We have reported that clinically isolated Prevotella nigrescens strain 22 (strain 22) produced large amounts of exopolysaccharide (EPS) and that this EPS might play an important role as a virulent ...factor. However, EPS production decreaces with repeated subcultures. In this study, we attempted to establish a method of animal passage that maintains the EPS production of strain 22 at high levels. We also examined the protein expression patterns of strain 22 before and after animal passage using 2-D electrophoresis. EPS production of strain 22 increased after the first animal passage, and continued to increase until the third passage. Mesh-like structures around the cells became significantly denser after animal passage. N-terminal sequence analysis of proteins revealed that three conspicuously up-regulated spots on a 2-D gel after animal passage were the homologs of bacterial heat shock proteins : chaperonin protein DnaK (HSP 70), 60kDa chaperonin (GroEL), and 10kDa chaperonin (GroES). We concluded that this method of animal passage could be used to maintain EPS production of P. nigrescens at a high level, and that the bacterial heat shock protein homologs up-regulated by the animal passage might be involved in a regulatory pathway of EPS production in P. nigrescens under stressful conditions.
Leishmaniasis is one of the major vectors borne parasitic disease recognized by World Health Organization (WHO) and this disease is endemic in tropical and sub-tropical countries of the world ...including Pakistan. There are three main clinical forms of Leishmaniasis caused by different species of parasite belonging to the genus Leishmania. The detection of Leishmania parasites can be confirmed by various methods like Direct Agglutination Test (DAT), Enzyme Linked Immuno-sorbent Assay (ELISA), Immunofluorescence Assay (IFA), Polymerase Chain Reaction (PCR), direct microscopy and culture technique. In vitro cultivation of parasite plays an important role in study and treatment of the disease. The objective of present study was to evaluate the positivity of direct microscopy and culture technique for detection of Leishmania parasite. The study and laboratory investigations were conducted at Centre of Excellence in Science and Technology (CESAT), Islamabad- Pakistan from January, 2001 to December, 2003. Parasites were isolated from suspected patients to Cutaneous Leishmaniasis (CL) referred by local hospital. Most of these patients were deployed in different regions of Baluchistan where Cutaneous Leishmaniasis is endemic. They were all males & age ranged from 21 - 52 years. They had one or more nodules/ lesions mainly on exposed areas of the body. History of visited in endemic area was one of the inclusion criteria. Organisms were isolated from material obtained by needle aspiration or from biopsy samples from the edges of the lesions using insulin syringe. The biopsy material was homogenized in sterile normal saline. Needle aspirates and tissue homogenates were inoculated into Novy, McNeal Nicole (NNN) Medium. The solid phase of the NNN media contained fresh, aseptically collected defabrinated rabbit blood, mixed with agar and gentamicin. The liquid phase NNN media comprised of 0.85% physiological saline. The Isolates were incubated at 22 - 25°C. Microscopy was done after 48 hours and repeated after 72 hours & than till 02 week for presence of promastigotes. For all patients, the questionnaire was filled. The recorded data included important clinical & social details. Saline aspirate was obtained from 232 patients suspected to CL. Out of 232 samples 87(37.5%) had positive results after microscopic analysis while 138(59.4%) samples showed negative results on microscopic examination and 07(3.017%) cultures were contaminated. These Local isolated Leishmania strains were identified as Leishmania major. The virulence of these organisms improves through animal passage. These Leishmania cultures are being maintained in NNN media for further R & D work on Cutaneous Leishmaniasis.
The closely related species Campylobacter jejuni and C. coli are now
recognized as important agents of diarrhea in both developed and
developing countries.
