Solid-phase reactive chromatography (SPRC): a sustainable method for the synthesis of benzimidazol-diphenyl-2-imino-thiazolidine-4-ols (hemiaminals) which are active against lung cancer.

The concept of a green approach has been applied in the reaction, separation and solidification of a single unit. The products, 2-benzimidazol-diphenyl-2-imino-thiazolidine-4-ols, are purely hemiaminals. They have a tendency to decompose at a high rate. The solid-phase reactive chromatographic technique has been applied to avoid extensive liquid-liquid extraction and stabilised the product by precipitation in solvents. Benzimidazole phenyl thiourea and phenacyl bromide have been used as starting materials in this study. Both the starting materials are adsorbed on dry silica and packed in a glass column with a systematic arrangement of layers. A directly solid product was precipitated in cold hexane. Anticancer activities have been recorded against four cell lines, human colon, prostate, lung and breast cancer, with reference to doxorubicin as a standard. These compounds show a promising effect on human lung cancer, products B9 (IC50 = 3.890 µM) and B10 (IC50 = 2.798 µM) and B13 (IC50 = 3.140 µM) which very close to doxorubicine (IC50 = 1.750 µM). It was observed that fluoro phenyl functionalities are effective compared to trifluoro methyl phenyl functionalities for anticancer activities. These small molecules lose their activities, except fluorination, on bulky substitution. This convenient metal-free approach is highly efficient for unstable hemiaminals such as the potent anticancer benzimidazol-diphenyl-2-imino-thiazolidine-4-ols.

Inertial effects in the dc+ac driven underdamped Frenkel-Kontorova model: Subharmonic steps, chaos, and hysteresis.

The effects of inertial terms on the dynamics of the dc+ac driven Frenkel-Kontorova model were examined. As the mass of particles was varied, the response of the system to the driving forces and appearance of the Shapiro steps were analyzed in detail. Unlike in the overdamped case, the increase of mass led to the appearance of the whole series of subharmonic steps in the staircase of the average velocity as a function of average driving force in any commensurate structure. At certain values of parameters, the subharmonic steps became separated by chaotic windows while the whole structure retained scaling similar to the original staircase. The mass of the particles also determined their sensitivity to the forces governing their dynamics. Depending on their mass, they were found to exhibit three types of dynamics, from dynamical mode-locking with chaotic windows, through to a typical dc response, to essentially a free-particle response. Examination of this dynamics in both the upforce and downforce directions showed that the system may not only exhibit hysteresis, but also that large Shapiro steps may appear in the downforce direction, even in cases for which no dynamical mode-locking occurred in the upforce direction.

Devil's staircase and the absence of chaos in the dc- and ac-driven overdamped Frenkel-Kontorova model.

The devil's staircase structure arising from the complete mode locking of an entirely nonchaotic system, the overdamped dc+ac driven Frenkel-Kontorova model with deformable substrate potential, was observed. Even though no chaos was found, a hierarchical ordering of the Shapiro steps was made possible through the use of a previously introduced continued fraction formula. The absence of chaos, deduced here from Lyapunov exponent analyses, can be attributed to the overdamped character and the Middleton no-passing rule. A comparative analysis of a one-dimensional stack of Josephson junctions confirmed the disappearance of chaos with increasing dissipation. Other common dynamic features were also identified through this comparison. A detailed analysis of the amplitude dependence of the Shapiro steps revealed that only for the case of a purely sinusoidal substrate potential did the relative sizes of the steps follow a Farey sequence. For nonsinusoidal (deformed) potentials, the symmetry of the Stern-Brocot tree, depicting all members of particular Farey sequence, was seen to be increasingly broken, with certain steps being more prominent and their relative sizes not following the Farey rule.

Are sex steroids essential for gonadal differentiation of the ornate frog, Microhyla ornata?

