Characterization of subtilisin-like serine proteases in trichomonas vaginalis

Trichomonas vaginalis is the etiologic agent causing human trichomoniasis, a sexually transmitted infection found worldwide. In this study, subtilisin-like serine proteases (subtilases) were examined for their putative role in cell viability. Published data of other eukaryotic protozoan parasites confirm the importance of subtilases for adhesion and invasion. Serine protease inhibitor assays show that 3,4 DIC and TPCK inhibited cell growth from 79.6 to 98.4% respectively. To examine subtilases in more detail, a number of putative subtilases were cloned from T vaginalis. Two proteases were expressed recombinantly for antiserum production. TvSUB-12 (SP12) was localized to the posterior cell surface as using immunofluorescence and a kexin homolog called TvSUB-6 (SP6), revealed perinuclear staining. This study illustrated an essential role for subtilases in trichomonad cell viability and preliminary examination of two specific serine proteases revealing two different locations in the cell. To date, both of these proteases have never been characterized in this important human parasite

Characterization of subtilisin-like serine proteases in trichomonas vaginalis

Trichomonas vaginalis is the etiologic agent causing human trichomoniasis, a sexually transmitted infection found worldwide. In this study, subtilisin-like serine proteases (subtilases) were examined for their putative role in cell viability. Published data of other eukaryotic protozoan parasites confirm the importance of subtilases for adhesion and invasion. Serine protease inhibitor assays show that 3,4 DIC and TPCK inhibited cell growth from 79.6 to 98.4% respectively. To examine subtilases in more detail, a number of putative subtilases were cloned from T vaginalis. Two proteases were expressed recombinantly for antiserum production. TvSUB-12 (SP12) was localized to the posterior cell surface as using immunofluorescence and a kexin homolog called TvSUB-6 (SP6), revealed perinuclear staining. This study illustrated an essential role for subtilases in trichomonad cell viability and preliminary examination of two specific serine proteases revealing two different locations in the cell. To date, both of these proteases have never been characterized in this important human parasite

Cloning and expression of a toxin-associated protein from Clostridium botulinum serotype E

Clostridium botulinum, the agent of botulism, infects several individuals each year as it raises concerns regarding bioterrorism and food safety. Current antitoxins can treat botulism’s toxic effects, but prevention is preferable. This can be achieved through a sensitive and accurate detection of the toxin and/or its accessory proteins in food. To support this effort, botulism accessory proteins were recombinantly expressed and purified to generate monoclonal antibodies which may serve as probes for detecting the toxin. We cloned by PCR one such accessory protein, called p48, from C. botulinum serotype E. We first ligated the PCR product into the PCR 2.1 and then into the expression vector PQE80. Regulated expression was analyzed and confirmed via western blotting prior to protein purification

In vitro Effect of Sanguinarine and Chelerythrine on Trichomonas vaginalis Trophozoites

Trichomonas vaginalis is a parasitic protozoan that causes the STD trichomoniasis showing physiological symptoms in women and asymptomatic symptoms in men. To date, there are two FDA approved treatments for this disease, metronidazole and tinidazole. Currently, patient strains are becoming increasingly resistant and implements for new drug targets are needed. In this study, sanguinarine (SANG) and chelerythrine (CHE) were analyzed as additional chemotherapeutic treatments for trichomoniasis. This experiment aimed to assess the inhibitory effects of SANG and CHE on human trichomonad strains T1 and G3. Multiple drug assays with varying concentrations of SANG and CHE were performed to estimate the IC50, the concentration of the drug that would inhibit the growth of the parasites by 50%. The cell lines were tested in three environmental conditions: aerobic, anaerobic, and anaerobic with no ascorbic acid. Ascorbic acid is an antioxidant with proposed negative effects on SANG and CHE potency. Its activity was used to help discover the SANG and CHE mechanism. Cells were incubated for 24 hours (assay counts +/- 1 hr.) at 37C. IC50 molarities were skewed towards a narrow degree ranging from 1-10M exemplifying the potency. The data showed a trend of decreasing percent survival parallel to the increase of concentration of SANG and CHE. Previous data showed G3 strains were slightly more virulent than T1. In aerobic and anaerobic conditions, T1 had higher survival rates than G3. In anaerobic no ascorbic acid conditions, T1 generally had less survival than G3. The absence of ascorbic acid demonstrated increased SANG and CHE potency. This experiment utilized very low concentrations of SANG and CHE with high effectiveness. The data supports the hypothesis that these are possible drugs to go further in analysis as alternative forms of treatment

Characterization of Serine Proteases in Trichomonas vaginalis

Trichomonas vaginalis, an anaerobic protozoan parasite, causes trichomoniasis the most prevalent non-viral STD. While males are usually asymptomatic, females may have symptoms such as inflammation and vaginal secretions. Trichomoniasis is also a cause of many complications during pregnancy. Infection is treated with metronidazole or tinidazole.However, there is a need to develop alternative chemotherapies because of drug resistance. Serine proteases have been shown to be virulence factors in various parasites such as Toxoplasma gondii and Entamoeba histolytica. T. gondii invasion of host cells has been shown to be blocked by serine protease inhibitors. T. vaginalis has been suspected to have serine protease activity, but this enzyme has not been studied in any detail. We have searched the T. vaginalis genome database and found potential serine protease candidate genes. We have cloned 10 of these genes using PCR with the goal of ultimately expressing these genes for recombinant protein expression. Currently we are also studying the effect of serine protease inhibitors on T. vaginalis growth

