Giardia and Cryptosporidium antibody prevalence and correlates of exposure among Alaska residents, 2007-2008.

Giardia duodenalis and Cryptosporidium spp. are common intestinal protozoa that can cause diarrhoeal disease. Although cases of infection with Giardia and Cryptosporidium have been reported in Alaska, the seroprevalence and correlates of exposure to these parasites have not been characterised. We conducted a seroprevalence survey among 887 residents of Alaska, including sport hunters, wildlife biologists, subsistence bird hunters and their families and non-exposed persons. We tested serum using a multiplex bead assay to evaluate antibodies to the Giardia duodenalis variant-specific surface protein conserved structural regions and to the Cryptosporidium parvum 17- and 27-kDa antigens. Approximately one third of participants in each group had evidence of exposure to Cryptosporidium. Prevalence of Giardia antibody was highest among subsistence hunters and their families (30%), among whom positivity was associated with lack of community access to in-home running water (adjusted prevalence ratio [aPR] 1.15, 95% confidence interval (CI) 1.02-1.28) or collecting rain, ice, or snow to use as drinking water (aPR 1.09, 95% CI 1.01-1.18). Improving in-home water access for entire communities could decrease the risk of exposure to Giardia.

Seroepidemiology of Toxoplasma in a coastal region of Haiti: multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm-blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children aged 0-11 years, the infection rate varied with age reaching a maximum of 0·131 infections/year in children aged 3 years [95% confidence interval (CI) 0·065-0·204]. The median time to seroconversion was estimated to be 9·7 years (95% CI 7·6-∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28·2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0·057 infections/year (95% CI 0·033-0·080) at age 2·6 years. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions.

The trypanosomatid Rieske iron-sulfur proteins have a cleaved presequence that may direct mitochondrial import.

We have cloned the gene that encodes subunit 4 of the T. brucei cytochrome-c reductase complex and a fragment of the C. fasciculata subunit 4 cDNA and have shown that subunit 4 is the Rieske iron-sulfur protein. The cleaved presequences of the trypanosomatid iron-sulfur proteins resemble conventional mitochondrial targeting presequences but are smaller than other eukaryotic iron-sulfur protein signal peptides.

Cytochromes c1 of kinetoplastid protozoa lack mitochondrial targeting presequences.

We have used the polymerase chain reaction to amplify cDNA fragments that encode the amino-terminal sequences of cytochrome c1 from two distantly related kinetoplastid species, Crithidia fasciculata and Bodo caudatus. Cloning and sequencing of these fragments have revealed that these proteins lack conventional mitochondrial targeting presequences.

Isolation of the gene coding for elongation factor-1alpha in Cryptosporidium parvum.

A gene encoding for Cryptosporidium parvum (C. parvum) elongation factor 1alpha (EF-1alpha) was isolated and sequenced from a cDNA expression library. The recombinant protein cross-reacted with a monoclonal antibody that was raised to a sporozoite cell surface antigen. The gene encoded a 435 amino acid protein with a predicted molecular weight of 48.1 kDa. The predicted C. parvum EF-1alpha protein sequence showed extensive homology with the EF-1alpha proteins of other eukaryotic organisms and included three conserved sequence motifs implicated in GTP binding.

Developmental regulation of Trypanosoma brucei cytochrome c reductase during bloodstream to procyclic differentiation.

The bloodstream forms of the protozoan parasite Trypanosoma brucei lack spectrally detectable cytochromes and satisfy energy requirements mainly by glycolysis. When infected blood is ingested by the tse-tse fly vector, the bloodstream form cells differentiate to procyclic forms that have fully functional mitochondria. Procyclic cells have cyanide-sensitive, cytochrome-mediated electron transport and the full complement of TCA cycle enzymes. The developmental regulation of the cytochrome c reductase complex was examined at the RNA and protein levels. RNase T1 protection studies and Northern blot analyses demonstrated that bloodstream and procyclic form cells constitutively expressed the genes for two nuclear encoded cytochrome c reductase subunits, cytochrome c1 and subunit 4. Polyadenylated transcripts of both genes were present in bloodstream form cells at up to 20% of the procyclic cell levels. These levels were significantly up-regulated sometime after the onset of differentiation to the procyclic form. Despite the presence of subunit mRNAs in bloodstream form cells, subunit proteins were not detected until the cells had been allowed to differentiate in vitro for 6 h. Procyclic cell levels of subunit proteins and holocytochromes were reached by 48 h. Our results suggest that cytochrome c reductase is developmentally regulated at multiple levels, some involving post-transcriptional mechanisms.

Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum.

Infection with Cryptosporidium parvum causes a self-limiting diarrheal illness in immunocompetent humans and is associated with the development of a serum IgG antibody response dominated by the 27-kDa and 17-kDa parasite surface antigens. Antibodies against the 27-kDa and 17-kDa antigens may serve as useful markers for past infection in population-based studies of the risk factors associated with Cryptosporidium infection. A recombinant form of the 17-kDa antigen would be useful both in epidemiologic studies and in studies of the role of the humoral response in immunity. We have partially purified and sequenced the immunodominant 17-kDa surface antigen from sporozoites, and we have cloned a 975 bp open reading frame from C. parvum that includes all of the 17-kDa antigen peptide sequences. We show immunologic identity between a recombinant form of the protein and the native 17-kDa antigen. We conclude that the carboxy-terminal fragment of the cloned protein is the authentic 17-kDa antigen.

The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored.

Cryptosporidium parvum is a protozoan parasite of the intestinal epithelium that has caused numerous outbreaks of diarrheal illness in humans. During our studies of the host immune response to C. parvum infection, we noted that two of the immunodominant surface antigens of the sporozoite stage of the parasite readily extract into Triton X-114. We recently cloned the immunodominant 17-kDa surface antigen and suggested that the carboxy-terminal peptide sequence may satisfy the requirements for GPI anchor addition. In the work presented here, we were able to show that the 17-kDa antigen could be metabolically labeled in vitro with tritiated ethanolamine and that the antigen contained myo-inositol. The antigen was cleaved by GPI-PLD but not by PI-PLC and it could be converted to a water soluble form by chemical deglycosylation. We suggest that the 17-kDa antigen is indeed GPI anchored and that the anchor contains an acylated inositol and either a lyso-acyl- or a diacyl-glycerol. We are currently working to determine what role the anchor may play in the human immune response to this antigen.

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg). T cell reactivity was greater against SAg (57%) than to Cp23 (34.3%) as measured by [3H]thymidine incorporation. Proliferative responses to Cp23 were significantly correlated with SAg responses. By enzyme-linked immunosorbent assay, most persons had IgG responses to both SAg (91.4%) and to recombinant Cp23 (88.5%). Antibody responses were greater among persons who exhibited T cell responses to SAg and Cp23. This study demonstrates that recombinant Cp23 antigen could be a useful antigen for detection of both antibody and cell-mediated responses in epidemiologic studies.

Developmental regulation of mitochondrial biogenesis in Trypanosoma brucei.

The metabolism of Trypanosoma brucei undergoes a significant change as the parasite differentiates from the mammalian bloodstream form to the form found in the tse-tse fly vector. Because the mitochondria of bloodstream form cells lack cytochromes and several key citric acid cycle enzymes, the metabolism of these cells is mostly limited to glycolysis. The reducing equivalents generated by this process are passed to oxygen by a plant-like alternative oxidase. As cells differentiate to the insect form, they begin to oxidatively metabolize proline. The mitochondria of insect form cells contain functional, cytochrome-mediated electron transport chains and have complete complements of citric acid cycle enzymes. Although the characterization is far from complete, the nuclear and mitochondrial genes involved in the expression of these mitochondrial functions appear to be developmentally regulated at posttranscriptional and posttranslational levels. This review outlines some of the molecular processes that are associated with the developmental regulation of mitochondrial biogenesis and suggests some possible mechanisms of regulation.

The effects of maximum steady state pace training on running performance.

