Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer

Myocardial glucose metabolism has been shown to be heterogeneous in patients with hypertrophic cardiomyopathy (HCM). We tested the hypothesis that myocardial glucose metabolism differs between patients with HCM and those with hypertensive heart disease (HHD) associated with asymmetrical septal hypertrophy. We studied 12 patients with HCM, 7 HHD patients associated with asymmetrical septal hypertrophy using 18F 2-deoxyglucose (FDG) and positron emission tomography. We calculated % FDG fractional uptake in the inter-ventricular septum and posterolateral wall. Heterogeneity of FDG uptake was evaluated by % interregional coefficient of variation of FDG fractional uptake in each wall segment. In both the interventricular septum and posterolateral wall, % FDG fractional uptake was not significantly different between the two groups. The % interregional coefficient of variation for both interventricular septum (10.6 +/- 1.6 vs. 4.1 +/- 0.5, p < 0.01) and posterolateral wall (5.9 +/- 0.7 vs. 3.8 +/- 0.5, p < 0.05) was significantly larger in patients with HCM than in HHD patients associated with asymmetrical septal hypertrophy. Echocardiography demonstrated that the degree of asymmetrical septal hypertrophy was similar between the two groups. These results suggest that myocardial glucose metabolism may be more heterogeneous in patients with HCM compared to HHD patients associated with asymmetrical septal hypertrophy, although the left ventricular shape is similar. The difference in the heterogeneity might have resulted from differences in the pathogeneses of the two diseases.     

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.     

共沉淀法合成三硅酸镁及其微观分析

由Na₂O·nSiO₂和Mg(NO₃)₂经沉淀法合成了三硅酸镁,用450℃煅烧或酸化方法对合成的样品进行改性.采用XRD、IR、TG/DTA和BET等表征手段,考察了原料加入顺序、酸化和煅烧过程对样品的结晶度和表面织构的影响规律,并对其影响机理进行了探讨.结果表明,不同滴定顺序和不同活化方法制得的样品均为非晶态物质.TG/DTA分析显示不同滴定顺序样品的组成相同.pH对样品的表面织构有明显的影响.BET分析表明,Mg(NO₃)₂滴加入泡花碱溶液合成的样品为微孔材料,以1~3nm和0.7~0.9nm的微孔为主,比表面积达568.93m²·g^-1,水合硅酸镁含量较高.泡花碱滴加入Mg(NO₃)₂合成的样品为大孔材料,比表面积为179.40m²·g^-1,水合硅酸镁含量降低.煅烧和酸处理增加样品的结晶度,减少样品比表面积,并改变样品的孔径分布.煅烧使中孔含量增加,形成中孔材料.酸处理使Mg²⁺被H⁺取代,表面形成硅羟基基团,材料以中孔为主.     

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.     

Comparison of the Amplicor HIV-1 monitor test and the nucleic acid sequence-based amplification assay for quantitation of human immunodeficiency virus RNA in plasma, serum, and plasma subjected to freeze-thaw cycles

The Amplicor HIV-1 Monitor test was compared to the nucleic acid sequence-based amplification (Nasba) assay system for the quantitation of human immunodeficiency virus (HIV) RNA in three different types of clinical samples: plasma, serum, and plasma subjected to freeze-and-thaw cycles. Each assay detected HIV RNA in the same 73 (90%) of 81 samples tested, and the quantitative results obtained with the two assays were significantly correlated. Both assays detected higher RNA levels in patients with CD4+ cell counts lower than 200 cells/mm3 than in patients with CD4+ cell counts higher than 200 cells/mm3. In addition, RNA levels in plasma higher than 5 logs predicted higher numbers of clinical events than did RNA levels in plasma lower than 5 logs. Quantitation of HIV RNA in paired plasma and serum samples showed lower HIV RNA content in serum than in the paired plasma sample, with mean differences between HIV RNA contents of plasma and serum of 0.54 and 0.28 log RNA copy/ml by the Nasba assay and the Amplicor HIV-1 Monitor assay, respectively. No significant loss of HIV RNA was detected with either assay in plasma samples subjected to multiple freeze-and-thaw cycles. These studies demonstrate that the Nasba and Amplicor assays perform similarly with plasma and serum samples. Further, the results indicate that freeze-and-thaw cycles do not result in significant loss of detectable HIV RNA.     

Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequence-based PCR with various typing methods and isolation of a novel epidemicity marker

OBJECTIVE: To detail the characteristics and management of rarely reported and incompletely described dermoid cysts originating in the temporal fossa. DESIGN: Retrospective case series. PARTICIPANTS: Five patients ranging from 2 to 38 years of age with a mass in the temporal region (posterior to the lateral orbital rim) participated. INTERVENTION: Computed tomography (CT) and excisional biopsy were performed. MAIN OUTCOME MEASURES: Clinical and CT characteristics and surgical outcomes were measured. RESULTS: Computed tomography showed cystic lesions, originating from the region anterior to the confluence of the greater wing of the sphenoid, frontal, and zygomatic bones. Displacement of the anteriormost portion of the temporalis muscle was common. Three cysts were isolated to the temporalis fossa, while two showed more extensive bony erosion and extension into the cranial and orbit cavities. At surgical excision, gross rupture of the cysts was noted in two cases, and two were completely liquefied. Histopathology showed variable inflammation surrounding all of the dermoid cysts. All patients did well after surgery. CONCLUSIONS: Dermoid cysts may infrequently occur "primarily" in the temporal fossa. Bone involvement and anterior temporalis muscle displacement are common. An origin from the area anterior to the confluence of the greater wing of the sphenoid, frontal, and zygomatic bones is seen. A coronal approach facilitates wide exposure and excision. When dural extension is suggested on CT, neurosurgical assistance may be required.     

