The impact of vitamin D on the innate immune response to uropathogenic Escherichia coli during pregnancy.

Urinary tract infections are highly common during pregnancy, and can cause serious complications for the mother and baby. Vitamin D, predominantly obtained from the sunlight, is known to have an effect on the urothelium, with immunomodulatory capacity against Escherichia coli infection. However, its influence at this site remains to be further explored. This study therefore investigated its impact during pregnancy in a population of women who have the possibility of adequate year-round sun exposure. Serum from pregnant Ugandan women (n = 32) in each trimester of pregnancy, from women after delivery (n = 29) and from never-pregnant controls (n = 25) was collected. 25-Hydroxyvitamin D (25-OHD), cathelicidin LL-37, human ß-defensin 2, interleukin (IL)-8 and soluble CD14 serum concentrations were measured by chemiluminescence immunoassay or ELISA. The ability of serum to inhibit E. coli growth was tested. The immunomodulatory capacities of these serum samples and 1,25-dihydroxyvitamin D3 were investigated in urothelial cells. Increases in 25-OHD and LL-37 levels were observed as pregnancy progressed, peaking in the third trimester. Serum 25-OHD levels were higher in multigravidae than in primigravidae, and correlated positively with maternal age. IL-8 levels were lower in the third trimester than in the first trimester, increased after delivery, but remained below those of never-pregnant women. Similarly, soluble CD14 concentrations increased after delivery. As gestation advanced, serum had an increased capacity to inhibit E. coli growth. In vitro, it modulated the IL-8 response to infection in a vitamin D concentration-dependent manner. Our findings demonstrate that increasing vitamin D levels as pregnancy advances modulate the innate immune system towards a protective response to infection.

Point Mutations in the folP Gene Partly Explain Sulfonamide Resistance of Streptococcus mutans.

Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens. Here, Streptococcus mutans isolates were obtained from saliva of HIV/AIDS patients taking cotrimoxazole prophylaxis in Uganda. The isolates were tested for resistance to cotrimoxazole and their folP DNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19. A set of recombinant plasmids carrying different point mutations in cloned folP were separately transformed into folP-deficient Escherichia coli. Using sulfonamide-containing media, we assessed the growth of folP-deficient bacteria harbouring plasmids with differing folP point mutations. Interestingly, cloned folP with three mutations (A37V, N172D, R193Q) derived from Streptococcus mutans 8 conferred substantial resistance against sulfonamide to folP-deficient bacteria. Indeed, change of any of the three residues (A37V, N172D, and R193Q) in plasmid-encoded folP diminished the bacterial resistance to sulfonamide while removal of all three mutations abolished the resistance. In contrast, plasmids carrying four other mutations (A46V, E80K, Q122H, and S146G) in folP did not similarly confer any sulfonamide resistance to folP-knockout bacteria. Nevertheless, sulfonamide resistance (MIC = 50 µ M) of folP-knockout bacteria transformed with plasmid-encoded folP was much less than the resistance (MIC = 4 mM) expressed by chromosomally-encoded folP. Therefore, folP point mutations only partially explain bacterial resistance to sulfonamide.

SCHISTOSOMA MANSONI INFECTION AND THE ASSOCIATED ANTIBODY IMMUNE RESPONSE AMONGST RESIDENTS OF KIGUNGU ENTEBBE, UGANDA.

UNLABELLED: BACNKGROUND: There are many foci endemic for Schistosoma (S.) mansoni in Uganda. The immune responses to infection with the parasites in these areas have been found to vary with host sex, age and infection intensity. OBJECTIVE: To determine the profile of antibody isotypes responses against S. mansoni crude soluble egg antigens (SEA) and soluble adult worm protein (SWAP) antigens that determine the host resistance or susceptibility to reinfection. DESIGN: Cross Sectional, cohort study. SETTING: Kigugu fishing village in Entebbe, Uganda. SUBJECTS: Nine hundred and forty five (945) Kigungu residents reported forpre-treatment screening and enrolment and 626 cohorts report for post-treatment screening and enrolment 18 months later. RESULTS: Pearson's Chi-sq2 showed thatincrease in titres of anti (SWAP IgE, SEA IgE, and SEA IgG2) was not significant, but increase in anti SEA IgG3 was significant. Decrease in titres of anti (SWAP IgG1, SEA IgG1, and SEA IgG4) was not significant but decrease of anti (SWAP IgG2, SWAP IgG3 and SWAP IgG4) was significant. Positive correlation existed between age and anti SWAP IgE in before and after treatment sera. On the contrary, age was positively correlated with anti SWAP IgG4 in pre-treatment sera but was negatively correlated with anti SWAP IgG4 in the post-treatment sera. In addition there were positive correlation between higher egg counts and the immunoglobulin levels of anti SWAP IgG4 and anti SEA IgG4 but negative correlations were observed between anti SWAP IgE and anti SEA IgE. Conversely low egg counts were associated with high levels of anti SWAP IgE. Furthermore, IgG1-4, IgE antibody to SEA and SWAP antigens did not differ significantly according to sex. CONCLUSION: We concluded that praziquantel treatment of S. mansoni infected persons alter the immune responses that are influenced by age and intensity. A phenomenon that is useful in the effort to produce vaccine against schistosome.