Maximum aerobic power (VO2 max), maximum anaerobic power (AP max), submaximal exercise heart rate (HRsub), and performance times for distances of 15m, 600 m, 3.22 km, and 10 km were evaluated in 12 male runners prior to and after 7 weeks of a running programme at each individual's maximum steady-state (MSS) pace. MSS pace, a running speed at which blood lactate is believed to equal 2.2 mmol . l-1, was calculated from weekly 3.22 km runs utilising the regression equation of LaFontaine et al (1981). During the training period, the mean MSS pace increased 11.3% from 3.76 to 4.19 m.s.-1. Body weight and maximal exercise heart rate were unaffected by MSS training. However, MSS training was associated with increases (p less than 0.05) in absolute VO2 max (8.9%) and VO2 max relative to body weight (8.1%), absolute AP max (3.7%) and AP max, relative to body weight (4.3%); decreases in resting HR (5.4%) and HRsub (6.9%); and decreases in performance times for runs of 15m (1.8%), 600 m (4.4%), 3.22 km (9.6%), and 10 km (12.1%). MSS paces determined prior to the pre- and post-training 10 km races were significantly related to the pre-training (r = 0.98) and post-training 10 km (r = 0.95) performance paces. Pretraining MSS pace, maximal aerobic power, and performance times for the 3.22 km and 10 km distances were highly related to improvements in MSS pace and performance times for the 3.22 km and 10 km runs. Our findings indicate that training at MSS pace is an effective method to increase maximal aerobic and anaerobic power, and decrease performance times for short- and middle-distance running events. Pre-training running performance may predict the magnitude of improvement due to MSS pace training.

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.

Patulin biosynthesis: epoxidation of toluquinol and gentisyl alcohol by particulate preparations from Penicillium patulum.

A crude extract that catalyzes the epoxidation of toluquinol and gentisyl alcohol was isolated from cultures of Penicillium patulum. About 60% of the activity sedimented from crude extract upon centrifugation at 105,000g for 2 h, and at 30,000g for 30 min after precipitation with 30% ammonium sulfate and resuspension in buffer. The quinone epoxide phyllostine, a product of gentisyl alcohol epoxidation, has previously been shown to be an intermediate in the biosynthesis of patulin [Sekiquchi, J., & Gaucher, G. M. (1978) Biochemistry 17, 1785-1791] and was shown to be further converted to neopatulin by the extract. The epoxide product of toluquinol, desoxyphyllostine (2-methyl-5,6-epoxy-1,4-benzoquinone), has not been reported previously from fungal cultures. Its structure was confirmed by GC-mass spectrometry and proton and 13C NMR. Its CD spectrum showed the same shape and signs as that of phyllostine, indicating that it too is an enzymatic product with a similar absolute configuration. Whereas chemical epoxidation of toluquinone and gentisyl quinone occurs with hydrogen peroxide, the enzymatic epoxidation utilized oxygen and the hydroquinone. The epoxidation was inhibited by 1,10-phenanthroline, EDTA, and p-(chloromercuri)benzenesulfonic acid and by degassing with nitrogen, but no inhibition was observed with KCN, catalase, or CO. The apparent Km's were similar for the two substrates (0.17 mM for toluquinol, 0.24 mM for gentisyl alcohol), with both substrates showing inhibition at 1.0 mM. The rate of desoxyphyllostine formation was more than 10 times that of phyllostine formation at equivalent substrate concentrations. Gentisaldehyde was not a substrate for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro import of the Rieske iron-sulfur protein by trypanosome mitochondria.

Most of the proteins present in the mitochondrion are imported to that location from the cytosol. While this process has been studied extensively in fungal and mammalian systems, little work has been done in other eukaryotic organisms. We are particularly interested in the Trypanosoma brucei system because this organism developmentally regulates mitochondrial function during its life cycle and because one of the imported proteins lacks a conventional targeting sequence. We report here the development of an in vitro import system using crude trypanosome mitochondria and a nuclear encoded, mitochondrial protein. Import of the Rieske iron-sulfur protein subunit of the cytochrome c reductase complex requires a membrane potential, ATP, and a protein component on the mitochondrial surface. The precursor protein is sequentially processed to the mature form in two steps by peptidases that require divalent metal ions for activity. As in other eukaryotic systems, the first processing event occurs inside the inner membrane and is probably catalyzed by a matrix-processing protease. Surprisingly, the second processing activity is located outside the inner membrane. Both processing steps require ATP but are independent of a membrane potential. We suggest that the trypanosome iron-sulfur protein is imported along a "conservative sorting pathway" but that the assembly mechanism of the reductase complex may be unique to trypanosomes.

Applications of high-performance liquid chromatography to quantitation of metabolites and enzymes of the patulin pathway from Penicillium patulum.

