Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Ionic iridium-gold and rhodium-gold perfluorobenzenethiolato binuclear complexes: syntheses and solution behavior

By reacting [(C₅Me₅)M(SR_F)₂] (forM = Ir, Rf = C₆F₅ (1a) or C₆F₄H-p (1b); for M = Rh, Rf = C₆F₅ (2a) or C₆F₄H-p (2a)) in toluene with Na[AuCl₄], ionic binuclear compounds with the general formula [(C₅Me₅)M(μ-SR_F)₂AuCl₂]Cl for M = Ir, R = C₆F₅ (3a) or C₆F₄H-p (3a); for M = Rh, R_F = C₆F₅ (4a) or C₆F₄H-p (4b) can be obtained, together with small amounts of [(C₅Me₅)₂Rh₂(μ-SR_F)(μ-Cl)₂]Cl (R_F = C₆F₅ (5a) or C₆F₄H-p (5b)) as by-products when 2a and 2b were used.     

Genetic and physiologic analysis of a formyl-tetrahydrofolate synthetase mutant of Streptococcus mutans.

Previously we reported that transposon Tn917 mutagenesis of Streptococcus mutans JH1005 yielded an isolate detective in its normal ability to produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the American Society for Microbiology 1995, 1995). In this report we describe the recovery of the mutated gene by shotgun cloning. Sequence analysis of insert DNA adjacent to Tn917 revealed homology to the gene encoding formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. Analysis of the fhs mutant grown anaerobically in a minimal medium demonstrated that the mutant had an absolute dependency only for adenine, although addition of methionine was necessary for normal growth. Coincidently it was discovered that the mutant was sensitive to acidic pH; it grew more slowly than the parent strain on complex medium at pH 5. Complementation of the mutant with an integration vector harboring a copy of fhs restored its ability to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptococcus.     

Airborne grass pollen in Granada (Spain)

We have carried out a study on the annual and daily pollen concentrations from Gramineae over four consecutive years in the atmosphere of Granada (Spain). Samples of pollen grains were collected by the volumetric method with the aid of a Burkard sporetrap. Gramineae, according both to their high sensitizing capacity and to data from allergologists, are responsible for many pollinoses diagnosed in this area. In this work, daily pollen levels from April to July are monitored and the variations identified are interpreted in relation to meteorological conditions. Results indicated that the highest airborne concentrations of Gramineae pollen were found in May and June, although the beginning and intensity of pollination have been variable during these 4 years.     

Anti-Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide

Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides.     

Effects of epidermal growth factor on deoxyribonucleic acid synthesis and stomach weight in hypophysectomized adult mice

Epidermal growth factor (EGF) or saline was administered intraperitonally to hypophysectomized adult male CD2F₁ mice or intact controls at 0700 hr. Subgroups of mice were killed at 4, 8, or 12 hr after injection. EGF was shown to stimulate [³H]TdR incorporation into DNA into several organs as previously reported. The response to EGF was found to be enhanced in both hypophysectomized and fasted mice. Differences in [³H]TdR incorporation into DNA, corneal epithelium mitotic index, RNA in pancreas and kidney of hypophysectomized and intact mice are reported. EGF was shown to result in stomach enlargement due to increased luminal contents in both hypophysectomized and intact mice.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

Summary The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (−2° C/min down to −30°C and then quick freezing to −196° C) and a fast freezing rate (FFR) (direct freezing of tubes to −196° C: −39° C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37° C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. The financial support for this work was from Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants 41/82 and 48/82.     

Cell-mediated immune responses in recurrent herpesvirus infections. I. Lymphocyte proliferation assay.

Studies in immunosuppressed and immunodeficient patients indicate that the cell-mediated immune response appears to be responsible for controlling reactivated herpesvirus infections. In this study, the various parameters of a herpesvirus (types 1 and 2) antigen specific lymphocyte proliferation assay were optimized and used to evaluate individuals with clinical, recurrent HSV-1 and HSV-2 infections. Normal individuals with neutralizing antibody to HSV-1 or HSV-2 responded to virus antigen in culture as well as individuals with recurrent disease. Normal individuals without neutralizing antibody responded with a significantly lower response. Specificity of the lymphocyte proliferation assay was observed most strikingly in normal individuals with a rare HSV-1 infection during the vesicular eruption. Specificity was also observed by determining the ratio of the response to HSV-1 as compared to the response to HSV-2. Evaluated in this manner, individuals with recurrent HSV-1 infections had significantly higher ratios than individuals with HSV-2 infections and vice versa. Data from individuals with recurrent disease was compared to that of normal individuals to determine whether the former demonstrated a specific alteration in this response. Individuals with recurrent disease were found to have higher neutralizing antibody titers than normals. The neutralizing antibody titers in normal individuals correlated well with the lymphocyte proliferation assay results, whereas a similar evaluation in individuals with recurrent disease gave a negative correlation. The ratio of HSV-1 response/HSV-2 response also demonstrated a suppressed response in recurrent infections to the homologous virus during active disease, which disappeared when the individual was convalescent. These studies indicate that individuals with recurrent HSV infections have virus antigen specific alterations of their cell-mediated immune response, which can be associated with their disease.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.