Characterization of the gene for apolipoprotein E5-Frankfurt (Gln81->Lys, Cys112->Arg) by polymerase chain reaction, restriction isotyping, and temperature gradient gel electrophoresis

A new apolipoprotein (apo) E variant, apoE5-Frankfurt, was identified in a 43-year-old male with moderate hypercholesterolemia. On isoelectric focusing in an immobilized pH gradient, apoE5-Frankfurt migrated to a position more cathodic than apoE4 (Cys112->Arg). On sodium dodecyl sulfate-gel electrophoresis, its apparent molecular weight could not be distinguished from that of the three common apoE isoforms (E2, E3 and E4). Restriction isotyping with CfoI (HhaI) showed that apoE5-Frankfurt had arginine in positions 112 and 158 of the mature protein, suggesting that the mutation accounting for the additional positive charge had occurred in an epsilon 4 allele. The third and the fourth exon of the apoE gene were amplified using the polymerase chain reaction and analyzed by temperature gradient gel electrophoresis. This suggested that there were two mutations in the fourth exon of the mutant allele. Cloning and sequencing disclosed that, apart from the exchange of arginine for cysteine in position 112, a C to A substitution replaced glutamine (CAA) in position 81 by lysine (AAA).     

Detection of southern African human immunodeficiency virus type 1 subtypes by polymerase chain reaction: evaluation of different primer pairs and conditions

The purpose of the study was to develop a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect HIV-1 subtypes present in the Western Cape, South Africa. Twenty-two virus strains, belonging to HIV-1 subtypes B, C and D, were randomly selected for PCR evaluation. Cell lysates prepared from these virus-infected cultured cells were tested using 5 different primer pairs: gag SK38/SK39; gag 22/SK39; gag a/b, gag c/d (nested); env SK68/SK69 and LTR SK29/SK30. Eight different PCR profiles were evaluated: one profile each for the 3 gag primer pairs, 3 profiles for the env and 2 profiles for the LTR primer pairs. The number of PCR cycles, time per cycle and/or annealing temperature were changed in each profile. The optimum PCR profile for a specific primer pair was defined as that which detected one copy of proviral plasmid DNA after dot-blot hybridisation. Gag primer pairs detected HIV-1 DNA in all 22 samples. With the env primer pair, suboptimal conditions failed to detect most of the HIV-1 subtype C samples. By increasing the number of cycles and time per cycle, a 100% sensitivity was achieved. With the LTR primer pair all samples were detected by decreasing the annealing temperature and increasing the individual cycle times. This confirms that once PCR conditions are optimised, all HIV-1 subtypes in our study could be detected using different PCR primer pairs.     

Nested polymerase chain reaction for detection of Theileria annulata and comparison with conventional diagnostic techniques: its use in epidemiology studies

In this work we studied the ability of a nested polymerase chain reaction (PCR) to detect Theileria annulata, the causative agent of Mediterranean theileriosis, in blood samples obtained from cattle on farms in different Spanish regions and its possible use in epidemiology studies. Of the 214 samples analyzed, 78.04%, 69.86%, and 62.26% were found to be positive by nested PCR, indirect immunofluorescent antibody test, and optical microscopy of Giemsa-stained smears, respectively. The three techniques were in agreement in 68.6% of the results. The observation that the prevalence of Mediterranean theileriosis estimated using nested PCR alone (70.3%) and that obtained using all three diagnostic techniques together (80.4%) did not significantly differ verifies the utility of this technique in epidemiology studies.     

