Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.     

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes.     

Ultrastructural differentiation of the epithelium and mucous glands of the esophagus in the chick embryo

Esophagi of White Leghron chick embryos, six days to hatching, were fixed in 5% glutaraldehyde in a cacodylate-sucrose buffer, postfixed in 1% OsO₄, dehydrated and embedded in Araldite. Tissues were sectioned with glass knives and viewed with a Philips EM 100C. The epithelium in early stages of development is characterized by small intercellular spaces, few cell processes and few desmosomes. In contrast the differentiated epithelium contains numerous cell processes, large intercellular spaces and numerous desmosomes. Mucin appeared in the mucous glands at 17 days.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Structure of the female reproductive tract of the apple snail. II. Scanning electron microscopy

The albumen gland of Pomacea paludosa, a prosobranch gastropod, contains two main ducts. The albumen gland duct consists of a single layer of secretory and non-secretory cells. The surface of the non-secretory cells is covered with cilia. Microvilli are associated with the luminal edges of the secretory cells. Globules of secretory products appear at the cell surfaces. The capsule gland duct coils through the albumen gland and is composed of two opposing faces each of two layers of cells. The upper layer consists of ciliated non-secretory cells and the microvilli covered necks of the goblet-shaped secretory cells. The bases of the secretory cells comprises the lower layer of cells. Differences in the arrangement of cellular processes and number exists between the duct epithelia.     

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.     

Binding of non-substrate nucleotides to a restriction endonuclease: a model for the interaction of bam HI with its recognition sequence

The kinetic constants of the site-specific endonuclease Bam HI for various substrates were determined and binding of non-substrate nucleotides to the enzyme was studied. Agarose gel assays in combination with an integrated Michaelis-Menten equation were used for the evaluation of data. The turnover number was 2.2 min-1 at 37 degrees C with pJC80 DNA as the substrate. It depends on the conformation and base composition of the substrate. Michaelis constants also depend on substrate conformation. Non-substrate polynucleotides were found to inhibit Bam competitively with KI ranging from 10(-6) to > 10(-3) M depending on base composition, base pairing, and helix conformation. Dinucleotides showed sequence-specific, competitive inhibition with KIs ranging from 10(-5) to > 10(-3) M. Mononucleotides and -nucleosides acted noncompetitively. Binding was influenced by the extent of phosphorylation, but not by the nature of the base. KIs varied between 10(-3) and 10(-2) M. The results are discussed with respect to the recognition requirements of Bam HI.