Interplay of H2A deubiquitinase 2A-DUB/Mysm1 and the p19(ARF)/p53 axis in hematopoiesis, early T-cell development and tissue differentiation.

Monoubiquitination of core histone 2A (H2A-K119u) has a critical role in gene regulation in hematopoietic differentiation and other developmental processes. To explore the interplay of histone H2A deubiquitinase Myb-like SWIRM and MPN domain containing1 (2A-DUB/Mysm1) with the p53 axis in the sequential differentiation of mature lymphocytes from progenitors, we systematically analyzed hematopoiesis and early T-cell development using Mysm1(-/-) and p53(-/-)Mysm1(-/-) mice. Mysm1(-/-) thymi were severely hypoplastic with <10% of wild-type cell numbers as a result of a reduction of early thymocyte progenitors in context with defective hematopoietic stem cells, a partial block at the double-negative (DN)1-DN2 transition and increased apoptosis of double-positive thymocytes. Increased rates of apoptosis were also detected in other tissues affected by Mysm1 deficiency, including the developing brain and the skin. By quantitative PCR and chromatin immunoprecipitation analyses, we identified p19(ARF), an important regulator of p53 tumor suppressor protein levels, as a potential Mysm1 target gene. In newly generated p53(-/-)Mysm1(-/-) double-deficient mice, anomalies of Mysm1(-/-) mice including reduction of lymphoid-primed multipotent progenitors, reduced thymocyte numbers and viability, and interestingly defective B-cell development, growth retardation, neurological defects, skin atrophy, and tail malformation were almost completely restored as well, substantiating the involvement of the p53 pathway in the alterations caused by Mysm1 deficiency. In conclusion, this investigation uncovers a novel link between H2A deubiquitinase 2A-DUB/Mysm1 and suppression of p53-mediated apoptotic programs during early lymphoid development and other developmental processes.

Relationship of body compositional and nutritional parameters with blood pressure in adults.

BACKGROUND: Obesity has been regarded as a single best predictor and major controllable contributor to hypertension. The present study aimed to assess the relationship between body compositional and nutritional parameters with blood pressure in rural Bengalee adults. METHODS: Anthropometric measures, blood pressure and nutritional parameters were measured in 522 rural Bengalee adults using standard protocols. Receiver operating characteristic analysis was carried out to identify cut-off values of body mass index (BMI) and percentage of body fat (BF%) as associated factors of hypertension and hypotension. RESULTS: Most of the subjects were normotensive. However, a notable percentage had hypertension (males: 21.86%; females: 15.27%), although the prevalence of hypotension was low (males: 10.53%; females: 8.73%). Obesity indicators were significantly higher in hypertensive individuals than hypotensive and normotensive individuals. All anthropometric parameters and obesity indicators were significantly correlated (P < 0.001) with blood pressure. Blood pressure increased steadily from being underweight through to normal and then to overweight/obese individuals. There were significant differences in the percentage of hypertension and hypotension between nutritional categories. Blood pressure had significant positive correlation with energy, carbohydrate and fat intake, whereas protein and calcium were negatively associated with blood pressure. The suggested cut-off values of BMI and BF%, which were taken as associated factors of hypertension, were 21.86 kg m⁻² and 20.31%, and those of hypotension were 18.18 kg m⁻² and 13.3%, respectively. CONCLUSIONS: In lean rural Bengalee populations, high BMI levels may be associated with an increased risk of hypertension. The cut-off values of BMI and BF% in the present study suggested associated factors for cardiovascular risk factors and these values may be of help with respect to reducing mean population blood pressure levels.

IKKß-I-κBɛ-c-Rel/p50: a new axis of NF-κB activation in lung epithelial cells.

