Regulation of CYP11B1 and CYP11B2 steroidogenic genes by hypoxia-inducible miR-10b in H295R cells.

Although numerous studies have shown that hypoxia affects cortisol and aldosterone production in vivo, the underlying molecular mechanisms regulating the steroidogenic genes of these steroid hormones are still poorly known. MicroRNAs are post-transcriptional regulators that control diverse biological processes and this study describes the identification and validation of the hypoxia-inducible microRNA, miR-10b, as a negative regulator of the CYP11B1 and CYP11B2 steroidogenic genes in H295R human adrenocortical cells. Using the human TaqMan Low Density miRNA Arrays, we determined the miRNA expression patterns in H295R cells under normoxic and hypoxic conditions, and in cells overexpressing the human HIF-1α. Computer analysis using three in silico algorithms predicted that the hypoxia-inducible miR-10b molecule targets CYP11B1 and CYP11B2 mRNAs. Gene transfection studies of luciferase constructs containing the 3'-untranslated region of CYP11B1 or CYP11B2, combined with miRNA overexpression and knockdown experiments provide compelling evidence that CYP11B1 and CYP11B2 mRNAs are likely targets of miR-10b.

Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel.

OBJECTIVE: Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel. MATERIALS AND METHODS: We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves. RESULTS: hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone. CONCLUSION: Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel.

Comparative analysis of the outer membrane protein profiles of isolates of the Pasteurella multocida (B:2) associated with haemorrhagic septicaemia.

Outer membrane proteins (OMP) of P. multocida (serotype B:2) field isolates (n = 6) and a vaccine strain (P-52) were extracted by a sarkosyl method and characterized using SDS-PAGE and immunoblotting. About 20 polypeptide bands were observed in the profile of the vaccine strain with MW ranging from 16 to 90 kDa and, based on band thickness and intensity of staining, three polypeptides of MW 31, 33 and 37 kDa were considered to be the major OMPs. The profiles of the field isolates showed minor differences when compared with that of the vaccine strain. The OMP of 33 kDa was only expressed by the vaccine strain. Four field isolates expressed an OMP of 39 kDa, which did not appear in the profiles of the remaining two field isolates and the P-52 strain. Similarly, an OMP of 25 kDa was exclusively seen in the profile of a single isolate. By immunoblotting studies, using anti-P. multocida (P-52) whole-cell hyperimmune serum raised in rabbits as well as buffalo immune sera, it became evident that the polypeptide of 37 kDa was the most antigenic OMP in the profiles of all the isolates, including the P-52 strain. Other polypeptides were either weakly antigenic or visible in the profile of only a few of the isolates. The study thus identified the major OMP of P. multocida (B:2) and suggested that this highly antigenic 37 kDa OMP has potential for further protective and immunodiagnostic studies.

Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2.

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.

Inhibition of Helicobacter pylori adherence by a peptide derived from neuraminyl lactose binding adhesin.

Helicobacterpylori, like many other gut colonizing bacteria, binds to sialic acid rich macromolecules present on the gastric epithelium. NLBH (neuraminyl lactose binding haemagglutinin) a 32 kDa adhesin located on the surface of H. pylori has been shown to have specific affinity towards NeuAcalpha2,3Galbeta1,4Gluc(3'SL). This sialic acid moiety is over-expressed in an atrophic stomach undergoing parietal cell depletion. Antibodies against a lysine rich peptide fragment of NLBH inhibit agglutination of human erythrocytes. This lysine rich sequence from NLBH was proposed to be the receptor-binding site. In order to elucidate the binding of NLBH to gastric epithelium, a peptide (D-P-K-R-T-I-Q-K-K-S) was synthesized. A series of experiments were performed involving adherence inhibition assays, 2D-NMR, molecular modelling and measurement of modulation in acid secretion. Results indicated that the peptide fragment could be involved in receptor recognition, which is important for the binding of H. pylori to gastric epithelium. The binding is possibly through hydrogen bonding. Two lysines and a threonine residue seem to be within the hydrogen bonding distance of NeuAcalpha2,3Galbeta1,4Gluc. Further, in vitro assays were performed to evaluate the role of the peptide on acid secretion by parietal cells isolated from human fundal biopsies. Interestingly, the peptide increases acid secretion only in H. pylori negative and in treated patients but not in H. pylori positive patients. This highlights the role of NLBH in acid secretion and could be of some consequence in the prognosis of the disease.

