Fc gamma receptor IIa-H131R polymorphism and malaria susceptibility in sympatric ethnic groups, Fulani and Dogon of Mali.

It has been previously shown that there are some interethnic differences in susceptibility to malaria between two sympatric ethnic groups of Mali, the Fulani and the Dogon. The lower susceptibility to Plasmodium falciparum malaria seen in the Fulani has not been fully explained by genetic polymorphisms previously known to be associated with malaria resistance, including haemoglobin S (HbS), haemoglobin C (HbC), alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Given the observed differences in the distribution of FcγRIIa allotypes among different ethnic groups and with malaria susceptibility that have been reported, we analysed the rs1801274-R131H polymorphism in the FcγRIIa gene in a study of Dogon and Fulani in Mali (n = 939). We confirm that the Fulani have less parasite densities, less parasite prevalence, more spleen enlargement and higher levels of total IgG antibodies (anti-CSP, anti-AMA1, anti-MSP1 and anti-MSP2) and more total IgE (P < 0.05) compared with the Dogon ethnic group. Furthermore, the Fulani exhibit higher frequencies of the blood group O (56.5%) compared with the Dogon (43.5%) (P < 0.001). With regard to the FcγRIIa polymorphism and allele frequency, the Fulani group have a higher frequency of the H allele (Fulani 0.474, Dogon 0.341, P < 0.0001), which was associated with greater total IgE production (P = 0.004). Our findings show that the FcγRIIa polymorphism might have an implication in the relative protection seen in the Fulani tribe, with confirmatory studies required in other malaria endemic settings.

Influence of infection on malaria-specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda.

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen-1 (AMA-1), merozoite surface protein-1 (MSP-119 ), merozoite surface protein-2 (MSP-2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA-1, MSP-119 , MSP-2 and gSG6 were related to concurrent (sub-)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re-infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission.

Interaction of malaria with a common form of severe thalassemia in an Asian population.

In many Asian populations, the commonest form of severe thalassemia results from the coinheritance of HbE and beta thalassemia. The management of this disease is particularly difficult because of its extreme clinical diversity; although some genetic and adaptive factors have been identified as phenotypic modifiers, the reasons remain unclear. Because the role of the environment in the course of severe thalassemia has been neglected completely and because malaria due to both Plasmodium falciparum and Plasmodium vivax has been prevalent in Sri Lanka, we carried out a pilot study of patients with HbE beta thalassemia that showed high frequencies of antibodies to both parasite species and that 28.6% of the children had DNA-based evidence of current infection with P. vivax. Malarial antibodies then were assessed in patients with HbE beta thalassemia compared with those in age-matched controls. There was a significant increase in the frequency of antibodies in the thalassemic patients, particularly against P. vivax and in young children. There was also a higher frequency in those who had been splenectomized compared with those with intact spleens, although in the latter it was still higher than that in the controls. The thalassemic patients showed significant correlations between malaria antibody status and phenotype. Patients with HbE beta thalassemia may be more prone to malaria, particularly P. vivax, which is reflected in their clinical severity. Because P. vivax malaria is widespread in Asia, further studies of its interaction with HbE beta thalassemia and related diseases are required urgently as a part of ongoing thalassemia control programs.

Multicenter collaborative study to calibrate salmon calcitonin by bioassay and high-performance liquid chromatography: establishment of the third international standard.

Salmon calcitonin (sCT) is widely used therapeutically in the treatment of patients with postmenopausal osteoporosis, Paget's disease, and some forms of hypercalcemia. Preparations of synthetic calcitonin peptides of high purity and reproducibility are now routinely produced and physicochemical methods, particularly reverse-phase high-performance liquid chromatography (RP-HPLC), are replacing the in vivo biological assay for monitoring and calibration. Although the bioassay is no longer required for routine batch control in Europe, calcitonin bioassays are still required in some countries and in the development of new products. Stocks of the Second International Standard (IS) for salmon calcitonin are now depleted and, to replace it with a new calibrant, an international collaborative study was organized in which the aims were to: determine the activity of the candidate sCT by in vivo bioassay in terms of the second IS; assess the stability of the preparation after accelerated thermal degradation; estimate the purity of the ampouled candidate preparation; and determine the sCT content in gravimetric units by HPLC. The HPLC data in terms of ampoule content were in good agreement giving an estimate of 23.1 (coefficient of variation [CV] 3.8%) microg per ampoule. The HPLC chromatograms revealed a small, but detectable, degree of heterogeneity, which possibly occurred during the formulating or ampouling procedures, resulting in a reduction in monocomponent content (purity) from 96% to 92%. The biological activity of the ampoule contents in international units (IU) was calculated from the mass value and the internationally agreed-upon figure of 6000 IU/per mg for the specific activity of salmon calcitonin. This gave a value of 138 IU per ampoule, which was in good agreement with the biological assay estimate (140 IU per ampoule). The preparation of sCT was subsequently adopted as the Third International Standard by the World Health Organization with an assigned content of 138 IU per ampoule.

