Population-based biochemistry, immunologic and hematological reference values for adolescents and young adults in a rural population in Western Kenya

Background

There is need for locally-derived age-specific clinical laboratory reference ranges of healthy Africans in sub-Saharan Africa. Reference values from North American and European populations are being used for African subjects despite previous studies showing significant differences. Our aim was to establish clinical laboratory reference values for African adolescents and young adults that can be used in clinical trials and for patient management.

Methods and Findings

A panel of 298, HIV-seronegative individuals aged 13–34 years was randomly selected from participants in two population-based cross-sectional surveys assessing HIV prevalence and other sexually transmitted infections in western Kenya. The adolescent (<18 years)-to-adults (≥18 years) ratio and the male-to-female ratio was 1∶1. Median and 95% reference ranges were calculated for immunohematological and biochemistry values. Compared with U.S-derived reference ranges, we detected lower hemoglobin (HB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), neutrophil, glucose, and blood urea nitrogen values but elevated eosinophil and total bilirubin values. Significant gender variation was observed in hematological parameters in addition to T-bilirubin and creatinine indices in all age groups, AST in the younger and neutrophil, platelet and CD4 indices among the older age group. Age variation was also observed, mainly in hematological parameters among males. Applying U.S. NIH Division of AIDS (DAIDS) toxicity grading to our results, 40% of otherwise healthy study participants were classified as having an abnormal laboratory parameter (grade 1–4) which would exclude them from participating in clinical trials.

Conclusion

Hematological and biochemistry reference values from African population differ from those derived from a North American population, showing the need to develop region-specific reference values. Our data also show variations in hematological indices between adolescent and adult males which should be considered when developing reference ranges. This study provides the first locally-derived clinical laboratory reference ranges for adolescents and young adults in western Kenya.     

A method for detecting centres of natural selection in protein structures: potential for predicting the location of functional areas

A simple method has been developed to detect protein microenvironments which are likely to be the focus of natural selection, and thereby important for function. It relies on distinguishing between the probability of an amino acid type arising by genetic mutation and the probability that it will be chosen by natural selection. When applied to proteins of known tertiary structure, the method revealed that major differences exist in the balance between neutral and selected change, and also that functional sites can be highlighted.     

Proteinase inhibitors and dendrotoxins. Sequence classification, structural prediction and structure/activity

The amino acid sequences of four presynaptically active toxins from mamba snake venom (termed 'dendrotoxins') were compared systematically with homologous sequences of members of the proteinase inhibitor family (Kunitz). A comparison based on the complete sequences revealed that relatively few amino acid changes are necessary to abolish antiprotease activity and convert a proteinase inhibitor into a dendrotoxin. When comparison centred only on the sequence segments known to comprise the antiprotease site of bovine pancreatic trypsin inhibitor, the dendrotoxins were clearly classified apart from all the known inhibitors. Since the mode of action of the bovine pancreatic trypsin/kallikrein inhibitor involves beta sheet formation with the enzyme, predictions were obtained for this secondary structure in the region of the 'antiprotease site' throughout the homologues. Again, the dendrotoxins were clearly distinguished from the inhibitors. Structure/activity analyses, based on the crystal structures of inhibitor/enzyme complexes, suggest that unlike proteinase inhibitors, dendrotoxins might specifically co-ordinate the active-site 'catalytic' histidine residues of serine proteases. Although the significance of this remains to be studied, the presynaptic target is expected to involve an as yet uncharacterised member of the serine protease family.     

The structural evolution of cobra venom cytotoxins

Summary In order to analyze the evolutionary behavior of the cobra venom cytotoxins, their probable tertiary structure was predicted using computer graphics. The 41 amino acid sequences known show that the major evolutionary changes have taken place in two particularly exposed areas of the molecular surface. In each area, neighboring residue positions seem to have evolved interdependently, but there is no obvious interdependence between the two areas. Indeed, the relative evolution of these two areas prompts a subdivision of the sequence set into four groups. According to the known cytotoxin circular dichroism spectra, one of these four groups could be characterized by a difference in molecular secondary structure. Sine the two variable areas have functional associations, it is suggested that their evolution may be governed by a target with several similar binding sites.     

