Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.     

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Leu-1+ (CD5+) B cells. A major lymphoid subpopulation in human fetal spleen: phenotypic and functional studies

Examination of the cell surface phenotype of fetal splenic lymphocytes demonstrated a major, novel subpopulation of B cells that co-express Leu-1 (CD5) in addition to B cell differentiation antigens (Leu-1+ B cells). These cells are similar to some conventional B cells in that they express HLA-DR, Leu-12, and B1, as well as both immunoglobulin (Ig) M and IgD. They comprise 40 to 60% of total splenic B cells in the fetus but are infrequent in fetal liver and adult spleen. Fetal Leu-1+ B cells do not respond to pokeweed mitogen with either proliferation or Ig secretion, and in contrast to the murine counterpart, Ly-1 B cells, they do not constitutively produce Ig. Leu-1+ B cells were incapable of augmenting Ig production of Leu-1- B cells when suboptimal numbers of T cells were present; however, they did require the presence of T cells to secrete antibody. They do not cap either the CD5 protein or surface Ig. These cells are a unique subpopulation of fetal splenic B cells that do not function as conventional B cells. Their role in the humoral immune response is unknown. They may represent the normal stage of B cell development, which is reflected in the phenotype of B cell CLL cells.     

CHEMICAL CHARACTERIZATION OF THE ISOLATED CELL SURFACE OF AMOEBA

The cell surface has been isolated from uninucleate, freshwater, phagocytic amoebae by a new procedure. Several criteria were employed to demonstrate purity of the cell surface fraction. All morphological components of the tripartite surface were present in the isolated surface and the weight of the isolated surface was quantitatively accounted for by the components analyzed. Chemical analyses showed the presence of lipid, protein, and carbohydrate. Mannose was the predominant neutral sugar. Analyses for three different strains of Amoeba were similar. Phosphate was found to be the major anionic group in the cell surface material. Sulfate, uronic acid, sialic acid, muramic acid, and nonamidated glutamic acid and aspartic acid were absent. Evidence is presented suggesting that the phosphate is associated with an unidentified nonreducing polyol.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Similarities between UDP-glucose and adenine nucleotide release in yeast: involvement of the secretory pathway

Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Delta was reduced by deletion of the YEA4 UDP- N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.     

Effect of Age-Stiffening Tissues and Intraocular Pressure on Optic Nerve Damages

Age-stiffening of ocular tissues is statistically linked to glaucoma in the elderly. In this study, the effects of age-stiffening on the lamina cribrosa, the primary site of glaucomatous nerve damages, were modeled using computational finite element analysis. We showed that glaucomatous nerve damages and peripheral vision loss behavior can be phenomenologically modeled by shear-based damage criterion. Using this damage criterion, the potential vision loss for 30 years old with mild hypertension of 25mmHg intraocular pressure (IOP) was estimated to be 4%. When the IOP was elevated to 35mmHg, the potential vision loss rose to 45%; and age-stiffening from 35 to 60 years old increased the potential vision loss to 52%. These results showed that while IOP plays a central role in glaucomatous damages, age-stiffening facilitates glaucomatous damages and may be the principal factor that resulted in a higher rate of glaucoma in the elderly than the general population.