Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Effects of stream order and season on mineralization of [14C]-phenol in streams

Mineralization of trace levels of [¹⁴C]-phenol by heterotrophic microorganisms was quantified at 4 sites along a river continuum in southwestern Virginia. Significant phenol mineralization rates were detected in surface sediment and seston samples at all sites from August 1985 through May 1986. Phenol degradation was strongly affected by season (ANOVA; P < 0.0001). From a baseline rate in August (range: 1.19 × 10^-5 to 897 × 10^-4 mg phenol mineralized mg AFDW^-1 h^-1) phenol mineralization rose to a yearly maximum in October (range: 1.21 × 10^-4 to 1.16 × 10^-3 mg phenol mineralized mg AFDW^-1 h^-1) despite decreasing stream temperatures. This autumnal peak in phenol degradation was attributed to the pulsed input of allochthonous detritus, especially leaf litter, which contains substantial quantities of phenols and related compounds. Although phenol mineralization was significant in these streams, phenols were metabolized at much slower rates than more labile compounds present in the dissolved organic matter (DOM) pool. Estimates of turnover rates for three major components of DOM revealed that glucose and glutamate turnover rates (0.064–0.140 h^-1 mg sediment AFDW^-1 and 0.140–0.610 h^-1 mg sediment AFDW^-1, respectively) were, respectively, 2.2–4.7 × and 9.6–16.9 × greater than phenol turnover rates (0.015–0.064 h^-1 mg sediment AFDW^-1). Although the relatively low rates of utilization of refractory phenolic materials suggest that these compounds may accumulate and become more prevalent components of the DOM pool, phenol concentrations at the 4 study sites remained below detectable levels (i.e., < 1 g 1^-1) throughout the study. Consequently, it seems that although phenolic materials are metabolized more slowly than labile DOM, phenols are degraded at rates which preclude accumulation in the water column.     

Disruption of pattern formation in palatal rugae in fetal mice heterozygous for First arch (Far)

The purpose of this study was to document the extent of disruption in the pattern of palatal rugae caused by the presence of one copy of the First arch mutation. The palatal ruga pattern was found to be disrupted in 86% of 15- to 17-day mouse fetuses that were heterozygous for the First arch mutation in the ICR/Bc strain, compared with 9% in ICR/Bc fetuses of normal (+/+) genotype. This new observation in First arch heterozygotes, together with the previously reported dominant effects of the First arch mutation, particularly the bifurcation of the maxillary nerve (100% in both BALB/cGaBc and ICR/Bc strains), the disruption of maxillary vibrissa pattern (80% in ICR/Bc), and the hemifacial deficiency (38% in ICR/Bc), has led us to redefine the First arch mutation as a semidominant, Far. Like the other defects caused by Far, the rugal defects are in tissue derived from the embryonic maxillary prominence. The rugal defects observed in +/Far palates were always asymmetrical and most often involved fragmentation and misalignment of two or more of rugae 4-7. The relatively large degree of variation in ruga pattern observed in fetuses of normal genotype suggests that it is a less well canalized trait than the normal pattern of maxillary vibrissae which varies only in a few very specific and minor ways. The First arch mutation, which in heterozygotes disrupts pattern formation in both palatal rugae and maxillary vibrissae, can be used to study genetic control of pattern formation in mammalian embryos.     

Collagen degradation in ischaemic rat hearts.

Myocardial extracellular matrix is organized into a complex arrangement of intercellular and pericellular fibres and fibrils that serves as a supporting framework for contracting cells. Recent evidence suggests that changes in ventricular shape and function occurring after ischaemic injury may be related to alterations of this matrix. In this report we describe the rapid and extensive loss of collagen in myocardial infarction produced by ligating the left anterior descending coronary artery of the rat for 1-3 h. The total collagen content in the myocardial infarct zones after 1, 2 and 3 h of ligation was 75 +/- 8%, 65 +/- 7% and 50 +/- 10% respectively (mean +/- S.D.) of that of either the non-infarcted tissue controls or of the same regions in sex- and age-matched normal left ventricles. A marked decrease also occurred in the residual collagens which were not extractable with 6 M-guanidine hydrochloride, suggesting that rapid degradation of insoluble collagen fibres may also occur. The decreased collagen content in the 3 h myocardial infarct coincided with the appearance of several enzyme activities. Collagenase, other neutral proteinase and presumed lysosomal serine proteinase activities were increased by 3, 3 and 2 times the control values respectively. These results suggest that the increased activities of collagenase and other neutral proteinases may be responsible for the rapid degradation of extracellular matrix collagen in myocardial infarct.     

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.     

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

Nitrate assimilation in Neurospora crassa: Enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures

Summary The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are ₂type homodimers. Enzymebound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. One, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. Three modes of regulation are imposed on the expression of nitrate assimilation, namely: nitrogen metabolite repression, nitrate induction and autogenous regulation by nitrate reductase. In this study, nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved proteins from crude extracts of the wild type and specific nitrate-nonutilizing (nit) mutants were examined for material cross-reactive with antibodies against nitrate reductase and nitrite reductase. The polyclonal antibody preparations used were rendered monospecific by reverse affinity chromatography. Growth conditions which alter the regulatory response of the organism were selected such that new insight could be made into the complex nature of the regulation imposed on this pathway. The results indicate that although nitrate reductase and nitrite reductase are coordinately expressed under specific nutritional conditions, the enzymes are differentially responsive to the regulatory signals.