Knowledge about sensitivities and responses of amphibian larvae to sex steroids and the chemicals alike is the first step towards understanding and assessing the effect of diverse chemicals that interfere with gonadal development and other endocrine functions. Herein, we used Microhyla ornata to determine the role of sex steroids on its gonad differentiation and sex ratio. Our results show that the exposure to increasing concentrations of estradiol-17ß throughout larval development did not affect gonad differentiation resulting in 1:1 sex ratio at metamorphosis. But, females emerging from estradiol-17ß treatment had larger ovaries with larger sized follicles. Further, testes of some males contained lumens, the number of which was dose dependent. Similarly, exposure to testosterone propionate had negligible effects on gonad differentiation. However, the mean diameter of the largest follicles was smaller in treated ovaries. Treatment of tadpoles with tamoxifen had no effect on gonad differentiation and ovary development while testicular development was accelerated at the highest concentration. Similarly, treatment of tadpoles with cyproterone acetate had little effect on gonad differentiation as well as development, hence the sex ratios at the end of metamorphosis. Further, in tadpoles exposed to increasing concentrations of formestane, gonad differentiation was normal resulting in 1:1 sex ratio. Thus, in M. ornata, both estradiol and testosterone are essential for the development of ovaries and testes respectively but, they are not critical to gonadal differentiation. Hence, the effects of sex steroids and other endocrine disrupting chemicals could be species-specific; different species may have differential sensitivities to such chemicals.

Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.

PURPOSE: Influenza epidemics and periodic pandemics occur worldwide resulting in significant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type influenza viruses, as cell culture has a long turnaround time. MATERIALS AND METHODS: Nasopharyngeal swabs were collected from patients presenting with influenza-like illness (ILI) at AIIMS OPD and Primary Health Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of 1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for influenza A using primers for matrix gene and for influenza B using non-structural gene (NS) primers. All influenza A positives were sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September 2009. RESULTS: Of the 544 samples, 136 (25%) were positive for influenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as influenza A and 47 (34.5%) as influenza B viruses and 3 (2%) samples showed dual infection with influenza A and B. Of the 86 influenza A positive samples 48 (55.8%) were identified as seasonal influenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of influenza positivity using virus culture revealed that only 97/136 (71.3%) were influenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and influenza B (38/47; 80.8%); all influenza A/H1N1pdm09 viruses were detected by both methods. CONCLUSION: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing influenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for influenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.

Agmatine in the hypothalamic paraventricular nucleus stimulates feeding in rats: involvement of neuropeptide Y.

BACKGROUND AND PURPOSE: Agmatine, a multifaceted neurotransmitter, is abundantly expressed in the hypothalamic paraventricular nucleus (PVN). Our aim was to assess (i) the effect of agmatine on feeding behaviour and (ii) its association, if any, with neuropeptide Y (NPY). EXPERIMENTAL APPROACH: Satiated rats fitted with intra-PVN cannulae were administered agmatine, alone or jointly with (i) α2-adrenoceptor agonist, clonidine, or antagonist, yohimbine; (ii) NPY, NPY Y1 receptor agonist, [Leu³¹, Pro³4]-NPY, or antagonist, BIBP3226; or (iii) yohimbine and NPY. Cumulative food intake was monitored at different post-injection time points. Furthermore, the expression of hypothalamic NPY following i.p. treatment with agmatine, alone or in combination with yohimbine (i.p.), was evaluated by immunocytochemistry. KEY RESULTS: Agmatine robustly increased feeding in a dose-dependent manner. While pretreatment with clonidine augmented, yohimbine attenuated the orexigenic response to agmatine. Similarly, NPY and [Leu³¹, Pro³4]-NPY potentiated the agmatine-induced hyperphagia, whereas BIBP3226 inhibited it. Moreover, yohimbine attenuated the synergistic orexigenic effect induced by the combination of NPY and agmatine. Agmatine increased NPY immunoreactivity in the PVN fibres and in the cells of the hypothalamic arcuate nucleus (ARC) and this effect was prevented by pretreatment with yohimbine. NPY immunoreactivity in the fibres of the ARC, dorsomedial, ventromedial and lateral nuclei of the hypothalamus was not affected by any of the above treatments. CONCLUSIONS AND IMPLICATIONS: The orexigenic effect of agmatine is coupled to increased NPY activity mediated by stimulation of α2-adrenoceptors within the PVN. This signifies the importance of agmatine or α2-adrenoceptor modulators in the development of novel therapeutic agents to treat feeding-related disorders.

Microleakage of restorative materials: an in vitro study.

