Emergence of a sylvatic enzootic formosan ferret badger-associated rabies in Taiwan and the geographical separation of two phylogenetic groups of rabies viruses.

Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation.

Traumatic brain injury causes frontotemporal dementia and TDP-43 proteolysis.

Traumatic brain injury (TBI) is a major risk factor for dementia. Recently, TBI has also been suggested as a risk factor for frontotemporal dementia (FTD), and plasma immunoreactivity to the TAR-DNA binding protein 43 (TDP-43) has been observed in both patients with acute TBI and long-term survivors of this condition. We used a population-based study to estimate and compare the risk of FTD in individuals with and without TBI. Furthermore, we used a rat model of TBI to show that increased TDP-43 proteolysis following TBI produces FTD-like impairments, including abnormal limb-clasping, and impaired performances in the Morris water maze. We recruited 24,585 patients who received ambulatory or hospital care for TBI and 122,925 patients without TBI for this study. Each individual was investigated for 4years to evaluate FTD development, and data were analyzed by Cox proportional hazard regression. In the TBI rat model, behavior and TDP-43 inclusions were assessed following intracranial administration of a caspase-3 inhibitor or vehicle. FTD was more likely to occur in the TBI group than in the group without TBI (adjusted hazard ratio, 4.43; 95% confidence interval, 3.85-5.10; P<0.001). Rats developed behavioral impairments similar to those in patients with FTD after TBI. Further, the behavioral impairments were likely associated with TDP-43 short fragment mislocalization and accumulation. Our findings suggest that in humans, TBI is associated with a greater occurrence of FTD. Moreover, clinical FTD manifestations may be associated with TDP-43 proteolysis, since impaired behaviors in TBI rats were reminiscent of those in humans with FTD.

Asymmetric expression patterns of brain transthyretin in normal mice and a transgenic mouse model of Alzheimer's disease.

Brain asymmetry is linked with several neurological diseases, and transthyretin (TTR) is a protein sequestering beta-amyloid (Abeta) and helping to prevent the Alzheimer's disease (AD). We show, by real time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting, that TTR exhibits a pattern of adult male-specific, leftward distribution in the mouse brain. This asymmetry appeared to be mainly due to the asymmetric distribution of the choroid plexus cells in the ventricles. Unlike the normal mice, however, the hemispheric levels of TTR transcripts of 2- and 6-month-old Tg2576 mice, a transgenic AD mouse model overexpressing Abeta, were symmetric in both sexes. Furthermore, at the age of 10 months when the pathological AD-like features had developed, the level of TTR transcripts in the left hemisphere of the male Tg2576 became significantly lower than the right one. This lowering of TTR transcript is accompanied with a higher Abeta level in the left hemisphere of the 10-month Tg2576 males. Finally, for both genders, the TTR transcript levels in the two hemispheres of aged Tg2576 mice were lower than either the adult Tg2576 or the aged nontransgenic controls. Based on the above, we suggest scenarios to correlate the changes in the levels and hemispheric patterns of TTR expression to the pathogenesis of AD.

The AdLMP-1 transfection in two different cells; AF cells, chondrocytes as potential cell therapy candidates for disc degeneration.

BACKGROUND: LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production. METHOD: We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining. AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression. The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 x 10(4) cells/chamber. FINDINGS: 1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression. CONCLUSIONS: The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration.

Stored of Hsp72/Hsp73 in germinal vesicle-stage mouse oocytes.

Heat-shock proteins (hsps) Hsp72 and Hsp73 are the stored maternal proteins found in mouse oocytes. Both hsps appear in mouse oocytes at germinal vesicle (GV) and metaphase II (M-II)-stages as previously demonstrated by immunoblotting analysis. In this report, we further determined the presences of Hsp72/Hsp73 proteins in mouse embryos at stages of 2-pronucleus, arrested 1-cell, 2-cell, arrested 2-cell, 4-cell, arrested 4-cell, 8-cell to morula and blastocyst. Except for the blastocyst stage, the Hsp72/Hsp73 proteins were detectable in most embryo stages. The concentration of Hsp72/Hsp73 in GV-stage oocytes was higher than that in M-II-stage oocytes, and in any stages of embryos before implantation. A dramatical increase in Hsp72/Hsp73 expression was found at the 2-cell stage. Together with these findings, we speculated that hsps accumulated or stored earlier in the GV-stage mouse oocytes to protect the oocytes against environmental influences acting on ovary, and hsps may be required for zygotic gene activation and provided a protective effect against apoptosis.

LeuO expression in response to starvation for branched-chain amino acids.

The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology. The cryptic nature of expression of leuO has previously limited such analysis. A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3',5'-bispyrophosphate-dependent. Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3',5'-bispyrophosphate. In this study, we tested whether LeuO was directly involved in the bacterial stringent response. LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E. coli K-12 relA1 strain. This result supports a functional role for LeuO in the bacterial stringent response.

ppGpp-dependent leuO expression in bacteria under stress.

