Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure

The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.     

A killer factor produced by the cellular slime mold Polysphondylium pallidum

A killer strain was discovered in cellular slime molds. The wild isolate CK-8 of Polysphondylium pallidum kills all other strains in Polysphondylium and Dictyostelium, as far as could be determined, except strain CK-8 itself and its complementary mating type strain CK-9. Growth-phase cells of CK-8 excrete a killer factor which is sensitive to heat, above 60°C for 5 min, and trypsin. The apparent molecular mass of the factor was determined at 10 000–12 000.Abbreviations BSS Bonner's salt solution - CM conditioned medium     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Thyrotropin-releasing hormone and insulin in chemically induced pancreatic islet cell tumors in rats

Thyrotropin-releasing hormone (TRH) and insulin were measured by radioimmunoassay in acetic-acid extracts of 19 pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. In addition, gel filtration properties of TRH-immunoreactivity and immunoreactive insulin (IRI) were examined in 5 and 14 tumors, respectively. TRH was demonstrated in 10 of 19 tumors, with a mean of 166 +/- 47 (SEM) pg/mg wet weight, whereas the concentration was less than 3 pg/mg wet weight in the other tumors. In contrast, all tumors contained IRI, with a mean of 11.0 +/- 1.6 micrograms/mg wet weight. Ten tumors in which TRH was demonstrated contained more IRI than those in which TRH was not detected (13.1 +/- 1.8 vs 6.5 +/- 1.7 micrograms/mg wet weight, P less than 0.02). After gel filtration, all TRH immunoreactivity was eluted at the same place as synthetic TRH in the 5 tumors. In addition, gel filtration elutes showed essentially the same pattern of IRI in the 14 tumors, with 3 peaks. The predominant IRI peak comigrated with marker insulin (95.7 +/- 0.8%), another prominent peak occurred coincident with proinsulin standard (3.3 +/- 0.5%), a third peak was present in the void volume (0.28 +/- 0.04%). These distributions of IRI were similar to those in extracts of normal pancreases. The present studies demonstrate TRH immunoreactivity in pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. Chemically induced insulinomas can serve as a model for insulin storage which is analogous to islet B cells.     

Regulation of urine marking in male and female mice: effects of sex steroids

Effects of sex steroids on urine-marking activity were studied in male, female, and neonatally androgenized female mice. Urine marking was estimated by suspending ceramic tubes that were connected in a horizontal row with a steel rod into the home cage of an isolated mouse. Intact males showed high marking activity, which was diminished after castration. Both testosterone propionate (TP) and estradiol benzoate (EB) were effective in restoring the marking activity of castrated males, while 5-alpha-dihydrotesterone (DHT) did not have any stimulative effects. Intact normal females showed quite low marking activity and ovariectomy further depressed it. TP and DHT enhanced the marking of ovariectomized females, but EB restored the activity only to the preovariectomy level. In intact females which were neonatally androgenized, the marking activity was much higher than that of normal females. The pattern of the change induced by gonadectomy and hormone treatment in these females resembled that in males. Thus, ovariectomy reduced the activity and both TP and EB restored the level. These results indicate that the sexual dimorphism in the urine marking in mice is primarily determined by hormonal environment during early postnatal age. Hormonal control of scent marking is discussed in relation to the studies in other rodents.     

Visible fibrinolysis by endothelial cells: Effect of vitamins and sterols

We have succeeded in corroborating the enhancing effect of vitamin A, vitamin C, sitosterol and fucosterol on the fibrinolytic activity of endothelial cells. The assay system consisted of anin situ dissolution of a fibrin layer coated onto a culture dish, over which endothelial cells were grown in a culture medium containing 10 % serum. The dissolution was enhanced by the addition of these vitamins and phytosterols to the culture medium.To whom correspondence should be addressed.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.