In vitro Effect of Sanguinarine and Chelerythrine on Trichomonas vaginalis Trophozoites

Trichomonas vaginalis is a parasitic protozoan that causes the STD trichomoniasis showing physiological symptoms in women and asymptomatic symptoms in men. To date, there are two FDA approved treatments for this disease, metronidazole and tinidazole. Currently, patient strains are becoming increasingly resistant and implements for new drug targets are needed. In this study, sanguinarine (SANG) and chelerythrine (CHE) were analyzed as additional chemotherapeutic treatments for trichomoniasis. This experiment aimed to assess the inhibitory effects of SANG and CHE on human trichomonad strains T1 and G3. Multiple drug assays with varying concentrations of SANG and CHE were performed to estimate the IC50, the concentration of the drug that would inhibit the growth of the parasites by 50%. The cell lines were tested in three environmental conditions: aerobic, anaerobic, and anaerobic with no ascorbic acid. Ascorbic acid is an antioxidant with proposed negative effects on SANG and CHE potency. Its activity was used to help discover the SANG and CHE mechanism. Cells were incubated for 24 hours (assay counts +/- 1 hr.) at 37C. IC50 molarities were skewed towards a narrow degree ranging from 1-10M exemplifying the potency. The data showed a trend of decreasing percent survival parallel to the increase of concentration of SANG and CHE. Previous data showed G3 strains were slightly more virulent than T1. In aerobic and anaerobic conditions, T1 had higher survival rates than G3. In anaerobic no ascorbic acid conditions, T1 generally had less survival than G3. The absence of ascorbic acid demonstrated increased SANG and CHE potency. This experiment utilized very low concentrations of SANG and CHE with high effectiveness. The data supports the hypothesis that these are possible drugs to go further in analysis as alternative forms of treatment

Cloning and expression of a toxin-associated protein from Clostridium botulinum serotype E

Clostridium botulinum, the agent of botulism, infects several individuals each year as it raises concerns regarding bioterrorism and food safety. Current antitoxins can treat botulism’s toxic effects, but prevention is preferable. This can be achieved through a sensitive and accurate detection of the toxin and/or its accessory proteins in food. To support this effort, botulism accessory proteins were recombinantly expressed and purified to generate monoclonal antibodies which may serve as probes for detecting the toxin. We cloned by PCR one such accessory protein, called p48, from C. botulinum serotype E. We first ligated the PCR product into the PCR 2.1 and then into the expression vector PQE80. Regulated expression was analyzed and confirmed via western blotting prior to protein purification

Characterization of Serine Proteases in Trichomonas vaginalis

Trichomonas vaginalis, an anaerobic protozoan parasite, causes trichomoniasis the most prevalent non-viral STD. While males are usually asymptomatic, females may have symptoms such as inflammation and vaginal secretions. Trichomoniasis is also a cause of many complications during pregnancy. Infection is treated with metronidazole or tinidazole.However, there is a need to develop alternative chemotherapies because of drug resistance. Serine proteases have been shown to be virulence factors in various parasites such as Toxoplasma gondii and Entamoeba histolytica. T. gondii invasion of host cells has been shown to be blocked by serine protease inhibitors. T. vaginalis has been suspected to have serine protease activity, but this enzyme has not been studied in any detail. We have searched the T. vaginalis genome database and found potential serine protease candidate genes. We have cloned 10 of these genes using PCR with the goal of ultimately expressing these genes for recombinant protein expression. Currently we are also studying the effect of serine protease inhibitors on T. vaginalis growth

Elevação de hormônio tireoestimulante (TSH) após as lobectomias: incidência e fatores associados

OBJETIVO: Determinar a freqüência de elevação da dosagem sérica do hormônio tireoestimulante (TSH) em pacientes submetidos à lobectomia da tireóide, em um período de até 12 semanas após a operação, buscando fatores associados à sua ocorrência. MÉTODO: Foram analisados retrospectivamente 88 pacientes submetidos à lobectomia da tireóide no Serviço de Cirurgia de Cabeça e Pescoço do Hospital das Clínicas da FMUSP, no período de setembro de 2002 a setembro de 2004. Realizaram-se dosagens de hormônios tireoideanos a partir de quatro semanas após a cirurgia. Excluíram-se os pacientes com dosagens hormonais pré-operatórias alteradas, os casos que necessitaram de totalização da tireoidectomia e também aqueles em que houve perda do seguimento pós-operatório. Foram analisados os dados quanto à idade e ao sexo dos pacientes, quanto à presença de tireoidite no estudo histopatológico da tireóide e quanto ao tempo de aparecimento do hipotireoidismo. A análise estatística dos dados obtidos foi realizada através do teste qui-quadrado de Pearson. RESULTADOS: Dos 88 pacientes, 71 (80,7%) eram mulheres. A idade média foi de 41,7 anos. Observou-se elevação do TSH em 20 (22,73%) dos 88 pacientes estudados. Não foi observada diferença estatisticamente significante na incidência de elevação do TSH, quando analisados quanto ao sexo, à idade ou à presença de tireoidite. CONCLUSÃO: A elevação do TSH é freqüente após lobectomias da tireóide e ocorre, muitas vezes, precocemente após a cirurgia. Não se encontraram, neste estudo, fatores que pudessem predizer sua ocorrência a curto prazo