Association of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Ureaplasma species infection and organism load with cervicitis in north Indian population.

Cervicitis is predominantly caused by Neisseria gonorrhoeae and Chlamydia trachomatis, which accounts for almost half of all the cases of cervicitis. The role of newer organisms like Mycoplasma genitalium and Ureaplasma sp. and association of bacterial load with cervicitis are also not well established. So the study aimed to determine the relative frequency of these organisms and their load in association with cervicitis cases from north India. A case-control study involving 300 women was conducted using quantitative real-time PCR from endocervical swabs for identification of organisms and quantification of bacterial load. Among 150 cervicitis cases, C. trachomatis, N. gonorrhoeae, M. genitalium and Ureaplasma parvum were detected in 5 (3·3%), 10 (6·6%), 37(24·6%) and 47 (31·3%) respectively. Old age (<0·001, chi-squared test) and irregular menstrual cycles (<0·001, chi-squared test) were significantly associated with cervicitis. M genitalium was the only organism to be associated significantly with cervicitis with regard to age (<0·031) and symptoms like discharge (P < 0·033, chi-squared test) and dysuria (P < 0·044, chi-squared test) in multivariate analysis. Our finding suggests that the bacterial load of these organisms is not significantly associated with cervicitis. However, we found significant association of M. genitalium infection with clinical characteristics of cervicitis cases.

Comparison of the diagnostic yield of transbronchial lung biopsies by forceps and cryoprobe in diffuse parenchymal lung disease.

Background: Transbronchial lung cryobiopsy (TBLC) in the diagnosis of diffuse parenchymal lung disease (DPLD) has shown a promising yield in recent times, with low post-procedural mortality and morbidity. Objectives: To compare the yield of TBLC and conventional transbronchial forceps lung biopsy (TBLB). Methods: A prospective study was carried out in patients with DPLD over a period of 1 year in a tertiary respiratory care institute in New Delhi, India. All 87 patients enrolled underwent both TBLB and TBLC. The procedures were performed in the bronchoscopy suite under conscious sedation and local anaesthesia, with an attempt to take a minimum of three biopsy specimens by conventional TBLB followed by TBLC. A 1.9 mm cryoprobe with a freezing time of 4 - 5 seconds was used. An Arndt endobronchial blocker was used to control bleeding along with locally administered medications. Results: TBLB and TBLC led to a definitive diagnosis in 27 (31.0%) and 69 (79.3%) cases, respectively. The commonest diagnoses were hypersensitivity pneumonitis, sarcoidosis and pulmonary tuberculosis. TBLC led to additional diagnoses in 42 cases (48.3%). Pneumothorax was observed in 12 cases (13.8%), and moderate bleeding occurred in 63 (72.4%). There were no procedure-related deaths. Conclusion: TBLC had a better diagnostic yield than conventional TBLB in DPLD. It has the potential to become a safe day-care procedure in a resource-limited setting, if certain precautions are taken. Study synopsis: What the study adds. Compared with transbronchial forceps lung biopsy, transbronchial lung cryobiopsy (TBLC) led to additional diagnoses in 42 (48.3%) of 87 patients with clinicoradiological features of diffuse parenchymal lung disease. Pneumothorax was observed in 12 cases (13.8%) and moderate bleeding in 63 (72.4%). TBLC without rigid bronchoscopy or advanced airway devices under conscious sedation had a good diagnostic yield with an acceptable adverse events profile.Implications of the findings. TBLC under conscious sedation is not resource intensive and can be carried out in settings with limited resources.

Current trends in nitric oxide research.

Nitric oxide (NO), a molecule with multidimensional effects has generated exponential amount of research since its identification as a biological messenger almost two decades back. The recent trend in NO research is to explore newer dimensions in the cellular and molecular mechanisms of actions and interactions of NO with various biomolecules and their implications in various pathophysiological states. Advances in our knowledge of the mechanisms by which this pleiotropic molecule regulates the expression of eukaryotic genes has generated considerable excitement and is paving the way for development of novel NO based therapeutic strategies. However, it is still a challenge to understand fully the paradox of beneficial and damaging effects of this exciting molecule. This review will discuss the current trends of research in this area especially highlighting the new insights gained from recent experimental and clinical studies. New approaches to reduce or augment the availability of NO to benefit a wide range of clinical conditions and avenues for future research are also briefly discussed.

Virological investigation of a hepatitis E epidemic in North India.

