A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

Protease-producing psychrotrophic bacteria isolated from Antarctica

The extracellular protease production capacity of 840 bacterial strains isolated during the austral summers of 1989/90 and 1991/92 from different sources of the Antarctic ecosystem was analysed in skim-milk agar plates. Thirty-four psychrotrophic strains were selected, classified at genus level and tested from proteolytic activity by the azocasein method from the cell-free supernatant of submerged cultures. Thirty-two of the selected strains were Gram-negative bacteria and Pseudomonas was the predominant genus. Three Pseudomonas maltophilia strains showed the highest levels of proteolytic activity at 20°C. No correlation was observed between the proteolytic activity estimated by the ring of hydrolysis in skim-milk agar plates and the activity measured by the azocasein method. The results suggest that these psychrotrophic strains are potentially useful for developing a biotechnological process to produce proteases with high activity at moderate temperatures.     

Chemical composition of throughfall,soil water,leaves and leaf litter in a beech forest receiving long term application of ammonium sulphate

Nitrogen deposition in the range of 10–40 kg N ha^-1 a^-1 is common in large parts of Europe. Substancial amounts are deposited as NH ₄ ⁺ having fertilization and acidification effects in ecosystems. In a long term experiment the reactions of different compartments of a forest ecosystem were studied when the system became N saturated by continuously applying (NH₄)₂SO₄. The experiment was conducted in a beech forest and the application of 10 kmol_c N ha^-1 a^-1 lasted 11 yr from 1983 till 1993.The results revealed that despite the high soil acidity, the applied NH ₄ ⁺ was quickly oxidized to NO ₃ ^- in the surface 10 cm soil layer and leached to deeper depths. The amount of NO ₃ ^- leached from the surface soil increased during the initial three years and remained constant on a high level for the rest of the experimental period. Nitrification was associated with acidification of the soil solution, causing high concentrations of Al and Mn²⁺ in soil solutions. More than 50% of total Al in solution occurred in non-phytotoxic form (Al–SO₄ complexes). Moreover, concentration of base cations and dissolved organic carbon increased. Concentrations of SO ₄ ^2- in soil solutions increased during the first few years approaching more or less constant values in the surface 40 cm depth, whereas in 40–100 cm depth it took about 10 yr to reach those levels of sulphate concentrations in soils, indicating its retention in the deeper soil layers. No significant change in the chemistry of throughfall water and leaves was observed, indicating to N-saturation of the trees.     

The Subcellular Localization of Isoperoxidases in Capsicum annuum Leaves and their Different Expression in Vegetative and Flowered Plants

The subcellular localization of leaf peroxidases (EC 1.11.1.7)and their expression in vegetative and flowered plants has beenstudied in Capsicum annuum (var. annuum) in order to assesswhether the expression of new peroxidase isoenzymes can characterizethe floral state which determines the beginning of reproductivedevelopment. The results showed that floral development is accompaniedby a significant increase in the level of soluble (non-sedimentable)leaf peroxidase, independently of leaf position along the internodes,and therefore independently of the leaf age. An analysis ofthe leaf peroxidase isoenzyme patterns along the internodesfor vegetative and flowered plants shows that the increase inperoxidase activity is due to a general increase in the activityof all the pre-existing peroxidase isoenzymes, although isoenzymeB₂ and, especially, isoenzyme A₁ showed a distinctive and majorincrease in activity. These two isoenzymes are mainly ionically-boundto cell walls, probably in equilibrium with the same isoenzymesmoving freely in the cell-wall free spaces. The differs fromother peroxidase isoenzymes, such as isoperoxidase B₆, whichis mainly located in the covalently-bound cell-wall fractionand in mesophyll vacuoles. These results are discussed in thelight of a possible role of cell wall peroxidases as markersof the floral state in Capsicum annuum morphogenesis.Copyright1993, 1999 Academic Press Capsicum, floral state, leaf peroxidases, subcellular localization, vegetative state     

Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes

The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units OAc 1 2/6 →3)-β-d -Galf-(1 →3)-α- d -Galp-(1→d -Galactan I-OAc →3)-α-d -Galp-(1 →3)-β-d -Galp-(1→d -Galactan II. K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only d -galactan I-OAc. The ¹H- and ¹³C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on d -galactan I and O-acetylation occurs only on the β- d -galactofuranose residues; 60% of the available β- d -galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to d -galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of d -galactan I. rfb_Kpo1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce d -galactan I. Six O1 strains were examined and all showed hybridization with rfb_KpO1 probes under conditions of high stringency. Three serotype O2 strains produce d -galactan I and these strains also contained DNA sequences recognized by rfb_KpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfb_KpO8 region from K. pneumoniae serotype O8 was only recognized by rfb_KpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.     

