Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Involvement of prostaglandin-induced proteins in the inhibition of herpes simplex virus replication

Cyclopentenone prostaglandin (PG), delta7-PGA1 was found to induce several polypeptides in human embryonic fibroblast (HEF) cells which were noticed to be dose-related and appeared after 1 h of treatment with a peak at around 5 h and gradual disappearance after 12 h. PG-induced proteins were almost identical in terms of molecular weights with those induced by heat-shock at 42 degrees C. Regarding the mechanism of inhibition of herpes simplex virus (HSV) replication by PG in cell culture, dot blot hybridization has revealed that the level of immediate early (IE) mRNA of the virus was reduced after PG treatment with time dependence. And this delayed inhibitory effect of delta7-PGA1 on HSV was shown to be associated with the production and accumulation of the induced polypeptides.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

Approach to the Involvement of Influenza B Neuraminidase in the Cleavage of HA by Host Cell Protease Using Low pH-Induced Cell Fusion Reaction

ts7, a temperature-sensitive mutant defective in neuraminidase (NA) of influenza B/Kanagawa/73, lacks NA enzymatic activity at the nonpermissive temperature (37.5 C). When MDCK cells were infected with the mutant at the permissive temperature (32 C) and exposed to pH 5.2 medium, extensive cell fusion occurred. In contrast, at the nonpermissive temperature cells did not show cell fusion at all unless they were pretreated with trypsin, suggesting that at 37.5 C the hemagglutinin (HA) of ts7 is expressed at the cell surface in an uncleaved form. It was also found that the replacement of RNA segment 6 of ts7 with that of wild-type B/Lee resulted in the emergence of low pH-induced fusion activity as well as NA enzymatic activity at the incubation temperature of 37.5 C and that the addition of bacterial NA to the cultures infected with ts7 at 37.5 C early in infection brought about low pH-induced cell fusion. We suggest that the removal of neuraminic acid from the carbohydrate moiety of HA by NA is essential for the cleavage of HA by cellular protease.     

Wave-separation in complex systems. Application to brain-signals

A departure from single-system dynamics, that may arise in characterizing self-organized dynamics of complex systems, is dealt with by using the Karhunen-Loève expansion of the trajectory matrix to decompose an experimental signal in a sum of spectral features. For an electroencephalographic -signal, a separation of waves and extraction of additive sub-signals are achieved, each sub-signal covering a well-bounded and physiologically meaningful frequency range. From the subsignals, an attractor that vanishes on phase-randomizing the data is characterized, under conditions where none was found for the recorded signal.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

The neuropeptide, neuromedin C, activates a potassium current in mouse macrophages.

Neuromedin C (NmC) induced an outward current (Io(NmC)) in macrophages. Reversal potentials were dependent on external K+ concentrations ([K+]o) and independent of [Cl-]o. Tetraethylammonium (TEA) and quinidine effectively suppressed Io(NmC). Charybdotoxin (ChTX) and apamin had little effect. Io(NmC) was abolished in Ca(2+)-free EGTA-containing solution. These results suggest that MnC activates a Ca(2+)-dependent K+ current (IK,Ca) and can modulate activities in macrophages.