Release of atriopeptin in the rat by vasoconstrictors or water immersion correlates with changes in right atrial pressure

Vasopressin, its 1-deamino analog (dAVP), angiotensin II, and phenylephrine, administered intravenously, increased plasma atriopeptin immunoreactivity in chloral hydrate-anesthetized rats. A continuous one hour infusion of either dAVP or phenylephrine caused a sustained elevation in: a) systemic blood pressure; b) right atrial pressure; c) left ventricular end diastolic pressure; and d) plasma atriopeptin immunoreactivity. While continuous infusion of angiotensin II also produced a sustained elevation in left ventricular end diastolic pressure, the changes in right atrial pressure and plasma atriopeptin were only transient. These data suggest that plasma atriopeptin most closely correlates with right atrial pressure. Consistent with this hypothesis, we found that the release of atriopeptin directly correlated with changes in right atrial pressure in anesthetized, water-immersed rats.     

The hemolymph coagulation system in invertebrate animals

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 3)--d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Structure of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus refined at 2.3 A resolution. Structural comparisons of bacterial ferredoxins

The structure of a low-potential ferredoxin isolated from Bacillus thermoproteolyticus has been refined by a restrained least-squares method. The final crystallographic R factor is 0.204 for 2906 reflections with F greater than 3 sigma F in the 6.0 to 2.3 A resolution range. The model contains 81 amino acid residues, one [4Fe-4S] cluster, and 59 water molecules. The root-mean-square deviation from ideal values for bond lengths is 0.018 A, and the mean coordinate error is estimated to be 0.25 A. The present ferredoxin is similar in the topology of the polypeptide backbone to the dicluster-type ferredoxins from Peptococcus aerogenes and Azotobacter vinelandii, but has considerable insertions and deletions of the peptide segments as well as different secondary structures. Although all but the C-terminal C zeta atoms of P. aerogenes ferredoxin superpose on the C alpha atoms of A. vinelandii ferredoxin, only 60% superpose on the C alpha atoms of B. thermoproteolyticus ferredoxin, with a root-mean-square distance of 0.82 A for each pair. The conformations of the peptide segments surrounding the [4Fe-4S] clusters in these three ferredoxins are all conserved. Moreover, the schemes for the NH...S hydrogen bonds in these ferredoxins are nearly identical. The site of the aromatic ring of Tyr27 in B. thermoproteolyticus ferredoxin is close spatially to that of Tyr28 in P. aerogenes ferredoxin with reference to the cluster, but these residues do not correspond in the spatial alignment of their polypeptide backbones. We infer that in monocluster-type ferredoxins, the side-chain at the 27th residue has a crucial effect on the stability of the cluster. Of the four cysteine residues that bind to the second Fe-S cluster in the dicluster-type ferredoxins, two are conserved in the monocluster-type ferredoxins from Desulfovibrio gigas. D. desulfuricans Norway, and Clostridium thermoaceticum. The tertiary structure of B. thermoproteolyticus ferredoxin suggests that in such monocluster-type ferredoxins these two cysteine residues, which in it correspond to Ala21 and Asp53, form a disulfide bridge.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Inactivation of bleomycin by an N-acetyltransferase in the bleomycin-producing strain Streptomyces verticillus

Abstract Bleomycin-producing Streptomyces verticillus ATCC15003 possesses a bleomycin acetyltransferase which inactivates the drug in the presence of acetyl coenzyme A. The site of acylation in enzymically prepared acetylbleomycin A2 was determined by nuclear magnetic resonance analysis; the primary amino group of the β-aminoalanine moiety of bleomycin was acetylated. Acetylbleomycin A2 had no detectable antibacterial activity and did not induce in vitro DNA degradation.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.