Sarafotoxin receptors mediate phosphoinositide hydrolysis in various rat brain regions

Sarafotoxin-b, a potent snake vasoconstrictor peptide homologous to the mammalian endothelial vasoconstrictor endothelin, induces phosphoinositide (PI) hydrolysis in various brain regions of the rat. Sarafotoxin-b induced PI hydrolysis is largely independent of extracellular Ca2+ and is detected in all brain regions where toxin-binding sites are found. These results point to the existence of a hitherto undetected neuroreceptor associated with the PI cycle.     

Immunological and structural characterization of sarafotoxin/endothelin family of peptides

A highly specific and sensitive radioimmunoassay (RIA) was developed for the potent vasoconstrictor peptides, sarafotoxin-b and human endothelin. The antigenic determinants of the antibodies employed in studies with these assays were found to be localized within the amino acid sequence at positions 4-7. This was confirmed by CNBr cleavage of the methionyl residue at position 6 in the sarafotoxin and at position 7 in the endothelin. The chemically characterized modified peptides showed very low cross reactivity in the RIAs. On the other hand, the binding properties as well as the ability to induce phosphoinositide hydrolysis were very similar in the modified and native peptides, indicating that despite cleavage of the peptide bond the biologically active conformation responsible for either binding or phosphoinositide hydrolysis is retained, probably because of the disulfide bonds. Thus, structural alteration might be a valuable means of curtailing some of the various activities induced by the sarafotoxin/endothelin family of peptides.     

Functional endothelin/sarafotoxin receptors in the rat uterus

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.     

Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ⁺ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ⁺ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of -galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ⁺ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.     

Mating behavior of male deer ticksIxodes dammini (Acari: Ixodidae)

To analyze the sexual behavior of male black-legged deer ticks Ixodes dammini,we collected ticks infesting 202 white-tailed deer. On average, 17.7 males and 8.8 females infested each deer. Field-collected males copulated with a mean of 2.25 females, and virgin males mated with 2.4 females. On experimental hosts, males established sexual contact with feeding females and repelled other males, and about half remained paired after their mate detached. Engorged females continue to be receptive, and males mate more readily with them than with nonfed females. We conclude that male I. damminiare endowed with a repertoire of behaviors which favor an opportunistic mating before seeking a host and a preference for mating with feeding females on the host accompanied by tenacious mate guarding.     

Uridine diphosphate D-glucose dehydrogenase of Aerobacter aerogenes

Uridine diphosphate d-glucose dehydrogenase (EC 1.1.1.22) from Aerobacter aerogenes has been partially purified and its properties have been investigated. The molecular weight of the enzyme is between 70,000 and 100,000. Uridine diphosphate d-glucose is a substrate; the diphosphoglucose derivatives of adenosine, cytidine, guanosine, and thymidine are not substrates. Nicotinamide adenine dinucleotide (NAD), but not nicotinamide adenine dinucleotide phosphate, is active as hydrogen acceptor. The pH optimum is between 9.4 and 9.7; the K(m) is 0.6 mm for uridine diphosphate d-glucose and 0.06 mm for NAD. Inhibition of the enzyme by uridine diphosphate d-xylose is noncooperative and of mixed type; the K(i) is 0.08 mm. Thus, uridine diphosphate d-glucose dehydrogenase from A. aerogenes differs from the enzyme from mammalian liver, higher plants, and Cryptococcus laurentii, in which uridine diphosphate d-xylose functions as a cooperative, allosteric feedback inhibitor.     

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.