Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Variability in tablet fragment weights when splitting unscored cyclobenzaprine 10 mg tablets.

OBJECTIVE: To determine the weight variation and calculated dosing variability of tablet fragments upon splitting unscored cyclobenzaprine hydrochloride 10 mg tablets using two common tablet splitting devices. DESIGN: Comparative pharmaceutics study. SETTING: Pharmacy school laboratory. PARTICIPANTS: Not applicable. INTERVENTIONS: Unscored cyclobenzaprine hydrochloride 10 mg tablets from one generic manufacturer were split with a tablet splitter or a kitchen knife by a licensed pharmacist and two doctor of pharmacy students (n = 15 tablets for each method per participant). MAIN OUTCOME MEASURES: Fragment weights (FWs) were compared with the theoretical weights (TWs), which were calculated as one half of the mean weight of the tablets used in each part of the experiment; means, relative standard deviations (RSDs), and percentages of TW were also calculated. RESULTS: The mean weight before splitting the 45 tablets with the tablet splitter was 136.6 +/- 2.1 mg (TW = 68.3 mg). The mean FW after splitting was 67.9 +/- 7.9 mg. The RSD of 11.6% corresponded to a range of 69.4% to 130.2% of the TW and an estimated drug content of the split fragments between 3.47 mg and 6.51 mg. The mean weight before splitting the 45 tablets cut with a kitchen knife was 136.6 +/- 2.0 mg (TW = 68.3 mg). The mean FW was 68.0 +/- 15.7 mg with a RSD of 23.2%, corresponding to a range of 49.9% to 149.5% of the TW and an estimated drug content of the split fragments between 2.49 mg and 7.48 CONCLUSION: Tablet fragments obtained after splitting this generic cyclobenzaprine 10 mg product varied considerably in weight and estimated drug content. Accordingly, splitting cyclobenzaprine 10 mg tablets to achieve 5 mg doses could result in unpredictable dosing and therapeutic response.     

An oral vaccine for type 1 diabetes based on live attenuated Salmonella

Type 1 diabetes (T1D) is a metabolic disease that is initiated by the autoimmune destruction of pancreatic insulin-producing beta cells that is accompanied by the development of antigen-specific antibodies and cytotoxic T lymphocytes (CTLs). Several studies have shown that vaccination with diabetic autoantigens provides some protection against this process. In this report we describe a new oral vaccine that utilizes live attenuated Salmonella for simultaneous delivery of autoantigens in conjunction with immunomodulatory cytokine genes to immune cells in the gut mucosa. Recent data showed that live attenuated Salmonella is a safe, simple and effective vector for expression of antigens and cytokines by antigen-presenting cells (APCs) of gut-associated lymphatic tissue (GALT). This novel strategy was tested by fusion of the diabetic autoantigen preproinsulin with Salmonella secretory effector protein (SseF) of pathogenicity island-2 (SPI2). In this way the autoantigen is only expressed inside the host immune cells and translocated to the host cell cytosol. In addition Salmonella was used to deliver the gene for the immunomodulatory cytokine transforming growth factor beta (TGFβ) for host cell expression. Oral co-vaccination of 8 week-old non-obese diabetic (NOD) mice with three weekly doses of both the autoantigen and cytokine significantly reduced the development of diabetes, improved the response to glucose challenge, preserved beta cell mass, and reduced the severity of insulitis compared with controls and autoantigen alone. Combination therapy also resulted in increased circulating levels of IL10 four weeks post-vaccination and IL2 for 12 weeks post-vaccination, but without effect on proinflammatory cytokines IL6, IL12(p70), IL17 and IFNγ. However, in non-responders there was a significant rise in IL12 compared with responders. Future studies will examine the mechanism of this vaccination strategy in more detail. In conclusion, Salmonella-based oral vaccines expressing autoantigens combined with imunomodulatory cytokines appears to be a promising therapy for prevention of T1D.     

Lower Frequency and Diversity of Antibiotic-Producing Fluorescent Pseudomonads in Rhizosphere of Indian Rapeseed–Mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L. Czern.)