Development and application of a new method for amplification and detection of human rhinovirus RNA

Motor nerve terminals on abdominal body-wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze-fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross-sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze-fractures through the presynaptic membrane showed particle-free areas indicative of synapses on the P-face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila.     

Structural analysis of the human RFC-1 gene encoding a folate transporter reveals multiple promoters and alternatively spliced transcripts with 5'' end heterogeneity

During embryonic life, renal morphogenesis is characterized by a defined period of intense cellular activity, inductive-transformation of undifferentiated cells to polarized epithelia, in-growth of capillaries into an intricate parenchymal epithelial-mesenchymal mass, and finally the maturation into an organ with diverse structural and biological functions. It should be emphasized that the interactions between various growth factors and their receptors, FCM glycoproteins and proto-oncogenes are required for proper epithelial: mesenchymal interactions essential to the process of nephrogenesis. A balance between the activities of these macromolecules, whether essential or redundant, is needed to orchestrate the proper cell signals and responses to assure the progression of normal organogenesis. Finally, in spite of the enormous wealth of data in the literature, the process of renal development is so complex that a clear picture has yet to emerge of the precise coordinated and sequential events that result in the formation of a mature functioning kidney.     

A new Combi test for simultaneous detection of antibodies to viral capsid, early and EBNA antigens of Epstein-Barr virus

In order to facilitate the differentiation between a recent (acute) and a past Epstein-Barr virus (EBV) infection, the Combi test was developed. This test is an anticomplement immunofluorescence test (ACIF) requiring only a single serum dilution to be tested on a single cellular spot. The cell line used expresses viral capsid antigen (VCA) and early antigen (EA) in about 5 to 10 percent of the cells as well as EBV nuclear antigens (EBNA) in more than 90 percent of cells. A satisfactory agreement between the Combi test and other tests for antibodies to EBV was obtained (IgG and IgM antibodies to VCA by IFA and EIA and antibodies to EBNA by ACIF including tests for heterophile and complement-fixing antibodies). When the standard serological tests gave negative results, the Combi test was also negative (absence of any fluorescence in the cells). Serologically confirmed recent (acute) infections lead to specific fluorescence in only 5 to 10 percent of the cells, while past infections result in fluorescence in 90 percent or more of the cells. For the diagnosis of a reactivated EBV infection or of EBV-associated malignancies, other tests should be employed. The test is based on the measurement of the activation and specific distribution of the C3 component of complement; the antibody class differentiation is therefore not necessary. The presence of rheumatoid factor (RF) and the IgG competition phenomenon do not influence the results of the Combi test. An introduction of the Combi test will enable a simplified, less expensive and more reliable serodiagnosis of EBV infections.     

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water

The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.     

Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.     

Detection of tumor virus infections with HPV 16, 18, and 33 from cytologic material in the uterine neck using the PCR method

A method for detection of oncogenic viruses HPV 16, 18, 33 based on enzymatic amplification (PCR) of specific viral DNA sequences is described. This technique has many advantages, including: specificity, reproducibility, relatively simple procedure and low cost.     

RNA binding analysis of yeast REF2 and its two-hybrid interaction with a new gene product, FIR1

The product of the REF2 gene is required for optimal levels of endonucleolytic cleavage at the 3' ends of yeast mRNA, prior to the addition of a poly(A) tail. To test the role of the previously demonstrated nonspecific affinity of REF2 for RNA in this process, we have identified RNA binding mutants in vitro and tested them for function within the cell. One REF2 variant, with an internal deletion of 82 amino acids (269-350), displays a 10-fold reduction in RNA binding, yet still retains full levels of processing activity in vivo. Conversely, a series of carboxyl-terminal deletions that maintain full RNA binding capability have progressively decreasing activity. These results rule out a major role for the central RNA binding domain of REF2 in mRNA 3' end processing and demonstrate the importance of the carboxyl-terminal region. To ask if the stimulatory role of REF2 depends on interactions with other proteins, we used a two-hybrid screen to identify a new protein termed FIR1 (Factor Interacting with REF) encoded on chromosome V. FIR1 interacts with two independent regions of REF2, one of which (amino acids 268-345) overlaps the RNA binding domain and is dispensible for REF2 function, whereas the other (amino acids 391-533) is located within the critical carboxyl-terminus. As with REF2, FIR1 has a small but detectable role in influencing the efficiency of poly(A) site use. Yeast strains containing a disrupted FIR1 gene are slightly less efficient in the use of cryptic poly(A) sites located within the lacZ portion of an ACT1-lacZ reporter construct. Likewise, a double delta ref2, delta fir1 mutant is more defective in processing of a reporter CYC1 poly(A) site than delta ref2 alone. This synergistic response provides additional support for the interaction of FIR1 with REF2 in vivo, and suggests that a number of gene products may be involved in regulating the cleavage reaction in yeast.