INTRODUCTION: Hepatitis E virus (HEV) infection is of major public health concern in the developing countries, including the Indian subcontinent, due to epidemics of large proportions, increased morbidity and high mortality, especially in pregnant women. This study shows the findings of two different epidemics that occurred due to HEV. METHODS: Blood samples were collected from 116 suspected HEV patients. Sera were separated and tested for hepatitis A virus HAV immunoglobulin M (IgM), hepatitis B virus surface antigen, hepatitis C virus (HCV) antibody and HEV IgM by Micro ELISA. 15 acute samples were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of HEV ribonucleic acid (RNA). RESULTS: Of the 116 blood samples collected, 68 (58.6 percent) were positive for HEV IgM antibodies. Mixed infections of HEV with HAV and HCV were detected in three (4.4 percent) and five (7.4 percent) cases, respectively. 15 HEV IgM-positive acute blood samples subjected to RT-PCR showed the presence of specific 343 bp amplified HEV ORF1 gene product in five cases. No untoward effects were observed in the five HEV-infected pregnant women during their follow-up. CONCLUSION: This study confirms the HEV aetiology and highlights a major disease outbreak that occurred due to mixing of drinking water with sewerage.

Vascular endothelial growth factor: Evidence for autocrine signaling in hepatocellular carcinoma cell lines affecting invasion.

BACKGROUND AND AIM: Vascular endothelial growth factor (VEGF) is a well-known pivotal regulator of tumor angiogenesis. Apart from endothelial cells, it is also expressed in nonendothelial cells, including tumor cells themselves. Hence the aim of this study was to investigate the autocrine effects of VEGF in hepatocellular carcinoma (HCC) -derived cell lines. MATERIALS AND METHODS: Two hepatocellular carcinoma cell lines (Hep3B and HepG2) were screened for expression of VEGF by quantitative real-time polymerase chain reaction (PCR) and its receptors VEGF-R1, VEGF-R2, and neuropilin-1 expression by reverse transcriptase-PCR, respectively. Furthermore, VEGF transcript was silenced by siRNA and the effects on cell migration, viability, and proliferation were determined by the wound healing assay, MTT assay, and propidium iodide staining, respectively. RESULTS: Both Hep3B and HepG2 cell lines expressed VEGF and all the three receptors at high levels. VEGF siRNA inhibited VEGF expression significantly in both Hep3B and HepG2 cell lines. Silencing of VEGF showed decreased migration in the Hep3B cell line. In both cell lines tested, there was decreased cell viability but no effect on cellular proliferation. CONCLUSION: Our data indicates that autocrine signaling of VEGF through its receptors exists in HCC cell lines, which has important implications for tumor invasion, metastasis, and for designing interventional strategies.

Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization.

An array of cis-, trans-, and dihydrostilbenes and some N-arylbenzylamines were synthesized and evaluated for their cytotoxicity in the five cancer cell cultures A-549 lung carcinoma, MCF-7 breast carcinoma, HT-29 colon adenocarcinoma, SKMEL-5 melanoma, and MLM melanoma. Several cis-stilbenes, structurally similar to combretastatins, were highly cytotoxic in all five cell lines and these were also found to be active as inhibitors of tubulin polymerization. The most active compounds also inhibited the binding of colchicine to tubulin. The most potent of the new compounds, both as a tubulin polymerization inhibitor and as a cytotoxic agent, was (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene (5a). This substance was almost as potent as combretastatin A-4 (1a), the most active of the combretastatins, as a tubulin polymerization inhibitor. Compound 5a was found to be approximately 140 times more cytotoxic against HT-29 colon adenocarcinoma cells and about 10 times more cytotoxic against MCF-7 breast carcinoma cells than combretastatin A-4. However, 5a was found to be about 20 times less cytotoxic against A-549 lung carcinoma cells, 30 times less cytotoxic against SKMEL-5 melanoma cells, and 7 times less cytotoxic against MLM melanoma cells than combretastatin A-4. The relative potencies 5a greater than 8a greater than 6a for the cis, dihydro, and trans compounds, respectively, as inhibitors of tubulin polymerization are in agreement with the relative potencies previously observed for combretastatin A-4 (1a), dihydrocombretastatin A-4 (1c), and trans-combretastatin A-4 (1b). The relative potencies 5a greater than 8a greater than 6a were also reflected in the results of the cytotoxicity assays. Structure-activity relationships of this group of compounds are also discussed.

Identification of human hepatocyte protein(s), which binds specifically to the recombinant envelope-2/non-structural-1 protein of hepatitis C virus.