Regulation of immune functions by sperm-specific LDH and its differences with somatic isozyme in primary and secondary lymphocyte cultures.

PROBLEM: Sperm-specific lactate dehydrogenase-C4 (LDH-C4) is an autoantigen that produces experimentally induced autoimmune orchitis in testes. In the present study, immunological functions of B and T cells have been examined and compared after immunization with sperm-specific LDH and the LDH from somatic cells. METHODS: Three sets of experiments were performed. In the first set, effects of Balb/C LDH isozymes at 10(-3) - 1 L microg/well were investigated: (i) by mixed lymphocyte cultures (MLC) using C-57 B1/6 female cells as responders and AKR lymphocytes (irradiated) as stimulators, (ii) for regulatory T cell activity in MLC co-cultured along with Con-A-induced AKR lymphoblasts and (iii) for modulation of lymphocyte activation by PHA in vitro. In the second set of experiments, female mice (C-57 B1/6) were distributed in six groups for various treatments: i) saline (as vehicle), ii) adjuvant, iii) LDH-B4 (20 x 3 microg), iv) LDH-B4 (40 x 3 microg), v) LDH-C4 (20 x 3 microg), and v) LDH-C4 (40 x 3 microg). Mice were hyperimmunized with -B4 or -C4 (Balb/c) with a primary dose of 20 or 40 microg of protein per mouse, emulsified in Freund's complete adjuvant (FCA) and two identical doses in Freund's incomplete adjuvant (s.c.) within 22 days. Saline (group i) or adjuvant treated dams (group ii) served as controls. One week after the second booster, sera were tested for IgG response and lymphocytes harvested for polyclonal activation in vitro using LPS and Con-A as mitogens. In the third set of experiments, female Balb/c mice were divided into six groups as in the second experiment and immunized with a single primary dose of isogenic LDH-B4 or LDH-C4 at 20 or 40 microg of protein in FCA. On day 5, after sensitization with LDH, lymphocytes were evaluated for mitogenesis and for IgM production in vitro using LPS and Con-A as mitogens. RESULTS: i) Primary MLC(s) were non-specifically suppressed in the presence of 10(-3)- 1 L x microg allogenic LDH-C4 or -B4, although LDH-C4 tended to abolish MLC completely. But MLC co-cultured with blast cells was suppressed by LDH-C4 alone, indicating that sperm LDH suppresses induced formation of regulatory T cells. ii) FCA primed lymphocytes in situ were significantly inhibited for Con-A stimulation in vitro. Since LPS stimulation remained unaffected, it appeared that FCA is immunosuppressive for T cell proliferation alone. iii) Cells primed with LDH increased mitogenic activity of LPS several fold, although LDH-C4 was less effective than LDH-B4 in sensitization of B lymphocytes. iv) However, effect of Con-A in mitogenesis was dose-dependent, viz. cells primed at 20 x 3 microg of each isozyme overcame the immunosuppressive nature of FCA by bringing back the SI ( x 25) equivalent to saline primed cells, while pre-treatment of cells with 40 x 3 microg LDH-C4 abolished SI completely, indicating that -C4 primed cells were immunologically suppressed for Con-A stimulation. Such a response was markedly visible when allogenic LDH-C4 was used for hyperimmunization; lymphocytes challenged with somatic LDH under similar conditions did not react. Loss of T cell functions by LDH-C4 was confirmed in the presence of PHA in primary cultures. v) For antibody responses, although sperm LDH was highly reactive and dose-dependent, somatic LDH was also immunogenic for IgG production in serum to a lesser degree. Besides, IgM antibody was also discernible by two isozymes in LPS-induced cultures. Significantly, -C4 primed cells at the higher dose, in comparison with the lower dose, were less responsive for IgM production. CONCLUSIONS: It is concluded that LDH(s) from sperm and somatic cells share functionally related antigenic epitopes that can generate/modify immune responses in vivo and in vitro with qualitative differences. However, immunosuppressive determinant of LDH-C4 is cell specific and dose selective.

Experimental salmonellosis in guinea-pigs: haematological and biochemical studies.