Despite the known potential transcription regulatory role of leuO gene product, LeuO, the condition when leuO expresses during bacterial growth cycle remains unclear. Mechanistically, leuO expression was shown to be part of promoter relay mechanism, however, the factor(s) responsible for the regulation of leuO expression is not known. Combining Northern and Western results, we demonstrate in the present communication that leuO expression is normally low and enhanced when bacteria are in transition from exponential growth to stationary phase. The stationary phase-associated leuO expression is ppGpp dependent and rpoS (sigma(s) factor) independent.

Induction of integrin-associated protein (IAP) mRNA expression during memory consolidation in rat hippocampus.

We have previously demonstrated that hippocampal integrin-associated protein (IAP) gene expression is associated with memory formation in a one-way inhibitory avoidance learning in rats. In the present study, we further investigated the role and mechanism of IAP involved in memory consolidation in rats. Because of the minute amount of IAP present in the brain, we have adopted the quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Our results revealed that hippocampal IAP mRNA expression is approximately fourfold higher in rats showing good memory retention (GM, retention score of 600 s) at 3 h, but not at other time points, after training when compared with the poor memory rats (retention score < 80 s). On the other hand, integrin alphav mRNA level was markedly increased ( approximately twofold), while integrin beta3 mRNA level was decreased ( approximately 50%) at 1 h post-training. Further, separate sets of RT-PCR analysis revealed that IAP5 and IAP6 mRNA expressions, but not that of IAP7, were markedly increased in GM rats 3 h post-training. Moreover, regional distribution studies revealed that different isoforms of the IAP gene are similarly distributed in different brain areas, while IAP7 has been the predominant form present in astrocyte cells. These results together suggest that IAP mRNA expression is indeed induced upon training, rather than that the GM rats have constitutively higher levels of IAP. The unparallel change of IAP and integrin mRNA expressions as far as time-course is concerned suggests that they are possibly involved in different forms and stages of memory processing. Further, IAP5 and IAP6 are more closely associated with memory consolidation, while IAP7 may constitute the major isotype for signal transduction in astrocyte cells.

Association of CYP1A1 and microsomal epoxide hydrolase polymorphisms with lung squamous cell carcinoma.

Lung cancer is the leading cause of death among cancers in Taiwan. Although the etiology of lung cancer has yet to be defined, genetic variability in activities of metabolic enzymes has been correlated with lung cancer. In the present study, the possibility of association of CYP1A1 and microsomal epoxide hydrolase (HYL1) genetic polymorphisms with lung cancer was examined among 132 lung cancer patients and 259 controls in Taiwan. No significant association was observed for either CYP1A1 or HYL1 polymorphism alone and the overall incidence of lung cancer after adjusting for age, gender and smoking status. When cases were stratified according to histological type, there was significant association between CYP1A1*2A homozygote and squamous cell carcinoma (SCC) (odds ratio (OR) 2.86; 95% confidence interval (CI) 1.33-6.12). Similarly, the proportion of HYL1 genotypes corresponding to high or normal enzyme activities was higher in SCC than in controls (OR 1.96; 95% CI 1.04-3.70). A combination of susceptible CYP1A1 and HYL1 genotypes was found to be highly associated with lung cancer, especially with SCC (OR 6.76; 95% CI 2.29-19.10). Our results suggest that the combination of CYP1A1 and HYL1 polymorphisms is an important risk factor for lung SCC.

NAD(P)H: quinone oxidoreductase polymorphism and lung cancer in Taiwan.

Lung cancer is one of the leading causes of death in Taiwan since 1996. Genetic variation in metabolic activation or detoxification enzymes has been associated with the occurrence of lung cancer. NAD(P)H:quinone oxidoreductase (NQO1) enzyme is a cytosolic two-electron reductase thought to be involved in bioactivation and detoxification of environmental carcinogens. The possible association between NQO1 genetic polymorphism and lung cancer risk was examined among 95 male smokers without cancer and 100 male smokers with lung cancer in Taiwan. There was no significant difference in the proportion of wild-type NQO1 among all cancer cases and controls. When cases were stratified according to histological subtypes, the wild-type NQO1 was more common in adenocarcinoma than in controls. The odds ratio was 2.93 (95% confidence interval, 1.23-7.02; p = .02). This is the first observation for the positive association of this locus with lung cancer in an Asian population. These results suggest that NQO1 polymorphism is an important genetic risk factor for lung adenocarcinoma among smokers in Taiwan.

Cytochrome P4502E1 genetic polymorphisms and lung cancer in a Taiwanese population.

Cytochrome P4502E1 (CYP2E1) is involved in metabolic activation of carcinogenic nitrosamines, benzene and low molecular weight halogenated hydrocarbons. In this study, we assessed the association between CYP2E1 RsaI and DraI genetic polymorphisms and lung cancer in a Taiwanese population. The RsaI genotype distribution was significantly different between 119 lung cancer patients and 231 non-cancer controls. The homozygote variants of RsaI genotypes were more common in controls (6.9%) than in lung cancer patients (0.8%). The estimated odds ratio (OR) was 0.11 (95% confidence interval (CI), 0.01-0.87). After adjusting for age, sex, and smoking status, the OR was 0.12 (95%, CI, 0.02-0.95). This is the first observation of a positive association between this locus and lung cancer in an Asian population. No significant differences in CYP2E1 DraI genotype distributions were found between cases and controls. The results of this study indicate that CYP2E1 RsaI polymorphism, but not DraI polymorphism, may contribute to the development of lung cancer in Taiwan.

