Arbovirological survey in Silica plateau area, Roznava District, Czechoslovakia

The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against Lipovník (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to Lipovník and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

Diurnal and circadian regulations by three melatonin receptors in the brain and retina of olive flounder Paralichthys olivaceus: profiles following exogenous melatonin

To establish the molecular basis of circadian rhythm control by melatonin receptors (MTs), we investigated the mitochondrial ribonucleic acid (mRNA) expressions of three types of MTs in different tissues of the olive flounder (Paralichthys olivaceus). All three types of MT mRNAs were expressed in the neural tissues, while MT1 mRNA was expressed in the peripheral tissues and MT2 and MT3 mRNAs were weakly expressed or undetected in these tissues. We observed increased MT mRNA expression in the neural tissues at night under both light–dark (LD) and constant dark (DD) conditions. Although the melatonin-treated cultured pineal gland samples showed similar diurnal variations with high-MT mRNA expression levels at night compared to those of untreated cultured pineal gland samples, the expression levels were considerably higher in the melatonin-treated samples. The plasma melatonin level also significantly increased at night. Under DD conditions, the expression patterns of MT mRNAs were similar to those under the LD photocycle, but the peak was lower and the circadian change patterns were less clear. These findings reinforce the hypothesis that MTs are active in processing light information, and that these genes are regulated by the circadian clock and light, thus suggesting that MTs play an important role in daily and circadian variations in the brain and retina of olive flounders.     

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Isolation and Characterization of Bacteria from Coal Mines of Dara Adam Khel,Pakistan

The coal fields of Pakistan and their microbiology have not been fully explored. Therefore, a study was conducted on the coal mines of Dara Adam Khel located in the Federally Administered Tribal Areas of Pakistan. For this purpose, sampling was done from nine different mines with varying depths. A total of 32 bacterial strains were isolated and their colony size, form, texture, color, margin, elevation and opacity were noted. The majority of the strains (75%) were found Gram negative. The bacterial strains were then characterized in detail by different biochemical tests including catalase, citrate, oxidase, indole, triple sugar iron, motility, methyl red-Vogues Proskeur, nitrate reduction and phenylalanine deaminase, and an enormous physiological diversity was observed. The Gram positive strains were further characterized on molecular level using 16S rRNA gene amplification and sequence analysis. Based on molecular analysis, seven strains were identified as Bacillus tequilensis, B. cereus, Janibacter melonis, Kocuria atrinae, B. anthracis, K. rosea and B. simplex. The other two strains (strains 6 and 41) had molecular similarity of only 98% and 97% with Brachybacterium spp. and Arthrobacter spp. respectively. The phylogenetic analysis further suggested that the strains 6 and 41 may be potential candidates for novel species; however, further work is needed for confirmation.     

Semilunar courtship rhythm of the fiddler crab<Emphasis Type="Italic"> Uca lactea</Emphasis> in a habitat with great tidal variation

Semilunar courtship rhythm is a widely distributed phenomenon among fiddler crabs in the genus Uca (Decapoda, Ocypodidae). Typically, synchronous courtship has been reported to peak near spring tides. To determine whether a region of large tidal variation shifts reproductive activity, we measured the frequency of specific courtship behaviors including claw-waving and semidome building for U. lactea males on Kanghwa Island, Korea. We found that synchronized courtship for U. lactea peaked near neap tides, whereas near the spring tides, seawater flooded the habitat and males predominantly fed on the mudflat. Although active females, which hold their burrows and usually feed on the mudflat, are abundant near to spring tides, males rarely claw-waved to attract females. This pattern is atypical for the species because other populations of U. lactea on Japan and Taiwan are synchronous around spring tides. We suggest that males invest most of their time in feeding during spring tides because foraging is limited during neap tides. During neap tides, males feed infrequently and thus expend stored energy on courtship signals. We conclude that patterns of reproductive synchrony may be dependent on food availability in periodically changing environments.