The present study was conducted with the aim of evaluating and comparing the microleakage of glass ionomer, composite resin and compomers. Class V cavities were made in thirty intact caries free premolars and restored with restorative materials to be tested respectively. The teeth were thermocycled and subjected to silver nitrate dye penetration. They were subsequently sectioned buccolingually. Microleakage was evaluated under a stereomicroscope and data subjected to statistical analysis. The study concluded that microleakage was evident in all restorative materials, with glass ionomer showing maximum leakage followed by composite resin. Compomer demonstrated the best results with minimum leakage.

Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.).

Aeromonas hydrophila causes disease under stress conditions or in concert with infection by other pathogens in goldfish. Sero-diagnostic and/or immunoprophylactic tools against Aeromonas infection in goldfish are not available so far. The present study was undertaken to fractionate and characterise the outer membrane proteins (OMP) of A. hydrophila and to identify suitable immunoreactive components. A total of 10 fractions were generated from crude OMP antigens upon gel permeation and subsequent ion-exchange chromatography. One of the fractionated antigens (GPID2), primarily a 57-kDa polypeptide, showed maximum sero-reactivity, even higher than the crude OMP. Suitability of GPID2 antigen for use in diagnostic preparations was assessed by dip-stick ELISA. In vitro goldfish lymphoproliferative ability of fractionated antigen, GPIID2 (primarily a 23-kDa polypeptide) was observed to be higher than all the fractionated antigens as well as crude OMP. It can be concluded that the 57 kDa and 23 kDa polypeptides of the OMP of A. hydrophila, possessing high immunoreactivity, should be given due attention while preparing immunodiagnostic and immunoprophylatic tools against Aeromonas infections in goldfish.

Oral vinorelbine given as monotherapy to advanced, elderly NSCLC patients: a multicentre phase II trial.

Vinorelbine intravenously (i.v.) demonstrated its efficacy and tolerability in advanced non-small cell lung cancer (NSCLC) patients, including elderly subjects. Since vinorelbine is now available as an oral formulation this phase II open study was designed to evaluate its activity and tolerability in advanced, elderly NSCLC patients. A total of 56 chemonaive patients were recruited from April 2001 through to March 2002. The dosage schedule, already tested in younger NSCLC patients, was 60 mg/m(2)once a week for three weeks (first cycle), followed by 80 mg/m(2) once a week until disease progression or development of unacceptable toxicity. A limited sampling scheme was used for performing pharmacokinetic analysis on 52 of 56 patients enrolled in the study. Treatment was well tolerated with grade 3/4 neutropenia in 11/17 patients (20/30%) and febrile neutropenia in 1 (2%). Six partial responses (11%) and 25 stable disease responses were recorded, with a disease control rate of 55%. Median overall survival was 8.2 months (95% Confidence Interval (CI) [6.2-11.3]). The clinical benefit response rate was 31% on 32 evaluable patients. Pharmacokinetic profiles appeared quite similar to the historical profiles recorded following i.v. administration. Oral vinorelbine appears to be a reasonable alternative to i.v. vinorelbine, both in terms of activity and tolerability, in advanced, elderly NSCLC patients.

Induction of reversible antifertility with a crude ethanol extract of Citrullus colocynthis Schrad fruit in male rats.

A crude 50% ethanol extract of Citrullus Colocynthis Schrad was administered orally to male albino rats for evaluation of antifertility effects. The animals were divided into five groups: group A was a vehicle-treated control group; treatment groups B, C, and D received 100 mg/kg/day C. Colocynthis extract for periods of 20, 40, and 60 days, respectively, and group E animals received the extract at 100 mg/kg/day for 60 days followed by 60 days of recovery. For androgenicity evaluation of the extract, the animals were divided into four groups: group F animals were castrated 30 days before the experiment to serve as controls, and group G, H, and I were subjected to castration 30 days before the experiments, followed by administration of fruit extract (100 mg/kg/day p.o.), testosterone propionate (0.01 mg/rat/alternate day s.c.), and fruit extract along with testosterone propionate, respectively, for 30 days. Significantly reduced cauda epididymis sperm motility and density, number of pups, fertility, and circulatory levels of testosterone were observed in all treatment groups. The weights of testes, epididymis, seminal vesicle, and prostate were significantly decreased in groups B, C, and D. The weights of all organs in the different groups of the androgenicity study were markedly decreased in group F when compared with group A, in group G when compared with group F, and in group I when compared with group H, and increased in group H when compared with group F. The serum testosterone levels also showed a similar pattern. The concentration of testicular cholesterol was significantly elevated, while protein, sialic acid, acid and alkaline phosphatase concentrations were decreased. The histoarchitecture of the testes showed degenerative changes in the seminiferous epithelium, arrest of spermatogenesis at the secondary spermatocyte stage, cytolysis, and the lumen filled with eosinophilic material. Histometric parameters except Sertoli cell nuclear area and number of round spermatids showed marked alterations. All altered parameters restored to normal in group E. No changes were observed in body weight, litter size, hematology, and serum biochemistry. In conclusion, a 50% ethanol extract of C. Colocynthis showed an antiandrogenic nature, thereby reduced reversible infertility in male albino rats.

