Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium.
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Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration. (+info)
A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.
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The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites. (+info)
Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.
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Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria. (+info)
A review of Gymnophalloides seoi (Digenea: Gymnophallidae) and human infections in the Republic of Korea.
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Studies on Gymnophalloides seoi (Digenea: Gymnophallidae) and human infections are briefly reviewed. This minute intestinal fluke was first discovered from a Korean woman suffering from acute pancreatitis and gastrointestinal troubles. It was described as a new species by Lee, Chai and Hong in 1993. The southwestern coastal village where the patient resided was found to be a highly endemic area, and additional endemic areas have been identified. The parasite is very small, 0.33-0.50 mm long and 0.23-0.33 mm wide, and characterized by the presence of a ventral pit. The first intermediate host remains unknown, but the second intermediate host has been found to be the oyster Crassostrea gigas. Man and the Palearctic oystercatcher Haematopus ostralegus have been shown to be natural definitive hosts, and wading birds including the Kentish plover Charadrius alexandrinus are highly susceptible to experimental infection. Gerbils, hamsters, cats, and several strains of mice were also susceptible laboratory hosts. In experimentally infected mice, the parasites inhabit the small intestine, pinching and sucking the root of villi with their large oral suckers, but they did not invade beyond the mucosa in immunocompetent mice. However, they were found to invade the submucosa in immunosuppressed mice. Human G. seoi infections have been found in at least 25 localities; 23 islands on the Yellow Sea or the South Sea, and 2 western coastal villages. The highest prevalence was found in a village on Aphaedo, Shinan-gun (49% egg positive rate); other areas showed 0.8-25.3% prevalence. Infected people complained of variable degrees of gastrointestinal troubles and indigestion. The infection can be diagnosed by recovery of eggs in the feces; however, an expert is needed to identify the eggs. Praziquantel, 10 mg/kg in single dose, is effective for treatment of human infections. Eating raw oysters in endemic areas should be avoided. (+info)
Antibodies that neutralize SIV(mac)251 in T lymphocytes cause interruption of the viral life cycle in macrophages by preventing nuclear import of viral DNA.
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Previous reports from our lab had shown that sera obtained from SIV(mac)-infected animals neutralized SIV(mac) infectivity in CD4(+) T cells but failed to protect monkey primary macrophages from infection with the virus. However, the antibodies could inhibit completion of the viral life cycle in the macrophages at the postentry stage(s). In this report we examined the mechanisms of the late effect of the antibodies. Using monoclonal antibodies (MAbs), we demonstrated that only antibodies to the SIV envelope protein (KK17 and KK42) but not antibody to the viral core protein (FA2) had the same inhibitory effect as that of the anti-SIV sera. To identify the stage of the viral replication cycle that was inhibited by anti-SIV antibodies in macrophages, we used various PCR techniques to study viral entry/reverse transcription (by amplifying the viral gag gene), viral genome nuclear transport (by amplifying 2-LTR circular forms), viral integration (by Alu-PCR assay), and viral protein expression (by RIPA). We found that in macrophage cultures inoculated with SIV(mac)251 that were preincubated with antienvelope MAbs, viral DNA was detected at 8 h postinoculation but the 2-LTR circular forms and integrated viral DNAs were undetectable, and viral proteins were not expressed in these infected macrophages. These results strongly suggested that anti-SIV antibodies inhibited SIV(mac) replication in macrophages by blocking nuclear transport of viral genomes since viral DNA could not be detected in the nuclei of treated cultures. Furthermore, we showed that although viral replication in macrophages was interrupted by the antibodies, when cocultured with permissive T cells, the viral genomes presented in the cytoplasm of the macrophages could readily transfer to T cells during cell-cell contact. Importantly, this transfer could not be prevented by the antibodies. These results might explain the failure of passive antibody immunization against SIV(mac)251--a critical obstacle in AIDS vaccine development. (+info)
Susceptibility of Schistosoma japonicum to praziquantel in China.
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To look for possible evidence of the development of resistance in Schistosoma japonicum to praziquantel, we conducted a field study in China. During the non-transmission period of schistosomiasis a random sample of 2860 individuals from six villages in three provinces of China were examined using a parasitological stool examination. Of the 372 stool-positive subjects, 363 subjects were treated with a single oral dose of 40 mg/kg of praziquantel. Six to Seven weeks after treatment, of 334 subjects examined using the same stool examination, stool-negative results were found in 319 patients which represents a 95.5% parasitologic cure rate. Fifteen subjects still excreting eggs were treated a second time with the same dose of praziquantel. All stool samples, including those from participants re-treated with praziquantel, were re-examined 12 weeks after the first treatment and no stool-positive subjects were found. The results indicate that there was no evidence for reduced susceptibility of S. japonicum to praziquantel despite its extensive use in the main endemic areas of China for more than 10 years. The in vitro responses to praziquantel of cercariae, miracidia and eggs of S. japonicum compared with S. mansoni demonstrate that the cercariae, miracidia and eggs of S. japonicum are more sensitive to praziquantel than those of S. mansoni. More sensitive worms would be less likely to develop resistance and this could explain why no evidence for resistance was found in S. japonicum in China. (+info)
Experimental evidence for a demographic cline in Panstrongylus megistus populations.
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The population biology of three populations of Panstrongylus megistus was compared to determine possible influence on the behaviour and epidemiological importance of this species. The results demonstrated differences in terms of egg eclosion time, nymphal mortality and development rates, and feeding and defaecation rates. These differences appeared to follow a geographical cline, primarily reflecting different degrees of adaptation to domestic habitats. (+info)
Biology of Triatoma flavida Neiva, 1911 (Hemiptera: Reduviidae) under laboratory conditions.
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The complete life cycle of Triatoma flavida, weekly fed on hens, was studied at 28+/-2 degrees C and 80+/-10% RH. Aspects related to hatching, life span, mortality and feeding behavior for each stage of its life cycle were evaluated. The hatching rate observed for 100 eggs was 93% with an average incubation period of 27.2 days. Sixty-two nymphs completed the cycle and the mean egg to adult development time was 230.4 days. Mean duration of 1st, 2nd, 3rd, 4th and 5th instar nymphs was 22.1, 25.3, 36.7, 49.7 and 69.4 days, respectively. The number of blood meals on each nymphal stage varied from 1 to 7. The mortality rate was 6.5% for NI, 23% for NIII and 7.5% for NV nymphs. Mean number of laid eggs per female was 283.1. Adult survival rates were 344.8 +/- 256.4 days for males and 285.3 +/- 201.8 days for females. (+info)