Hepatitis C virus (HCV), which is the major pathogen responsible for human chronic liver disease, has special tropism for hepatocytes. Although, low-density lipoprotein receptor, CD81 and negatively charged glycosaminoglycans have been proposed as candidate receptors for HCV, no confirmed receptor(s) on the hepatocytes have been identified to date. It is also suggested that additional, yet unidentified, cellular proteins may be involved in the host-viral interaction. Therefore, this study was conducted with the main aim to identify hepatocyte protein(s) that may have affinity for the HCV structural protein, envelope-2/non-structural-1 (E2/NS1) protein. For the binding studies, hepatocytes were isolated from fresh normal human liver tissues. The hepatocyte proteins on the nitrocellulose paper were reacted with recombinant E2/NS1 protein and anti-E2 (rabbit). In another approach, to rule out the possibility of binding of rec-E2/NS1 with the hepatocyte cytoplasmic proteins, hepatocyte plasma membrane proteins were passed through CNBr-activated and recombinant E2/NS1 bound sepharose-4B column. The recombinant E2/NS1 binding hepatocyte plasma membrane protein(s) were eluted and were then analyzed. Altogether, our data suggest that E2/NS1 protein of HCV binds to two hepatocyte proteins of molecular weights 25-28 kDa and 59-60 kDa. These results indicate the possible role of the above proteins (25-28 kDa and 59-60 kDa) in the viral binding to the hepatocytes.

Biological and growth properties of a 20-methyl-cholanthrene transformed murine fibroblast cell line (PMM-14) established from primary culture.

Some major transformation related biological and growth properties have been analysed in an in vitro, system to characterize the PMM-14 cell line, a 20-methylcholanthrene induced transformed mouse embryonic fibroblast cell line. The population doubling time of this cell line was 19 h with moderately high saturation density and plating efficiency. Attachment detachment properties showed reduced adhesion to substratum. Cytogenetic study revealed the existence of a large number of Robertsonian biarmed chromosomes with hypertriploid modal range (60-69) represented by about 21% cells. These cells showed enhanced concanavalin A agglutinability which was reduced by trypsin treatment. Its neoplastic nature was finally established by its ability to grow in agar with a high plating efficiency.

Disruption of functions of wild-type p53 by hetero-oligomerization.

We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which p53 mutants may disrupt functions of wild-type p53.

Outer-membrane-protein subtypes of Haemophilus influenzae isolates from North India.

Haemophilus influenzae serotype b and non-typable isolates from blood, cerebrospinal fluid, sputum and throat swabs of patients and carriers in North India were analysed by outer-membrane protein (OMP) profiling. OMP analysis could differentiate the samples into 18 different subtypes. The non-typable isolates were more variable than the serotype b samples. OMP subtypes 1-6 were found only among the serotype b isolates and subtypes 7-18 among the non-typable isolates, while subtypes 2 and 8 were exhibited by both. The OMP profiles of isolates from blood, cerebrospinal fluid and sputum are in complete agreement with their ribotypes and RAPD fingerprints. The present study demonstrates for the first time the subtyping of Indian H. influenzae isolates by an easy and less-expensive method that is applicable to developing countries like India.

Subtype distribution of Haemophilus influenzae isolates from north India.

A total of 120 Haemophilus influenzae isolates from blood, cerebrospinal fluid, sputum and throat swabs of patients and carriers in North India was characterised by biotyping, ribotyping and random amplification of polymorphic DNA (RAPD)-PCR. Of these, 77 isolates (64%) were serotype b; the other 43 (36%) were non-typable. Biotype I was the most predominant among the typable strains and biotype II among the non-typable strains. Ribotyping with restriction endonucleases HaeIII and EcoRI differentiated the isolates into three and six ribotypes, respectively. However, RAPD fingerprints generated by the application of arbitrary primers AP1 and AP2 provided a higher level of discrimination. RAPD typing revealed distinct polymorphism among the serologically typable isolates. This study is the first report that stratifies the subtypes of H. influenzae strains from India by molecular techniques.

Intracameral amphotericin B: initial experience in severe keratomycosis.

