Comparison of an antiviral activity of recombinant consensus interferon with recombinant interferon-alpha-2b

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.     

Dynamic perceptual changes in audiovisual simultaneity

Background

The timing at which sensory input reaches the level of conscious perception is an intriguing question still awaiting an answer. It is often assumed that both visual and auditory percepts have a modality specific processing delay and their difference determines perceptual temporal offset.

Methodology/Principal Findings

Here, we show that the perception of audiovisual simultaneity can change flexibly and fluctuates over a short period of time while subjects observe a constant stimulus. We investigated the mechanisms underlying the spontaneous alternations in this audiovisual illusion and found that attention plays a crucial role. When attention was distracted from the stimulus, the perceptual transitions disappeared. When attention was directed to a visual event, the perceived timing of an auditory event was attracted towards that event.

Conclusions/Significance

This multistable display illustrates how flexible perceived timing can be, and at the same time offers a paradigm to dissociate perceptual from stimulus-driven factors in crossmodal feature binding. Our findings suggest that the perception of crossmodal synchrony depends on perceptual binding of audiovisual stimuli as a common event.     

Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.     

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation.

Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Amine Oxidases of Microorganisms

The amine oxidase was found to be formed in mycelia of fungi when they were grown on monoamines or diamines as sole nitrogen sources. The maximal formation of enzyme was observed in the initial stage of growth, then the enzyme disappeared semilogarithmically. Other sources of nitrogen, such as ammonia, nitrate, urea and amino acids, were fully inactive for the enzyme formation. Furthermore, ammonia repressed the enzyme formation by fungi. The amine oxidase of fungi resembled in substrate specificity the monoamine oxidase of animal tissues. The enzyme oxidized preferentially aliphatic monoamines of C₃–C₆. Agmatine and histamine were also oxidized but in lower rates. Benzylamine was well oxidized by the enzymes of Aspergillus niger and Penicillium chrysogenum, but not by the enzymes of Monascus anka and Fusarium bulbigenum. Polyamines were not oxidized by the fungal enzymes.     

Salmonid olfactory system-specific protein (N24) exhibits glutathione S-transferase class pi-like structure

A salmonid olfactory system-specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as approximately 43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S-transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity-purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.     

Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.