1-Thiomethoxsalen and 1-thiopsoralen: synthesis, photobiological properties, and site specific reaction of thiopsoralens with DNA

The sulfur analogues of psoralen and 8-methoxypsoralen (8-MOP) in the pyrone moiety were synthesized and compared to the parent compounds in terms of photoreactivity with viral M13mp19 RF DNA. The damaged viral DNA was transfected into Escherichia coli and scored for infectivity toward Ca-treated wild-type E. coli. This allowed a comparative study of the sulfur and oxygen analogues to be made in terms of photoreactivity. Furthermore, the DNA sequence specificity for the formation of monoadducts and cross-links of the four analogues was determined with 32P-labeled oligonucleotides containing thymidine in different sequences. The most site specific of the studied psoralens is 8-MOP, while 1-thiopsoralen is the most reactive analogue. This new thio analogue of psoralen leads to the efficient formation of monoadducts and cross-links in any pyrimidine-purine site.     

Effects of preservation methods, parasites, and gut contents of black flies (Diptera: Simuliidae) on polymerase chain reaction products

The vesicle to micelle transition which results in the interaction of the Triton X-100 surfactant with phosphatidylcholine vesicles was studied by means of dynamic light scattering (at different reading angles) and by freeze-fracture electron microscopy techniques. Vesicle solubilization was produced by the direct formation of mixed micelles without the formation of complex intermediate aggregates. Thus, vesicle to micelle transformation was mainly governed by the progressive formation of mixed micelles within the bilayer. A subsequent separation of these micelles from the liposome surface (vesicle perforation by the formation of surfactant-stabilized holes on the vesicle surface) led to a complete solubilization of liposomes.     

Demonstration of Chlamydia trachomatis in inguinal lymphadenitis of lymphogranuloma venereum: a light microscopy, electron microscopy and polymerase chain reaction study

Intravacuolar organisms in vacuolated macrophages were associated with areas of necrosis and suppuration in 12 patients with suppurative inguinal lymphadenitis. The intravacuolar organisms measured 0.2 to 2.0 micrometers in diameter, stained Gram negative with the Brown-Hopp's tissue Gram stain, faintly blue with hematoxylin and eosin stain, and black with the Warthin-Starry silver impregnation stain. The organisms lined vacuolar membranes and/or clumped in centers of vacuoles. Electron microscopy revealed elementary and reticulate bodies and intermediate forms characteristic of the genus Chlamydia. Cultures of three lymph nodes in McCoy cells grew Chlamydia trachomatis, lymphogranuloma venereum (LGV) serovars. Polymerase chain reaction using primers for chlamydial 16S ribosomal DNA confirmed the organisms as Chlamydia in lymph nodes from nine patients. Recognition of chlamydial organisms by light microscopy in tissue sections of lymph nodes allows a definitive diagnosis of lymphogranuloma venereum.     

An investigation of genital ulcers in Jackson, Mississippi, with use of a multiplex polymerase chain reaction assay: high prevalence of chancroid and human immunodeficiency virus infection

To test the hypothesis that the frequencies of normal alleles (ANs) with a relatively large number of CAG repeats (large ANs) are related to the prevalences of the dominant spinocerebellar ataxias (SCAs)-SCA types 1, 2, 3 (Machado-Joseph disease), 6, and dentatorubral-pallidoluysian atrophy (DRPLA)-we investigated the relative prevalences of these diseases in 202 Japanese and 177 Caucasian families and distributions of the number of CAG repeats of ANs at these disease loci in normal individuals in each population. The relative prevalences of SCA1 and SCA2 were significantly higher in Caucasian pedigrees (15% and 14%, respectively) than in Japanese pedigrees (3% and 5%, respectively), corresponding to the observation that the frequencies of large ANs of SCA1 (alleles >30 repeats) and of SCA2 (alleles >22 repeats) were significantly higher in Caucasians than in Japanese. The relative prevalences of MJD/SCA3, SCA6, and DRPLA were significantly higher in Japanese pedigrees (43%, 11%, and 20%, respectively) than in Caucasian pedigrees (30%, 5%, and 0%, respectively), corresponding to the observation that the frequencies of large ANs of MJD/SCA3 (>27 repeats), SCA6 (>13 repeats), and DRPLA (>17 repeats) were significantly higher in Japanese than in Caucasians. The close correlations of the relative prevalences of the dominant SCAs with the distributions of large ANs strongly support the assumption that large ANs contribute to generation of expanded alleles (AEs) and the relative prevalences of the dominant SCAs.     

