Second‐derivative synchronous spectrofluorimetric determination of nebivolol hydrochloride and amlodipine besylate in their combined dosage form

A rapid, simple, accurate and highly sensitive spectrofluorimetric method was developed for the simultaneous analysis of nebivolol hydrochloride (NEB) and amlodipine besylate (AML). The method was based on measuring the synchronous fluorescence intensity of the drugs at Δλ = 40 nm in methanol. Various experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully studied and optimized. The calibration plots were rectilinear over concentration ranges of 0.05–1.5 µg/mL and 0.5–10 µg/mL for NEB and AML with limits of detection (LOD) of 0.010 and 0.051 µg/mL and limits of quantitation (LOQ) of 0.031 and 0.156, respectively. The peak amplitudes (²D) of the second derivative synchronous fluorimetry (SDSF) were estimated at 282 nm for NEB and at 393 nm for AML. Good linearity was obtained over the concentration ranges. The proposed method was successfully applied to the determination of the studied compounds in laboratory‐prepared mixtures, commercial single and laboratory‐prepared tablets. The results were in good agreement with those obtained using the comparison method. The mean percent recoveries were found to be 100.12 ± 0.77 and 99.91 ± 0.77 for NEB and AML, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Phagocytosis of degenerating myelin in transected feline optic nerve: an immunohistochemical study

The phagocytic activity of neuroglial cells in adult feline degenerating optic nerve was investigated by immunocytochemistry at both light and electron microscopy levels. Degeneration was initiated by unilateral eye enucleation and the segment distal to the transection showing true Wallerian degeneration was examined. Following enucleation, twelve adult domestic cats were examined over a period of seven to 215 days. All cases showed slow clearance of myelin debris and absence of proliferating monocytes throughout the post-enucleation period. All phagocytic cells present were neuroglial cells, and many of these cells expressed oligodendroglial antigens. These findings demonstrate the persistence of an active population of oligodendrocytes that might play an additional functional role during Wallerian degeneration of feline optic nerve.     

Serum dihydroceramides correlate with insulin sensitivity in humans and decrease insulin sensitivity in vitro

Serum ceramides, especially C16:0 and C18:0 species, are linked to CVD risk and insulin resistance, but details of this association are not well understood. We performed this study to quantify a broad range of serum sphingolipids in individuals spanning the physiologic range of insulin sensitivity and to determine if dihydroceramides cause insulin resistance in vitro. As expected, we found that serum triglycerides were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals. Serum ceramides were not significantly different within groups but, using all ceramide data relative to insulin sensitivity as a continuous variable, we observed significant inverse relationships between C18:0, C20:0, and C22:0 species and insulin sensitivity. Interestingly, we found that total serum dihydroceramides and individual species were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals, with C18:0 species showing the strongest inverse relationship to insulin sensitivity. Finally, we administered a physiological mix of dihydroceramides to primary myotubes and found decreased insulin sensitivity in vitro without changing the overall intracellular sphingolipid content, suggesting a direct effect on insulin resistance. These data extend what is known regarding serum sphingolipids and insulin resistance and show the importance of serum dihydroceramides to predict and promote insulin resistance in humans.Supplementary key words: sphingolipids, circulating ceramides, serum, insulin resistance, lipidomics, CVD, T2D, obesity, myotube

Circulating ceramides, especially specific saturated ceramide species, and other sphingolipids are linked to CVD risk and insulin resistance (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In fact, circulating ceramide and sphingolipid contents predict development of CVD better than some common risk factors such as plasma cholesterol, LDLs, and triglycerides (6, 9, 11, 12). As a result, it was recently proposed that plasma ceramide could be the new cholesterol for assessing risk of CVD (11). Beyond the cross-sectional studies referenced above, there are several lines of evidence supporting the link between ceramides, CVD, and insulin resistance. Plasma ceramide content decreases after insulin-sensitizing gastric bypass surgery and weight loss interventions (13, 14, 15). Animal studies show that ceramides accumulate in atherosclerotic lesions, which may explain the increased risk associated with plasma content (16). However, the relationship of circulating sphingolipids to insulin resistance is not absolute, as insulin-sensitizing treatments do not always change plasma sphingolipid content (17). Combined, most data from epidemiology studies, as well as human interventions and animal models, support the concept that circulating ceramides and sphingolipids are related to insulin resistance and CVD risk.Ceramides circulate primarily bound to lipoproteins and are secreted predominately by the liver. Circulating ceramides are mainly increased in LDL in individuals with obesity (15). Obese rodents have increased hepatic ceramide secretion, which may explain increased plasma ceramide content in individuals with obesity (15). In one mechanistic study, an LDL-ceramide mixture was infused in mice to recapitulate increased plasma ceramide content in obesity, which caused membrane ceramide accumulation, decreased insulin signaling, and a decrease in insulin sensitivity specifically in skeletal muscle, providing evidence for a direct effect of circulating ceramides on tissues (15). Similarly, LDL-ceramide administration to myotubes caused ceramide accumulation, decreased insulin sensitivity, and signaling independent of inflammation. These data indicate that plasma ceramides are not simply markers of insulin resistance but play mechanistic roles in decreasing insulin sensitivity.Ceramides are only one member of the sphingolipid family, and other sphingolipids may also be related to insulin resistance and CVD risk. Lactosylceramides and glucosylceramides are sphingolipids that also accumulate in atherosclerotic plaques and therefore may be involved in the CVD process (18). Sphingomyelins are the most abundant sphingolipids circulating in lipoproteins and, while they are positively related to obesity and waist circumference, they are not correlated to insulin sensitivity in cross-sectional human studies (5, 19). Dihydroceramides are immediate precursors to ceramide synthesis and are negatively related to insulin sensitivity (20, 21) and insulin secretion (21), are positively related to waist circumference (22), are elevated in plasma of individuals with prediabetes and T2D compared with controls (23), and predict development of diabetes 9 years before onset (21). Despite strong evidence linking plasma dihydroceramides to decreased insulin sensitivity, mechanistic studies to determine if circulating dihydroceramides cause insulin resistance are lacking.To address this knowledge gap, we performed the current study to assess serum sphingolipids in humans across the metabolic spectrum as well as determine if dihydroceramides induce insulin resistance in vitro.     

Detection of a secreted metalloprotease within the nuclei of liver cells

ADAMTS13 is a secreted zinc metalloprotease expressed by various cell types. Here, we investigate its cellular pathway in endogenously expressing liver cell lines and after transient transfection with ADAMTS13. Besides compartmentalizations of the cellular secretory system, we detected an appreciable level of endogenous ADAMTS13 within the nucleus. A positively charged amino acid cluster (R-Q-R-Q-R-Q-R-R) present in the ADAMTS13 propeptide may act as a nuclear localization signal (NLS). Fusing this NLS-containing region to eGFP greatly potentiated its nuclear localization. Bioinformatics analysis suggests that the ADAMTS13 CUB-2 domain has a double-stranded beta helix (DSBH) structural architecture characteristic of various protein-protein interaction modules like nucleoplasmins, class I collagenase, tumor necrosis factor ligand superfamily, supernatant protein factor (SPF) and the B1 domain of neuropilin-2. Based on this contextual evidence and that largely conserved polar residues could be mapped on to a template CUB domain homolog, we hypothesize that a region in the ADAMTS13 CUB-2 domain with conserved polar residues might be involved in protein-protein interaction within the nucleus.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).