Cigarette smoke (CS), a major risk factor for developing lung cancer, is known to activate transcriptional activator nuclear factor kappa B (NF-κB). However, the underlying mechanism of this activation remains unclear because of conflicting reports. As NF-κB has a pivotal role in the generation and maintenance of malignancies, efforts were targeted towards understanding its activation mechanism using both ex vivo and in vivo studies. The results show that CS-induced NF-κB activation mechanism is different from that of other pro-inflammatory signals such as lipopolysaccharide (LPS). The NF-κB dimer that translocates to the nucleus upon stimulation with CS is predominantly composed of c-Rel/p50 and this translocation involves degradation of I-κBɛ and not I-κBα. This degradation of I-κBɛ depends on IKKß activity, which preferentially targets I-κBɛ. Consistently, CS-activated form of IKKß was found to be different from that involved in LPS activation as neither Ser177 nor Ser181 of IKKß is crucial for CS-induced NF-κB activation. Thus, unlike other pro-inflammatory stimulations where p65 and I-κBα have a central role, the predominantly active signaling cascade in CS-induced NF-κB activation in the lung epithelial cells comprises of IKKß-I-κBɛ-c-Rel/p50. Thus, this study uncovers a new axis of NF-κB activation wherein I-κBɛ and c-Rel have the central role.

Potentiation by cigarette smoke of macrophage function against Leishmania donovani infection.

OBJECTIVE: Cigarette smoke is able to induce the generation of reactive oxygen species and pro-inflammatory cytokines, which are mediators of macrophage function and therefore, we have investigated the ability of cigarette smoke to activate Leishmania donovani infected peritoneal macrophage. MATERIALS AND METHODS: Cultured peritoneal macrophages were either left untreated or treated with aqueous cigarette smoke extract prior to L. donovani infection. Parasite burden was assessed by giemsa staining. The level of intracellular reactive oxygen species was determined by FACS analysis. PCR was performed to analyze mRNA levels of cytokines. NF- kappaB activity was assessed by EMSA and reporter assay. RESULTS: A pre-treatment with cigarette smoke extract (CSE) causes a decrease in parasite burden, an increase in intracellular ROS level, up-regulation of pro-inflammatory cytokines, reduced expression of immuno-suppressive cytokine and boosting of NF-kappaB activity in L. donovani-infected macrophage. CONCLUSION: Low concentration of cigarette smoke extract (CSE) counteracts L. donovani infection-mediated suppression of macrophage function without affecting host cell viability. This study reveals a new role of cigarette smoke extract (CSE) as an activator of macrophage function.

Adsorption of cadmium on husk of Lathyrus sativus: physico-chemical study.

Adsorption of cadmium (II) from aqueous solution by low-cost biosorbents was investigated. Husk of Lathyrus sativus (HLS) was found to be the most efficient in this respect and removed approximately 95% of the metal. The influence of pH, temperature, contact time and metal ion concentration on the adsorption process by HLS was studied. Hydrogen ion concentration of the solution greatly influenced the process with an optimum at pH 5.0-6.0, whereas temperature had no significant effect. The process was very fast and more than 90% of the total adsorption took place within the first 5 min and was found to follow pseudo-second order rate kinetics. The adsorption data can better be explained by Langmuir isotherm model and the calculated maximum adsorption capacity was 35 mg/g of HLS at pH 5.0 and 30 degrees C. Scanning electron micrographs showed that cadmium was present as micro precipitate on the surface of the adsorbent. Cadmium replaced calcium of the biomass as revealed from the EDX analysis indicating that the adsorption proceeds through ion exchange mechanism. Cadmium could be desorbed from the loaded biomass by lowering pH approximately 1.0 with mineral acid.

Modulation of tumor induced angiogenesis in Ehrlich ascites tumor.