The International Reference Preparation of Gonadorelin for Bioassay: a comparison with different preparations of synthetic luteinizing hormone releasing hormone using physicochemical methods of analysis, different bioassays and immunoassay.

The preparation and nature of the International Reference Preparation of Gonadorelin for Bioassay (IRP; coded 77/596) are described. The IRP was studied by four laboratories in four countries and compared, using physicochemical methods of analysis, various bioassay procedures and immunoassay, with preparations of synthetic luteinizing hormone releasing hormone (LH-RH) produced by different manufacturers. Analyses by thin-layer chromatography and by reverse-phase high-performance liquid chromatography (HPLC) indicated some heterogeneity of the peptide present in most of these preparations of synthetic LH-RH, including that of the IRP; the latter preparation appeared to be 88.3% (w/w) pure, judged by HPLC. The data from the collaborative study suggested that each ampoule of the IRP contains approximately 31 nmol LH-RH. The IRP appeared to be suitable to serve as an international reference preparation for bioassay since its behaviour was similar in different bioassays, so far as this could be examined, to that of the other preparations of LH-RH with which it was compared. Furthermore, the biological activities of different preparations of LH-RH, assessed in terms of the IRP, appeared to correlate with their degrees of purify assessed by physicochemical methods, suggesting that the peptides other than LH-RH present in the IRP did not contribute significantly to the biological activity of the preparation in these assay procedures. The limited data available suggested that the IRP might also be suitable as a reference preparation for immunoassay. The ampouled preparation, coded 77/596, was therefore established by the World Health Organization as the International Reference Preparation of Gonadorelin for Bioassay and assigned a unitage of 31 i.u./ampoule on the basis that the i.u. is represented by 1 nmole of LH-RH.

Characterisation of the chains of human fibrinogen isolated by perfusion chromatography using fibrin specific monoclonal antibodies.

In order to study the epitopes on fibrin to which monoclonal antibodies are directed, we required pure individual polypeptide chains of human fibrinogen in milligram quantities. High purity chains of human fibrinogen were rapidly obtained, in under 3 minutes, by the novel procedure of reversed-phase perfusion chromatography and these chains were subjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these antibodies in both immunoblotting and enzyme linked immunospecific assay (ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were employed to initiate studies to define the epitopes in fibrin to which four fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are targeted. Two of these antibodies, B10 and A11, were shown to be directed to a linear sequence on the A alpha chain, although the binding profiles for the two antibodies suggested that different epitopes may be involved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that conformational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.

Differences between neonates and adults in tissue-type-plasminogen activator (t-PA)-catalyzed plasminogen activation with various effectors and in carbohydrate sequences of fibrinogen chains.

Our study investigates the effect of fetal and adult soluble fibrin (SF), fetal and adult fibrinogen Aalpha- and gamma-chains, as well as adult CNBr-fibrinogen fragments on tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation of both fetal and adult Glu-plasminogen types 1 and 2. In addition, we determined carbohydrate sequences of fetal and adult Bbeta- and gamma-chains by mass spectrometric analysis. In the absence of an effector, no substantial differences in the rate of plasmin formation could be seen between the fetal and adult plasminogen types. In the presence of an effector, both fetal Glu-plasminogen types revealed lower values for k(cat app) than the respective adult types. No differences could be seen in the values for K(m app). The resulting differences in catalytic efficiencies between the fetal and adult plasminogen types were much less than previously reported. No differences could be seen between fetal and adult effectors in stimulating t-PA-catalyzed plasminogen activation. Detailed analyses of the activation kinetics revealed a longer initial phase of slow plasmin formation of both fetal Glu-plasminogen types compared to their respective adult types, indicating a slower plasmin-induced modification of CNBr-fibrinogen fragments or SF by fetal plasmin. Mass spectrometric analysis of the N-glycans present on adult and fetal Bbeta- and gamma-fibrinogen chains showed the presence of a major monosialylated biantennary structure with lesser amounts of the disialylated form. In contrast to previous data, we conclude that catalytic efficiency of t-PA-catalyzed plasminogen activation in neonates is only slightly lower than in adults.

Lysine vasopressin undergoes rapid glycation in the presence of reducing sugars.

Lysine vasopressin (LVP) readily reacts with reducing saccharides both in lyophilized preparations and in aqueous solution. Incubation of LVP with, for example, lactose over a pH range of 3.0-8.5 in phosphate buffer or simply in water, gives rise to a number of reaction products, some of which form rapidly (in a matter of hours) even in the frozen state. Reaction mixtures were analysed by reversed-phase HPLC and the structures of the products were deduced from the amino-acid composition of isolated components, by comparison with product profiles obtained with analogues under similar conditions and by FAB mass-spectral analysis of derivatives isolated after reduction with cyanoborohydride. The primary products arise from the formation of Schiff's bases at one or both of the two amino functions. The alpha-amino group of the N-terminal cystine is considerably more reactive than is the epsilon-amino group of lysine and it is the N-terminal adduct which rapidly forms even at -20 degrees C. It is concluded that caution must be shown in using reducing sugars in formulations containing peptides and proteins, particularly the vasopressins and oxytocin.

