Studies on Scenedesmus acutus growth. The incorporation of 14C-glucose by Scenedesmus acutus under light and dark periods

Qualitative and quantitative estimations of alcohol-extractable compounds from (14)C-glucose-incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with (14)C-glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.     

Cannabinoid induced degranulation of rabbit neutrophils

We have examined the effects of various cannabinoids on the degranulation of rabbit peritoneal neutrophils. Several cannabinoids were found to cause a dose-dependent and noncytotoxic release of lysosomal enzymes from the neutrophils. The degranulation induced by cannabidiol is rapid ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3 min}$), and enhanced by extracellular calcium and cytochalasin B. In addition to their intrinsic activity, cannabinoids also modulate the neutrophils' responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. This investigation represents the initial step toward the characterization of the effect of cannabinoids on the excitation-activation coupling sequence of hormonally responsive cells.     

Reduced inhibition of replicon initiation and chain elongation by neocarzinostatin in skin fibroblasts from patients with ataxia telangiectasia

Cells from patients with the genetic disease ataxia telangiectasia are hypersensitive to the DNA-breaking agents X-rays, bleomycin and neocarzinostatin, and show reduced inhibition of DNA synthesis after treatment with these agents, as compared to normal cells. The rate of replicon initiation and chain elongation was measured shortly after brief exposure of two normal and two ataxia telangiectasia fibroblast strains to low doses (0.10-0.30 microgram/ml) of neocarzinostatin, by means of alkaline sucrose gradient analysis. Neocarzinostatin was found to inhibit both initiation and elongation, and both components of DNA synthesis were more resistant to this inhibition in the A-T strains.     

Facilitation of parental behavior in ring doves by systemic or intracranial injections of prolactin

Systemic administration of ovine prolactin (PRL) has been previously reported to stimulate parental feeding behavior toward 7-day-old foster squabs by nonbreeding ring doves with previous breeding experience. The first of the present experiments reexamined this claim in experienced, nonbreeding birds given test squabs of different ages. Each visually isolated male and female dove was given twice-daily subcutaneous injections of ovine PRL or vehicle for 7 days and then tested for parental responses toward a single 1-3, 6-8, or 11- to 13-day-old foster squab. Prolactin significantly increased the incidence or frequency of parental regurgitation-feeding episodes in tests with all three squab age groups and, in addition, increased the incidence of parental feeding invitations (squab-oriented bill openings) in tests with 6- to 8-day-old squabs. A second study explored the degree to which PRL can act directly on the central nervous system to facilitate parental activity in the absence of peripheral cues generated from PRL-induced changes in other target organs, such as those associated with crop sac growth and distension. In this experiment, 6- to 8-day-old test squabs were used to determine if parental behavior is enhanced by twice-daily intracerebroventricular (ICV) injections of PRL in doses below those required to stimulate peripheral target organs. Injection schedules and behavior testing procedures were the same as those used in Experiment 1. However, half of the ICV PRL-treated and ICV vehicle-treated birds were food deprived for 16 hr before and during the test in order to control for PRL-induced hyperphagia and resulting crop sac distension, which could confound the results by generating peripheral stimuli conductive to the display of regurgitation-feeding behavior. Intracranial injections of prolactin significantly increased the incidence of feeding behavior, parental feeding invitations, and crouching or sitting in the nest in food-deprived doves but not in freely fed animals. Empty crop sac weights of freely fed and food-deprived PRL-treated birds were not increased above control values, thus indicating that ICV PRL treatment did not result in significant stimulation of peripheral target organs. These results demonstrate a facilitative action of PRL on regurgitation-feeding responses and associated parental behaviors that is not restricted to squabs of one particular age range. They also indicate that PRL is capable of acting directly on the brain to promote these activities in the absence of PRL-induced changes in the crop sac and other peripheral target organs.     

Biphasic 24-Hour Variations in Cyclic GMP Accumulation in the Rat Pineal Gland Are Due to Corresponding Changes in the Activity of Cytosolic and Particulate Guanylate Cyclase

Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.