Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification

Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community. Strain identification of M. tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance. To research this problem, strain identification must be reliable and accurate. Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M. tuberculosis strains. We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M. tuberculosis strains. This technique may be particularly useful in cases in which M. tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking.     

Oligonucleotide (GTG)5 as an epidemiological tool in the study of nontuberculous mycobacteria

Analysis of restriction fragment length polymorphisms in the genome of Mycobacterium tuberculosis (DNA fingerprinting) has proved to be a useful epidemiological tool in the study of tuberculosis within populations or communities. However, to date, no similar method has been developed to study the epidemiology of nontuberculous mycobacteria (NTM). In this communication, we report that a simple oligonucleotide repeat, (GTG)5, can be used to accurately genotype all species and strains of NTM tested. We suggest that this technology is an easily applied and accurate tool which can be used for the study of the epidemiology of NTM.     

Detection of microsatellite instability in cancers by arbitrarily primed-PCR fingerprinting using a fluorescently labeled primer (FAP-PCR)

The microsatellite mutator phenotype (MMP), detected as a change in the number of repeating units in hundreds of thousands of microsatellite sequences in the tumor cell genome, underlies the carcinogenesis of a variety of tumors including sporadic and hereditary nonpolyposis colon cancers. This enhanced microsatellite instability was discovered using arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting of DNA from colon cancers. In this study, we found an arbitrary primer that can amplify multiple DNA fragments containing repeated sequences, including the poly A tracts found in the Alu repeats of the human genome. The combined use of primer labeling with fluorescence and an automated DNA sequencing analysis of AP-PCR products (FAP-PCR) detected alterations in fingerprint bands in all DNA samples previously determined to belong to the MMP. Fluorescent AP-PCR fingerprinting using this single arbitrary primer provides a convenient and efficient method for detecting tumor specific fingerprint alterations that are usually undetectable by conventional fingerprinting.     

Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a defined fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system using fluorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10(3) bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of fluorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a [corrected] small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.     

A method to increase the sensitivity of mutation specific oligonucleotide hybridization using asymmetric polymerase-chain reaction (PCR)

We propose a simple and reliable method to increase the sensitivity of mutation specific oligonucleotide hybridization (MSOH) at least 2.5 times, when it is used to detect mutations in samples of DNA from tumor tissues. The method is based on using single stranded (ss) DNA, amplified by asymmetric PCR, as a target for MSOH analysis. During the first step, genomic DNA, isolated from tissue samples, has to be amplified by "standard", symmetric PCR, with sense and antisense primers in equimolar concentration. This amplification can be performed in a diminished volume of reaction mixture. In the second step obtained double stranded (ds) PCR DNA-product can be used as a template for asymmetric PCR, using only a single primer. The ss DNA must be complementary to the set of mutation specific oligonucleotides. By this innovation we have been able to clarify questionable results of MSOH using ds DNA as a target. Comparing MSOH from ss DNA to that from ds DNA, the observed rate of Gs-alpha mutations in thyroid tumor tissue samples increased to 16.7% (14/66) from 6% (4/66).     

Enterobacteriaceae identification compared by MORLOC (a new system) and API 20E

Identifications of 201 Enterobacteriaceae isolates were compared by MORLOC (a new system) and the API 20E system. Identifications agreed for 192 isolates (95.5 per cent). Of the nine discrepancies, MORLOC was correct for four, API for three; neither for one; and for one, MORLOC agreed with the reference laboratory but apparently all three systems were incorrect. The MORLOC system requires an extra day for a TSI but is smaller and simpler and can be recommended as easier to use, more reproducible, less expensive, and, in this study, more accurate than the API 20E.     

Rapid identification of up to three Candida species in a single reaction tube by a 5'' exonuclease assay using fluorescent DNA probes

OBJECTIVE: The objective of our investigation was to determine whether an MR imaging system designed to obtain images of the extremities affects the safety and functionality of pacemakers or that of implantable cardioverter defibrillators (ICDs). MATERIALS AND METHODS: Ex vivo experiments were conducted in which seven pacemakers and seven ICDs were exposed to a 0.2-T extremity MR imaging system. Magnetic field attraction was assessed at three positions relative to the MR imaging system. In addition, the devices were placed into a test apparatus that was oriented parallel and perpendicular relative to the MR imaging system while imaging was performed on a phantom using T1-weighted spin-echo and gradient-echo sequences. Various functional aspects of the pacemakers and ICDs were evaluated before, during (pacemakers only), and after MR imaging. RESULTS: Magnetic field attraction was relatively minor for all devices. The quality of the MR images was unaffected by the devices. Operation of this MR system did not alter any of the functional aspects of the pacemakers or ICDs evaluated in this study. CONCLUSION: According to these data and in consideration of how patients are positioned during examinations--that is, positioned so that the thorax (where the pacemaker or ICD and the corresponding leads are located) does not enter the magnet bore--the results suggest that it should be safe to perform MR imaging in patients with the pacemakers and ICDs evaluated in this study.     

