Compartmental analysis of 45Ca2+ efflux in perfused rat liver: effects of hormonal stimulation.

The kinetics of calcium movements in the isolated perfused rat liver were examined using compartmental analysis of the efflux profiles of 45Ca2+ from 45Ca(2+)-equilibrated livers under a variety of calcium concentrations and hormonal treatments. From the 45Ca2+ efflux profiles, we determined that a three compartment model was appropriate to describe the movements of calcium in the liver on the time scale of the experiments. Hormonal treatment with the alpha-adrenergic agonist, phenylephrine, or the vasoactive peptide, vasopressin, during the efflux period lowered significantly the rate of transfer of Ca2+ between the internal compartments at all of the calcium concentrations employed. Also, phenylephrine treatment leads to increased transfer of Ca2+ into the liver from the perfusate. The temporal characteristics of the phenylephrine and vasopressin sensitive Ca2+ pools were examined by pulsing livers, loaded for variable periods of time with 45Ca2+, with the two hormones during the efflux of 45Ca2+ to measure the kinetics of Ca2+ exchange in the hormone-sensitive pools. Results from these experiments indicate that the rate of unstimulated Ca2+ efflux, k2, for the phenylephrine and vasopressin sensitive Ca2+ pools, modeled as a one compartment system, are the same, 0.074 and 0.078 min-1 for phenylephrine and vasopressin respectively, corresponding to half times for turnover of the pool(s) of 9.3 and 8.9 min, respectively.     

Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats.

Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats.     

A pathway of lysosomal proteolysis mediated by the 73-kilodalton heat shock cognate protein.

Cultured IMR-90 diploid human lung fibroblasts respond to withdrawal of serum or growth factors by increasing protein degradation. This increase, due to enhanced transfer of proteins into lysosomes, is specific for a class of intracellular proteins containing peptide sequences biochemically related to Lysine-Phenylalanine-Glutamate-Arginine-Glutamine (KFERQ). This peptide motif is recognized by an intracellular protein which facilitates its transfer into lysosomes in vitro and presumably, in vivo. We called this protein the peptide recognition protein of 73-kilodaltons (prp73). We have shown prp73 to be the constitutive member of the heat shock 70kD family (hsc73) by a variety of criteria. Furthermore, our reconstitution of this pathway of lysosomal degradation in vitro has provided insight in to the molecular mechanisms and requisite biochemical components.     

Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair.

The human chromosomal assignments of genes of the creatine kinase (CK) family--loci for brain (CKBB), muscle (CKMM), and mitochondrial (CKMT) forms--were studied by Southern filter hybridization analysis of DNAs isolated from a human x rodent somatic cell hybrid clone panel. Probes for the 3'-noncoding sequences of human CKBB and CKMM hybridized concordantly only to DNAs from somatic cell hybrids containing chromosomes 14 and 19, respectively. Thus the earlier assignment of the gene coding for the CKBB isozyme to chromosome 14 was confirmed by molecular means, as was the provisional assignment of CKMM to the long arm of chromosome 19. A probe containing canine sequences for CKMM cross-hybridized with human sequences on chromosomes 14 and 19, a result consistent with the assignments of CKBB and CKMM. A probe containing human sequences for CKMT enabled the provisional assignment of CKMT to human chromosome 15. Independent hybrids with portions of the long arm of chromosome 19 missing indicated the order of genes on the long arm of chromosome 19 as being cen-GPI-(TGFB, CYP1)-[CKMM, (APOC2-ERCC1)]-(CGB, FTL). The unexpectedly more distal location of APOC2 among the genes on the long arm--and APOC2's close association with CKMM--is discussed with respect to the close linkage relationship of APOC2 to myotonic muscular dystrophy.     

Transposon Tn4556 of Streptomyces fradiae: nucleotide sequence of the ends and the target sites.

A transposon, Tn4556, has recently been isolated from Streptomyces fradiae (S.-T. Chung, J. Bacteriol. 169:4436-4441, 1987). The ends of Tn4556 were found to contain inverted repeats of 38 base pairs with 70% sequence identity with the ends of Tn3. Insertion of Tn4556 into a Streptomyces plasmid resulted in a 5-base-pair duplication of the target site.     

Characterization of matrix domains of the hamster acrosome

In this study we describe the purification and the structural and biochemical properties of a detergent-stable complex of the hamster sperm acrosome. This complex consists of two distinct acrosomal matrix domains and a layer of electron-dense material, termed the acrosomal lamina, derived from the luminal surface of the outer acrosomal membrane. This complex has been isolated by centrifugation of detergent-extracted sperm suspensions on Percoll density gradients. The complex contains two major polypeptides of Mr 29,000 and Mr 22,000 and minor polypeptides of Mr 64,000-62,000, 56,000 and 35,000. Gelatin-containing sodium dodecyl sulfate-polyacrylamide gels demonstrate that bands of proteinase activity are not the major polypeptide components of the complex. These data demonstrate that the matrix of the acrosome is compartmentalized into domains of differing structural properties that occupy specific locations in the intact acrosome and that matrix components are physically associated with the outer acrosomal membrane. These data indicate that a structural framework is present within the acrosome and we speculate that it may be involved in sequestering hydrolases into specific spatial domains and could affect the temporal release of activity of selected hydrolases during the acrosome reaction.     

Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.