Once-daily tablet formulation and in vitro release evaluation of cefpodoxime using hydroxypropyl methylcellulose: A technical note

Summary and Conclusions  Decreasing the dose frequency of cefpodoxime proxetil increases patient compliance; patients prefer to take the drug once daily. It also improves the rate of bacterial killing and hastens the cure from the indications, and therefore increases compliance. The hydrophilic matrix of HPMC controlled the cefpodoxime proxetil release effectively for 24 hours; hence, the formulation can be considered as a once-daily sustained-release tablet of cefpodoxime proxetil. The formulation showed acceptable pharmacotechnical properties and assay requirements. In vitro dissolution studies indicated a sustained-release pattern throughout 24 hours of the study that was comparable to the theoretical release profile. Drug release kinetics indicated that drug release was best explained by Higuchi’s equation, as these plots showed the highest linearity (r ²=0.9734), but a close relationship was also noted with zero-order kinetics (r ²=0.9708). Korsmeyer’s plots indicated ann value of 0.57, which was indicative of an anomalous diffusion mechanism or diffusion coupled with erosion; hence, the drug release was controlled by more than one process. Hixson-Crowell plots indicated a change in surface area and diameter of the tablets with the progressive dissolution of the matrix as a function of time. Published: September 22, 2006     

Chromium tolerance,oxidative stress response,morphological characteristics,and FTIR studies of phytopathogenic fungus <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>

Sclerotium rolfsii is one of the most destructive fungal plant pathogens that can infect over 500 plants and can adapt to diverse environmental conditions. The present research work was carried out to evaluate the impact of both hexa- and trivalent chromium (Cr) on growth, morphology, enzymatic characteristics, and metal accumulation in S. rolfsii under laboratory conditions. Experiments were performed in both malt extract broth and agar growth medium amended with six different concentrations (10, 20, 40, 60, 80, and 100 ppm) of each Cr(III) and Cr(VI) ions inoculated with fungus and incubated for 6–7 days at 25 ± 3 °C. In broth medium, the total protein content was declined and activities of antioxidant enzymes were increased with an increase in metal concentrations. Lower concentrations (10 ppm) of the metal ions stimulated the growth of fungus and higher concentrations (60–100) inhibited it. The Fourier transform infrared spectroscopy (FTIR) assessment showed hydroxyl, carboxyl, and amine groups as major metal binding sites. In agar medium, tolerance index was decreased up to 0.56 at 10–80 ppm of Cr(III) and up to 0.62 at 10–60 ppm of Cr(VI). Considerable modifications were observed in hyphal and sclerotial morphology with an increase in concentration of metal ions. The current study concluded that interference of Cr with growth and physiological process of S. rolfsii could affect its infection level on its host plant. This study provides important information regarding cultivation of susceptible plant varieties in Cr-polluted soil as evidenced by pathogen growth up to 50 ppm of Cr(III) and Cr(VI) ions.     

Differential effects of gastric bypass and banding on circulating gut hormone and leptin levels

Objective: To quantify plasma concentrations of hormones that regulate energy homeostasis in order to establish possible mechanisms for greater weight loss after Roux‐en‐Y gastric bypass (RYGBP) compared with gastric banding (BND). Research Methods and Procedures: Four groups of women were studied: lean (n = 8; mean BMI, 21.6 kg/m²); BND (n = 9; BMI, 35.8; 25% weight loss), RYGBP (n = 9; BMI, 34.2; 36% weight loss), and controls matched for BMI to the surgical groups (n = 11; BMI, 34.4). Results: Fasting total peptide YY (PYY) and PYY_(3–36) immunoreactivity were similar among all groups, but the postprandial response in the RYGBP group was exaggerated, such that 30 minutes after the meal, total and PYY_(3–36) levels were 2‐ to 4‐fold greater compared with all other groups. Maximal postprandial suppression of total ghrelin was blunted in the BND group (13%) compared with RYGBP (27%). Postprandial suppression of octanoylated ghrelin was also less in BND (29%) compared with RYGBP (56%). Fasting insulin was lower in RYGBP (6.6 μU/mL) compared with BND (10.0 μU/mL). Compared with lean controls, leptin concentrations were significantly higher in BND but not in RYGBP. There was a greater increase in post‐meal satiety in the RYGBP group compared with BND and overweight controls. Discussion: The differences between RYGBP and BND subjects in postprandial concentrations of PYY and ghrelin would be expected to promote increased satiety and earlier meal termination in RYGBP and may aid in greater weight loss. The differences in insulin and leptin concentrations associated with these procedures may also reflect differences in insulin sensitivity and energy partitioning.     

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.     

A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1

Autophagic flux can be measured by determining the declining abundance of autophagic substrates such as sequestosome 1 (SQSTM1, better known as p62), which is sequestered in autophagosomes upon its direct interaction with LC3. However, the total amount of p62 results from two opposed processes, namely its synthesis (which can be modulated by some cellular stressors including autophagy inducers) and its degradation. To avoid this problem, we generated a stable cell line expressing a chimeric protein composed by p62 and the HaloTag (?) protein, which serves as a receptor for fluorescent HaloTag (?) ligands. Upon labeling with HaloTag (?) ligands (which form covalent, near-to-undissociable bonds with the Halotag (?) receptor) and washing, the resulting fluorescent labeling is not influenced by de novo protein synthesis, therefore allowing for the specific monitoring of the fusion protein decline without any interference by protein synthesis. We demonstrate that a HaloTag (?) -p62 fusion protein stably expressed in suitable cell lines can be used to monitor autophagy by flow cytometry and automated fluorescence microscopy. We surmise that this system could be adapted to high-throughput applications.     

Soluble Flt-1 and PlGF: New Markers of Early Pregnancy Loss?

Recent data have indicated a relationship between placental oxygen and angiogenic protein levels in the first trimester of normal pregnancies. Our objective was to investigate if maternal serum levels of angiogenic factors Soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1), soluble Endoglin and placental growth factor (PlGF) are altered in women with symptoms of threatened miscarriage (TM) and if they are predictive of a subsequent miscarriage. Blood samples were collected at 6–10 weeks from women presenting with TM (n = 40), from asymptomatic controls (n = 32) and from non- pregnant women in their luteal phase (n = 14). All samples were assayed for serum level of sFLT-1, PlGF, sEndoglin and HSP70 using commercial ELISAs. Samples were analysed retrospectively on the basis of pregnancy outcome. TM group included 21 women with a normal pregnancy outcome and 19 with subsequent complete miscarriage. The latter subgroup had significantly lower mean maternal serum (MS) sFlt-1 (83%, P<0.001) and PlGF (44%, P<0.001) compared to those with a normal pregnancy outcome. Asymptomatic control pregnant women had similar MS levels of sFlt-1 and PlGF compared to the TM patients with a normal outcome. The mean MS sFlt-1 (>10 fold) and MS PlGF (∼2 fold) levels were significantly (P<0.001) higher in control pregnant women compared to the non-pregnant group in the luteal phase of the menstrual cycle. Soluble Endoglin was not altered in the normal pregnant women compared to non pregnant women, although lower in the TM subgroup with a subsequent miscarriage (∼25%, P<0.001) compared to TM with a live birth. There was no significant difference in the mean MS HSP 70 levels between the different groups. This study shows that sFlt1 and PlGF MS levels are increased by several folds in early pregnancy and that MS sFlt-1 and MS PlGF are markedly decreased in threatened miscarriage patients who subsequently have a miscarriage suggesting these proteins are sensitive predictive markers of subsequent pregnancy loss.     

Population attributable risk of unintentional childhood poisoning in Karachi Pakistan

Background

The percentage of unintentional childhood poisoning cases in a given population attributable to specific risk factors (i.e., the population attributable risk) which can be calculated; determination of such risk factors associated with potentially modifiable risk factors, are necessary to focus on the prevention strategies.

Methods

We calculated PARs, using 120 cases with unintentional poisoning and 360 controls in a hospital based matched case- control study. The risk factors were accessibility to hazardous chemicals and medicines due to unsafe storage, child behavior reported as hyperactive, storage of kerosene and petroleum in soft drink bottles, low socioeconomic class, less education of the mother and the history of previous poisoning.

Results

The following attributed risks were observed: 12% (95% confidence interval [CI] = 8%–16%) for both chemicals and medicines stored unsafe, 19% (15%–23%) for child reported as hyperactive, 40% (38%–42%) for storage of kerosene and petroleum in soft drink bottles, 48% (42%–54%) for low socioeconomic status, 38% (32%–42%) for no formal mothers education and 5.8% (2%–10%) for history of previous poisoning. 48% of cases for overall study population which could be attributed to at least one of the six risk factors. Among girls, this proportion was 23% and 43% among boys. About half of the unintentional childhood poisoning cases in this Pakistani population could be avoided.

Conclusion

Exposure to potentially modifiable risk indicators explained about half of the cases of unintentional poisoning among children under five years of age in this Pakistani population, indicating the theoretical scope for prevention of the disease.     

H₂O₂ stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4

In fission yeast, the stress-activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H(2)O(2) but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H(2)O(2), resulting in the full-length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4.