Histologic and clinical characteristics associated with rapidly progressive invasive cervical cancer: a preliminary report from the Yale Cancer Control Research Unit

Histologic and clinical characteristics associated with rapidly progressive invasive cervical cancer are presented in this preliminary report from a population-based study involving all patients in Connecticut diagnosed with cervical cancer from March 1, 1985. Rapidly progressive invasive cervical cancer, i.e., invasive cancer diagnosed within three years of a true negative Pap smear, is more likely to occur in younger women with high annual incomes (61 percent greater than $40,000) who report a greater frequency of benign gynecologic conditions (uterine leiomyomata, vaginitis) compared to a control cervical cancer group. These preliminary data suggest that as many as 35 percent of the rapidly progressive cervical cancers are likely to be adenocarcinomas. Because they are mostly endocervical in origin, they may not be detected cytologically if scrapers or cotton swabs are used to sample the endocervical canal. New cytologic screening techniques using brushes may identify these lesions earlier and should routinely be employed in cytologic screening for cervical neoplasia. The difficulty in early detection of this form of the disease requires that physicians rapidly assess patients with unexplained pelvic and lower abdominal pain, vaginal discharge, or abnormal vaginal bleeding since early recognition is the only chance for cure. Further analyses of this population of women will be made to identify additional risk factors when the study data are complete.     

Uncoupling the MgATP-induced inhibition and aggregation of Escherichia coli phosphofructokinase-2 by C-terminal mutations

Binding of MgATP to an allosteric site of Escherichia coli phosphofructokinase-2 (Pfk-2) provoked inhibition and a dimer-tetramer (D-T) conversion of the enzyme. Successive deletions of up to 10 residues and point mutations at the C-terminal end led to mutants with elevated K(Mapp) values for MgATP which failed to show the D-T conversion, but were still inhibited by the nucleotide. Y306 was required for the quaternary packing involved in the D-T conversion and the next residue, L307, was crucial for the ternary packing necessary for the catalytic MgATP-binding site. These results show that the D-T conversion could be uncoupled from the conformational changes that lead to the MgATP-induced allosteric inhibition.     

Evidence for the signaling function of egg color in the pied flycatcher Ficedula hypoleuca

A recent hypothesis proposes that the bright colors, especiallyblue and green, of many avian eggs may function as signals offemale or offspring phenotypic quality or condition to malesin species with biparental care, inducing them to allocate moreeffort to their offspring. The pigment determining blue andgreen egg colors is an antioxidant whose availability for eggshellcoloring may be limited. To test the signaling function on aspecies with blue eggs, the pied flycatcher Ficedula hypoleuca,we measured egg color with a spectrophotometer on the day oflaying and obtained two principal components from their reflectancespectra that together explained 99% of variation and representedshell lightness, and hue and saturation, respectively. We alsomeasured female immunocompetence during the nestling periodthrough the response to phytohemagglutinin as a measure of cell-mediatedimmunity and the response to a tetanus vaccination as a measureof humoral immunity. The total amount of immunoglobulins inblood of females and of nestlings before fledging was also estimated.Mean within-clutch egg darkness was positively associated withboth measures of female immunocompetence, while better femalecondition was associated with colors tending away from intermediateand toward short wavelengths. Ageing female laid lighter eggs.The mean within-brood level of nestling IgY was also associatedwith mean within-clutch egg colors tending away from intermediateand toward short wavelengths. Mean egg darkness decreased linearlyduring the laying sequence, suggesting pigment limitation. Maleswere observed frequently visiting nests during the laying period,allowing them to observe eggs before the start of incubation.These results support the signaling hypothesis for explainingbright colors of avian eggs.     

Coupling of endothelial injury and repair: an analysis using an in vivo experimental model

The repair of the endothelium after inflammatory injury is essential to maintaining homeostasis. The link between inflammation-induced endothelial damage and repair has not been fully characterized in vivo. We have developed a rat model to evaluate the coupling of lipopolysaccharide (LPS)-induced endothelial injury and repair. Aortic endothelium injury was analyzed by both inmunohistochemistry and flow cytometry to quantify the number of endothelial cells and the percentage of apoptotic endothelial cells. We have also identified the percentage of circulating angiogenic cells capable of repairing the damaged endothelium. Erythropoietin was administered to inhibit LPS-induced endothelial apoptosis. Loss of the normal endothelial structure was observed in the aorta of the animals treated with LPS. Eight hours after LPS administration, the number of endothelial cells decreased by 40%, returning to normal after 24 h. There was a threefold increase in the percentage of circulating angiogenic cells, which did not return to normal levels until 48 h after LPS administration. Circulating angiogenic cell levels did not change when LPS-induced endothelial damage was prevented by erythropoietin. The endothelial injury caused by inflammation activates the mobilization of circulating angiogenic cells, thus completing endothelial repair. Inflammation without endothelial injury does not trigger the mobilization of circulating angiogenic cells.