Conditions for extraction and high-performance liquid chromatographic (HPLC) analysis for fourteen of the patulin pathway metabolites from Penicillium patulum are described which allow quantitation of the metabolite content of cultures at hourly intervals. The HPLC analysis is more sensitive than gas-liquid chromatographic analysis and is more quantitative than thin-layer chromatographic analysis. Separations on a preparative column allow for the collection and identification of new metabolites. The column elution program can be varied to optimize analysis time for individual metabolites, allowing individual enzymes of the pathway to be assayed by following the conversion of substrate to product. Analysis of product formation in crude enzyme mixtures can be used to assay an enzyme in the presence of subsequent enzymes of the pathway and to establish the pathway reaction sequence.

Myosin heavy chain isoform and ubiquitin protease mRNA expression after passive leg cycling in persons with spinal cord injury.

OBJECTIVE: To determine the effects of passive leg cycling exercise on myosin heavy chain (MHC) isoform and ubiquitin (UBI) protease mRNA expression in patients with spinal cord injury (SCI). STUDY DESIGN: Case series. INTERVENTION: Eight SCI subjects (5 men, 3 women) participated in a 12-week exercise program involving the Psycle ergometer. Training occurred 2 days a week at 75% of each subject's maximum heart rate. Anthropometric measures (body weight, thigh girth, and body mass index) and muscle biopsy specimens were obtained before and after training. Analyses were performed to determine the mRNA expression of types I, IIa, and IIx MHC, as well as UBI, a UBI-conjugating enzyme (E2), and 20S proteasome (20S). RESULTS: Despite small increases, paired t tests (p < .05) to assess changes from pretraining to posttraining failed to locate significant differences for the three anthropometric measures. For mRNA expression, there were significant increases in expression of MHC types IIa and IIx and significant decreases in expression for UBI, E2, and 20S. CONCLUSION: Exercise using passive leg cycling increases the expression of fast MHC isoforms while concomitantly decreasing proteolytic activity associated with muscle degradation, thus helping to possibly ameliorate muscle atrophy in patients with SCI.

In vitro evidence that covalent crosslinking of neurofilaments occurs in gamma-diketone neuropathy.

We have postulated that the toxic neuropathies associated with neurofilament-filled axonal swellings have a common pathogenesis, the covalent crosslinking of neurofilaments during anterograde transport. The newly described gamma-diketone, 3,4-dimethyl-2,5-hexanedione (DMHD), is a more potent analogue of the toxic metabolite of n-hexane, 2,5-hexanedione. The axonal swellings observed in DMHD toxicity are in the proximal axon, as seen in intoxication with beta, beta'-iminodipropionitrile, rather than in the distal axon, where neurofilamentous swellings are observed in n-hexane, carbon disulfide, and acrylamide neurotoxicity. In these studies, 14C-labeled DMHD and 2-butanone were synthesized and allowed to react with peripheral nerve. Only 14C-labeled DMHD resulted in stable radiolabeled protein polymers, which were retained by nitrocellulose filters with pore sizes as large as 12 microns. More specific evidence for covalent crosslinking of neurofilaments was obtained when DMHD was allowed to react with peripheral nerve in which the neurofilaments had been pulse-labeled with L-[35S]methionine.

The Serologic response to Cryptosporidium in HIV-infected persons: implications for epidemiologic research.

Advances in serologic assays for Cryptosporidium parvum have made serology an attractive surveillance tool. The sensitivity, specificity, and predictive value of these new assays for surveillance of immunocompromised populations, however, have not been reported. Using stored serum specimens collected for the San Francisco Men's Health Study, we conducted a case-control study with 11 clinically confirmed cases of cryptosporidiosis. Based on assays using a 27-kDa antigen (CP23), the serum specimens from cases had a median response immunoglobulin (Ig) G level following clinical diagnosis (1,334) and a net response (433, change in IgG level from baseline) that were significantly higher than their respective control values (329 and -32, Wilcoxon p value = 0.01). Receiver operator curves estimated a cutoff of 625 U as the optimal sensitivity (0.86 [0.37, 1.0]) and specificity (0.86 [0.37, 1.0]) for predicting Cryptosporidium infection. These data suggest that the enzyme-linked immunosorbent assay technique can be an effective epidemiologic tool to monitor Cryptosporidium infection in immunocompromised populations.

Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens.

Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.