The transepithelial potential difference in the gills of the fiddler crab,Uca tangeri: Influence of some inhibitors

Summary Euryhaline Crustacea living in dilute media, counterbalance the salt loss by active absorption of NaCl across the gill epithelium. To investigate the mechanisms involved in salt absorption, transeptithelial potential difference (PD_te) was measured in isolated, perfused gills of the fiddler crab,Uca tangeri. The influence of some specific inhibitors of epithelial ion transport on the PD_te was tested.With symmetrical conditions on both sides of the epithelium, the posterior gills ofUca tangeri showed a spontaneous PD_te of +5 to +10 mV, that is an active transport potential which was positive on the bath side as referred to the hemolymph side. This potential decreased considerably after application of KCN or 2,4-dinitrophenol (DNP) to the perfusion saline.Omission of K⁺ from the perfusion saline or addition of ouabain led to a reversible drop of the PD_te, suggesting that the absorption of Na⁺ and also of Cl^– is driven by the (Na⁺+K⁺)ATPase located in the basolateral membrane of the epithelial cells.Perfusion of the hemolymph space with saline containing diphenylamine-2-carboxylate (DPC) or the loop diuretic furosemide resulted in a decrease of the PD_te.After application of amiloride to the bath saline the PD_te increased. Half-maximum response to amiloride was reached at a concentration of about 10^–5 mol·l^–1. This suggests that one of the Na⁺ pathways across the apical membrane may consist of Na⁺ channels.Abbreviations PD _te transepithelial potential difference - DPC diphenylamine-2-carboxylate - R _ps resistance of perfusate shunt - R _te transepithelial resistance - R _in input resistance - DNP 2,4-dinitrophenol Parts of this study have been reported at the 1st Congress of Comparative Physiology and Biochemistry, Liège 1984, and at the Vth European Colloquium on Renal Physiology, Frankfurt, 1985     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

Tagging of a maize gene involved in kernel development by an activated Uq transposable element

Summary A quiescent Uq transposable element has been activated in a maize plant treated with 5-aza-2-deoxycyti-dine. This activated Uq cosegregates with a heritable dominant miniature (Mn) kernel phenotype, indicating its physical association with a maize miniature locus (Mn:: Uq). The Mn:: Uq mutant is dominant in producing a miniature seed phenotype of variable size and in reducing seedling vigor in the early growth stage. Genetic experiments indicate that the Mn:: Uq mutant also affects the activity of the male gametophyte, whereby pollen germination is inhibited, thus lacking pollen tube growth resulting in the male nontransmissibility of this mutant. Proof for the Uq element in this mutant is derived by its ability to transactivate the standard a-ruq reporter allele to yield spotted aleurone tissue. However, the Mn:: Uq mutant does not transactivate a normally Uq-responsive c-ruq allele, suggesting a structural difference between the two ruq receptors at the A1 and C1 loci. It is anticipated that cloning of the Uq transposable element would facilitate the molecular cloning and characterization of the maize miniature gene.Journal Paper No. J-13425 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011, USA, Project No. 2850     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Australine [(1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrrolizid ine] is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis [Molyneux et al. (1988) J. Nat. Prod. (in press)]. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, we tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the alpha-glucosidase amyloglucosidase (50% inhibition at 5.8 microM), but it did not inhibit beta-glucosidase, alpha- or beta-mannosidase, or alpha- or beta-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc3Man7-9(GlcNAc)2-oligosaccharides.