Tenax-GC Extraction Technique for Residual Polychlorinated Biphenyl and Polyaromatic Hydrocarbon Analysis in Biodegradation Assays

A rapid Tenax-GC extraction technique has been evaluated for use in conjunction with aqueous biodegradation assays for polyaromatic hydrocarbons and polychlorinated biphenyls. The method was quantitatively efficient and reproducible for phenanthrene, but variable and not quantitative for Aroclor 1254 (polychlorinated biphenyls). Aqueous sample volumes and varying concentrations of organic matter influenced polychlorinated biphenyl and polyaromatic hydrocarbon extraction efficiency. Phenanthrene recovery was decreased by soil extract but unaffected by spent bacteriological culture medium. Both types of organic matter caused significant reduction of Aroclor 1254 recovery. Polyaromatic hydrocarbon and polychlorinated biphenyl biodegradation assays, performed with reservoir samples, supported the laboratory evaluation. The study demonstrated the utility of the Tenax-GC extraction technique for phenanthrene analysis in biodegradation assessment; however, Tenax-GC extraction was not appropriate for Aroclor 1254 biodegradation studies.     

Radiation-induced aberration in aneuploid vs. diploid swine leukocytes

Swine leukocytes were cultured for 48 h after receiving γ-ray exposures up to 400 R. Cells with one or two less than the diploid number of centromeres consistently contained more chromosome deletions than did diploid cells. This effect was not evident for dicentric and ring aberrations. Both aneuploid frequencies and the proportion of aberrations in aneuploid cells increased in irradiated samples but showed no dose-response relationship.     

Inhibitors of sterol synthesis. Dietary administration of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one inhibits the intestinal absorption of cholesterol in lymph-cannulated rats

The effect of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol synthesis with marked hypocholesterolemic activity, on the intestinal absorption of exogenous cholesterol has been studied in lymph-cannulated rats. Administration of the 15-ketosterol at a level of 0.05% in a rat chow diet for 10 days was associated with a marked decrease (-64%) in the absorption of cholesterol.     

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009. © 2009 Wiley‐Liss, Inc.     

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010. © 2009 Wiley‐Liss, Inc.     

Kinetics of activation of the tyrosine kinase of a deletion mutant of epidermal growth factor receptor missing the sites of self-phosphorylation

The addition of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) dissolved in a solution of the detergent Triton X-100 results in the activation of its protein tyrosine kinase. To investigate the importance of the sites for self-phosphorylation on the enzyme in this process, the kinetics of activation of a deletion mutant missing the last 195 amino acids of the protein, including all of the sites for self-phosphorylation, were followed by monitoring the initial velocity at which the enzyme catalyzes the phosphorylation of the exogenous substrate RRKGSTAENAEYLRV. The activation of the enzymatic activity of this deletion mutant of EGF receptor displays kinetics that are second-order with respect to the concentration of the enzyme as does wild-type EGF receptor. The second-order rate constant for its activation is 36 +/- 10 microM-1 min-1, which is only 3-fold greater than the second-order rate constant for the activation of wild-type EGF receptor under the same conditions (13 +/- 2 microM-1 min-1). These results suggest that the mechanism by which the protein tyrosine kinase of the deletion mutant is activated is the same as that for the activation of the wild-type receptor and that the sites of self-phosphorylation in the wild-type EGF receptor do not participate in the mechanism of activation of the enzyme.     

Comparison of the β-lactamases ofBacteroides species

-Lactam antibiotic susceptibility and the presence of -lactamase were examined in clinical strains ofBacteroides species. All strains produced a noninducible, cell-associated cephalosporinase. Based on isoelectric focusing, molecular weight determinations, substrate profiles, and inhibition studies, it was concluded that allBacteroides strains examined produced a very similar, if not identical, -lactamase in terms of these enzymatic and physical characteristics.     

Insulin resistance and decreased spexin in Indian Patients with Type 2 Diabetes Mellitus

Spexin is novel biomarker, which plays a potential role in glucose and lipid metabolisms. However, there was paucity of serum spexin levels in obesity and diabetes mellitus subjects. Hence the current study was aimed to find the relationship between the serum spexin levels in type 2 Diabetes mellitus (type 2 DM) with extrapolation of cardiovascular disease (CVD) risk. A cross-sectional study included 330 participants, subdivided as control (n=110), type 2 DM (n=110) and type 2 DM with CVD groups (n=110). HbA1c, insulin, lipid profile, spexin & leptin including blood pressure and body mass index were analyzed from all the participants. The serum spexin levels (ng/ml) were significantly decreased in type 2 DM (mean ± sd: 0.65 ± 0.03) and type 2 DM with CVD (0.48 ± 0.02) groups compared to the control (0.79 ± 0.03) group (p<0.001). The decreased spexin levels were observed in type 2 DM, and further more decreased in type 2 DM with CVD patients compared to controls indicating that spexin levels could be served as an early prediction of obesity-induced T2DM with CVD risk.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.