they break open and many substances are released. Something in this mixture can be absorbed by living bacteria, leading to a genetic transformation. But because the mixture contains protein, RNA, DNA, lipids, and carbohydrates, the question remained – which molecule is the “transforming agent?”
This question was examined in several ways, most famously by three scientists working at The Rockefeller Institute (now Rockefeller University) in New York: Oswald Avery, Colin MacLeod, and Maclyn McCarty. These scientists did almost exactly what Griffith did in his experiments but with the following changes. First, after heat-killing the S strain of bacteria, the mixture was separated into six test tubes. Thus, each of the test tubes would contain the unknown “transforming agent.” A different enzyme was then added to each tube except one – the control – which received nothing. To the other five tubes, one of the following enzymes was added: RNase, an enzyme that destroys RNA; protease, an enzyme that destroys protein; DNase, an enzyme that destroys DNA; lipase, an enzyme that destroys lipids; or a combination of enzymes that break down carbohydrates. The theory behind this experiment was that if the “transforming agent” was, for example, protein – the transforming agent would be destroyed in the test tube containing protease, but not the others. Thus, whatever the transforming agents was, the liquid in one of the tubes would no longer be able to transform the S. pneumonia strains. When they did this, the result was both dramatic and clear. The liquid from the tubes that received RNase, protease, lipase, and the carbohydrate-digesting enzymes was still able to transform the R strain of pneumonia into the S strain. However, the liquid that was treated with DNase completely lost the ability to transform the bacteria .
Thus, it was apparent that the “transforming agent” in the liquid was DNA. To further demonstrate this, the scientists took liquid extracted from heat-killed S. pneumoniae (S strain) and subjected it to extensive preparation and purification, isolating only the pure DNA from the mixture. This pure DNA was also able to transform the R strain into the S strain and generate pathogenic S. pneumoniae. These results provided powerful evidence that DNA, and not protein, was actually the genetic material inside of living cells.
PART-B
Do the two strands of DNA duplex carry the same genetic information? Explain.
Ans:- No,the two strands of dna duplex carry different information ,because complementary base pairs binding to form a double helix.The two chains are wound round each other and linked together by hydrogen bonds between specific complementary bases to form a spiral ladder-shaped moleculeThe stabilization of duplex (double-stranded) DNA is also dependent on base stacking. The planar, rigid bases stack on top of one another, much like a stack of coins. Since the two purine.pyrimidine pairs (A.T and C.G) have the same width, the bases stack in a rather uniform fashion. Stacking near the center of the helix affords protection from chemical and environmental attack. Both hydrophobic interactions andvan der Waal’s forces hold bases together in stacking interactions. About half the stability of the DNA helix comes from hydrogen bonding, while base stacking provides much of the rest.
What is the difference between Z and B- DNAs?
ANS:- Z-DNA is one of the many possible double helical structures of DNA. It is a left-handed double helical structure in which the double helix winds to the left in a zig-zag pattern. alternating purine-pyrimidine sequence (especially poly(dGC)2), negative DNA supercoiling or high salt and some cations (all at physiological temperature, 37°C, and pH 7.3-7.4). Z-DNA can form a junction (called a “B-to-Z junction box”) in a structure which involves the extrusion of a base pair. The Z-DNA conformation has been difficult to study because it does not exist as a stable feature of the double helix. Instead, it is a transient structure that is occasionally induced by biological activity and then quickly disappears.
B-DNA
It is an antiparallel double helix.It is a right-handed helix. The base-pairs are perpendicular to the axis of the helix. (Actually, they are very slightly tilted – at an angle of 4 degrees)The axis of the helix passes through the centre of the base pairs.Each base pair is rotated by 36 degrees from the adjacent base pair.The base-pairs are stacked 0.34 nm apart from one another.The double helix repeats every 3.4 nm, i.e. the pitch of the double helix is 3.4 nm.B-DNA has two distinct grooves: a MAJOR groove; and, a MINOR groove. These grooves form as a consequence of the fact that the beta-glycosidic bonds of the two bases in each base pair are attached on the same edge. However, because the axis of the helix passes through the centre of the base pairs, both grooves are similar in depth.
6. What is the role of RNA in DNA replication?
ANS:- RNA WAS NEED TO INTIATE THE TRANSCRIPTION PROCESS. On the lagging strand, primase builds an RNA primer in short bursts. DNA polymerase is then able to use the free 3′ OH group on the RNA primer to synthesize DNA in the 5′ †’ 3′ direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase is then able to ligate the deoxyribonucleotides together, completing the synthesis of the lagging strand. This rna primer was a short strand of RNA that is synthesized along single-stranded DNA during replication, initiating DNA polymerase-catalyzed synthesis of the complementarystrand.

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