Epirubicin/docetaxel regimen in progressive breast cancer-a phase II study.

The purpose of this investigation was to evaluate the efficacy and toxicity of 6 months' treatment with the combination of epirubicin and docetaxel in metastatic breast cancer. Thirty-eight women (mean age 51 years, range 35-72) with metastatic breast cancer were treated with a regimen of epirubicin 75 mg/m and docetaxel 75 mg/m every 3 weeks, given 4 times if progression was seen upon evaluation after 4 courses or 8 times in responding/stable patients. The patients received 285 cycles of combination treatment and two treatments with docetaxel or epirubicin alone. When neutropenia with fever was observed, further cycles were given with dose reduction. The median cumulative docetaxel dose was 462 mg/m (range 199-600) and that of epirubicin 476 mg/m (range 199-740). The overall response rate was 54% (95% CI 37-71), with a median duration of response of 14.8 months (95% CI 8.8-27.8). Median time to progression was 12 months, median survival 26 months. Neutropenia below 0.5 x 10 /l occurred following 113 (39%) of the total of 285 cycles given; 21 patients (55%) were hospitalized for febrile neutropenia. We conclude that dose tailoring is required in treatment with an epirubicin and docetaxel regimen to avoid grade 3/4 adverse effects in a significant number of patients treated for metastatic breast cancer.

Antifertility effect of chronically administered Martynia annua root extract on male rats.

The effects of 50% ethanol extract of Martynia annua L. root on reproduction was studied on male rats. The study was divided into four groups of five animals each. The first group (I) received vehicle alone to serve as control. The second, third and fourth groups (II, III and IV) of animals were administered the root extract daily at 50 mg/kg body weight, po, 100 mg/kg body weight, po, and 200 mg/kg body weight, po, respectively, for a period of 60 days. Significant decreases in the weights of testes, epididymides, seminal vesicle and ventral prostate were observed. A dose related reduction in the testicular sperm count, epididymal sperm count and motility, number of fertile males, ratio between delivered and inseminated females and number of pups were observed. The testis showed a clear correlation between the dose and severity of lesions of seminiferous epithelium. In general, the seminiferous tubules appear reduced in size with a frequently filled eosinophilic material. Spermatogenesis arrested at the secondary spermatocyte stage. Pachytene spermatocytes were undergoing degeneration. Disorganisation and sloughing of immature germ cells were visible. Leydig cells were atrophied. No morphological changes were observed in Sertoli cells. Significant reduction in serum concentration of luteinising hormone and testosterone were observed. No distinct change in serum FSII concentration was recorded. The final body weights of all groups were elevated markedly. No alterations were recorded in any hematological parameters. It is concluded that the 50% ethanol extract of M. annua root produced dose related effects on male reproduction without altering general body metabolism.

Docetaxel and irinotecan (CPT-11) in the treatment of malignant pleural mesothelioma--a feasibility study.

We chose to treat malignant pleural mesothelioma with a combination of docetaxel and irinotecan (CPT-11), because there have been preliminary reports that CPT-11 is active against mesothelioma, and docetaxel and CPT-11 were the most active agents in our in vitro experiments in human mesothelioma cell lines. Fifteen previously untreated patients with pleural mesothelioma (IMIG Stage III-IV) were given docetaxel 60 mg/m2 followed by CPT-11 190 mg/m2 on day 1, repeated every 3 weeks. All the patients were evaluable for toxicity and 13 patients were evaluated for response. No objective responses (complete or partial) were achieved, but there were two minor responses (overall response rate 15%) each of a duration of 4 months. Three patients had stable disease (23%); median time to progression was 7 months. Median survival in all the patients was 8.5 months from the first chemotherapy cycle and 11 months from diagnosis. Toxicity was severe with seven of 15 patients suffering neutropenic fever and six of 15 patients grade 3-4 diarrhea. The trial was discontinued because of toxicity and lack of activity. We do not recommend the combination of docetaxel and CPT-11 using the schedule presented here for further investigation in malignant mesothelioma. However, CPT-11 and docetaxel, individually, still warrant further study in this disease, especially in combination with cisplatin.

