Rat atrial natriuretic factor: Isolation,structure and biological activities of four major peptides

Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H₂N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COOH) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four pepdides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 × 10^?9 to 2.5 × 10^?9 M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.     

Evidence for intracellular formation of angiotensins: coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis

Leydig cells were purified from rat testes by discontinuous metrizamide density gradient and were shown to contain renin (EC 3.4.99.1), angiotensin-converting enzyme (dipeptidyl carboxypeptidase, (EC 3.4.15.1), and the peptide hormone angiotensins I, II and III as determined by the combined HPLC and radioimmunoassay. In germinal cells only angiotensin II (AII) was found at a significant level. These findings provide evidence for intracellular formation of AII in testicular cells and demonstrate that an intracellular renin-angiotensin system exists in normal non-transformed cells.     

Sleep Profile and Longevity in Three Generations of a Family of Captive Bolivian Aotus

The daily total sleep time (TST) of the only nocturnal simian primate, Aotus spp., remains little studied under controlled conditions. We conducted 3 experiments in 4 owl monkeys (Aotus sp.), aged 1–27+ yr, to determine the daily TST. We housed 3 their—a nuclear family—in 1 cage and the remaining senescent female in an adjacent separate cage. We monitored their activity-sleep pattern longitudinally for 15–20 d via actigraphy: by tagging an acclerometer-type miniature transmitter (Actiwatch-MINIMITTER) sensitive to omnidirectional movement, to the owl monkey's neck. The TST (9.5–12.5 h) was ca. 4.5–7 h less than the 17 h Perachio reported for owl monkeys in 1971 by polysomnography, under similar 12-h-light; 12-h dark conditions. Our finding corroborates well with the TST for other nonhuman primates. Four members of the Aotus colony at our facility reached 20 yr in captivity; the oldest (wild-born female) is still living at >27 yr, and the second oldest (captive-born male), is 23 years now.     

Atrial natriuretic factor receptor on cultured Leydig tumor cells: ligand binding and photoaffinity labeling

Atrial natriuretic factor (ANF) is a peptide hormone discovered recently from the heart atrium that possesses potent natriuretic and vasorelaxant activities. Recently we found that ANF markedly stimulates intracellular cGMP and almost completely inhibits cAMP accumulation in testicular interstitial tumor cells [Pandey, K. N., Kovacs, W. J., & Inagami, T. (1985) Biochem. Biophys. Res. Commun. 133, 800-806]. These actions of ANF suggest the presence of ANF receptors in testicular interstitial cells. In this study, cultured murine Leydig tumor cells have been shown to contain specific binding sites for ANF. Saturation binding studies indicated a single class of binding sites with a Kd of 5 X 10(-9) M at a density of 2 X 10(6) sites/cell. The binding of mono[125I]iodo-ANF (125I-ANF) was competed by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensin I, bovine luteinizing hormone, and human chorionic gonadotropin were not able to compete against 125I-ANF. The binding of 125I-ANF was rapid, reaching maximum levels in 15 min at 4 degrees C. At 37 degrees C, the cell-bound 125I label was quickly decreased. Pretreatment of cells with NH4Cl, chloroquine, or NaN3 resulted in significant increases in maximum levels of the cell-bound 125I radioactivity. A photoaffinity reagent for ANF receptor was prepared by reacting ANF with succinimido 4-azidobenzoate, and resultant 4-azidobenzoyl- (AZB-) ANF was purified by high-performance liquid chromatography (HPLC). AZB-ANF was radioiodinated by use of chloramine T and purified again by HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of elevated antibody against calreticulin by ELISA in aged cynomolgus monkey plasma

Calreticulin (Crt) is a molecular chaperone ubiquitously present in the endoplasmic reticulum. In non-human primates, age-related occurrence of anti-Crt antibody has not been reported. We developed an ELISA assay for an anti-Crt antibody and determined the age-related increase in the levels of anti-Crt antibody in three groups of cynomolgus monkeys: juvenile (1.5 yr), young adults (5-10 yr) and aged adults (20-34 yr). Mean ± SD auto-antibody levels at 450 nm in juvenile, young adults and aged groups were 0.23 ± 0.18, 0.30 ± 0.28, and 0.55 ± 0.33, respectively. Statistically significant differences were noted in the autoantibody levels to Crt among the aged group and juvenile or young adults. This is the first report to demonstrate the expression of anti-Crt autoantibody in aged monkeys and indicates that cynomologous monkeys may serve as an appropriate nonhuman primate model for studies of age-related alteration of immune function in elderly humans. Though preliminary, this finding merits further investigation to determine the relationship between immunosenescence and expression of antibodies to Crt.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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Properties of taurine: alpha-ketoglutarate aminotransferase of Achromobacter superficialis. Inactivation and reactivation of enzyme

The activity of taurine: alpha-ketoglutarate aminotransferase (taurine: 2-oxoglutarate aminotransferase, EC 2.6.1.55) from Achromobacter superficialis is significantly diminished by treatment of the enzyme with (NH4)2SO4 in the course of purification, and recovered by incubation with pyridoxal phosphate at high temperatures such as 60 degrees C. The inactive form of enzyme absorbing at 280 and 345 nm contains 3 mol of pyridoxal phosphate per mol. The activated enzyme contains additional 1 mol of pyridoxal phosphate with a maximum at 430 nm. This peak is shifted to about 400 nm as a shoulder by dialysis of the enzyme, but the activity is not influenced. The inactive form is regarded as a partially resolved form, i.e. a semiapoenzyme. The enzyme catalyzes transamination of various omega-amino aicds with alpha-ketoglutarate, which is the exclusive amino acceptor. Hypotaurine, DL-beta-aminoisobutyrate, beta-alanine and taurine are the preferred amino donors. The apparent Michaelis constants are as follows; taurine 12 mM, hypotaurine 16 mM, DL-beta-aminoisobutyrate 11 mM, beta-alanine 17 mM, alpha ketoglutarate 11 mM and pyridoxal phosphate 5 micron.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.