Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.     

Detection of Xeljanz Enantiomers in Diethyl Amine Active Pharmaceutical Ingredients and Tablets

A high‐performance liquid chromatography (HPLC) method was established to detect Xeljanz enantiomers in active pharmaceutical ingredients (APIs) and tablets. The separation was achieved on a Chiralpak IC column using a mobile phase of hexane‐ethanol‐diethylamine (65:35:0.1, v/v). The detection wavelength was 289 nm. The peak areas and the enantiomer concentrations in the range of 0.15–2.25 μg?mL^?1 were in high linearity, with correlation coefficients higher than 0.999. The recoveries were 86.44% at the concentrations of 7.5, 18.75, and 37.5 μg?mL^?1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.042 and 0.14 μg?mL^?1, respectively. This HPLC method is suitable for detecting the enantiomers of Xeljanz in its APIs and tablets. Chirality 27:235–238, 2015. © 2014 Wiley Periodicals, Inc.     

Increased Risks of Mortality and Atherosclerotic Complications in Incident Hemodialysis Patients Subsequently with Bone Fractures: A Nationwide Case-Matched Cohort Study

Background

Hemodialysis (HD) patients with bone fractures have an increased risk for death. However, the risks for mortality and atherosclerotic complications in incident HD patients subsequently with bone fractures are unknown.

Methods

Data derived from the Taiwan National Health Institute Research Database between January 1997 and December 2008 was analyzed. The enrolled patients included 3,008 incident HD patients subsequently with a single long bone fracture (LB Fx) and 2,070 incident HD patients subsequently with a single non-long bone fracture (NLB Fx). These patients were matched (1:5 ratio) for age, sex, and same duration of HD with incident HD patients who had no fractures and outcomes were measured over a 3-year follow-up.

Results

After demographic and co-morbidity adjustment, LB Fx increased the risk for overall mortality (HR = 1.59, p < 0.001) and stroke (HR = 1.09, p = 0.028) in incident HD patients. NLB Fx increased the risk for overall mortality (HR = 1.52, p < 0.001), stroke (HR = 1.19, p < 0.001), coronary artery disease (CAD), (HR = 1.13, p = 0.003), and peripheral arterial occlusive disease (PAOD), (HR = 1.41, p < 0.001) in incident HD patients. Moreover, incident patients subsequently with NLB Fx had significantly higher risks of CAD and PAOD than those subsequently with LB Fx.

Conclusions

The rates of mortality and stroke were significantly higher in incident HD patients subsequently with bone fractures than in matched patients without bone fractures. Incident HD patients subsequently with NLB Fx had significantly higher risks of CAD and PAOD than those subsequently with LB Fx and without bone fractures. Thus, incident HD patients subsequently with bone fractures should be closely followed for a higher mortality and possible development of atherosclerotic complications.     

Crystal structure of human S100A8 in complex with zinc and calcium

Background

S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.

Results

Here we report two crystallographic structures of calcium and zinc-loaded human S100A8. S100A8 binds two zinc ions per homodimer, through two symmetrical, all-His tetracoordination sites, revealing a classical His-Zn binding mode for the protein. Furthermore, the presence of a (Zn)₂-cacodylate complex in our second crystal form induces ligand swapping within the canonical His₄ zinc binding motif, thereby creating two new Zn-sites, one of which involves residues from symmetry-related molecules. Finally, we describe the calcium-induced S100A8 tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.

Conclusions

Our structures of Zn²⁺/Ca²⁺-bound hS100A8 demonstrate that S100A8 is a genuine His-Zn S100 protein. Furthermore, they show how zinc stabilizes S100A8 tetramerization and potentially mediates the formation of novel interdimer interactions. We propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo.

     

Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.     

Exhaled Aerosol Pattern Discloses Lung Structural Abnormality: A Sensitivity Study Using Computational Modeling and Fractal Analysis

Background

Exhaled aerosol patterns, also called aerosol fingerprints, provide clues to the health of the lung and can be used to detect disease-modified airway structures. The key is how to decode the exhaled aerosol fingerprints and retrieve the lung structural information for a non-invasive identification of respiratory diseases.

Objective and Methods

In this study, a CFD-fractal analysis method was developed to quantify exhaled aerosol fingerprints and applied it to one benign and three malign conditions: a tracheal carina tumor, a bronchial tumor, and asthma. Respirations of tracer aerosols of 1 µm at a flow rate of 30 L/min were simulated, with exhaled distributions recorded at the mouth. Large eddy simulations and a Lagrangian tracking approach were used to simulate respiratory airflows and aerosol dynamics. Aerosol morphometric measures such as concentration disparity, spatial distributions, and fractal analysis were applied to distinguish various exhaled aerosol patterns.

Findings

Utilizing physiology-based modeling, we demonstrated substantial differences in exhaled aerosol distributions among normal and pathological airways, which were suggestive of the disease location and extent. With fractal analysis, we also demonstrated that exhaled aerosol patterns exhibited fractal behavior in both the entire image and selected regions of interest. Each exhaled aerosol fingerprint exhibited distinct pattern parameters such as spatial probability, fractal dimension, lacunarity, and multifractal spectrum. Furthermore, a correlation of the diseased location and exhaled aerosol spatial distribution was established for asthma.

Conclusion

Aerosol-fingerprint-based breath tests disclose clues about the site and severity of lung diseases and appear to be sensitive enough to be a practical tool for diagnosis and prognosis of respiratory diseases with structural abnormalities.     

Tolerance of soil algae and cyanobacteria to drought stress

Tolerance to drought stress in soil crust microorganisms is essential for exploiting suitable organisms for restoring soil. In this study, the responses to drought stress of two drought‐tolerant species, a green alga and a cyanobacterium, were compared with those of two non‐tolerant green algae. In response to drought stress, induced by treatment with polyethylene glycol, the intracellular proline levels increased and were associated with increases in malondialdehye, pigment contents, and enzyme activities such as superoxide dismutase (SOD) and peroxidase (POD). Our results suggest that tolerance to drought stress could be indicated by the intracellular levels of proline, SOD, and carotenoids. This study provides insights into the drought physiology of the photosynthetic microorganisms and suggests that Leptolyngbya boryana and Chlorella vulgaris are suitable pioneer organisms for soil restoration.     

The potato amylase inhibitor gene SbAI regulates cold‐induced sweetening in potato tubers by modulating amylase activity

Potato cold‐induced sweetening (CIS) is critical for the postharvest quality of potato tubers. Starch degradation is considered to be one of the key pathways in the CIS process. However, the functions of the genes that encode enzymes related to starch degradation in CIS and the activity regulation of these enzymes have received less attention. A potato amylase inhibitor gene known as SbAI was cloned from the wild potato species Solanum berthaultii. This genetic transformation confirmed that in contrast to the SbAI suppression in CIS‐resistant potatoes, overexpressing SbAI in CIS‐sensitive potatoes resulted in less amylase activity and a lower rate of starch degradation accompanied by a lower reducing sugar (RS) content in cold‐stored tubers. This finding suggested that the SbAI gene may play crucial roles in potato CIS by modulating the amylase activity. Further investigations indicated that pairwise protein–protein interactions occurred between SbAI and α‐amylase StAmy23, β‐amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both α‐amylase and β‐amylase in potato tubers primarily by repressing StAmy23 and StBAM1, respectively. These findings provide the first evidence that SbAI is a key regulator of the amylases that confer starch degradation and RS accumulation in cold‐stored potato tubers.