Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

Anti-galactocerebroside antibodiesin human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA)

The standard ELISA technique was improved for the detection of antigalactocerebroside antibody in biological fluid. Mouse monoclonal antigalactocerebroside antibody was used to demonstrate specificity and sensitivity of the technique. After optimization of the assay, the usefulness of this measurement for the evaluation of patients with multiple sclerosis was assessed. The presence of antigalactocerebroside antibodies in the cerebrospinal fluid of 20 patients with multiple sclerosis, 10 with other neurological diseases and 10 normal individuals was determined. All the CSF samples from normal individuals were negative. In patients with multiple sclerosis 14 of the 20 samples had elevated levels of antigalactocerebroside antibody, whereas with other neurological diseases 5 out of 10 were positive. Antigalactocerebroside levels were lower in samples from patients during an acute relapse than in those from more chronic cases. These results indicate that the presence of anti-galactocerebroside antibody in cerebrospinal fluid is not specific to MS but may reflect previous damage to myelin.Abbreviations and trivial names used ELISA Enzyme-Linked Immunosorbent Assay - CSF cerebrospinal fluid; galacto- or glucocerebroside, ceramide-1-0-beta-galactoside or-glucoside     

DEVELOPMENTAL PATTERN OF THE BONY FALX AND BONY TENTORIUM OF SPOTTED DOLPHINS (STENELLA ATTENUATA) AND THE RELATIONSHIP BETWEEN DEGREE OF DEVELOPMENT AND AGE

The bony falx (BF) and bony tentorium (BT) of spotted dolphins ( Stenella attenuata ) at different growth stages were observed. The BF and BT were observed to have the shape of the entire falx cerebri (FC) and tentorium cerebelli (TC), respectively, in adults, but were not seen in fetuses at all. Partially formed BF and BT were observed in the young. The BF and BT of spotted dolphins are formed by ossification in the FC and TC in the course of aging. Based on age determination by means of dentinal growth layers of the maxillary teeth, the ossification in the FC and TC of spotted dolphins begins by about 1 yr of age, and is completed, except for the passage of nerves and blood vessels, by about 9 yr of age. The BF and BT of spotted dolphins are formed possibly by continual dietary intake of large amounts of calcium, phosphorus and vitamin D.     

Mechanism of protein folding: I. General considerations and refolding of myoglobin

To explain the rapidity of the process of protein folding, we cite two aspects of hydrophobic interaction: its long-range nature and the specificity of pairing after the formation of secondary structures. These two factors, when incorporated with the growth-type mechanism, can determine the folding pathway of proteins. This mechanism is applied to myoglobin. Appropriate introduction of side chains of amino acid residues and the heme group attached to His 93 yield a refolded tertiary structure that is in good agreement with the native structure.     

Cardiovascular anomalies produced by nimustine hydrochloride in the rat fetus

The cardiovascular teratogenicity of nimustine hydrochloride (ACNU) was studied in rat fetuses. This drug is a nitrosourea derivative anticancer agent and produces alkylation of DNA. Pregnant Donryu rats were treated with single doses of 10, 11 or 13 mg/kg of the teratogen at various stages during gestation. Examination of the hearts was performed by microdissection after sacrificing the animals on the 20th day of gestation. The highest frequency of cardiovascular anomalies was found in the groups treated on the 8th day of gestation, but there was no difference in the rates induced by the three dosages of ACNU administered. The most common cardiovascular anomalies observed were ventricular septal defect (76.8%) and double outlet right ventricle (10.3%). A considerable number of affected fetuses (37/263) showed complex cardiac anomalies with atrioventricular (AV) malalignment and other AV valve anomalies. These anomalies include: double inlet left ventricle, straddling AV valve, atresia or stenosis of the AV valve, and dysplastic AV valve. ACNU appears to be a useful teratogenic agent for inducing complexes of cardiac anomalies which include AV malalignment.     