The effects of ellagic acid on arylamine N-acetyltransferase activity in the bacterium Pseudomonas aeruginosa.

Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by ellagic acid (EA), a naturally occurring dietary plant phenol. By measuring the acetylation of 2-aminofluorene (2-AF), the NAT activity was determined. In P. aeruginosa ATCC 27853, a NAT activity of 1.37 +/- 0.25 nmol/min/10(10) CFU for intact cell and a NAT activity of 5.92 +/- 0.20 nmol/min/mg protein for cytosolic preparation were measured. EA (ranging from 1 to 0.125 mM) showed a dose-dependent inhibition of NAT activities in the analysis of both intact cell and cytosolic preparations. Enzymatic kinetics were determined and found that EA was a potent non-competitive inhibitor of NAT activity in P. aeruginosa ATCC 27853. EA inhibition of NAT activities in P. aeruginosa ATCC 27853 was time-dependent for at least 4 hrs. These data strongly indicated that EA could suppress NAT activity in P. aeruginosa.

Impaired production of nitric oxide, superoxide, and hydrogen peroxide in glucose 6-phosphate-dehydrogenase-deficient granulocytes.

Since the generation of superoxide and hydrogen peroxide by NADPH oxidase and nitric oxide (NO) by NO synthase (NOS) in granulocytes is NADPH-dependent, we investigated the production of NO, superoxide and H2O2 in glucose 6-phosphate dehydrogenase (G6PD)-deficient human granulocytes. Our results showed that upon stimulation with either 5 microg/ml of lipopolysaccharide (LPS) or 10 microM of phorbol 12-myristate 13-acetate (PMA), the production of nitrite in normal granulocytes was elevated, 252 +/- 135% and 239 +/- 72%, respectively, compared to the resting stage. In contrast, G6PD-deficient granulocytes did not produce more nitrite upon stimulation with either LPS or PMA compared to the resting stage. Western blot analysis indicated a normal expression pattern of inducible NOS in G6PD-deficient granulocytes. In addition, the production of H2O2 and superoxide was also significantly impaired in G6PD-deficient granulocytes compared to control cells. These data demonstrate that G6PD deficiency causes an impairment in the production of NO, superoxide and H2O2.

Arecoline desensitizes carbachol-stimulated phosphatidylinositol breakdown in rat brain cortices.

To understand the effects of arecoline administration on the muscarinic cholinergic signaling pathway, rats were injected with arecoline, 10 mg/kg i.p., and the carbachol-stimulated phosphoinositide breakdown in rat brain cortical slices was examined. In vivo administration of arecoline resulted in inhibition of carbachol-stimulated phosphoinositide turnover in rat brain cortical slices. Arecoline was a partial agonist with peak effects of 30% of the maximum as obtained with carbachol. Coaddition of arecoline inhibited the carbachol-stimulated phosphoinositide breakdown. Pretreatment of rat brain cortical slices with arecoline in vitro resulted in a dose-dependent inhibition of carbachol-stimulated [3H]inositol monophosphate accumulation. The inhibition occurred rapidly, with half-maximal inhibition occurring at 15 min and maximal inhibition achieved within 60 min. The inhibition of phosphoinositide breakdown was recovered 1 h after arecoline was removed. When synaptoneurosomes were used for the ligand binding studies, arecoline pretreatment was found to have decreased the maximal ligand binding (Bmax) without inducing any marked change in binding affinity (K(D)). The influence could be recovered by incubating the synaptoneurosomes in the absence of arecoline for 2 h. Taken together, these data suggest that the underlying mechanism by which phosphoinositide turnover is inhibited is arecoline-induced receptor sequestration.

Palladium or platinum exacerbates hydroxyl radical mediated DNA damage.

Strand breakage of supercoiled pBR322 DNA by a Fenton system is increased in the presence of palladium or platinum (Pt) ions. Neither Pd nor Pt ions can substitute for iron in the Fenton system. We have obtained several lines of evidence that Pd and Pt ions in the presence of a Fenton system can augment the production of OH., as monitored by a spectrophotometric method quantifying hydroxylated salicylate or by a fluorometric method quantifying catechol production. Furthermore, the promoting effect of both metal ions on OH. production was substantiated by the identification of multiple hydroxylated products of salicylate [2,3-dihydroxybenzoate (A), 2,5-dihydroxybenzoate (B), and catechol (C)] using HPLC. The concentrations of A, B, and C produced in the control were 4.5, 8.0, and 2.0 microM, respectively; whereas, their respective concentrations increased to 23.6, 42.0 and 10.0 microM with the addition of Pd ions. The observed phenomenon was further confirmed by the identification of HO-DMPO spin adducts using ESR spectroscopy. Taken together, our data suggest that the mechanism of Pd or Pt ion-mediated exacerbation of DNA damage by a Fenton system is due to the promotion of OH. production by these metal ions.