Application of an in vitro endopeptidase assay for detection of residual toxin activity in tetanus toxoids.

Tetanus vaccine is composed of chemically denatured tetanus toxin (TeNT), thus safety testing requires confirmation of freedom from residual and reversible toxicity. Currently, TeNT activity is estimated using in vivo assay models. Information that TeNT acts by selectively inactivating protein leading to the blocking of release of neurotransmitters has provided the opportunity to develop in vitro biochemical assay for toxin activity. In this study we describe development and use of an in vitro endopeptidase assay for detection of TeNT activity in toxoid vaccine formulations.

Estimating medium- and long-term trends in malaria transmission by using serological markers of malaria exposure.

The implementation and evaluation of malaria control programs would be greatly facilitated by new tools for the rapid assessment of malaria transmission intensity. Because acquisition and maintenance of antimalarial antibodies depend on exposure to malaria infection, such antibodies might be used as proxy measures of transmission intensity. We have compared the prevalence of IgG antibodies with three Plasmodium falciparum asexual stage antigens in individuals of all ages living at varying altitudes encompassing a range of transmission intensities from hyper- to hypoendemic in northeastern Tanzania, with alternative measures of transmission intensity. The prevalence of antibodies to merozoite surface protein-1(19) was significantly more closely correlated with altitude than either point-prevalence malaria parasitemia or single measures of hemoglobin concentration. Analysis of age-specific seroprevalence rates enabled differentiation of recent (seasonal) changes in transmission intensity from longer-term transmission trends and, using a mathematical model of the annual rate of seroconversion, estimation of the longevity of the antibody response. Thus, serological tools allow us to detect variations in malaria transmission over time. Such tools will be invaluable for monitoring trends in malaria endemicity and the effectiveness of malaria control programs.

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.

Identification of gonadorelin (LHRH) derivatives: comparison of reversed-phase high-performance liquid chromatography and micellar electrokinetic chromatography.

A number of reversed-phase (RP) HPLC systems for the separation of gonadorelin (gonadoliberin, LHRH) and five therapeutically important analogues have been systematically examined. The selectivity of RP-HPLC has been compared with several micellar electrokinetic chromatographic (MEKC) systems and free solution capillary electrophoresis. RP-HPLC exhibits greater selectivity towards structural differences, but complete separation of the peptides in one isocratic analytical run is tedious due to the large differences in retention. Gradient elution gives satisfactory separation in an acceptable time span. Of the micellar systems examined (sodium dodecyl sulphate, cetrimide, 3-[(cholamidopropyl)dimethylamino]-1-propanesulphonate and Triton X-100) only MEKC with cetrimide micelles gave a complete separation showing selectivity similar, but not identical, to RP-HPLC, and providing a complete separation of all six compounds as rapidly as gradient RP-HPLC.

Comparison of selectivities of reversed-phase high-performance liquid chromatography, capillary zone electrophoresis and micellar capillary electrophoresis in the separation of neurohypophyseal peptides and analogues.

A number of RP-HPLC systems, including those prescribed in several official monographs, have been evaluated for separating oxytocin, the vasopressins, some clinically important analogues and two additional neurohypophyseal nonapeptides. The separation has been compared with capillary zone electrophoresis and micellar electrophoresis in four micellar systems: cationic, anionic, zwitterionic and neutral. Complete separation was achieved by both RP-HPLC and micellar CE but the importance of charge as a major parameter of separation in CE confers a distinct and useful selectivity to micellar CE based separations.

Amino acid sequences around the cysteine residue of calf lens -crystallin.

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.

N-terminal sequences of alpha-crystallin.

The amino acid sequences at the N-terminal ends of the chains of the lens protein, alpha-crystallin, were studied. Both the main kinds of chain in bovine alpha-crystallin (A chains and B chains) have an N-terminal methionine residue, and the amino group is acetylated. Selective purification of the peptides in a tryptic digest of bovine alpha-crystallin gave a preparation consisting largely of the N-terminal peptide from the A chains, and the sequence of this peptide was elucidated. Subsequently, the N-terminal peptides were prepared from separated A and B chains. The proposed sequences are: A chain, acetyl-Met-Asp-Ile-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys; B chain, acetyl-Met-Asp-Ile-Ala-Ile-His-(Pro,Trp)-Ile-Arg. The similarity between the sequences supports the hypothesis that the A and B chains are derived evolutionarily from a common precursor.

The amino acid sequence of rabbit muscle triose phosphate isomerase.

The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.

The tryptic peptides of rabbit muscle triose phosphate isomerase.

1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.