In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal administration of this enzyme reduces the number of tumor directed capillaries in solid and ascites tumor bearing Swiss mice induced by transplantation of Ehrlich ascites cells. The enzyme has a critical role in regulating the secretion of vascular endothelial growth factor (VEGF) from tumor cell and in turn tumor growth. Glutamine analogue like 6-diazo, 5- oxo L-norleucine (DON) is also found to be effective in regulating vascular endothelial growth factor (VEGF) secretion from tumor cells in vitro. Treatment with enzyme reduced serum VEGF levels of the tumor induced animals. In vitro VEGF production by EAC cells was reduced in a concentration dependent manner in presence of glutamine analogue.

Physiological characters of Sporothrix schenckii isolates.

Sporotrichosis is endemic in three regions (east, north and south) in India. The colony morphology and physiological characteristics of 49 clinical isolates from these three regions (25 from north India, 17 from east India and seven from south India) were analysed in both mycelial and yeast forms. No difference in colony character was seen among the 49 isolates on three different media. Growth of all isolates was inhibited at 40 degrees C. The yeast forms were found to be more tolerant to osmotic pressure and salt concentrations. Most mycelial forms grew well between pH 3-12.0 whereas most yeast forms could tolerate a pH range of 2.4 to 9.5. Variations in assimilation of arabinose, dextrin, raffinose, rhamnose and starch was observed among strains from different geographical regions. The yeast forms did not show any urease activity but the mycelial forms of all isolates could split urea. Phenol oxidase and potassium nitrate assimilation were positive and gelatinase activity and casein hydrolysis were negative for all isolates.

Efficacy of topical and systemic itraconazole as a broad-spectrum antifungal agent in mycotic corneal ulcer. A preliminary study.

PURPOSE: To evaluate the efficacy of topical (1%) and systemic itraconazole against common fungi such as Aspergillus and other filamentous fungi that cause mycotic corneal ulcer. METHODS: A prospective randomised, controlled study was done in 54 clinically suspected cases of fungal keratitis of which 44 were culture proven. Half the cases (n=27) with superficial involvement were treated with only topical itraconazole (1%) and the other half were treated with both topical and systemic itraconazole. RESULTS: Aspergillus, Penicillium and Fusarium were the most common fungi isolated. The ulcer resolved in 42 eyes (77%) and 12 eyes (23%) did not respond well to treatment. Four of 12 non-responding eyes were caused by Fusarium species. CONCLUSION: Itraconazole, given either topically or systemically, is effective in treating mycotic corneal ulcers.

Effect of purified glutaminase from human ascites fluid on experimental tumor bearing mice.

Glutamine is the major respiratory fuel and energy source of the rapidly proliferating tumor cells and that is why glutamine clearance by glutaminase therapy provides an opportunity to fight against the neoplasm. Glutaminase from bacterial source was tried on experimental models but had to be excluded because of its limited efficacy. Search for a better glutaminase continued exploiting the mammalian sources. In the present study, glutaminase purified from human ovarian cancer ascites fluid was used in experimental solid and ascites mice model alone and in combination with Cu-Sulphate and heparin. Cumulative findings indicate that the enzyme alone is quite effective in lowering tumor burden and reducing not only the tumor induced angiogenesis, but also an angiogenic inducer, heparin mediated angiogenesis. However, the presence of Cu with the enzyme, amplified the antineoplastic response by improving anti-angiogenic potential and hematological status of the tumor bearing host. Therefore, Cu-glutaminase combination strengthened the hypothesis that together they may provide a better therapeutic regimen in experimental mice tumor model.

Localization of phosphate dependent glutaminase in ascites fluid of ovarian cancer patient.

