Use of Ultrafiltration To Isolate Viruses from Seawater Which Are Pathogens of Marine Phytoplankton

Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (Micromonas pusilla), a pennate diatom (likely a Navicula sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a Synechococcus sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a Rhodomonas sp.) and a chlorophyte (Nannochloropsis oculata) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting M. pusilla and the Navicula sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.     

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [¹⁴C]methylamine. The pH of unfractionated blood cells is 7.39±0.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.     

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.     

NOTE—THE STA-1 MUTATION PREVENTS ASSEMBLY OF STARCH GRANULES IN NITROGEN-STARVED CELLS AND SERVES AS A USEFUL MORPHOLOGICAL MARKER DURING SEXUAL REPRODUCTION IN CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE)

Iodine staining of clones of nitrogen-starved Chlamydomonas cells was used to screen for mutants with altered levels or altered composition of storage starch. Mutations leading to defects in quantity or morphology of starch granules not only can provide information on storage starch biosynthesis and granule assembly but can also be used as morphological markers in genetic and cell biological studies. A mutant of Chlamydomonas monoica Strehlow devoid of starch granules was obtained following ultraviolet mutagenesis. Nitrogen-starved cells of the sta-1 strain lacked pyrenoidal starch granules and granules normally associated with thylakoid membranes. The mutant phenotype was the consequence of a single Mendelian mutation that appeared to affect granule assembly rather than starch biosynthesis per se and that had no effect on vegetative growth, sexual reproduction, or zygospore viability.     

MHC Class I Antigen Presentation of DRiP-Derived Peptides from a Model Antigen Is Not Dependent on the AAA ATPase p97

CD8⁺ T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8⁺ T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.     

Skp1-Cul1-F-box Ubiquitin Ligase (SCFβTrCP)-mediated Destruction of the Ubiquitin-specific Protease USP37 during G2-phase Promotes Mitotic Entry

Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. The Skp1-Cul1-F-box (SCF) and anaphase-promoting complex (APC) ubiquitin ligases target numerous components of the cell cycle machinery for destruction. Throughout the cell cycle, these ligases cooperate to maintain precise levels of key regulatory proteins, and indirectly, each other. Recently, we have identified the deubiquitinase USP37 as a regulator of the cell cycle. USP37 expression is cell cycle-regulated, being expressed in late G₁ and ubiquitinated by APC^Cdh1 in early G₁. Here we report that in addition to destruction at G₁, a major fraction of USP37 is degraded at the G₂/M transition, prior to APC substrates and similar to SCF^βTrCP substrates. Consistent with this hypothesis, USP37 interacts with components of the SCF in a βTrCP-dependent manner. Interaction with βTrCP and subsequent degradation is phosphorylation-dependent and is mediated by the Polo-like kinase (Plk1). USP37 is stabilized in G₂ by depletion of βTrCP as well as chemical or genetic manipulation of Plk1. Similarly, mutation of the phospho-sites abolishes βTrCP binding and renders USP37 resistant to Plk1 activity. Expression of this mutant hinders the G₂/M transition. Our data demonstrate that tight regulation of USP37 levels is required for proper cell cycle progression.     

Wood construction more strongly shapes deadwood microbial communities than spatial location over 5 years of decay

Diverse communities of fungi and bacteria in deadwood mediate wood decay. While rates of decomposition vary greatly among woody species and spatially distinct habitats, the relative importance of these factors in structuring microbial communities and whether these shift over time remains largely unknown. We characterized fungal and bacterial diversity within pieces of deadwood that experienced 6.3–98.8% mass loss while decaying in common garden ‘rotplots’ in a temperate oak-hickory forest in the Ozark Highlands, MO, USA. Communities were isolated from 21 woody species that had been decomposing for 1–5 years in spatially distinct habitats at the landscape scale (top and bottom of watersheds) and within stems (top and bottom of stems). Microbial community structure varied more strongly with wood traits than with spatial locations, mirroring the relative role of these factors on decay rates on the same pieces of wood even after 5 years. Co-occurring fungal and bacterial communities persistently influenced one another independently from their shared environmental conditions. However, the relative influence of wood construction versus spatial locations differed between fungi and bacteria, suggesting that life history characteristics of these clades structure diversity differently across space and time in decomposing wood.