PURPOSE: Fungal keratitis is a significant cause of ocular morbidity in India. The most commonly implicated fungi are Aspergillus spp. Patients often present with hypopyon, which usually contains fungal elements. The treatment is difficult owing to poor intraocular penetration of most available antifungal agents. This study evaluated the results of intracameral injection of amphotericin B in natamycin resistant cases of severe keratomycosis. METHODS: Three patients of culture proven Aspergillus flavus corneal ulcer with hypopyon not responding to topical natamycin 5%, amphotericin B 0.15%, and oral itraconazole were administered intracameral amphotericin B. The first case received 7.5 microg in 0.1 mL followed by two subsequent injections of 10 microg in 0.1 mL each, the second case received two injections of 10 microg in 0.1 mL, and the third patient received a single dose of 10 microg in 0.1 mL. Culture of the aqueous sample also grew A. flavus in all three cases. RESULTS: All three cases responded favorably, with the ulcer and hypopyon clearing completely. There was no clinical evidence of corneal or lenticular toxicity in any patient. CONCLUSIONS: Intracameral amphotericin B may be a useful modality in the treatment of severe keratomycosis not responding to topical natamycin. It ensures adequate drug delivery into the anterior chamber and may be especially useful to avoid surgical intervention in the acute stage of the disease.

Studies on the interaction of hematoporphyrin with hemoglobin.

Spectrophotometric and spectrofluorimetric studies reveal that an interaction occurs between hemoglobin and hematoporphyrin, a photosensitizing drug used in photodynamic therapy. Two concentration ranges of hematoporphyrin, 0.4-0.9 microM and 1.8-3.6 microM, representing significantly monomeric and aggregated (dimeric) state, respectively, have been used in the binding studies. The binding affinity constant (K) decreases, while the possible number of binding sites (p) increases as the concentration range of the porphyrin is increased. The nature of interaction has been studied by fluorescence quenching titration method under different ionic strengths and temperature conditions. It appears to be predominantly electrostatic and enthalpy-driven in the lower range of porphyrin concentration. However, the interaction follows mostly hydrophobic and entropy-driven modality in the higher concentration range of the ligand. The porphyrin-hemoglobin interaction results in release of oxygen from the protein. The extent of oxygen release depends on the stoichiometric ratio of hematoporphyrin:hemoglobin.

Dynamics of market correlations: taxonomy and portfolio analysis.

The time dependence of the recently introduced minimum spanning tree description of correlations between stocks, called the "asset tree" has been studied in order to reflect the financial market taxonomy. The nodes of the tree are identified with stocks and the distance between them is a unique function of the corresponding element of the correlation matrix. By using the concept of a central vertex, chosen as the most strongly connected node of the tree, an important characteristic is defined by the mean occupation layer. During crashes, due to the strong global correlation in the market, the tree shrinks topologically, and this is shown by a low value of the mean occupation layer. The tree seems to have a scale-free structure where the scaling exponent of the degree distribution is different for "business as usual" and "crash" periods. The basic structure of the tree topology is very robust with respect to time. We also point out that the diversification aspect of portfolio optimization results in the fact that the assets of the classic Markowitz portfolio are always located on the outer leaves of the tree. Technical aspects such as the window size dependence of the investigated quantities are also discussed.

Isolation of emetine resistant clones of Entamoeba histolytica by petri dish agar method.

Emetine resistant clones of Entamoeba histolytica strain HM1:IMSS were isolated by using petri dish agar method after mutation with ethyl-methanesulphonate. Two emetine resistant clones were obtained and both were resistant to emetine at a concentration of 24 micrograms/ml of emetine. The 50 per cent inhibitory concentration (IC50) for both emetine sensitive and resistant clones was 5 and 14 micrograms/ml respectively. The colony forming efficiency of E. histolytica strain HM1:IMSS varied from 44 to 54 per cent. This method is useful for isolating clones from different strains of the parasite for molecular and immunological studies.

Trifluoperazine is more effective than chlorpromazine in releasing oxygen from haemoglobin and myoglobin.

The extent of oxygen release from two heme proteins, haemoglobin and myoglobin have been studied in the presence of trifluoperazine and chlorpromazine (5-1000 microM). At a molar ratio (drug:protein) of 1.5, the release of oxygen from haemoglobin was 4 and 15% in the presence of chlorpromazine and trifluoperazine respectively, while from myoglobin the corresponding values were 20 and 40%. The findings were attributed to the greater extent of local conformational change around tryptophan moieties of each of the proteins induced by trifluoperazine.

Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines.

Binding modalities of chlorpromazine and trifluoperazine, two widely used antipsychotic phenothiazine drugs with hemoglobin and myoglobin have been studied to understand how the quaternary, tertiary and secondary structural organisations of the proteins regulate the binding process. NaCl-induced alteration in the quaternary structure of hemoglobin influences its binding modality with phenothiazines. Minor alterations in the tertiary structure of thermally denatured myoglobin (denaturation temperature ranging between 30-70 degrees C) do not affect its affinity and the modality of binding with the drugs, but alterations in the secondary structure of the protein denatured at temperatures between 70-80 degrees C influence its binding.

Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus.

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.