The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

STESYS2: extended STESYS software for MS Windows

The STESYS2 software is a new version of the IBM PC software supporting interactive stereological measurements. In comparison with the previous STESYS, it is enhanced by a number of useful options, e.g. on-line image input via a TV camera coupled with a microscope operating under MS Windows OS. The main advantage, when compared with other such software packages, is the design of the STESYS2 as a module of the freeware image processing system Image Tool which provides a user-friendly environment including a number of image processing and preprocessing routines. Capabilities of the STESYS2 are illustrated by a practical example: estimation of the surface area of capillaries in the terminal villi of human placenta by the Sandau spatial grid method.     

Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.     

Cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation

Summary The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1β and tumor necrosis factor α induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor β had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.     

Aerobic exercise maintains regional bone mineral density during weight loss in postmenopausal women

This studyexamines the effects of weight loss by caloric restriction (WL) andaerobic exercise plus weight loss (AEx+WL) on total and regional bonemineral density (BMD) in older women. Healthy,postmenopausal women [age 63 ± 1 (SE) yr] not onhormone-replacement therapy underwent 6 mo of WL(n = 15) consisting of dietarycounseling one time per week with a caloric deficit (250-350kcal/day) or AEx+WL (n = 15)consisting of treadmill exercise three times per week in addition tothe weight loss. Maximal aerobic capacity increased only in the AEx+WLgroup (P < 0.001). Body weight,percent fat, and fat mass decreased similarly in both groups(P < 0.005), with no changesin fat-free mass. Total body BMD (by dual-energy X-rayabsorptiometry) decreased in both groups(P < 0.05). Femoral neck, Ward'striangle, and greater trochanter BMD decreased in the WL group(P  0.05) but were not significantlydifferent after AEx+WL.L₂-L₄BMD did not significantly change in either group. Thus WL andAEx+WL both result in losses of totalbody BMD; however, AEx+WL appears to prevent the loss in regional BMDseen with WL alone in healthy, older women. This suggests that theaddition of exercise to weight-loss programs may reduce the risk forbone loss.

     

Immune systems biology: immunoprofiling of cells and molecules

White blood cells and their secreted products are key elements of immune systems biology that are important indicators of patient health and disease. We have developed the SurroScan microvolume laser scanning cytometer to immunoprofile hundreds of variables, including cell populations, cell surface antigens, and intracellular molecules in antibody-based assays on small samples (about 1 mL) of whole blood, processed blood, or other fluids without cell purification or washing steps. The system enables high-throughput, robust and automated data capture and analysis. We demonstrate the utility of this immunoprofiling technology platform by surveying patient samples before and after glucocorticosteroid administration and show both the expected and novel response characteristics. This system complements recent advances in genomic and proteomic approaches to disease prediction and monitoring.     

Pantolactams as androgen receptor antagonists for the topical suppression of sebum production

A series of pantolactam based compounds were identified as potent antagonists for the androgen receptor (AR). Those that possessed properties suitable for topical delivery were evaluated in the validated Hamster Ear Model. Several compounds were found to be efficacious in reducing wax esters, a major component of sebum, initiating further preclinical work on these compounds.     

Oestrous red deer hinds prefer male roars with higher fundamental frequencies

Across vertebrates, the observation that lower-pitched vocalizations are typically associated with larger and/or higher quality males has lead to the widespread belief that inter- and intra-sexual selection will produce male calls with low fundamental frequencies (F0). Here we investigated the response of oestrous red deer hinds to playback of re-synthesized male roars characterized by either higher than average or lower than average F0. We found that hinds prefer higher rather than lower ‘pitched’ roars, providing, to our knowledge, the first evidence of such a bias in nonhuman mammals. Our findings can be interpreted in relation to previous observations that the minimum F0 of roars is positively correlated with male reproductive success in free-ranging red deer stags, and that across Cervids the F0 of male mating calls shows extreme variability. Females showing preferences for higher-pitched roars might derive genetic benefits through more competitive male offspring. Our results emphasize the need for further investigations of female preferences in mammals in order to better understand the extreme variation of F0 values observed in male sexual calls.