An experimental study of five isolates of
Aeromonas jandaei and 12 of
A. trota was carried out to examine if they produced an enterotoxic substance, and if so, to characterise that factor and to see ...if it caused any mucosal damage. Only two of the
A. trota strains caused fluid accumulation in the initial rabbit ileal loop (RIL) tests. The remaining strains did so only after one to five sequential passages through RILs and once they caused a secretory response they showed a gradual enhancement of fluid outpouring after each subsequent passage. Inocula of ∼1×10
5 viable cells and 0.25 ml of culture filtrate caused fluid accumulations comparable to those of toxigenic
V. cholerae 569B. The enterotoxic factors of both organisms were inactivated when held at 56°C for 20 min or 65°C for 10 min and showed biological activity over a wide range of pH. The only histopathological change observed in the ileal loop was depletion of mucus from the goblet cells. These data thus indicate that strains of
A. jandaei and
A. trota may produce a heat-labile and pH-stable diarrhoeagenic substance that causes little or no damage to the intestinal mucosa, like that of other known heat-labile enterotoxins.
Red raspberry (Rubus idaeus L.) seeds germinate only after seed coats are degraded. In nature this happens slowly. Seeds from recently collected fruit (fresh to four years old) germinated only after ...scarification of the seed coat by 20-minute soaking in concentrated sulfuric acid. Germination was not enhanced by: (1) short-term intermittent soaking, up to 81 hours, in dilute (0.01 normal) hydrochloric acid; (2) passage through the digestive tracts of bears, coyotes, or birds; (3) physical perturbations such as nicking, mechanical scarification, repeated freezing and thawing and/or four years of exposure in the field; (4) exposure to light; (5) increased temperatures or temperature fluctuations; or (6) addition of nitrogen (ammonium nitrate, urea).
Rat2 cells are thymidine kinase-deficient derivatives from the immortalized rat embryo cell line Rat1. They show no phenotypic correlates of malignancy in vitro and produce tumors in syngeneic ...Fischer rats after long latency periods. We have investigated how transfection with oncogenes would alter the in vitro and in vivo behavior of Rat2 cells. Thus we have manipulated Rat2 cultures in various ways. The cell lines obtained were categorized as parental, in vitro subclones, untransfected in vivo derivatives, non-oncogene (neor and tk) transfectants, oncogene (mutated c-Ha-ras, polyoma middle-T, FBR v-gag-fos-fox) transfectants, and in vivo derivatives of transfectants. They were tested in vitro for morphotype, colony formation in soft agar, growth in organ culture, invasion in organ culture, and in vivo for latency period of tumor formation, tumor growth rate, invasiveness, and metastasis. Differences between the consequences of various manipulations were found in the number of malignancy-related phenotypic alterations. The following trend could be deduced from our data: induction of invasiveness in organ culture by all manipulations; morphotypic transformation and shortening of tumor-latency period by all oncogene transfections and by passage with tumor formation in vivo; growth in organ culture and increased tumor growth rate in vivo by transfection with ras-, or fos-oncogenes and by passage in vivo. Metastatic capability (present in parental Rat2 cell tumors) and colony formation in soft agar (absent in Rat2 cells) were not affected by the present manipulations. We concluded that differences between the oncogene-transfectants and the untransfected in vivo derivatives do not lie in the expression of malignancy-related phenotypes but in the time needed to acquire them.
Abstract
An experimental study of five isolates of Aeromonas jandaei and 12 of A. trota was carried out to examine if they produced an enterotoxic substance, and if so, to characterise that factor ...and to see if it caused any mucosal damage. Only two of the A. trota strains caused fluid accumulation in the initial rabbit ileal loop (RIL) tests. The remaining strains did so only after one to five sequential passages through RILs and once they caused a secretory response they showed a gradual enhancement of fluid outpouring after each subsequent passage. Inocula of ∼1×105 viable cells and 0.25 ml of culture filtrate caused fluid accumulations comparable to those of toxigenic V. cholerae 569B. The enterotoxic factors of both organisms were inactivated when held at 56°C for 20 min or 65°C for 10 min and showed biological activity over a wide range of pH. The only histopathological change observed in the ileal loop was depletion of mucus from the goblet cells. These data thus indicate that strains of A. jandaei and A. trota may produce a heat-labile and pH-stable diarrhoeagenic substance that causes little or no damage to the intestinal mucosa, like that of other known heat-labile enterotoxins.
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