Evidence of Long Term Benefit of Praziquantel Treatment Against Schistosoma mansoni in Kigungu Fishing Village of Entebbe, Uganda

Praziquantel (PZQ) is efficacious against all species of schistosome: Schistosoma mansoni; Schistosoma haematobium; Schistosoma japonicum and other parasites like the Taenia species. This cross-sectional cohorts study was carried out in Kigungu fishing village along Lake Victoria shore in Entebbe Uganda. Our analysis was based on examining microscopically three slides from a single stool specimen from each of base line cohorts 945.These included children and adults; participants from both sexes in Kigungu fishing village in Entebbe Uganda. Nine hundred and one (901) of the cohorts were re-examined after six months and 625 of the same cohorts who were examined at the baseline and after six months were re-examined 18 months later. The slides were prepared using modified Kato/Katz (Odongo-Aginya) method. The infection proportion with Schistosoma mansoni at baseline was 448 (47.5) but this was reduced to 244 (25.8) 18 months after treatment with a single oral dose of praziquantel at 40mg/kg. However 495 (52.5) were negative at the baseline study. The cure proportion after six was significant ///{(P=0.00); (OR4.63) CI at 95(3.53-6.06)///}. Similarly the cure proportion after 18 months was significant ///{(P=0.00); (OR2.2) CI at 95(1.87-3.34)///}. The force of re-infection after six months was significant ///{(P=0.0001); (OR 0.47) CI at 95(0.31-0.71)///}. Nevertheless the force of re-infection was not significant after 18 months ///{(P=0.766); (OR 0.95) CI at 95(0.68-1.34)///} eggs excretion did not reach the level of the pre-treatment intensity. The egg reduction was 69.3. This was associated with age and pre-treatment intensity 400 eggs per gram (epg) of faeces and age groups ? 30 years. The egg reduction also resulted in marked decrease in clinical symptoms in the participants. Our study suggests evidence of long-term benefit of praziquantel in Kigungu and that the re-infection occurred more commonly in younger age group than in the older patients

Evidence of long term benefit of morbidity reduction due to praziquantel treatment against schistosoma mansoni in Kigungu fishing village in Entebbe, Uganda

Praziquantel (PZQ) is efficacious against Schistosoma mansoni. This was prospective cohort study. This study was carried out at Kigungu fishing village, Entebbe, Uganda. The goal of the study was to establish cost effective regiment for mass drug administration (MDA) of Praziquentel in the morbidity reduction of S.mansoni infection. In January 2004, nine hundred and forty five (945) participants were registered in this study. Our analysis was based on examining microscopically three slides prepared from each of 945 stool specimens delivered by each of the participant using modified Kato/Katz method. These included male and female, children and adults living in Kigungu fishing village in Entebbe Uganda. In total 901, cohorts were re-examined for infections clearance six months later in July 2004 and 18 months later in June 2005, 625 cohorts were again re-evaluated for S.mansoni infections after the baseline study. At baseline, (448) of 945 (47.5%) cohorts were S. mansoni positive. All these participants were treatment with a single oral dose of praziquantel at 40mg/kg. At the same time, 495 (52.5%) were S. mansoni negative. Of the 625 (66.3%) cohorts who came back for final review, 80 (12.8%) were still positive for S. mansoni while 210 (33.6%) remained negative after the base line treatment with praziquantel. On the other hand 103 (16.3%) of cohorts who were initially negative at the base line became S.mansoni positive after 18 months and 213(34.1%) remained negative for S.mansoni. The force of re-infection after six months was significant {(P=0.0001),(OR 0.47) CI at 95% (0.31-0.71)}. Nevertheless the force of reinfection was not significant after 18 months {(P=0.766), (OR 0.95) CI at 95% (0.68-1.34)}.The geometric mean eggs excretion of the 80 cohorts who were S.mansoni positive at 18 months was 151.967.This did not reach the geometric mean egg excreted by the same cohorts at baseline which was 285.05. The egg excretion was reduced by 46.8%. Similarly there was marked decrease in clinical symptoms amongst the cohorts. Our study suggests evidence of long-term benefit of praziquantel in Kigungu and that a yearly administration of praziquantel to the community could be a regiment for mass drug administration (MAD) for this community to control schistosomiasis morbidity

Effect of seasonal rainfall and other environmental changes, on snail density and infection rates with Schistosoma mansoni fifteen years after the last snails' study in Kigungu, Entebbe, Uganda.

