Pregnancy in women undergoing hemodialysis: case series in a Southeast Brazilian reference center / Gestação em mulheres em tratamento hemodialítico: série de casos em um centro de referência do Sudeste do Brasil

PURPOSE: To describe maternal and neonatal outcomes in pregnant women undergoing hemodialysis in a referral center in Brazilian Southeast side. METHODS: Retrospective and descriptive study, with chart review of all pregnancies undergoing hemodialysis that were followed-up at an outpatient clinic of high- risk prenatal care in Southeast Brazil. RESULTS: Among the 16 women identified, 2 were excluded due to follow-up loss. In 14 women described, hypertension was the most frequent cause of chronic renal failure (half of cases). The majority (71.4%) had performed hemodialysis treatment for more than one year and all of them underwent 5 to 6 hemodialysis sessions per week. Eleven participants had chronic hypertension, 1 of which was also diabetic, and 6 of them were smokers. Regarding pregnancy complications, 1 of the hypertensive women developed malignant hypertension (with fetal growth restriction and preterm delivery at 29 weeks), 2 had acute pulmonary edema and 2 had abruption placenta. The mode of delivery was cesarean section in 9 women (64.3%). All neonates had Apgar score at five minutes above 7. CONCLUSIONS: To improve perinatal and maternal outcomes of women undergoing hemodialysis, it is important to ensure multidisciplinary approach in referral center, strict control of serum urea, hemoglobin and maternal blood pressure, as well as close monitoring of fetal well-being and maternal morbidities. Another important strategy is suitable guidance for contraception in these women. .

OBJETIVOS: Descrever os resultados maternos e neonatais de mulheres grávidas que estavam em tratamento de hemodiálise em um centro de referência no Sudeste brasileiro. MÉTODOS: Estudo retrospectivo e descritivo, com revisão de prontuários de todas as gestações em hemodiálise, acompanhadas no pré-natal especializado da região Sudeste do Brasil. RESULTADOS: Entre as 16 mulheres identificadas, 2 foram excluídas devido à perda de seguimento. Das 14 descritas, a hipertensão foi a causa mais frequente de insuficiência renal crônica (50% dos casos). A maioria (71,4%) realizava tratamento de hemodiálise há mais de um ano e todas elas foram submetidas a 5 ou 6 sessões por semana. Onze mulheres tinham hipertensão crônica, 1 das quais também era diabética, e 6 eram fumantes. Em relação às complicações da gravidez, 1 das mulheres hipertensas desenvolveu hipertensão maligna (com restrição de crescimento fetal e parto prematuro com 29 semanas), 2 tiveram edema pulmonar agudo e 2 apresentaram descolamento prematuro de placenta. O tipo de parto foi cesariana em 9 mulheres (64,3%). Todos os recém-nascidos tiveram Apgar aos cinco minutos maior que 7. CONCLUSÕES: Para melhorar os resultados perinatais e maternos de mulheres em hemodiálise, é importante ter uma abordagem multidisciplinar em centro de referência, um controle rigoroso da uremia, hemoglobina e pressão arterial materna, bem como acompanhar de perto o bem-estar fetal e a morbidade materna. Outra estratégia importante é a orientação adequada para contracepção nessas mulheres. .

A socialização da medicina na era do adhemarismo / The socialization of medicine in the era of São Paulo Governor Adhemar de Barros

A análise observa as conexões entre o processo de profissionalização dos médicos paulistas e políticas de saúde do governo estadual de Adhemar de Barros em São Paulo (1947-1951), em meio a amplas mudanças na área de saúde denominadas pelos médicos paulistas “socialização da medicina”. Reconhecemos aspectos ambivalentes para o profissionalismo médico diante desse governo populista, como: a luta médica pela equiparação ante os advogados servidores públicos estaduais; a criação de uma secretaria de saúde estadual; e certos elos contraditórios entre a área de saúde adhemarista e a ideologia e a organização profissionais da medicina paulista. Nesse particular, o artigo aprofunda a análise de manifestações ideológicas de importantes lideranças médicas paulistas.

