Expression of gap junctions in cultured rat L6 cells during myogenesis

Although gap junctions are absent from adult skeletal muscle, they have been described in embryonic and neonatal rat skeletal muscle and in cultured rat myoblasts. In order to determine the precise developmental expression and molecular composition of gap junctions during myogenesis, RNA was isolated from cultures of rat L6 myoblasts and examined using Northern blot analysis with cDNA probes specific for connexin32 and connexin43. Connexin32 mRNA could not be detected in rat myoblast and myotube samples. However, connexin43 mRNA was expressed at high levels in cycling L6 myoblasts and this expression decreased by approximately 60% in L6 myotubes following fusion. Immunofluorescent localization with an antibody specific for connexin43 confirmed the accumulation of connexin43 protein in membranes shared between adjacent myoblasts at 12 hr of culture. By 24 hr of culture, connexin43 disappeared from most cells, only to reappear at 36 hr at a low level that was maintained through 72 hr in culture. Although most myoblasts in these cultures expressed connexin43, myotubes expressed little or no membrane-associated connexin43. Dye transfer experiments established that, at 12 hr of culture, the majority of myoblasts were dye coupled suggesting that connexin43 protein is assembled into functional gap junctions. At 24 hr, the number of coupled cells decreased slightly, while at 48 hr, most of the myoblasts were not dye coupled. These results demonstrate that the expression of connexin43 is temporally correlated with myoblast fusion and may play a role in this process.     

Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies

During angiogenesis in the chorioallantoic membrane (CAM) of the chick, capillary proliferation occurs primarily by intussusceptive growth. Previously, we reported that such growth in the CAM proceeded without substantial macromolecular extravasation. Neovascularization involving capillary sprout formation, on the other hand, has been associated with a concomitant loss of endothelial selectivity. Thus, the present study tested the hypothesis that endothelial selectivity during angiogenesis is dependent on the mode of microvascular growth. Capillary sprout formation occurs in peripheral regions of the CAM, in addition to the more centrally located areas of intussuceptive growth. In this study, angiogenic endothelial permselectivities were evaluated in these respective areas of CAM microvascular growth by intravital fluorescent microscopy of a graded series of FITC-dextrans. In both cases, the angiogenic endothelia restricted extravasation of macromolecules > or = 20 kDa. Furthermore, capillary sprout endothelia, like the intussusceptive CAM endothelia, remained tightly sealed at the junctional clefts. Thus, angiogenic endothelial permselectivity in the CAM is not dependent on the mode of microvascular growth. Whether distinct cellular mechanisms are operable in capillary endothelial sprouts of the CAM, relative to those of other proliferating sprout endothelia, remains to be tested.     

The effect of formulation of parenteral solutions on microbial growth--measurement of D- and z-values

In order to assess the sterilization conditions for parenteral solutions, the microbial inactivation kinetics of steam resistant spores of Clostridium sporogenes were evaluated. The D-value or death rate kinetics as well as z-values were obtained for several formulation categories of solutions employing a miniature steam retort. Amino acid and heparin solutions provided appreciable microbial resistance as demonstrated by resulting high D- and low z-values. The addition of electrolytes to carbohydrate solutions resulted in increased microbial resistance compared to carbohydrate solutions without electrolytes. A categorization of solutions and their potential impact on microbial thermal resistance will be presented.     

The precision of 3-D parameters in correspondence-based techniques:the case of uniform translational motion in a rigid environment

A general method of calculating the precision of environmental parameters which differs from conventional methods by finding analytical results, and which does so using only information in the image is discussed. This method called uncertainty-analysis, is applied to the case of first-order-error-based motion algorithms for determining environmental depth by matching corresponding points between two or more frames of a dynamic image sequence. How to calculate the uncertainty of in predicted values for the depth is shown, as well as how to calculate the search region for further instances of the point in subsequent frames, based only on information present in the image. The analysis is applied to a real image sequence with good results     

Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization

Acidic phosphorylated proteins have been shown to be prominent constituents of the extracellular matrix of bone and dentin. The acidic phosphoproteins of bone contain more glutamic acid than aspartic acid and a lower serine content than either. On the other hand, the major dentin acidic phosphoproteins, phosphophoryns, have been defined as aspartic acid- and serine-rich proteins, with a lesser content of glutamic acid. Both sets of phosphoproteins have been implicated as key participants in regulating mineralization, but it has been difficult to unify their mechanisms of action. We have now identified, by cDNA cloning, a new serine-rich acidic protein of the dentin matrix, AG1, with a composition intermediate between the bone acidic proteins and dentin phosphophoryns. AG1 has numerous acidic consensus sites for phosphorylation by both casein kinases I and II. Immunochemical and organ culture biosynthetic studies show that AG1 is present in phosphorylated form at low levels in the dentin matrix. If fully phosphorylated, AG1 would bear a net charge of -175/molecule of 473 residues. AG1 contains single RGD integrin binding and N-glycosylation sequences. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.