BACKGROUND: The last study on snail population density in relation to rainfall pattern in Kigungu canoe landing and recreational sites on Lake Victoria shore was earlier carried out about fifteen years ago. This study also reviewed the influence of other environmental factors on the snails' infection rate. OBJECTIVE: To reassess the density dynamic of Biomphalaria (B) choanomphala and Biomphalaria (B) pfeifferi, which act as the intermediate host for S. mansoni and Bulinus (B) globosus, and Bulinus (B) tropicus, which act as intermediate host for S. haematobium. DESIGN: Retrospective study. SETTING: Busy canoe landing sites along Lake Victoria in Kigungu fishing village were selected for the snail sampling. RESULTS: Nine thousand one hundred and ninety four B. choanomphala were collected over the study period. The numbers of B. choanomphala collected in each yearwas 4742 (51.6%) and 4452 (48.4%) in 2004 and 2005 respectively. Of the 4742 B. Choanomphala collected in 2004, 82 (1.7%) shed human cercariae and 329 (6.7%) shed non-human cercariae. Whereas in 2005, out of 4452 B. choanomphala collected 302 (6.85%) shed non-human cercariae and 82 (1.8%) shed human cercariae. Similarly, 4173 B. pfeifferi were also collected in the same period. Out of which 2224 (53.3%) were collected in 2004 and 1949 (46.7%) in 2005. For B. pfeifferi, 42 (1.9%) out of 2224 snails collected in 2004 shed human cercariae and 246 (11.1%) shed non-human cercariae. While in 2005, 33 out of 1949 snails (1.7%) shed human cercariae and 159 (8.2%) shed non-human cercariae. Other snails of medical importance collected included 292 B. globosus and 3094 B. tropicus. None of the Bulinus spp. collected shed any human cercariae but 37 (2.1%) and 30 (2.3%) B. tropicus shed non-human cercariae in 2004 and 2005 respectively. In 2004 and 2005, the area received, 1729mm and 1959mm of rainfall respectively, The mean rainfall during the year was 144.05 mm and 163.3 mm in 2004 and 2005 respectively. There was a negative correlation between rainfalls and snail density dynamic. CONCLUSION: We have found in this study that in spite of the bush clearing of the papyrus swamps which originally was the major habitats for B. choanomphala, B. pfeifferi and the Bulinus spp the intermediate host for schistosome at all canoe landing sites at Kigungu, these snails are still present. Moreover, that their population density dynamic and infection rate are inversely proportional to the rainfall pattern.

Isolation and genotyping of Toxoplasma gondii from Ugandan chickens reveals frequent multiple infections.

The genetic make-up of an infecting Toxoplasma gondii strain may be important for the outcome of infection and the risk of reactivation of chronic disease. In order to survey the distribution of different genotypes within an area, free-range chickens act as a good model species. In this study 85 chickens were used to investigate the prevalence, genotype and mouse virulence of T. gondii in Kampala, Uganda. Antibodies were detected in 40 chickens, of which 20 had MAT-titres of 1:20 or higher and were also positive by PCR. Genotyping of 5 loci (SAG1, SAG2, SAG3, BTUB and GRA6) showed that 6 strains belonged to genotype I, 8 to Type II and 1 to Type III. Five chickens had multiple infections; 3 individuals with Type I plus Type II and a further 2 harbouring Types I, II and III. Isolates were obtained from 9 chickens via bioassay in mice, 6 were Type II strains and 3 were from animals with mixed infection. This is the first set of African T. gondii strains to be genotyped at multiple loci and in addition to the 3 predominant lineages we found a small number of new polymorphisms and a high frequency of multiple infections.

Evaluation of quantitative buffy coat (QBC) assay and polymerase chain reaction (PCR) for diagnosis of malaria.