Sequential expression of nucleoproteins during rat spermiogenesis.

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX-XI, followed by high levels at stages XII-XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII-I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9-14, TP1 in steps 12-15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.

Haploid gene expression: temporal onset and storage patterns of 13 novel transcripts during rat and mouse spermiogenesis.

The temporal and spatial expression of thirteen novel spermatid-specific genes corresponding to cDNA clones isolated from an adult mouse testis library was analyzed. Northern analysis of RNA from seminiferous tubules at defined stages of the rat and mouse seminiferous epithelial cycle and in situ hybridization of testis sections revealed that these mRNAs were expressed in a stage-specific manner. The expression of all mRNAs was first detected in early round spermatids, and it increased to abundance during stages VII-VIII of the epithelial cycle. Twelve out of thirteen mRNAs were found not only in round spermatids but also in transcriptionally inactive elongated spermatids, suggesting that they are stored and regulated at the translational level. The variation in the length of the poly(A) tail was detected for four of the transcripts, represented by cDNA clones MTEST70, MTEST627, MTEST641, and MTEST643 at defined stages of the cycle. Similarity in the stage-specific expression pattern displayed by this group of haploid-specific genes strongly suggests the presence of common regulatory mechanisms that act during spermiogenesis, and these genes also provide a means for further studies of these mechanisms.

Follicle-stimulating hormone regulates the expression of cyclic protein-2/cathepsin L messenger ribonucleic acid in rat Sertoli cells in a stage-specific manner.

Cyclic protein-2/cathepsin L (CP-2) is secreted by Sertoli cells in a highly stage-specific manner, maximally during stages VI-VII of the rat seminiferous epithelial cycle. We investigated FSH regulation of CP-2 mRNA expression of its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA expression and its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA levels in stages IX-I, whereas in stages II-VIII, the levels of CP-2 mRNA were reduced. A similar effect was produced by two cAMP analogs, dbcAMP (0.2 mM) and Sp cAMP (20 microM). FSH and cAMP did not affect on the levels of SGP-2 mRNA during the seminiferous epithelial cycle. The magnitude of the response was time- and dose-dependent; the maximum was obtained with 100 ng/ml of FSH. It is likely that FSH regulates Cp-2 gene transcription, since de novo RNA synthesis was required for the stimulatory FSH effect on CP-2 mRNA levels, while ongoing protein synthesis was not. In conclusion, the data suggest that FSH, via cAMP-mediated pathway, regulates CP-2/cathepsin L gene transcription in rat Sertoli cells and modulated the stage-specific expression pattern.

Localization of urokinase- and tissue-type plasminogen activator mRNAs in rat testes.

The expressions of urokinase (uPA) and tissue-type plasminogen activators (tPA) in different stages of the rat seminiferous epithelial cycle were analyzed by in situ and Northern hybridizations combined with zymographic analysis. Irradiated rat testes were used to assess the cell localization. Both of the plasminogen activators were expressed in a strictly stage specific manner. Maximal expression of uPA mRNA was seen in Sertoli cells during stages VII-VIII of the cycle. The same expression in the basal compartment of the tubules was detected at 7 days post-irradiation (p-i), during a selective reduction of spermatogonia and preleptotene spermatocytes. Levels of tPA mRNA started to accumulate in Sertoli cells at stage VIII and were high during stages IX-XII and detectable during stages XIII-XIV. At 26 days p-i, reduction of pachytene spermatocytes, which are shown to be immunoreactive for tPA, did not have an effect on tPA mRNA expression. Catalytic activities of uPA and tPA changed concomitantly to their RNA levels in different stages of the cycle. However, at 7 days p-i, uPA activity was decreased at stages VII-VIII of the cycle suggesting that germ cell Sertoli cell interaction is important for uPA activity.

Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.