The article analyzes how the process of the professionalization of physicians in São Paulo related to healthcare policy under the administration of São Paulo governor Adhemar de Barros (1947-1951) during a period of broad change in the realm of health known by São Paulo physicians as the “socialization of medicine.” Medical professionalism confronted certain ambivalences under this populist administration, including doctors’ struggle to achieve pay equal to that of state public attorneys; the establishment of a state health department; and some contradictory ties between the area of health under Adhemar and the professional ideology and organization of medicine in São Paulo. The article undertakes a more in-depth analysis of the ideological manifestations of important leaders in the state’s medical community.

Predictive factors of alcohol and tobacco use in adolescents / Fatores preditivos do uso de álcool e tabaco em adolescentes / Factores predictivos del uso de alcohol y tabaco en los adolescentes

OBJECTIVES: to analyze the effect of self-esteem, assertiveness, self-efficacy and resiliency on alcohol and tobacco consumption in adolescents. METHOD: a descriptive and correlational study was undertaken with 575 adolescents in 2010. The Self-Esteem Scale, the Situational Confidence Scale, the Assertiveness Questionnaire and the Resiliency Scale were used. RESULTS: the adjustment of the logistic regression model, considering age, sex, self-esteem, assertiveness, self-efficacy and resiliency, demonstrates significance in the consumption of alcohol and tobacco. Age, resiliency and assertiveness predict alcohol consumption in the lifetime and assertiveness predicts alcohol consumption in the last year. Similarly, age and sex predict tobacco consumption in the lifetime and age in the last year. CONCLUSION: this study can offer important information to plan nursing interventions involving adolescent alcohol and tobacco users. .

OBJETIVOS: analisar o efeito da autoestima, assertividade, autoeficácia e resiliência sobre o consumo de álcool e tabaco em adolescentes. MÉTODO: estudo descritivo correlacional com 575 adolescentes, realizado no ano 2010. Foram utilizadas a Escala de Autoestima, o Questionário de Confiança Situacional, o Questionário de Assertividade e a Escala de Resiliência. RESULTADOS: o ajuste do modelo de regressão logística, considerando a idade, sexo, autoestima, assertividade, autoeficácia e resiliência foi significante em relação ao consumo de álcool e tabaco. A idade, resiliência e assertividade foram preditores do consumo de álcool em algum momento na vida e a idade e a assertividade foram preditores no último ano. Para o consumo de tabaco, a idade e o sexo foram preditores em algum momento na vida e a idade no último ano. CONCLUSÃO: este estudo pode proporcionar informações importantes para o planejamento de intervenções de enfermagem em adolescentes usuários de álcool e tabaco .

OBJETIVOS: analizar el efecto de la autoestima, asertividad, autoeficacia y resiliencia sobre el consumo de alcohol y tabaco en adolescentes. MÉTODO: descritivo correlacional con 575 adolescentes, en 2010. Se utilizaron la Escala de Autoestima, el Cuestionario de Confianza Situacional, el Cuestionario de Asertividad y la Escala de Resiliencia. RESULTADOS: el ajuste del modelo de regresión logística, considerando la edad, sexo, autoestima, asertividad, autoeficacia y resiliencia, muestra significancia en el consumo de alcohol y tabaco. La edad, resiliencia y asertividad predicen el consumo de alcohol alguna vez en la vida y la edad y asertividad en el último año. De la misma forma la edad y sexo predicen el consumo de tabaco alguna vez en la vida y la edad en el último año. CONCLUSIÓN: este estudio puede proporcionar información importante para la planificación de intervenciones en enfermería de los adolecentes usuarios de alcohol y tabaco. .

Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems / Association between embB mutations and ethambutol resistance in Mycobacterium tuberculosis isolates from Cuba and the Dominican Republic: reproducible patterns and problems.

The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.

Enhanced biohydrogen production by homologous over-expression of fnr, pncB, fdhF in Klebsiella sp. HQ-3 / 生物工程学报

To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDH1) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 / 生物工程学报

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing β1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.

A novel double expression shuttle vector to get marker-free recombinant modified vaccinia virus Ankara / 生物工程学报

<p><b>UNLABELLED</b>A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted.</p><p><b>CONCLUSIONS</b>double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.</p>

Enzymes related with NAD synthesis promote conversion of 1,4-butanediol to 4-hydroxybutyrate / 生物工程学报

Besides medical application, 4-hydroxybutyrate (4-HB) is a precursor of P3HB4HB, a bioplastic showing excellent physical properties and degradability. Escherichia coli S17-1 (pZL-dhaT-aldD) can transform 1, 4-butanediol (1,4-BD) into 4HB with participation of cofactor NAD. To enhance productivity, nicotinic acid phosphoribosyltransferase (PncB) and nicotinamide adenine dinucleotide synthetase (NadE) were overexpressed to increase intracellular nicotinamide adenine dinucleotide concentration and promote reaction process. The shake flask fermentation result showed that the conversion rate increased by 13.03% with help of PncB-NadE, leading to 4.87 g/L 4HB from 10 g/L 1,4-BD, and productivity was increased by 40.91% to 1.86 g/g. These results demonstrated that expression of PncB and NadE is beneficial for conversion of 1,4-BD to 4HB.