A prospective study was undertaken to compare the Polymerase Chain Reaction (PCR) and Quantitative Buffy Coat (QBC) assay with conventional Giemsa technique for diagnosis of malaria. A total of 104 samples were taken for the purpose. They comprised of fever cases suggestive of malaria (n=74) and control group, fever cases other than malaria (n=30). Peripheral blood smears were prepared by Giemsa staining and QBC assay was performed as per standard protocol. From the stored blood samples, parasite DNA was extracted and PCR was performed using P. falciparum and P. vivax specific sets of primers. The QBC assay was 100% in agreement with the Giemsa stain. Specificity of the PCR detection of P. falciparum parasites was 100%. However, sensitivity for detection of P. falciparum and P. vivax by PCR was 64.28% and 82.35% respectively. In mixed cases of malaria (n=2), PCR results were in 100% agreement with that of Giemsa. The lower sensitivity of PCR for P. falciparum could probably be due to inaccessibility of target DNA, presence of PCR inhibitors in samples and parasite strain variations.

A novel 70-kDa Triton X-114-soluble antigen of Plasmodium falciparum that contains interspecies-conserved epitopes.

In order to identify novel conserved integral membrane and other membrane-associated proteins of Plasmodium falciparum, lambda gt11-P. falciparum DNA library phages were immunoscreened with convalescent-phase mouse sera and rabbit antiserum against Triton X-114-soluble proteins of P. falciparum. One recombinant phage clone, L857, reacted with both of the antibody probes. Insert DNA (857 bp long) in L857 was 69% dA+dT rich and hybridized to a fragment of 1800 bp from mung bean nuclease-digested P. falciparum genomic DNA. The cloned parasite DNA did not show notable sequence homology with any known protein gene. The L857-encoded polypeptide, p34 (M(r) 34 kDa) was expressed in bacteria, fused to glutathione S-transferase (GST). The fusion peptide, GST-p34 (M(r) 62 kDa), was recognized by immune serum against Triton X-114-soluble antigens of P. falciparum and was reactive with anti-P. falciparum, anti-Plasmodium yoelii, and anti-GST sera. Rabbit antiserum raised against the fusion peptide recognized a 70-kDa protein from lysates of P. falciparum cells and a putative homologous 100-kDa protein from lysates of P. yoelii. The rabbit serum anti-fusion peptide antibodies bound to acetone-fixed P. falciparum-infected erythrocytes and, in immunofluorescent antibody tests, produced a punctate pattern of fluorescence suggesting that the 70-kDa native protein is associated with an apical organelle of the parasite.

Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth.

We demonstrated that antibodies in the serum of BALB/c mice convalescent from Plasmodium yoelii infection inhibit the in vitro growth of Plasmodium falciparum. Blood stage P. falciparum antigens that cross-react with the convalescent-phase mouse serum antibodies were identified and partially characterized. Convalescent-phase mouse serum immunoglobulin G (IgG) reacted with P. falciparum lysates at up to a 1:15,000 dilution of the immune sera and bound to P. falciparum-parasitized erythrocytes at up to a 1:5,000 dilution of the sera. The cross-reactive moieties of antigens in parasite lysates were resistant to oxidation by periodate but sensitive to trypsinization. About 15 polypeptides (M(r)s of 15,000 to 110,000) of P. falciparum blood stages were recognized by the convalescent-phase mouse anti-P. yoelii sera; many of these antigens were metabolically 35S labeled and specifically immunoprecipitated. Also, virtually all of the cross-reactive antigens were recognized by human malaria-immune sera. The anti-P. yoelii serum antibodies bound, with high affinity, to at least five of the cross-reactive antigens (M(r)s of 107,000, 84,000, 53,000, 36,000, and 30,000). By phase separation in Triton X-114, eight interspecies cross-reactive antigens (M(r)s of 84,000, 76,000, 51,000, 31,000, 29,000, 28,000, 23,000, and 22,000) were found to be integral membrane proteins. Convalescent-phase mouse serum IgG strongly inhibited the differentiation of P. falciparum from schizonts to rings; 75 micrograms of IgG per ml caused 80% inhibition of release of merozoites and their invasion into erythrocytes. On the other hand, the anti-P. yoelii serum antibodies also inhibited intracellular development of P. falciparum from rings to schizonts; 25 micrograms of IgG per ml caused 50% inhibition. Inhibition of P. falciparum growth by anti-P. yoelii serum IgG suggests that some of the interspecies cross-reactive antigens contain important conserved epitopes and induce protective antibodies against P. falciparum.

Antibody recognition and isoelectrofocusing of antigens of the malaria parasite Plasmodium yoelii.

