Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Radiation effects on diamine oxidase activities in intestine and plasma of the rat

Diamine oxidase (DAO; EC 1.4.3.6) activity was measured in plasma and in ileal tissue homogenates prepared from male Sprague-Dawley rats euthanized at 1-15 days after acute whole-body irradiation with 14.5-MeV electrons. Animals irradiated with 1 Gy showed no diminution in plasma and ileal DAO activities through Day 13 relative to nonirradiated controls. Animals irradiated with 5, 10, and 12 Gy displayed marked declines in ileal DAO activity, with levels reaching a nadir on Day 3. This was paralleled by a decrease in plasma DAO activity in all three dose groups. Recovery of ileal and plasma DAO levels was later seen as early as Day 4 in animals irradiated with 5- and 10-Gy doses, but animals receiving 12 Gy did not survive beyond Day 3. The relationship between radiation dose and levels of plasma and ileal DAO on Day 3, the time of maximum decrease at all doses, was also investigated. Ileal DAO activity decreased almost linearly between 2 and 8 Gy. Plasma DAO activity closely paralleled the dose dependency of the ileal levels. These data suggest that plasma DAO activity might be useful as a biologic marker of intestinal epithelial injury and recovery after acute radiation exposure.     

Effects of tissue preservation on murine bone mechanical properties

Murine bone specimens are used extensively in skeletal research to assess the effects of environmental, physiologic and pathologic factors on their mechanical properties. Given the destructive nature of mechanical testing, it is normally performed as a terminal procedure, where specimens must be preserved without affecting their mechanical properties. To this end, we aimed to study the effects of tissue preservation (freezing and formalin fixation) on the elastic and viscoelastic mechanical properties of murine femur and vertebrae. A total of 120 femurs and 180 vertebral bodies (L3–L5) underwent non-destructive cyclic loading to assess their viscoelastic properties followed by mono-cyclic loading to failure to assess their elastic properties. All specimens underwent re-hydration in 0.9% saline for 30 min prior to mechanical testing. Analysis indicated that stiffness, modulus of elasticity, yield load, yield strength, ultimate load and ultimate strength of frozen and formalin-fixed femurs and vertebrae were not different from fresh specimens. Cyclic loading of both femurs and vertebrae indicated that loss, storage and dynamic moduli were not affected by freezing. However, formalin fixation altered their viscoelastic properties. Our findings suggest that freezing and formalin fixation over a 2-week period do not alter the elastic mechanical properties of murine femurs and vertebrae, provided that specimens are re-hydrated for at least half an hour prior to testing. However, formalin fixation weakened the viscoelastic properties of murine bone by reducing its ability to dissipate viscous energy. Future studies should address the long-term effects of both formalin fixation and freezing on the mechanical properties of murine bone.     

A protein processing filter method for bacterial identification by mass spectrometry-based proteomics

A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.     

Inhibition of angiotensin-converting enzyme by des-Leu10-angiotensin I: a potential mechanism of endogenous angiotensin-converting enzyme regulation

Des-Leu10-angiotensin I is a nonapeptide generated from angiotensin I by the action of carboxypeptidase-like activities residing in the human platelet and mast cell. This nonapeptide was found to inhibit rabbit lung angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) with a Ki of 3.1 X 10(-7) M. The mechanism of inhibition was competitive. Inhibition of human serum angiotensin-converting enzyme by des-Leu10-angiotensin I was comparable in magnitude to inhibition by bradykinin and angiotensin III. These results suggest that limited proteolysis of angiotensin I by cells resident in vascular tissue may result in the generation of an endogenous inhibitor of angiotensin-converting enzyme. Such pathways may play roles in controlling levels of vasoactive peptides at local vascular sites.     

Densitometric, morphometric and mechanical distributions in the human proximal femur

With the prevalent use of DXA-measured BMD to assess pathologic hip fractures and its recently reported lack of reliability to predict fracture or account for efficacy of anti-resorptive therapy, it is reasonable to assess whether variations in the primary and secondary tensile and compressive trabecular microstructure can account for variations in proximal femur strength in comparison to DXA-measured BMD. To that end, microstructural and densitometric measures of trabecular bone specimens, from discrete sites within the proximal femur, were correlated with their mechanical properties. We hypothesize that accounting for regional variations in trabecular microstructure will improve predictions of proximal femur strength and stiffness compared to bone density measured by DXA. Forty-seven samples (seven donors) from seven distinct sites of human proximal femur underwent DXA and muCT imaging and mechanical testing. The results revealed significant variations in BMC, morphometric indices and mechanical properties within the proximal femur. This work has demonstrated that the mechanical performance of each sub-region is highly dependent on the corresponding trabecular microstructure. BMD measured by DXA at standard regions of interest cannot resolve the variations in trabecular density and microstructure that govern the mechanical behavior of the proximal femur. This work suggests that a quantitative Singh index that uses high resolution QCT to monitor the trabecular microstructure at specific sub-regions of the proximal femur may allow better predictions of hip fracture risk in individual patients and an improved assessment of changing bone structure in response to pharmacological interventions.     

Invasibility in a spatiotemporally fluctuating environment is determined by the periodicity of fluctuations and resident turnover rates

The ability of a species to invade a community is influenced by the traits of the invader, the resident community and the environment. However, qualitative generalizations are possible. Using a model of perennial plants in a spatiotemporally fluctuating environment, we find that fluctuating environments may be more or less invasible than static environments. Invasibility is strongly dependent on the interaction of the difference in turnover rates of resident and invader populations and the rate of temporal change of the environment. If resident population turnover is faster than the invader's, then invasibility is an initially positive, decreasing function of the period temporal variation, such that invasibility is increased by rapid temporal fluctuations but slightly reduced in slowly fluctuating environments. If resident turnover is slower than the invader's, then invasibility is an initially negative, increasing function of temporal period, such that invasibility is reduced in rapidly changing environment facilitated by slow temporal fluctuations. These results are explained by the relative abilities of resident and invader populations to successfully respond to environmental variation at different temporal scales.