GROWTH OF HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE) ON NITRATE AND AMMONIUM AT THREE PHOTON FLUX DENSITIES: EVIDENCE FOR N STRESS IN NITRATE-GROWING CELLS

The marine alga Heterosigma carterae Hulburt (Raphidophyta) was grown in N-limiting batch cultures using either nitrate or ammonium as the N source, at photon flux densities (PFDs) of 50, 200, and 350 μmol·m^-2·s^-1 in a 12:12 h LD cycle. Carbon content could be estimated from biovolume (μg C = 0.278 × nL; R = 0.98) but not reliably from pigment content. During exponential growth, ammonium-grown cells (in comparison with nitrate-grown cells at the same PFD) attained higher growth rates by at least 20%, contained more N, and had a lower C:N ratio, higher concentrations of intracellular free amino acids, and higher ratios of glutamine: glutamate (Gln: Glu) and asparagine: aspartate (Asn:Asp). Growth was nearly light-saturated on ammonium at 200 μmol·m^-2·s^-1 (cell-specific growth rate of 1.2 d^-1) but probably not saturated in nitrate-grown cells at 350 μmol·m^-2·s^-1. PFD did not affect Gln: Glu or Asn: Asp for a given N source. These results indicate that the nitrate-growing cells were more N-stressed than those using ammonium (which in contrast were relatively C-stressed) and that this organism would show an enhanced competitive advantage against other species when supplied with a transient supply of ammonium rather than nitrate .     

Rapid Glutamate Decarboxylase Assay for Detection of Escherichia coli

Volume 59, no. 12, p. 4347, column 1, line 3 from bottom: "500 x g" should read "2,500 x g." [This corrects the article on p. 4347 in vol. 59.].     

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.     

Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons.

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.     

Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA.

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3'-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3'-terminal 46 bases. In contrast, the 5' 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3' 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element.     

A predicted consensus structure for the protein kinase C2 homology (C2H) domain,the repeating unit of synaptotagmin

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred. © 1995 Wiley-Liss, Inc.     

An ultra-pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting

We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.     

Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.