Phosphate dependent glutaminase was purified from ascites fluid of ovarian cancer patients. The purified enzyme showed a final specific activity of 110 unit / mg protein with 72 fold purification and 21% yield. Purified enzyme gives one dark band of Mr approximately 65.5 KD and two light bands of Mr approximately 47.5 KD and approximately 45 KD respectively on 10% SDS-PAGE. One major immunoreactive band was found in trans-immunoblot analysis using antibodies against rat kidney and ascites fluid glutaminase raised in rabbit and mice respectively. Phosphate dependent glutaminase enzyme purified from mitochondria of malignant and non malignant ovarian tissue also showed bands of same molecular weight on 10% SDS-PAGE and gave same immunoreactive bands in trans-immunoblot like the purified glutaminase from ascites fluid. This result was confirmed by using the specific activity stain for glutaminase, which indicates that same enzyme activity is probably due to leakage of the same enzyme from malignant tissue into the ascites fluid. The purified enzyme from human peritoneal fluid showed a high specificity toward glutamine, therefore is a true glutaminase. Moreover, ascites fluid taken from patients of different age group with different stages of ovarian carcinoma revealed the presence of same glutaminase on 10% SDS-PAGE, and exhibited immunoreaction on ELISA, trans-immunoblot and dot immunoblot analysis.

A general survey of glutamine level in different tissues of murine solid tumor bearing mice before and after therapy with purified glutaminase.

Distribution of glutamine level in different tissues of tumor bearing mice such as brain, liver, kidney, spleen, large and small intestine and the tumor itself were studied in three solid tumor models, viz, Ehrlich ascites carcinoma, Sarcoma-180 and methylcholanthrene induced carcinoma. Tumor bearing mice were subjected to therapy for 7 days with the glutaminase purified from malignant S-180 cell. The results exhibit a significant decrease in tumor burden after enzyme therapy. Host tissue glutamine levels were significantly elevated in tumor bearing untreated mice in comparison to the normal ones, while significant lower values were obtained after enzyme therapy. It therefore appears that elevated levels of glutamine in host tissue are associated with the tumor burden.

Neovascularisation offers a new perspective to glutamine related therapy.

Angiogenesis or the generation of new blood vessel, is an important factor in the growth of a solid tumor. Hence, it becomes a necessary parameter of any kind of therapeutic study. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. Therefore, using different murine solid tumor models, the present study was undertaken to find out whether the S-180 cell glutaminase has any effect on angiogenesis of solid tumor, or not. Result indicates that the purified S-180 cell glutaminase reduces tumor volume and restrict the generation of neo blood vessels. Therefore, it can be concluded that this enzyme may be an effective device against the cancer metastasis.

Angiogenesis a putative new approach in glutamine related therapy.

Angiogenesis or the generation of new blood vessels, is an important factor regarding the growth of a tumor. Hence, it becomes a necessary parameter of any kind in therapeutic studies. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. So, whether this enzyme has any effect on angiogenesis of a tumor or not becomes an obvious question. To address this question, this study has been carried out with different murine tumor models. The results indicate that purified glutaminase reduces tumor volume as well as restricts the generation of new blood vessels. Glutaminase is effective in the case of solid as well as ascites tumor models. In the case of induced cancer, the host exhibits delayed onset of neoplasia following enzyme treatment and tumor host interactions determine the intensity of the neovascularisation process. Therefore, it can be concluded that this enzyme might be an effective agent against cancer metastasis.

Isolation and purification of phosphate dependent glutaminase from sarcoma-180 tumor and its antineoplastic effects on murine model system.

High rate of glutamine use is a characteristic of tumor cell both in vivo and in vitro and experimental cancer therapies have developed by depriving tumor cells of glutamine. In several investigations, bacterial glutaminase was found to be a potent therapeutic agent against varieties of tumor, but it showed suppressive effects on haematopoietic systems and inhibitory effects on normal lymphocytic blastogenesis. No antineoplastic study has nevertheless been undertaken with glutaminase enzyme purified from mammalian source. In the present study we report the purification of glutaminase enzyme from mitochondria of highly malignant S-180 cell using ion exchange chromatography and affinity column chromatography of glutamine. Purified enzyme is a kidney type phosphate dependent glutaminase with Mr 64 KD. Effect of enzyme therapy has been investigated in transplantable as well as induced tumor model in both ascites and solid form. It has been observed that the enzyme at the total dose of 10 unit/mouse successfully inhibited the tumor burden both in ascitic and solid tumor and subsequently increases the host's life span. There was no significant toxic effect on the peripheral blood cells.

