NAD attenuates oxidative DNA damages induced by amyloid beta-peptide in primary rat cortical neurons.

One major pathological hallmark of Alzheimer's disease (AD) is accumulation of senile plaques in patients' brains, mainly composed of amyloid beta-peptide (Aß). Nicotinamide adenine dinucleotide (NAD) has emerged as a common mediator regulating energy metabolism, mitochondrial function, aging, and cell death, all of which are critically involved in neuronal demise observed in AD. In this work, we tested the hypothesis that NAD may attenuate Aß-induced DNA damages, thereby conferring neuronal resistance to primary rat cortical cultures. We found that co-incubation of NAD dose-dependently attenuated neurotoxicity mediated by Aß25-35 and Aß1-42 in cultured rat cortical neurons, with the optimal protective dosage at 50 mM. NAD also abolished the formation of reactive oxygen species (ROS) induced by Aß25-35. Furthermore, Aßs were capable of inducing oxidative DNA damages by increasing the extents of 8-hydroxy-2´-deoxyguanosine (8-OH-dG), numbers of apurinic/apyrimidinic (AP) sites, genomic DNA single-stranded breaks (SSBs), as well as DNA double-stranded breaks (DSBs)/fragmentation, which can all be attenuated upon co-incubation with NAD. Our results thus reveal a novel finding that NAD is protective against DNA damage induced by existing Aß, leading ultimately to neuroprotection in primary cortical culture.

Expression and clinical significance of lung-specific X protein mRNA in bronchial brushing specimens from patients with or without lung cancer.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the diagnostic utility of lung-specific X protein (LUNX) mRNA expression in bronchial brushing specimens from patients with lung cancer. METHODS: LUNX mRNA levels were assessed by performing RT-PCR on liquid-based cytology bronchial brushing specimens from patients with lung cancer (n=104) or benign lung disease (n=91). RESULTS: LUNX mRNA expression was significantly more frequent in patients with all carcinomas, squamous cell carcinomas, adenocarcinomas, as well as patients with central, peripheral and diffuse carcinomas (P<0.01), and non-small cell lung carcinomas (P<0.05) compared with patients with benign disease. The diagnostic performance of RT-PCR analysis of LUNX mRNA was significantly better than that of cytology in terms of sensitivity (93.3±4.8% vs 64.4±9.2%), negative predictive value (91.6±6.0% vs 71.1±7.9%) and accuracy (88.7±4.4% vs 81.0±5.5%), whereas specificity (83.5±7.6%) and positive predictive value (86.6±6.3%) were lower than those of cytology (100%). CONCLUSIONS: Liquid-based cytology and RT-PCR can be performed to detect LUNX mRNA expression in bronchial brushing specimens, and this technique may be a useful adjunct to cytological diagnosis of lung cancer. The sensitivity of the technique was greater than that of cytology but its lower specificity should be taken into account.

Reduction and restoration of mitochondrial dna content after focal cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Oxidative damage of mitochondrial DNA (mtDNA) in the ischemic brain is expected after ischemia/reperfusion injury. A recent study demonstrated limited patterns of mtDNA deletion in the brain after ischemia/reperfusion. We studied the ischemia/reperfusion-induced global changes of mtDNA integrity and its restoration in a rat model of transient focal ischemia in vivo. METHODS: Changes in mtDNA content in the ischemic brain were assessed with the use of a rat stroke model featuring transient severe ischemia confined to the cerebral cortex of the right middle cerebral artery territory for 30 or 90 minutes. A new long polymerase chain reaction method, using mouse DNA as an internal standard, was applied to measure the relative content of intact rat mtDNA. Southern hybridization following alkaline gel electrophoresis was conducted in a parallel study to confirm long polymerase chain reaction results. RESULTS: A reduction in mtDNA content was found after ischemia for 30 and 90 minutes. The mtDNA was restored to near nonischemic levels 24 hours after 30- but not 90-minute ischemia. CONCLUSIONS: These results confirm that ischemia/reperfusion causes mtDNA damages. Restoration of the mtDNA content to nonischemic levels after 30-minute ischemia raises the possibility that mtDNA repair or repletion occurs after brief ischemia.

Inducible nitric oxide synthase neutralizes carbamoylating potential of 1,3-bis(2-chloroethyl)-1-nitrosourea in c6 glioma cells.

Expression of iNOS in glioma and other tumors has been extensively documented but the effects of NO derived from iNOS on tumor-killing mechanisms of chemotherapy drugs remain to be fully defined. We note that increased NO synthesis by cytokine exposure or iNOS overexpression neutralized the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), but not cisplatin, in rat C6 glioma cells. Suppression of BCNU cytotoxicity associated with iNOS overexpression could be abolished by pharmacological inhibition of NOS or coexpression of an antisense RNA against iNOS. Both BCNU and CCNU are chloroethylnitrosoureas that kill tumor cells via carbamoylating and alkylating actions. Further studies using compounds that each carry these different activities indicate that iNOS neutralized carbamoylating, but not alkylating, action of chloroethylnitrosoureas. Temozolomide, a novel chemotherapy drug recently available for treating brain tumors, carries only alkylating, but not carbamoylating, action. Overexpression of iNOS in C6 cells failed to neutralize temozolomide cytotoxicity. Results from the present study demonstrate the ability of iNOS-derived NO to confer chemoresistance against the carbamoylating potential of chloroethylnitrosoureas in vitro. Further investigation is needed to test whether iNOS expression, frequently noted in malignant brain tumors, also enhances chemoresistance against chloroethylnitrosoureas in vivo.

iNOS expression inhibits hypoxia-inducible factor-1 activity.

Hypoxia-inducible factor-1 (HIF-1) activates genes important in vascular function such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a free radical gas that mediates a number of cellular processes, including regulation of gene expression, vasodilatation, and neurotransmission. Here we demonstrate that iNOS expression inhibits HIF-1 activity under hypoxia in C6 glioma cells transfected with an iNOS gene and a VEGF promoter-driven luciferase gene. HIF-1 induction of VEGF-luciferase activity in C6 cell is also inhibited by sodium nitroprusside (SNP). Furthermore, pretreatment of C6 cells with N-acetyl-l-cysteine (NAC), an antioxidant, nullified the inhibitory effect of iNOS on HIF-1 binding. These results demonstrate that NO generated by iNOS expression inhibits HIF-1 activity in hypoxic C6 cells and suggest a negative feedback loop in the HIF-1 --> iNOS cascade.

Molecular cloning of ovine connexin44 and temporal expression of gap junction proteins in a lens cell culture.

PURPOSE: The lens plasma membranes of several mammalian species have been shown to contain three different connexin proteins. The goal of this study was to clone the sheep homologue of rat connexin46 identified as sheep connexin44 and to determine the temporal changes in the expression of the three sheep connexin proteins in a lens primary cell culture system. METHODS: A sheep genomic library was screened with a rat lens connexin46 cDNA probe. Lens junctional protein and mRNA levels were determined in a sheep primary cell culture system by Western and Northern blot analyses, respectively. RESULTS: Sheep connexin44, the homologue of rat lens connexin46, was identified as a single-copy gene with a predicted molecular weight of 43,989 Daltons that is contained within a single exon. Northern blot analysis detected a 2.2-kb connexin44 transcript in RNA isolated from lens but not that isolated from heart, kidney, liver, or lung. During the in vitro differentiation of lens epithelial cells from 5 to 20 days in culture, connexin43 mRNA levels declined approximately 75%, whereas connexin49 RNA levels increased approximately 24 fold. The 40% decrease in the level of connexin43 protein and the 21-fold increase in the level of connexin49 protein did not directly correlate with the changes in mRNA levels encoding these proteins during this same period. Although detectable, the amount of connexin44 mRNA and protein remained low throughout the 20-day period during which lens cells were grown in culture. Neither mRNA nor protein encoding MP20 or MP26 transcripts could be detected in even the oldest 20-day lens cultures. CONCLUSIONS: Steady state mRNA levels of sheep connexin43 and connexin49 do not appear to be the only factor regulating the expression of these genes during in vitro differentiation of lens cells in culture. Although a decreased level of expression of connexin43 was accompanied by an increased level of expression of connexin49 over the 20-day period in culture, connexin44 mRNA and protein levels remained low throughout this 20-day period. Overall, these results suggest that these junctional proteins have a unique temporal pattern of expression during differentiation, and this lens primary cell culture system provides a valuable tool to better understand this process.

Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants.

OBJECTIVES: To compare the accuracy of standard and hemocytometer white blood cell (WBC) counts and urinalyses for predicting urinary tract infection (UTI) in febrile infants. METHODS: Enrolled were 230 febrile infants < 12 months of age. All urine specimens were obtained by suprapubic bladder aspiration and microscopically analyzed by the standard urinalysis (UA) and by hemocytometer WBC counts simultaneously, and quantitative urine cultures were performed. Receiver-operating characteristic (ROC) curves were constructed for each method of UA. The optimal cutoff point of the UA test in predicting UTI was determined by ROC analysis. RESULTS: There were 37 positive urine cultures of at least 1,000 CFU/ml. Of these 37 patients, 9 females and 28 males, 1 had a positive blood culture (Escherichia coli). Thirty (81%) of the positive urine cultures had a bacterial colony count > or = 100,000 colony-forming units/ml, whereas the remaining had between 1,000 and 50,000 colony-forming units/ml. The area under the ROC curve for standard UA was 0.790 +/- 0.053, compared with 0.900 +/- 0.039 for hemocytometer WBC counts (P < 0.05). For hemocytometer WBC counts, the presence of < or =10 WBC/microl appeared to be the most useful cutoff point, yielding a high sensitivity (83.8%) and specificity (89.6%). Standard UA, with a cutoff point of 5 WBC/high power field, had a lower sensitivity (64.9%) and similar specificity (88.1%). The hemocytometer WBC counts showed significantly greater sensitivity and positive predictive value (83.8 and 60.8%, respectively) than the standard urinalysis (64.9 and 51.1%, respectively) (P < 0.05). The accuracy, specificity and likelihood ratio of hemocytometer WBC counts were also greater than that of standard UA (88.7, 89.6 and 8.08% vs. 84.3, 88.1 and 5.44%). CONCLUSION: Hemocytometer WBC counts provide more valid and precise prediction of UTI in febrile infants than standard UA. The presence of > or =10 WBC/microl in suprapubic aspiration specimens is the optimum cutoff value for identifying febrile infants for whom urine culture is warranted.

Evaluation of autoSCAN-W/A and the Vitek GNI+ AutoMicrobic system for identification of non-glucose-fermenting gram-negative bacilli.

The autoSCAN-W/A (W/A; Dade Behring Microscan Inc., West Sacramento, Calif.) and Vitek AutoMicrobic System (Vitek AMS; bioMérieux Vitek Systems, Inc., Hazelwood, Mo.) are both fully automated microbiology systems. We evaluated the accuracy of these two systems in identifying nonglucose-fermenting gram-negative bacilli. We used the W/A with conventional-panel Neg Combo type 12 and Vitek GNI+ identification systems. A total of 301 isolates from 25 different species were tested. Of these, 299 isolates were identified in the databases of both systems. The conventional biochemical methods were used for reference. The W/A correctly identified 215 isolates (71. 4%) to the species level at initial testing with a high probability of >/=85%. The Vitek GNI+ correctly identified 216 isolates (71.8%) to the species level at initial testing with a high probability of >/=90%. After additional testing that was recommended by the manufacturer's protocol, the correct identifications of the W/A and Vitek GNI+ improved to 96.0 and 92.3%, respectively. The major misidentified species were Sphingomonas paucimobilis and Agrobacterium radiobacter in the W/A system and Acinetobacter lwoffii, Chryseobacterium indologenes, and Comamonas acidovorans in the Vitek GNI+ system. The error rates were 4.0 and 7.6%, respectively. The overall accuracy for both systems was above 90% if the supplemental tests were applied. There was no significant difference in accuracy (P > 0.05) between the two systems.

Urinary tract infection in febrile infants younger than eight weeks of Age.

OBJECTIVE: To assess the usefulness of laboratory parameters, including peripheral white blood cell (WBC) count, C-reactive protein (CRP) concentration, erythrocyte sedimentation rate (ESR), and microscopic urinalysis (UA), for identifying febrile infants younger than 8 weeks of age at risk for urinary tract infection (UTI), and comparison of standard UA and hemocytometer WBC counts for predicting the presence of UTI. METHODS: A total of 162 febrile children <8 weeks of age were enrolled in this prospective study. All underwent clinical evaluation and laboratory investigation, including WBC count and differential; ESR; CRP; blood culture; a lumbar puncture for cell count and differential, glucose level, protein level, Gram stain, and culture; and a UA and urine culture. All urine specimens were obtained by suprapubic aspiration and microscopically analyzed with standard UA as well as with hemocytometer WBC counts. Quantitative urine cultures were performed. Sensitivity, specificity, accuracy, likelihood ratios, and receiver operating characteristic (ROC) curves were determined for each of the screening tests. RESULTS: There were 22 positive urine culture results of at least 100 colony-forming unit/mL. Eighteen of these 22 patients were males, and all were uncircumcised. There were significant differences for pyuria >/=5 WBCs/hpf, pyuria >/=10 WBC/microL, CRP >20 mg/L, and ESR >30 mm/hour between culture-positive and culture-negative groups (P <.05). The ROC area for hemocytometer WBC count, standard UA, peripheral WBC count, ESR, and CRP concentration were.909 +/-.045,.791 +/-.065,.544 +/-.074,. 787 +/-.060, and.822 +/-.036, respectively. The ROC curve analysis indicates that the CRP, ESR, and standard UA were powerful but imperfect tools with which to discriminate for UTI in potentially infected neonates. Hemocytometer WBC counts had the highest sensitivity, specificity, accuracy, and likelihood ratios for identifying very young infants with positive urine culture results. For all assessments, hemocytometer WBC counts were significantly different, compared with the standard urinalysis. ESR, CRP, and peripheral WBC counts were not helpful in identifying UTI in febrile infants. CONCLUSION: UTI had a prevalence of 13.6% in febrile infants <8 weeks of age. The CRP, ESR, and standard UA were imperfect tools in discriminating for UTI, and the sensitivity of these laboratory parameters was relatively low. Hemocytometer WBC count was a significantly better predictor of UTI in febrile infants.

Bacillus popilliae endocarditis with prolonged complete heart block.

Bacillus popilliae, a fastidious, aerobic, gram-positive, spore-forming bacillus, has never been reported as a pathogen in human infectious diseases. We report the first case of a human infected by the pathogen B. popilliae, which presented as endocarditis involving the bicuspid aortic valve and complicated with prolonged (> 30 days; to our knowledge, the longest in the literature) complete heart block. Although surgery may be warranted by previous reports, the patient was successfully managed by medical treatment instead, because of the absence of evidence from various approaches that support the existence of perivalvular extension of infection.

Characterization of group D1 non-typhoid Salmonella isolates by serotyping and pulsed field gel electrophoresis.

Non-typhoid salmonella infection is not uncommon in immunocompetent patients in Taiwan. Bacterial factors may play an important role in the pathogenesis of such infections. In a previous study, Salmonella group D1 was found to have the tendency to cause bacteremia with a higher frequency than other serotypes. In the present study, we prospectively collected 94 Salmonella group D1 isolates for serotyping and molecular typing. Salmonella panama and Salmonella dublin seemed more invasive than other serotypes. Pulsed field gel electrophoresis was also done to characterize of Salmonella enteritidis and Salmonella dublin. PFGE type "a" of Salmonella dublin appeared to be more invasive than the other two PFGE types. All six Salmonella dublin isolates were Vi antigen negative. Further study using a larger number of isolates is needed to identify the tendency to invade blood stream of Salmonella dublin and Salmonella panama.

Investigation of an outbreak of Pseudomonas putida using antimicrobial susceptibility patterns, pulsed-field gel electrophoresis of genomic DNA and restriction fragment length polymorphism of PCR-amplified rRNA operons.

Seventeen Pseudomonas putida isolates were investigated which were collected from the urine specimens of 14 patients and one reflectrometer by comparing antimicrobial susceptibility patterns, pulsed-field gel electrophoresis (PFGE) of genomic DNA, and restriction fragment length polymorphism (RFLP) of PCR-amplified rRNA operons. Three susceptibility patterns were defined by testing 22 antimicrobial agents, with 14 isolates resistant to all agents. PFGE of XbaI-genomic DNA fragments divided the 17 isolates into 9 distinct types. One type, seen in 6 isolates showing identical patterns of approximately 35 fragments of 10 to 350 kb, was defined as the outbreak strain. Another 4 types, in a total of 6 isolates, were considered closely related to the outbreak strain; 2 types in 1 isolate each were possibly related to the outbreak strain; and 2 types in a total of 3 isolates were different from the outbreak strain. All 12 outbreak or closely related isolates were from patients in the surgical intensive care unit and a surgical ward, and were different from isolates in other wards, clearly indicating an outbreak of P. putida. Only two types were defined by the RFLP of 4.5 kb PCR-amplified rRNA operons; one type was seen in 15 isolates, while the other was seen in only 2 isolates. In conclusion, PFGE of genomic DNA is a highly discriminatory and reproducible method for epidemiological typing of P. putida.

Molecular cloning of sheep connexin49 and its identity with MP70.

The nucleotide sequence of the sheep homologue of the lens-specific mouse connexin50, chicken connexin45.6, and human connexin50 has been obtained following screening of a sheep genomic library. This connexin comprises 1323 nucleotides, coding for a protein of 440 amino acid residues and a predicted molecular weight of 49,160 daltons, so by convention is termed sheep connexin49. A connexin49 cDNA probe detected a single major band with a mobility of 6.8 kb in sheep lens RNA, but not in RNA isolated from five other sheep organs. The N-terminal amino acid sequence of sheep connexin49 is identical to that of mouse connexin50 and closely matches that of MP70, indicating the identity of sheep connexin49 with MP70. The nucleotide and translated amino acid sequences of connexin49 have 69-87% and 76%-87% identity respectively with chicken connexin45.6, human connexin50 and mouse connexin50. Like other members of this lens connexin family, sheep connexin49 coding region is completely contained within one exon, and the sequence of the N-terminal region, the four transmembrane domains and the two extracellular loops are highly conserved.

Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor.

The skeletal muscle ryanodine receptor of malignant hyperthermia-susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86-99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86-99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure.

The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.

Enteric group 17 in clinical isolates.

The Enteric Group 17, a new group of Enterobacteriaceae, has been classified (by the Centers for Disease Control, Atlanta, Georgia, U.S.A.) based on its positive methyl-red test as well as its negative response to Voges-Proskauer, motility, rhamnose and melibiose fermentation tests. The isolation rate of Enteric Group 17 among 500 clinical isolates of Enterobacteriaceae in Taiwan is 1.8% (9/500). This finding recommends that Taiwan's hospital laboratories should pay particular attention to the possible presence of this bacteria when an isolate has reactions similar to that of Enterobacter cloacae or other members of the Enterobacter species.