Relationship between proteoglycans and proliferation of human salivary adencid cystic carcinoma / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of down-regulated proteoglycans on the proliferation of human salivary adenoid cystic carcinoma (SACC).</p><p><b>METHODS</b>The short hairpin RNA (shRNA) plasmid silencing human xylosyltransferase-I (XT-I) gene was constructed and named shRNA-WJ3. Adenoid cystic carcinoma cells with high metastatic tendency (ACC-M) were transfected by shRNA-WJ3. The plasmid shRNA-HK not targeting any human gene was transfected into ACC-M cells used as negative control. After 48 h of transfection, the positive cells were screened by G418 to isolate the stable transfected cells. Real-time PCR and Western blotting were used to test the gene silence, and the proteoglycans contents of the cells were detected. The stable cell line silenced XT-I was named ACC-M-WJ3. MTT assay was performed to detect the cell proliferation. The cell cycle was analyzed by flow cytometry.</p><p><b>RESULTS</b>ShRNA-WJ3 showed powerful RNA interference and gene silence of XT-I. The inhibition rate was 83.70% of mRNA expression and 79.60% of protein expression respectively. The content of proteoglycans in ACC-M-WJ3 was down-regulated by 49.71%-54.59%. The results of MTT assay showed that the cell growth was inhibited significantly. S phrase decreased and G₁-G₀ phrase increased in group ACC-M-WJ3 compared with that of group ACC-M-HK (P < 0.05).</p><p><b>CONCLUSIONS</b>The down-regulated proteoglycans could inhibit the proliferation of human ACC-M cells.</p>

Pharmacokinetics and the bystander effect in CD::UPRT/5-FC bi-gene therapy of glioma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.</p><p><b>METHODS</b>C6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA).</p><p><b>RESULTS</b>(19)F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased.</p><p><b>CONCLUSIONS</b>(19)F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes. The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.</p>

Molecular characterization of embB306 gene in Mycobacterium tuberculosis isolates from tuberculosis patients in Chongqing municipality / 中华预防医学杂志

<p><b>OBJECTIVE</b>To understand the characteristics of embB gene mutation of Mycobacterium tuberculosis (MTB) isolates from tuberculosis patients in Chongqing, and the value of embB306 as a molecular marker used to diagnose ethambutol (EMB)-resistant MTB strains.</p><p><b>METHODS</b>Direct sequencing was used to analyze the polymorphism of embB mutation in 51 EMB-resistant MTB strains and 50 EMB-sensitive MTB strains. And diagnostic testing was used to evaluate the value of embB306 as a molecular marker of EMB -resistant MTB strains as compared with the traditional sensitivity test.</p><p><b>RESULTS</b>All 34 of 51 EMB-resistant strains (66.7%) and 3 of 51 EMB-sensitive strains (6%) had had embB306 mutation. The embB306 mutation rate in EMB-resistant strains coming from previously treated case was 87.5%, showing significantly higher than that from new cases (48.1%, P < 0.01); embB306 mutation rate was increased with the number of the resistant drugs; embB306 mutation serving as a marker to diagnose EMB-resistant MTB strains comparing with the traditional sensitivity test, had the rate of sensitivity = 66.7%, specificity = 94.0%, accuracy = 80.2% and Youden index = 60.7%.</p><p><b>CONCLUSION</b>embB306 mutation should be the main mechanism of MTB resistance to EMB in Chongqing, showing an association with the history of the treated and numbers of the resistant drugs. embB306 mutation should be a good marker to diagnose EMB-resistant MTB strains.</p>

Novel mutations in emb B gene of ethambutol resistant isolates of Mycobacterium tuberculosis: a preliminary report.