Classification of phospholipases A2 according to sequence. Evolutionary and pharmacological implications

The inhibition of elongation of Bacillus megaterium KM growing in the presence of low concentrations of nocardicin A resulted in the production of osmotically stable, actively dividing coccal-shaped cells. Saturation of penicillin-binding proteins 3a and 3b with nocardicin A in vivo at these concentrations was correlated with the inhibition of cell elongation. Analysis of the DD-carboxypeptidase activity of isolated vegetative membranes of B. megaterium KM in vitro indicated that penicillin-binding protein 4 is not a DD-carboxypeptidase under the assay conditions used. Penicillin-binding proteins were analysed by two-dimensional gel electrophoresis and the suitability of lysozyme treatment of cells as a method of membrane preparation was investigated with regard to the detection of proteins with highly labile penicillin-binding activities in vitro.     

Snake toxin secondary structure predictions. Structure activity relationships

Modified Chou &; Fasman (1974a,b) secondary structure prediction rules have been successfully applied to the 57 snake venom toxins described as being neurotoxic or cytotoxic. Despite the different toxicities involved, a common distribution of secondary structure was detected throughout these toxins. The results also highlight the contrasts between short and long neurotoxins, and neurotoxins and cytotoxins. From comparisons of the typical structure of each toxin group with the known X-ray data an erabutoxin b and Philippines sea-snake toxin b, regions that dictate neurotoxicity or cytotoxicity can be tentatively identified. These deductions are discussed with regard to known chemical properties of these molecules. Similarly, the relevance of the differences between short and long neurotoxins to the superior binding of the latter to the cholinergic receptor is considered.It appears that the cytotoxins and neurotoxins are variations on a central toxic theme, but have differing specificities, whose origin can be traced to certain regions of the toxin in question.     

Simple intrasequence difference (SID) analysis: an original method to highlight and rank sub-structural interfaces in protein folds. Application to the folds of bovine pancreatic trypsin inhibitor,phospholipase A2, chymotrypsin and carboxypeptidase A

We present Simple Intrasequence Difference (SID) analysis, a novel bioinformatic technique designed to help comprehend the properties of protein fold topologies. The analysis grades numerically every residue position in a given protein 3D structure according to the topological situation of the position in the folded chain. This results in an expression of the potential contribution of each residue position and its vicinity towards the integrity of the molecular conformation. Contiguous highly graded residues delineate the sub-structural interfaces that arise from the presence within the molecular fold of discrete domains and sub-domains. This comprehensive rendering of the internal arrangement of chain interfacing helps predict the potential for site-specific inductions (e.g. via mutations or ligand binding) of conformational change in the fold. Whereas SID analysis of single folds can convey an idea of the basic potential for topological adjustment in the protein family, comparative SID analysis of related folds focuses attention on those areas of the family fold where evolutionary changes, activation events and ligand binding have had the most topological impact. For demonstration, SID analysis is applied to the folds of pancreatic trypsin inhibitor (Kunitz), phospholipase A(2), chymotrypsin and carboxypeptidase A. We find that many of the potentially vulnerable sub-structural interfaces tend to be protected in the fold interior, in many cases stabilised by disulfide bridges spanning the interface. However, the most prominent interfaces tend to be externally accessible, without remedial stabilisation by disulfide bridges. These latter interfaces are associated so closely with the known functional sites that alterations to the interfacial juxtapositions should influence recognition and catalytic behaviour directly. This shows how side chain mutations, chemical modifications and binding events remote from the sites can nevertheless adjust, via interfacial realignment, the conformations and emergent properties of the sites. The close association also provides clear opportunities for interfacial rearrangements to follow intermolecular recognition events in the sites, facilitating translation of the binding into adjustment of the molecular conformation in areas distant from the sites. As a direct consequence of the topological arrangements, a large proportion of the molecular structure has the capacity to shape the character of the functional sites and, conversely, binding at these sites has the potential to radiate influence to the rest of the molecule. For the enzymes considered, the evidence is consistent with the possibility that primary and secondary binding by the substrate enhances catalytic efficiency by imposing conformational change upon the catalytic centre via adjustments to the fold. This influence may be expressed as favourable adjustment of the catalytic geometry, transition state ensemble, energy propagation pathway, or as a physical strain exerted on the substrate bond to be cleaved. The scale of the adjustments, and their importance to the mechanisms, may have been seriously underestimated.