Fluorescent pseudomonads are one of the most important microbial communities which play a key role in rhizosphere to enhance plant growth-promotion and protection. The diverse groups of antibiotics viz. 2,4-diacetylphloroglucinol (DAPG), phenazine-1-carboxylic acid (PCA) and pyoluteorin (PLT) are produced by fluorescent pseudomonads inhibiting growth of fungal pathogens which results in health upliftment of plants. The present study, discusses about frequency and diversity of 138 antibiotic-producing fluorescent pseudomonads isolated from eight genotypes of rapeseed mustard rhizosphere (Brassica juncea L. Czern.). The plant growth promoting traits and antibiotics (DAPG, PCA and PLT) production of isolates were examined by using polymerase chain reaction (PCR), thin layer chromatography (TLC) and dot blot-hybridization. Among 138 isolates, 47, 25 and 9 % of isolates were positive in indole production, phosphate solubilization and antagonism potential against Sclerotinia sclerotiorum (causal agent of white mold disease in rapeseed mustard), respectively. PCR amplifications showed that none of the isolates had phlD (DAPG) and phzC (PCA) genes, but four isolates (UKA-2, UKA-8, UKA-11, UKA-66) had pltB (PLT) gene, which was further confirmed by TLC and DNA dot-blot hybridization. BOX profiles of pltB positive isolates were distinct, showing unique genetic diversity in the small population. The four pltB positive fluorescent pseudomonad isolates could be used as promising bio-control and plant growth-promoting inoculants for Indian rapeseed mustard.     

Weighted Coherence: A More Effective Measure Than Average Coherence

In this study, we evaluated the effectiveness of the statistic, Weighted Coherence in relation to the average or mean coherence in a particular frequency band after cross-spectral analysis. In time domain, correlation coefficient explains the relationship between two variables of interest, and a similar statistic in frequency domain using cross-spectral analysis is referred to as coherence, which is equivalent to the squared correlation coefficient and ranges from zero to unity. In this study, we show that weighted coherence is much more sensitive to various challenges within the same subject and also may be a more sensitive indicator of group differences in different frequency bands. Our data on normal subjects and patients with panic disorder during supine and standing postures during normal breathing, and also at different breathing rates throw new light on the sensitivity of this previously described novel index to quantify coherence between two related time series such as beat-to-beat heart rate and QT interval.     

Adulticidal, repellent, and ovicidal properties of indigenous plant extracts against the malarial vector, Anopheles stephensi (Diptera: Culicidae)

Mosquito-borne diseases with an economic impact create loss in commercial and labor outputs, particularly in countries with tropical and subtropical climates. Mosquito control is facing a threat because of the emergence of resistance to synthetic insecticides. Extracts from plants may be alternative sources of mosquito control agents because they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products and potentially suitable for use to control mosquitoes. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. In view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the adulticidal, repellent, and ovicidal potential of the crude hexane, ethyl acetate, benzene, aqueous, and methanol solvent extracts from the medicinal plants Andrographis paniculata, Cassia occidentalis, and Euphorbia hirta against the medically important mosquito vector, Anopheles stephensi (Diptera: Culicidae).The adult mortality was observed after 24 h of exposure. All extracts showed moderate adulticide effects; however, the highest adult mortality was found in methanol extract of A. paniculata followed by C. occidentalis and E. hirta against the adults of A. stephensi with LC₅₀ and LC₉₀ values of 210.30, 225.91, and 263.91 ppm and 527.31, 586.36, and 621.91 ppm, respectively. The results of the repellent activity of hexane, ethyl acetate, benzene, aqueous, and methanol extract of A. paniculata, C. occidentalis, and E. hirta plants at three different concentrations of 1.0, 3.0, and 6.0 mg/cm² were applied on skin of forearm in man and exposed against adult female mosquitoes. In this observation, these three plant crude extracts gave protection against mosquito bites without any allergic reaction to the test person, and also, the repellent activity is dependent on the strength of the plant extracts. Mean percent hatchability of the ovicidal activity was observed 48 h post-treatment. The percent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. Mortality of 100 % with methanol extract of A. paniculata exerted at 150 ppm and aqueous, methanol extract of C. occidentalis and E. hirta were exerted at 300 ppm. These results suggest that the leaf extracts of A. paniculata, C. occidentalis, and E. hirta have the potential to be used as an ideal eco-friendly approach for the control of the A. stephensi. Further detailed research is needed to identify the active ingredient in the extracts and implement the effective mosquito management program.