Some haematological and biochemical parameters were studied in guinea-pigs infected intraperitoneally with Salmonella dublin 493 at 1 x 10(6) viable cells per animal. The infected animals showed a rise in temperature within 24 h, followed by depression and loss of body weight. On the 15th day post infection, haematological studies revealed a significant increase in the total leukocyte count due to both lymphocytosis and neutrophilia, and a decrease in the total erythrocyte count and haemoglobin concentration. There was also a significantly higher mean corpuscular volume and lower mean corpuscular haemoglobin concentration, indicating a macrocytic hypochromic anaemia. The infection caused a significant increase in alanine aminotransferase activity and creatinine, blood urea nitrogen and globulin concentrations, and a decrease in albumin and triiodothyronine. There was no significant effect on serum total protein or on thyroxine, or in the activity of aspartate aminotransferase in the serum.

Sex dependent immune responses by allogenic LDH isozymes.

Regulatory effects on polyclonal activation of primed splenocytes have been studied following immunization through the intrarectal route with allogenic sperm specific lactate dehydrogenase (LDH-C4) and somatic LDH from kidney. Results indicate that LDH primed cell proliferation by mitogens is dependent on the nature of the isozyme and sex of donor cells. Compared to somatic LDH, LDH-C4 was immunosuppressive for T cell proliferation in vitro and the effect was more significant with female splenocytes as compared to male spleen cells. However, the suppressive effect of LDH-C4, on B cell function was identical in both males and females. In contrast to the somatic LDH which did not produce alloantibody in significant amount, LDH-C4 was highly immunogenic in production of humoral antibody in female mice. Alloantibody formation in dams was substantiated with a similar degree of immune regulation of B cell functions as shown by lipopolysaccharide stimulation. The role of LDH-C4 in protection of allogenic sperm in the female genital tract has been suggested. However, it is concluded that recipients of sperm constituents through the intrarectal route are at greater risk for immune suppression and bacterial/viral infection.

Affinities of methylphenidate derivatives for dopamine, norepinephrine and serotonin transporters.

We have synthesized several derivative of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC50 values for binding these compounds to rat brain monoamine transporters were assessed using [3H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [3H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [3H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET >> 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC50 values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of o-bromomethylphenidate did not bind to the DAT (IC50 > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r2=0.90). Abilities of several methylphenidate derivatives to inhibit [3H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [3H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC50 > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [3H]SCH 23390 or [123I]epidepride, respectively, to striatal membranes.

Clinical survey of cannabis users in varanasi.

A survey of Cannabis users reveal that long-term use of the drug does not produce any harmful effect. The findings of this scientific study is reported here minutely.

The effect of nuclear magnetic resonance imaging on metallic middle ear prostheses.

Nuclear magnetic resonance imaging, a relatively new diagnostic instrument, is a noninvasive imaging method which, among its many advantages, uses no ionizing radiation. There are a few limitations and contraindications to its use. There may be displacement of intracerebral aneurysm clips and metallic implants, and cardiac pacemakers can be disabled because of the high magnetic field created by this device. We studied the effects of a magnetic field on metallic middle ear prosthetics and concluded that nuclear magnetic resonance imaging should offer no risks to hearing or otologic function in patients with nonferromagnetic metallic middle ear prosthetics. Nuclear magnetic resonance imaging is contraindicated in patients with cochlear implants.

Experimental and clinical studies on diabetes mellitus evaluating the efficacy of an indigenous oral hypoglycaemic drug - arani (clerodendron phlomidis).

Diabetes mellitus is a common problem in clinical practice. An indigenous herbal drug Arani (Clerodendron phlomidis) was selected for this experimental and clinical study. The experimental study was conducted on albino rats. Arani inhibited the adrenaline induced hyperglycaemia effectively. The alcoholic extractive of Arani produced a well comparable fall in blood sugar to that of tolbutamide. Moreover, Arani caused a significant fall in hyperglycaemia ofalloxan diabetic patients along with clinical improvement. The results were found quite comparable to tolbutamide.

Clinical trial of adhatoda vasica syrup (vasa) in the patients of non-ulcer dyspepsia (amlapitta).

The efficacy of Vasa (Adhatoda vasica) in the form of syrup against Non-ulcer Dyspepsia (Amiapitta) through a clinical trial is attempted here. This trial showed hopeful results. The drug has reduced the total and free HCL in the patients of hyperacidity and hyperchlorhydria.