BACKGROUND & OBJECTIVE: Ethambutol (EMB) resistance, thought to be occurring due to mutations in embB gene of Mycobacterium tuberculosis on the rise is a cause of grave concern. The present study was planned to investigate the presence of EMB resistance in M. tuberculosis isolates and to look for prevalent mutations in embB gene. METHODS: A total of 591(283 from new and 308 from previously treated cases) sputum samples from the same number of pulmonary tuberculosis cases were cultured. Isolates were tested by 1 per cent proportion method for resistance to isoniazid, rifampicin streptomycin and ethambutol. Minimum inhibitory concentration (MIC) of EMB was measured by absolute concentration method. Ten randomly selected isolates were subjected to single strand conformational polymorphism (SSCP) and direct DNA sequencing to look for mutation in 364 bp segments of embB gene. RESULTS: Of 353 isolates of M. tuberculosis from 591 sputum samples, 62 (17.58%) were resistant to EMB, of which, 16 (25.8%) showed initial resistance and 46 (74.2%) acquired. Mono resistance to EMB was rare. Only two isolates showed resistance to EMB alone. From 62 EMB resistant isolates, 88.7 per cent (55) were resistant to INH, 82.2 per cent (51) to rifampicin and 61.2 per cent (38) were resistant to streptomycin. Co-resistance to isoniazid and rifampicin (multidrug resistant, MDR-TB) with EMB resistance was seen in 41(66.1%) isolates. High level of EMB resistance was seen in 16.5 per cent isolates. SSCP showed altered mobility in 8 of 10 isolates tested. Among the 8 mutants, 4 had known mutations at codon Met 306 being replaced by Val/ Leu. The second most frequent mutation encountered was at codon Phe 287 being replaced by Val, Cys or Leu (novel mutations). Sequence analysis revealed 10 novel mutations in codon 221, 225, 227, 271, 272, 281, 282, 287, 293 and 294 within embB gene. INTERPRETATION & CONCLUSION: Presence of high frequency of EMB resistance, occurrence of high level EMB resistance, co-existence of MDR-TB with EMB resistance and novel mutations in emb B gene of M. tuberculosis clinical isolates reported highlight the need to work on larger samples to identify the diagnostic marker of EMB resistance in mycobacteria.

Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To design and construct the plasmids expressing short hairpin RNA (shRNA) targeting human xylosyltransferase- I (XT- I) which is the initiating enzyme in the biosynthesis of proteoglycans (PC).</p><p><b>METHODS</b>Short chain oligonucleotides were designed according to the sequence of XT-I provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil-1 vector. The recombinant XT- I shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT-I expression vectors were transfected into salivary adenoid cystic carcinoma cell line (ACC-M) by Lipofectomine 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT-PCR was used to detect the expression of mRNA level of XT- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot.</p><p><b>RESULTS</b>The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed GFP successfully. And by RT-PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of mRNA level and 70.18% of protein level respectively.</p><p><b>CONCLUSION</b>The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.</p>

Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y322C, D331Y double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.

Reference to the Mutations in the embB Locus among Korean Clinical Isolates of Mycobacterium tuberculosis Resistant to Ethambutol

NO Abstract Available

Tiempo mínimo para detectar calidad proteica usando indicadores del catabolismo proteico en pollos / Minimal time for detecting protein quality using indicators of protein catabolism in chickens

Previous studies shown that in chickens the hepatic activities of the purine enzymes Xanthine Dehydrogenase and Nucleoside Phosphorylase and the uric acid excretion can predict the quality of the protein consumed in a very short time. In these studies even though the experimental time was short, the time used for the conditioning of the chickens was long and included five days with six chickens per cage and then five to six days for progressively changing the chickens to individual cages in order to avoid the stress associated with the isolation of the animals. Thus the purpose of this study was to determine the minimal time required to detect differences in these parameters after feeding a soy-met and a gelatin diet and eliminating completely the time required for the isolation of the chickens. Thus, 76 one day old Warren male chickens were placed in groups of six on a soy-met powdered diet during five days and on day six all the chicken were placed in individual cages and one halve was offered the same diet while the rest received a gelatin diet. Then on day 1, 2, 3, 5, 7, 10 and 15 after the diet change five chickens on each diet were sacrificed and the activity of the liver purine enzymes as well as the uric acid excreted were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of inhibitors of pyrimidine metabolism

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.

Development of inhibitors of pyrimidine metabolism

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.