Central catecholaminergic control of ACTH secretion

Plasma adrenocorticotropic hormone (ACTH) has been measured after an intra-third ventricular administration of noradrenaline, an adrenergic agonist or an adrenergic antagonist. Centrally administered noradrenaline caused a significant increase in ACTH secretion. The alpha-agonist phenylephrine also increased the ACTH level. However, neither the alpha-antagonist phentolamine nor beta-agonist isoproterenol affected the ACTH level. The beta-antagonist propranolol evoked a significant elevation in ACTH. Passive immunoneutralization was examined with anti-rat corticotropin-releasing factor (CRF) rabbit serum, anti-arginine vasopressin (AVP) rabbit serum and normal rabbit serum (NRS) on the intra-third ventricular noradrenaline-induced ACTH secretion to study the involvement of endogenous CRF. An intra-third ventricular administration of noradrenaline caused a significant increase of ACTH levels in NRS-injected rats and anti-AVP-injected rats, whereas an i.v. anti-rat CRF injection significantly reduced the intra-third ventricular noradrenaline-induced ACTH secretion. These results suggest that central catecholamine stimulated ACTH secretion via the alpha-adrenergic mechanism and that endogenous CRF is at least partly involved in the noradrenaline-induced ACTH secretion.     

Bovine factor VII. Its purification and complete amino acid sequence

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).     

Substitution of gamma Arg-275 by Cys in an abnormal fibrinogen, "fibrinogen Osaka II". Evidence for a unique solitary cystine structure at the mutation site

In an abnormal fibrinogen with impaired fibrin monomer polymerization designed as fibrinogen Osaka II, we have identified substitution of Arg by Cys at position 275 of the gamma chain. This Cys is linked to a free cysteine molecule by a disulfide link as evidenced by fast atom bombardment mass spectrometry. This finding was supported by identification of a single cysteine released from isolated abnormal fragment D1 upon reduction. This unique cystine structure at the mutation site has not been reported heretofore in any abnormal protein including fibrinogen. The substitution may well perturb the structure required for fibrin monomer polymerization, specifically that assigned to the carboxyl-terminal D domain of fibrinogen. Indeed, isolated fragment D1 with the Cys substitution failed to inhibit thrombin-mediated clotting of normal fibrinogen and normal fibrin monomer polymerization, while normal fragment D1 inhibited them markedly. Our data seem to provide supporting evidence that the putative polymerization site(s) assigned to the D domain of fibrinogen may be structure-dependent, including the carboxyl-terminal segment of the gamma chain as well as a contiguous region that contains the gamma 275 residue.     

Effects of lithium on the hypothalamo-pituitary-adrenal axis

The effect of lithium on the hypothalamo-pituitary-adrenal axis was studied in vivo and in vitro. The levels of plasma vasopressin, ACTH and corticosterone increased after the administration of lithium (LiCl 4 mmol/kg BW, 11 days) in rats, while the tissue vasopressin concentration in the median eminence, the rest of the hypothalamus and the posterior pituitary was decreased. The CRF concentration in the posterior pituitary increased markedly, but it did not change significantly in the median eminence or the rest of the hypothalamus. The elevated plasma ACTH level might be at least partly due to the increased vasopression secretion. Lithium stimulated ACTH secretion per se and also enhanced vasopressin-induced ACTH secretion in cultured pituitary cells and in half pituitary incubations, while it did not affect CRF-induced ACTH secretion. Lithium inhibited CRF-induced cAMP accumulation in half pituitary incubations, while lithium and vasopressin did not affect cAMP accumulation per se or even when administered together. The results suggest that lithium-induced ACTH release is via a cAMP-independent mechanism. Thus, it is possible that lithium stimulates ACTH release by acting directly on the corticotroph, stimulating vasopressin release and potentiating vasopressin-induced ACTH release.     

Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity

Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker ([¹⁴C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100–200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets.