CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Identification of Rotavirus Strains Causing Diarrhoea in Children under Five Years of Age in Yogyakarta, Indonesia

Background: Rotavirus is an important cause of severe diarrhoea in children. The aims of this study were to identify the rotavirus strains that cause diarrhoea in children in Yogyakarta and to determine the association between rotavirus positivity and its clinical manifestations. Methods: Clinical data and stool samples were collected from children hospitalised at Kodya Yogyakarta Hospital, Indonesia. Rotavirus was detected in stool samples using an enzyme immunoassay (EIA), which was followed by genotyping using reverse transcriptase polymerase chain reaction (RT-PCR). Electropherotyping was performed for the rotavirus-positive samples. Results: In total, 104 cases were included in the study, 57 (54.8%) of which were rotavirus-positive. Based on a multiple logistic regression analysis, age group, vomiting and stool mucous were associated with rotavirus positivity. Most of the 56 samples subjected to genotyping were classified as G1 (80.36%) and P[8] (69.64%) genotypes. The genotype combination G1P[8] was identified as the most prevalent strain (66.07%). Of the 19 samples subjected to electropherotyping, 17 G1 isolates and 1 G3 isolate had long patterns, and 1 G1 isolate had a short pattern. Conclusion: G1P[8] was the most dominant strain of rotavirus causing diarrhoea in children in Yogyakarta. Age group, vomiting and stool mucous were associated with rotavirus positivity.

Prevalence of rotavirus genotypes in South Korea in 1989-2009: implications for a nationwide rotavirus vaccine program / 소아과

The epidemiology of human group A rotavirus was analyzed by examining genotypic data acquired from 1989 to 2009 in South Korea. This information was derived from all the available published articles on rotavirus studies in South Korea, retrieved from both the PubMed and KoreaMed databases. Four common G types (G1, G2, G3, and G4) and three common P types (P[8], P[4], and P[6]) accounted for approximately 93% and 99% of the rotavirus reports, respectively. The G9 type was frequently detected after 2000, and because of this prevalence, it is considered to be the fifth most important G type rotavirus after the G1.G4 genotypes. Less common G types of the virus such as G12, G11, and G10 were detected in some geographic settings, and it is important to consider the context of these subtypes and their epidemiological significance. The P[9] virus genotype was observed in the study and has been discussed in many other studies; however, the P[3], P[10] and P[25] genotypes were rarely detected in the epidemiological research. In general, the distributions of the G and P genotypes showed temporal and geographical fluctuations, and a nationwide rotavirus vaccine program that targeted these genotypes demonstrated effectiveness in protecting against the circulating rotavirus strains. However, further analysis is needed to determine the true long-term effectiveness of these vaccines; the analysis should also consider the unexpected effects of vaccinations, such as vaccine-induced diseases, herd immunity, and changes in host susceptibilities.

Comparison of the effect between two methods of digestive tract reconstruction after gastrectomy / 中国基层医药

ObjectiveTo compare the advantages,disadvantages and clinical value of the improved loop the jejunum behalf of the stomach surgery and the P - type jejunum on behalf of stomach surgery.Methods56 patients with gastric cancer were divided into two groups by different ways of gastrectomy alimentary tract after gartrectomy.Patients in observation group( n =35 ) were given the modified loop jejunum on behalf of gastric surgery and patients in control group( n =31 ) received P-type jejunum on behalf of the stomach surgery.The clinical effects were compared between two groups.ResultsThere were no significant differences in surgical time and bleeding volume in 2 groups ( P ＞ 0.05 ).Emptying time [(61 ± 3 ) min] of observation group was longer than that of control group (37 ± 19) min]( t =3.03,P ＜ 0.05 ) ; Each food intake [( 308 ± 44 ) ml] in observation group was significiantly improved compared with control group [(262 ± 34) ml (t =2.55,P ＜ 0.05) ; The times of daily diet [(4.2 ± 1.2) times] in observation group was lower than that of control group [( 5.7 ± 2.3 )] ( t =2.46,P ＜ 0.05 ).The incidence of postoperative complications( 14.3％ ) of the observation group was significantly lower than the control group (42.9％) ( x2 =5.71,P ＜ 0.05).ConclusionOn the terms of 2 ways of digestive tract reconstruction,the improved loop the jejunum behalf of the stomach surgery is superior to the P-type jejunum on behalf of the stomach surgery,which can effectively improve the quality of life of patients and reduce the incidence of complications,but have no complex surgical procedures.

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

The expression of copper-transporting P-type adenosine triphosphatase in ovarian cancer and its correlation with chemotherapy resistance / 肿瘤

Objective: To investigate the expression of copper-transporting P-type adenosine triphosphatase (ATP 7B) in the ovarian cancer and evaluate its relationship with chemo-resistance and the clinicopathologic parameters of ovarian carcinoma. Methods: Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of ATP 7B in 36 cases of ovarian carcinoma, 10 cases of benign ovarian tumor, and 10 cases of normal ovarian tissue. Results: The positive expression rate of ATP 7B in ovarian carcinoma was 38.9% significantly higher than that in benign ovarian tumor (0%) and normal ovarian tissue (0%) (P < 0.01). There was a significant correlation between ATP 7B expression and differentiation degree. ATP 7B expression was significantly higher in poorly differentiated carcinoma than that in well and moderate differentiated carcinoma (P < 0.05). The expression of ATP 7B was more positive in patients who received chemotherapy before surgery than those who did not receive chemotherapy. The difference was significant (P < 0.05). The response rate of ATP 7B-positive patients to chemotherapy was significantly lower than the ATP 7B-negative patients (28.6% vs 72.7%, P < 0.05). Conclusion: These data suggested that ATP 7B plays an important role in the multidrug resistance of the ovarian carcinoma to Pt-based chemotherapy.

Identification and Analysis of Afe0329 Transcripton Relative to Copper Homeostasis in Acidithiobacillus ferrooxidans / 微生物学通报

With reverse transcriptase PCR, the transcripton of copper homeostasis relative gene Afe0329 in Acidithiobacillus ferrooxians standard strain ATCC23270 was investigated. The further analysis of genes in this transcripton was analyzed employed by Vector NTI, Blast, TMHMM Server, PSORTb software and so on. From the DNA of different strains, the transcripton of Afe0329 was amplified using special primer pairs to identify the universality of it in the genome of A.ferrooxidans strains. The results showed that gene Afe0330 and Afe0331 were cotranscribed with Afe0329, and they were in a single transcripton. Gene Afe0329 was supported to express a P1b3-type ATPase which is a heavy metal ion pumping transmembrane protein, protein AFE0330 which expressed by gene Afe0330 was a cytoplasmic protein, no significant ho- mologous sequences of Afe0330 or Afe0331 had been obtained by Blast analysis. And the transcripton of Afe0329 was universal in genome of A. ferrooxidans strains.

Experimental study on the relationship between p/Q-type calcium channel and migraine / 中华神经科杂志

Objective To investigate the mechanism of P/Q-type calcium channel in nitroglycerin induced migraine experimental model Methods Twenty Sprague-Dawley rats ( half female and half male) were randomly divided into control group and model group. The model of migraine rats was reproduced using Tasserelli Cristina method that wassubcutaneou injection of GTN of 10 mg/kg, once a week for four weeks. After the model of migraine had been established, trigeminal ganglion and trigeminocervical complex and cortex of frontal lobe were removed and the expressions of P/Q-type calcium channel were detected by RT- PCR and Western-blot, and at the same time the concentration of intracellular Ca2+ was investigated by Fluo- 3/AM fluorometric method. Results Compared with the control group, the expression of mRNA and protein of P/Q-type calcium channel of trigeminal ganglion and trigeminocervical complex (mRNA: 0. 472 36± 0. 049 54; protein: 0. 337 25 ± 0. 035 93 ) and cortex of frontal lobe ( mRNA: 0. 547 45 ± 0. 044 39 ; protein : 0. 402 13 ± 0. 029 83) in model group all upregulated (t = 2. 6697, 3. 1993, 3. 4398 and 3. 7661, all P <0. 05), at the same time the concentration of intracellular Ca2+ in model group increased (211 182 ± 12 973 vs 135 243 ± 18 105 in trigeminal ganglion and trigeminocervical complex; 186 511 ± 18 297 vs 143 289±25 175 in cortex of frontal lobe. t =10.7819 and 4. 3917, beth P<0.05). Conclusions P/Q- type calcium channel may play a role in the pathogenesis of migraine via its upregulated expression.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25°C in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40 percent dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Genotype of rotavirus isolated from patients with rotaviral enteritis and neurological complications / 소아과

PURPOSE: This study was undertaken to determine the differences in genotypes of rotavirus and their incidence between patients with acute rotaviral enteritis who suffered neurologic complications and those who did not suffer neurologic complications. METHODS: Among the 82 patients with rotaviral enteritis whose genotype was analyzed, 71 patients were not associated with neurologic complications(neurology(-) group), and eleven patients were associated with neurologic complications(neurology(+) group). Four G genotypes and four P genotypes were determined by reverse trans cription and multiplex polymerase chain reaction. RESULTS: In the neurology(-) group, the number of G4, G3, G2 and G1 was 24(35.3 percent), 23 (33.8 percent), 17(25.0 percent) and 4(5.6 percent), respectively and the number of P6, P4, P8 and P9 was 26(36.1 percent), 23(31.9 percent), 22(30.6 percent) and 1(1.4 percent), respectively. G2P4, and G4P6 were the most frequently found com bination genotypes, respectively. In the neurology(+) group, the number of G2, G3 and G4 was 9(75.0 percent), 2(16.7 percent) and 1(8.3 percent), respectively and the number of P4, P6, P8 and P9 was 8(66.7 percent), 2(16.7 percent), 1(8.3 percent) and 1(8.3 percent), respectively. G2P4 was the most frequently found combination genotype. The incidence of each of the G2, P4 and G2P4 was significantly higher in the neurology(+) group when compared to that of each of the G2, P4, and G2P4 in the neurology(-) group. CONCLUSION: In the patients with acute rotavirus gastroenteritis associated with neurologic complications, the G1 genotype was not found and genotypes of the G2, P4, and G2P4 were more frequently found.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.

Determination of Porcine Rotavirus Serotypes by RT-PCR and RFLP Analysis

G and P tying of group A porcine rotaviruses (P(o)RV) from field fecal samples were performed using reversetranscriptase polymerization chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis. After amplifying full length VP7 and partial length VP4 genes, restriction endonucleases were used to digest and analyze the cutting pattern of the gene products. After analysis of digests with restriction endonucleases, seven and six RFLP types were observed for VP7 and VP4, respectively. The G typing analysis of 50 fecal samples revealed that 68% (34/50) were G4, which included G4-like (22/50); 22% (11/50) were G5; 6% (3/50) were G4 and G5 mixed types. The P typing analysis of the same fecal samples revealed that 36% (18/50) were P2B, 52% (26/50) were P9, 1 sample (2%) was a mixture of P2B and P9. Combinations of G and P types, the G4P2B and G4P9 types including G4-like accounted for 26% (13/50) and 32% (16/50), respectively. The G5P2B and G5P9 type also represented 4% (2/50) and 18% (9/50) of the samples. No G3 and G11 or other new P types were identified from the samples tested. Information on the G and P types and G/P combinations in the field fecal samples is useful for developing more effective PoRV vaccines and understanding the epidemiology of PoRV infections in the field.

Distribution of Human Rotavirus G and P Types and G9 VP7 Sequences Circulating in Seoul, Korea

Genotyping of human rotaviruses was performed using 55 rotavirus-positive samples collected from 90 young children with diarrhea at Chung-Ang University Yongsan Hospital between December 2001 and May 2002. G typing of the VP7 protein showed that G2 was the most dominant circulating genotype (32.8%) followed by G1 (21.8%), G4 (12.7%), G3 (10.9%), G9 (1.8%) and a combination of G2/G3 (7.3%). P typing of the VP4 protein revealed that P[4] (60.0%) was the most prevalent strain, followed by P[6] (25.5%), P[8] (3.6%) and P[9] (1.8%). Seven samples (12.7%) for G type and 5 samples (9.1%) for P type remained untypable. The most predominant G-P combination was G2P[4] (29.1%), which is one of the most commonly observed type worldwide. A G9 serotype strain identified in this study shared more than 97% nucleotide homologies with 18 foreign G9 isolates. Therefore, incorporation of G9 rotavirus into current vaccine formula should be considered.

Study of level and functions of P-type ATPase7B in hepatic cells of patients with hepatolenticular degeneration / 临床神经病学杂志

Objective To explore the level and main functions of P-type ATPase7B in hepatic cells of patients with hepatolenticular degeneration(HLD).Methods The hepatic cells from 5 normal controls and 9 patients with HLD were cultured in vitro. P-type ATPase7B levels in hepatic cells were examined and compared by SDS-PAGE and Western-blot techniques.Results Compared with the controls, 9 patients displayed various changes of electrophoresis strip. Almost normal strips at 155?103 were found in 3 cases, no strip was found in 1 case, and thinner and lighter strips were showed in the remain 5 cases. 6 cases presented abnormal specific reaction strips.Conclusion Mutations of gene ATPase7B in HLD patients cause change of P-type ATPase7B in quantity and quality, thus leads to dysmetabolism of copper.