Correlation of ribonucleic acid polymerase chain reaction, acid dissociated p24 antigen, and neopterin with progression of disease. A retrospective, longitudinal study of vertically acquired human immunodeficiency virus type 1 infection in children

OBJECTIVE: We investigated the relationship between cell-free viral load, neopterin, age-adjusted CD4+ cell concentration, and clinical events in 49 children with vertically acquired human immunodeficiency virus type 1 infection. STUDY DESIGN: Viral load was measured by quantitating viral ribonucleic acid in serum by polymerase chain reaction and measurement of immune complex dissociated p24 antigen in serum and plasma. Children were followed for an average of 2 1/2 years, with an average of 6 samples per child. Medical records were reviewed for weight, CD4+ cell count and clinical events. RESULTS: High virus copy number in serum was predictive of a decrease in weight-for-age zscore during the subsequent 6 months. High viral load, low CD4+ cell count, and high neopterin level were correlated with encephalopathy. High viral load correlated with opportunistic infections. All of these relationships held regardless of treatment status, although viral load decreased significantly after treatment was begun. CONCLUSIONS: Measurements of viral load were useful prognostic indicators for poor weight gain. Elevated serum virus levels and neopterin values and low CD4+ cell counts were all associated with encephalopathy.     

Detection of Epstein-Barr virus in the L & H cells of nodular lymphocyte predominance Hodgkin''s disease: report of a case documented by immunohistochemical, in situ hybridization, and polymerase chain reaction methods

The pharmacological response with tilt-table testing predicts long-term efficacy in neurocardiogenic syncope. However, beta-blockers for neurocardiogenic syncope are often not tolerated or are ineffective. Since cholinergic tone is important in the efferent part of the neurocardiogenic reflex, we investigated the pharmacodynamics and efficacy of propantheline bromide in preventing neurocardiogenic syncope. We studied 16 patients (11 males) with a mean age of 48.8 (+/- 15.1) years with presyncope or syncope and who had positive baseline tilt-table studies at a mean of 15.8 (+/- 10.3) minutes into the upright 60 degrees tilt. They were given propantheline bromide orally, an anticholinergic agent, at a dose of 64.3 (+/- 21.8) mg/day for 7 days, and tilt-table testing was repeated 1 hour after readministration of propantheline bromide, 30 mg orally. After propantheline bromide treatment, 13 of 16 patients (81%) had no inducible presyncope or syncope on repeat tilt-table testing. In this group of responders, the mean minimum heart rate during upright tilt-table testing increased from 43.2 (+/- 77.3) beats/min to 77.3 (+/- 17.2) beats/min after propantheline bromide (p < 0.005). More significantly, the minimum mean arterial blood pressure increased from 42.2 (+/- 25) mmHg to 81.3 (+/- 16.7) mmHg (p < 0.0005) during upright tilt. At a follow-up of 15.2 (+/- 7.4) months, in the responder group (12 patients with long-term follow-up), the average dose of propantheline bromide was 32.5 (+/- 23.8) mg/day, which was significantly reduced from the initial dose (p < 0.05). A clinical recurrence of symptoms occurred in only 4 out of 12 patients on propantheline bromide (33%), none of which were directly attributable to drug failure. It was concluded from this study that propantheline bromide is highly effective in preventing neurocardiogenic syncope. In addition, propantheline bromide's effectiveness is more than would be expected by prevention of cardioinhibition in neurocardiogenic syncope and would support a role for direct cholinergic control of vascular tone.     

Structural analyses of polymorphic transitions of sn-1, 3-distearoyl-2-oleoylglycerol (SOS) and sn-1, 3-dioleoyl-2-stearoylglycerol (OSO): assessment on steric hindrance of unsaturated and saturated acyl chain interactions

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.