Pt-ATP as an antineoplastic agent in an experimental mice model system.

The antineoplastic activity of a platinum complex K4 (Pt Cl2 ATP) (Pt-ATP) has been investigated in transplantable tumors, both in an ascitic and solid (Ehrlich ascites carcinoma) tumor model. It has been observed that the drug at the total dose of 10 mg/kg body weight successfully inhibited the tumor burden both in ascitic and solid tumor system and subsequently increased the host's life span. An assessment of the in vitro (3H) thymidine incorporation into TCA precipitable material of EAC tumor cells done in the presence of Pt-ATP indicates that the drug inhibits (3H) thymidine incorporation in tumor cells. The drug has no appreciable toxic effect on the peripheral blood cells as well as bone marrow and spleenic cellularity.

A comparative study on expression of mucin related antigens in preneoplastic and neoplastic rat colorectal mucosa.

A polyclonal antibody (PAb35) defined antigen (Ag) was characterized in association with two monoclonal antibody (MAbM1 and MAb660) defined mucin M1 and 660 Ags in colorectal mucosa during 20-methylcholanthrene-induced rat carcinogenesis in the same organ. Immunohistochemistry and ELISA revealed that these three antibodies were reactive with most of the colorectal carcinomas. MAbM1 and MAb660 were reactive with preneoplastic colorectal mucosa in rats with no detectable carcinoma, in contrast to non-reactivity with PAb35. PAb35 reacted with preneoplastic mucosa, present adjacent to the cancerous tissues only. Staining was mainly localized in the cytoplasm of cancer cells, goblet cells and luminal mucous deposits. In control rats, M1 and 660 Ags were present in gastric mucosa, but not in colorectum. PAb35 defined Ag was absent in gastrointestinal mucosa of controls. ELISA revealed 82% reduction in reactivity, when PAb35 was reacted with 2-mercaptoethanol (2ME) treated colorectal mucosal extracts. Ninety percent reduction was seen in the case of MAbM1. However, 50% reduction was demonstrated when MAb660 was reacted with 2ME treated extracts.

Anti-tumor efficacy of glutaminase-copper-ATP combination in mice bearing Ehrlich ascites carcinoma.

Glutaminase is a hematotoxic anti-tumor agent, and copper-ATP complex (Cu-ATP) is both anti-neoplastic and hematostimulatory. Combination chemotherapy with these two agents has been performed in mice bearing Ehrlich ascites carcinoma, to elucidate whether this could result in augmented tumor inhibition with reduced hematotoxicity. Glutaminase-Cu-ATP combination (glutaminase 250 IU/kg per day intraperitoneally for 10 days and Cu-ATP 2.5 mg/kg per day intraperitoneally for 10 days) was observed to be more effective in inhibiting tumor growth and in increasing the life span of the tumor hosts, compared with the individual efficacies of these two agents. Moreover, addition of Cu-ATP successfully prevented the hematotoxic effects of glutaminase in normal and in tumor-bearing animals. Thus glutaminase in combination with Cu-ATP holds promise for an effective cancer chemotherapeutic regimen.

Alterations in large gut associated lymphatic tissues (LGALT) during experimental colorectal carcinogenesis.

Alterations in large gut associated lymphatic tissues (LGALT) were studied histologically during 20-methylcholanthrene (MCA) induced colorectal carcinogenesis. Precancerous changes in LGALT included hyperplasia, hyperchromasia of lymphocytes and enlargement of lymphoid follicles. In addition, follicular invasion in muscular layer and cellular disorganization of diffuse lymphatic tissues were observed in neoplasia. Since, LGALT showed remarkable changes during carcinogenesis, this aspect may be considered during assessment of preneoplastic lesions, along with other histologic features of early neoplasia.