以2-(5-溴-2-吡啶偶氮)-5-二甲氨基苯胺作螯合剂浊点萃取-激光热透镜光谱法测定水中痕量钴

钴是生命体必需的微量元素,研究建立水样中钴的测定方法具有重要意义。在pH5.0的醋酸-醋酸钠缓冲介质中,于60℃水浴中加热10min,螯合剂2-(5-溴-2-吡啶偶氮)-5-二甲氨基苯胺(5-Br-PADMA)与钴(Ⅱ)反应生成配位比为2∶1的疏水性螯合物Co(Ⅱ)-5-BrPADMA,其最大吸收波长位于611nm,用非离子表面活性剂TritonX-114为萃取剂进行浊点萃取(CPE),将该疏水性螯合物萃取到表面活性剂胶束相中,在相分离之后,以0.45mL2mol/L HCl-乙醇溶液溶解胶束相,转入光程为5mm的自制石英吸收池中,以单模He-Ne激光器(λ=632.8nm)做激发和探测光束进行热透镜光谱法(TLS)测定,建立了浊点萃取-激光热透镜光谱法(CPE-TLS)测定超痕量钴的方法。在优化条件下,热透镜光谱信号强度与钴(Ⅱ)质量浓度在0.40～6.0ng/mL范围内呈良好的线性关系,相关系数为0.997 8,检出限为0.05ng/mL。按富集前后溶液体积比值计算其浓缩因子为22。将实验方法用于湖水和温泉水中钴含量的测定,结果的相对标准偏差(RSD,n=6)小于4%;结果与电感耦...     

微波消解-氯磺酚偶氮硫代若丹宁分光光度法测定催化剂中的铂

研究了用微波消解样品,氯磺酚偶氮硫代若丹宁(HSCT)分光光度法测定催化剂中铂的方法。在盐酸介质中,HSCT与铂反应生成2∶1稳定络合物,λmax=540nm,ε=6.15×104。在25mL溶液中,铂质量在0～50μg内符合比尔定律,催化剂样品经微波消解后用分光光度法测定,结果令人满意。     

Metabolism of 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human neutrophils

We have previously shown that human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to 6,11-dihydro metabolites (Powell and Gravelle (1988) J. Biol. Chem. 263, 2170-2177). In the present study, we have shown that the first step in the formation of these dihydro metabolites is oxidation of the 5-hydroxyl group to a 5-oxo group, which is catalyzed by an NADP(+)-dependent microsomal dehydrogenase enzyme. All the dihydroxyeicosanoids we investigated which contained a 5(S)-hydroxyl group followed by a 6-trans double bond were good substrates for this reaction. However, LTB4, which contains a 6-cis double bond, was not metabolized to any detectable 5-oxo products. The preferred substrate for the dehydrogenase reaction is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE), which has a Km of about 0.2 microM, compared to approx. 0.9 microM for 12-epi-6-trans-LTB4. In contrast to 5(S)-HETE, 5(R)-HETE as well as a variety of positional isomers of 5(S)-HETE are not metabolized to significant extents by the PMNL dehydrogenase. 5-Oxo-ETE and 5-oxo-15-hydroxy-ETE, which are formed from 5(S)-HETE and 5,15-diHETE, respectively, by this pathway, are potent chemotactic agents for human neutrophils, and raise intracellular calcium levels in these cells.     

Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806-3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.     

Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): current clinical and development status

Although low levels of social support have been related to mortality from coronary heart disease, little is known about the role of social support among Mexican Americans. The authors therefore examined the relationship between social support and long-term survival in the Corpus Christi Heart Project. They developed a social support scale that used data collected during in-hospital interviews of 292 Mexican Americans and 304 non-Hispanic Whites who survived a myocardial infarction for more than 28 days. The scale incorporated three measures: marital status; if not married, whether living alone; and whether advised to seek help. During an average follow-up period of 43 months, 115 participants died. Survival following myocardial infarction was greater for those with high or medium social support than for those with low social support. With age, gender, ethnicity, education, employment, smoking, diabetes, hypertension, and hypercholesterolemia included in a proportional hazards regression model, the relative risk of mortality was 1.89 (95% CI, 1.20-2.97) for those with low social support. But when the two ethnic groups were analyzed separately, low social support was no longer a significant predictor of mortality for non-Hispanic Whites, whereas for Mexican Americans, the relative risk of mortality was 3.38 (95% CI, 1.73-6.62) for those with low social support.     