Inbred BALB/c mice were either immunized with Triton X-100-extracted antigens of blood-stage Plasmodium yoelii or infected with P. yoelii and cured in three successive schedules. Whereas the immunized BALB/c became only partially protected from subsequent challenge infection with blood-stage P. yoelii, the convalescent mice acquired total immunity. When total P. yoelii antigen extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-P. yoelii serum, five major protein bands of 150, 84, 40, 19, and 16 kDa were recognized by the sera of fully protected convalescent mice but not by the sera of partially protected mice. The utility of comparing reactivities of sera from fully protected and partially protected malaria hosts and the possibility that antigens uniquely recognized by the convalescent mouse sera may contribute to immunity against P. yoelii infection are discussed. Although previously reported to be an effective adjuvant for immunization against P. yoelii infection in (BALB/c x C57BL)F1 hybrid mice, saponin did not promote protection any better than did Freund adjuvant in BALB/c mice immunized with detergent-extracted P. yoelii antigen. Most of the P. yoelii proteins (14 to 250 kDa) found in Triton X-100 extracts of P. yoelii-parasitized erythrocytes isoelectrofocused as a single peak in the pH region 4.4 to 4.6, suggesting a rationale for previous findings that the most anti-P. yoelii protective and T-helper activities are induced by antigens isoelectrically focused in a fraction of similar pH.

Towards the design of heterovalent anti-malaria vaccines: a hybrid immunogen capable of eliciting immune responses to epitopes of circumsporozoite antigens from two different species of the malaria parasite, Plasmodium.

The peptide CS.T3, corresponding to residues 378-398 of the Plasmodium falciparum (Pf) circumsporozoite (CS) protein sequence (except with cysteines 384 and 389 replaced by alanines), has been found to be almost universally recognized by human and mouse T lymphocytes. When colinearly linked to the repetitive B-lymphocyte-specific epitope (Asn-Ala-Asn-Pro)n of Pf CS protein, CS.T3 induces T-helper activity for an anti-(Asn-Ala-Asn-Pro)n antibody response in mice of different haplotypes. We constructed a double-epitope peptide, CS.T3-R3, by co-linearly joining a truncated 18-mer form (IEKKIAKMEKASSVFNVV) of CS.T3 to three tandem repeats (R3) of a B-cell-specific epitope, QGPGAP, of Plasmodium yoelii (Py) CS protein, via a two-glycine spacer. Whereas CS.T3 and R3 did not induce specific antibodies, CS.T3-R3 elicited anti-CS.T3 and anti-R3 antibodies in different mouse strains. Some human anti-Pf sera from malaria-endemic areas contained high-titred anti-CS.T3 antibody IgG, indicating that parasite-derived CS.T3 contains a B-cell determinant which is maintained in the alanine-substituted synthetic CS.T3. Antibody absorption experiments showed that CS.T3-R3 contains no new B-cell-specific determinants other than R3 and CS.T3. That the Pf CS protein epitope, CS.T3, supports T-cell help for antibody responses against the Py CS protein repeat epitope, QGPGAP, implies the possible use of CS.T3 in anti-sporozoite multiple-epitope vaccines against different species of Plasmodium. Colinearly linking CS.T3 to R3, via a two-glycine spacer, appears to be a useful model by which different T- and B-cell-specific determinants can be jointed into a heterovalent immunogen while retaining their distinct immunological properties.

Random mutagenesis of the gene for the beta-subunit of F1-ATPase from Escherichia coli.

ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.

The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly.

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location.

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites.

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.

Bovine immune response to African ;trypanosomes: specific antibodies to variable surface glycoproteins of Trypanosoma brucei.

Cattle were infected with two different clones of Trypanosoma brucei (MITat 1.2 and ILTat 1.3) and antibody response was followed by radioimmunoassay. In four of the seven animals there were at least two peaks of antibody activity to the infecting clones, with the second peak much higher than the first. Specific antibodies (IgG1 and IgM but not IgG2) were eluted from the immunoabsorbent columns on which the variant surface glycoproteins (VSGs) were coupled. By neutralization of infectivity tests, IgM antibodies from the first peak of antibody activity were more efficient in neutralizing trypanosomes than IgG1 but the reverse was true of the antibodies isolated from the second peak. By absorption with multiple variable antigen types isolated during the course of infection, all the IgM and IgG1 in the first 3 weeks of infection were shown to be specific. It is suggested that polyclonal B cell stimulation leading to dysfunction in the control of IgM and IgG production may not be responsible for the high levels of these immunoglobulins in bovine trypanosomiasis.