Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC₅₀ = 0.35 × 10⁻⁹ M).     

Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd²⁺ sequence than to P-type ATPases from animal sources. Menkes syndrome is an X-chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P-type ATPase, whose properties satisfactorily explain the phenotype of the disease. P-type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K⁺ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca²⁺ ATPase), or for cation exchange (e.g. the animal cell Na⁺/K⁺ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P-type ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

Histories and Stories from Chiapas: Border Identities in Southern Mexico.

Histories and Stories from Chiapas: Border Identities in Southern Mexico. R. Aída Hernández Castillo. Texas: University of Texas Press, 2001. 317 pp.     

Mutants in a yeast Ran binding protein are defective in nuclear transport.

Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.     

Cloned DNA probes distinguish endemic and exotic Entomophaga grylli fungal pathotype infections in grasshopper life stages

Entomophaga grylli is a fungal pathogen of grasshoppers and at least three pathotypes are recognized world-wide. Pathotypes 1 and 2 are endemic to North America while the Australian pathotype 3 had been released into two field sites in North Dakota between 1989 and 1991. Grasshoppers were collected over the summer at the field sites in 1992 and assessed for pathotype infection by cloned DNA probe analysis. The three most predominant grasshopper species that were infected ( Melanoplus sanguinipes, M. bivittatus and Camnula pellucida ) were assessed for pathotype infection with respect to their life stages (nymphal instars and adult males and females). Pathotype 1 predominantly infected grasshoppers in the subfamilies Oedipodinae and Gomphocerinae and pathotype 2 predominantly infected grasshoppers in the subfamily Melanoplinae. Early-instar M. sanguinipes and M. bivittatus had higher pathotype 2 infection frequencies, while late-instar and adult C. pellucida had higher pathotype 1 infection frequencies. Cross-infection by the pathotypes did occur in up to 3% of the individuals, on a per species basis, and primarily in later instar and adult grasshoppers. Pathotype 3 infections occurred in later instar and adults of the three grasshopper species. Infection of grasshoppers by E. grylli pathotypes is discussed with reference to the fungal life cycles.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10–14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 M 2,4-D and 5.0 M TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (120 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DKW Driver and Kuniyuki (1984) walnut medium - IBA indolebutyric acid - LP Long and Preece medium described herein - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM Woody Plant Medium of Lloyd and McCown (1980)     

Isolation of a cDNA encoding a growth-arrest associated gene and characterization of its regulation

We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted ³²P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G₂ phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α₁(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt₂cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.