Evaluation of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as a new sulfurizing reagent in combination with labile exocyclic amino protecting groups for solid-phase oligonucleotide synthesis

3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) was recently introduced as an efficient sulfurizing reagent for solid-phase oligonucleotide synthesis. The successful syntheses were performed using standard base protecting groups (i.e. benzoyl for A and C, isobutyryl for G), which required deprotection in concentrated ammonium hydroxide at 55 degrees C for 15-18 h. We have explored the possibility of using EDITH in combination with fast deprotection chemistry(e.g. Expedite Chemistry using tert -butylphenoxy acetyl as a base protecting group). Surprisingly, poor synthesis performance was observed when syntheses were conducted with EDITH, Expedite Chemistry and standard synthesis cycle (i.e. Coupling-Thio-Cap). Potential G modification seemed to be the source of incompatibility since sequences containing no G or carrying isobutyryl- protected G residues could be synthesized with high efficiency. However, the deleterious G modification can be readily eliminated by inserting a capping step before the sulfurization reaction. Oligomers prepared with the Coupling-Cap-Thio-Cap cycle contained few phosphodiester contaminants as measured by31P-NMR, anion-exchange HPLC and MALDI-TOF mass spectrometry. In addition to reducing deprotection time, this new combination also provides a mild method for the preparation of certain phosphorothioate oligomers that may be sensitive to prolonged ammonia treatment (e.g. thioated RNAs).     

Frequency and severity of headache after lumbar myelography using a 25-gauge pencil-point (Whitacre) spinal needle

A 77-year-old man had been suffering from stress urinary incontinence for 2 years since he had received transurethral resection of prostate (TUR-P) for benign prostatic hypertrophy. A 60-min pad test yielded 3 g of urine. Prostatic urethra was widely open and the external sphincteric injury was suggested because of the short membraneous urethra on the urethrogram. Urethral pressure profile indicated his maximal urethral closing pressure (MUCP) of 24 cmH2O and functional urethral length of 1.6 cm and cystometry demonstrated an underactive bladder, indicating that his incontinence was caused by sphincteric injury. Autologous fat injection therapy was performed in the lithotomy position under spinal anesthesia. Fifteen ml of subcutaneous fat was obtained from his lower abdomen by liposuction through a 15G needle, and 10 ml was injected submucosally from the perineum at 6 o'clock area of the prostatic apex under the guidance of transrectal echography using a 15G needle. The patient became completely dry after the procedure. MUCP and FUL increased to 35 cmH2O and 1.9 cm, respectively, although longer follow up is necessary. The advantage of autologous fat injection to the prostate for post-TUR-P SUI patients is briefly discussed.     

Chemoprevention of azoxymethane-induced intestinal carcinogenesis by a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone, in rats

The present study was designed to investigate the modifying effects of dietary 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone (KYN-54), a new synthetic retinoidal butenolide, during the post-initiation phase on azoxymethane (AOM)-induced rat intestinal carcinogenesis. The number of aberrant crypt foci (ACF) in rat colon, colonic ornithine decarboxylase (ODC) activity and bromodeoxy-uridine (BrdUrd) labeling index in rat colonic epithelium were also assessed. At 7 weeks of age, male F344 rats (except the KYN-54 alone and control groups) were given weekly s.c. injections of AOM at 15 mg/kg body wt for 3 weeks. Starting 1 week after the last injection of AOM, rats (except the control group) were fed a diet containing KYN-54 at concentrations of 100 or 200 p.p.m. throughout the experiment. All animals were necropsied at 32 weeks after the start of the experiment. Compared with the AOM alone group, KYN-54 at both doses reduced the incidence and multiplicity of tumors in entire intestine (small and large intestines). In the 200 p.p.m. KYN-54 fed group especially, tumor incidence and multiplicity in the entire intestine were lower compared with the AOM alone group (P < 0.005 and P < 0.05 respectively). Also, the number of ACF/cm2 colon in the groups of rats treated with AOM and KYN-54 at both doses were significantly lower than that of rats treated with AOM alone (P < 0.05). Colonic ODC activity and BrdUrd labeling index in the groups of rats treated with AOM and KYN-54 at both doses were slightly lower than those treated with AOM alone. KYN-54 at 200 p.p.m. significantly lowered BrdUrd labeling index induced by AOM (P < 0.005). These results suggest that KYN-54 might be a promising chemopreventive agent for intestinal neoplasia.     

The reconstruction of inflammatory biliary duct stenoses by using a ring-drainage (author's transl)

In treating the inflammatory closures and stenoses of the biliary ducts surgically, the bilio-biliary and bilio-digestive anastomoses were splinted by inserting an annular drainage. In this way it was possible to ensure the bileflow into the intestine and to prevent recurrent stenoses. The ring-drainage is suggested to be kept for 2 years at least. In our experience this method has been validated in 13 patients. Therefore it is recommended for use in reconstructive operations with inflammatory obstructions of the biliary tract.