Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.     

Determination of anti-Aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha

The dynamic growth rate of a single hypha of Aspergillus niger was analysed using an automatic system. A colony of A. niger was in contact with saline, saline containing an antifungal agent, and flushing saline, in sequence. The growth rate of a test hypha selected arbitrarily from the colony responded dynamically to the antifungal agent. The minimum concentration that caused the complete inhibition of hyphal growth was defined as the minimum inhibitory concentration (MIC). The MIC values obtained were compared with those determined by conventional methods based on increasing rate of colony diameter or dry matter weight.     

A model for neural representation of binocular disparity in striate cortex: distributed representation and veto mechanism

A model in striate cortex is proposed for a distributed neural representation of binocular disparity with a simple cell. In the model, disparity is represented by far, near and tuned inhibitory simple cells. However, the representation will be vetoed by model cells where disparity is excessively large. The veto mechanism consists of a neural network of the model cell which received output from simple cells and which interacts with neighbors. The mechanism is necessary, the model cell responds like a simple cell, and the network is physiologically plausible in the brain. Computer simulation on the neural network model with random dot stereography indicates reasonable performance.     

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

PCR method for genotyping and zygosity-testing of RasH2 transgenic mice.

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.