Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA™) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA™ and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

A polymerase chain reaction-based assay to identify genotype F of hepatitis B virus

We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4%) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America.

Staircase in mammalian muscle without light chain phosphorylation

In disuse atrophied skeletal muscle, the staircase response is virtually absent and light chain phosphorylation does not occur. The purpose of the present study was to determine if staircase could be restored in atrophied muscle with continued absence of myosin light chain phosphorylation, by reducing what appears to be an otherwise enhanced calcium release. Control (untreated) and sham-operated female Sprague-Dawley rats were compared with animals after 2 weeks of complete inactivity induced by tetrodotoxin (TTX) application to the left sciatic nerve. In situ isometric contractile responses of rat gastrocnemius muscle were analyzed before and after administration of dantrolene sodium (DS), a drug which is known to inhibit Ca2+ release in skeletal muscle. Twitch active force (AF) was attenuated by DS from 2.2 ± 0.2 N, 2.7 ± 0.1 N and 2.4 ± 0.2 N to 0.77 ± 0.2 N, 1.05 ± 0.1 N and 1.01 ± 0.2 N in TTX (N = 5), sham (N = 11) and control (N = 7) muscles, respectively. Following dantrolene treatment, 10 s of 10-Hz stimulation increased AF to 1.32 ± 0.2 N, 1.52 ± 0.1 N and 1.45 ± 0.2 N for the TTX, sham and control groups, respectively, demonstrating a positive staircase response. Regulatory light chain (R-LC) phosphorylation was lower for TTX-treated (5.5 ± 5.5%) than for control (26.1 ± 5.3%) and sham (20.0 ± 5%) groups. There was no significant change from resting levels for any of the groups after DS treatment (P = 0.88). This study shows that treatment with dantrolene permits staircase in atrophied muscle as well as control muscle, by a mechanism which appears to be independent of R-LC phosphorylation.

Hunting for differentially expressed genes

Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Several methods have been used for this purpose, including differential hybridization, cDNA subtraction, differential display and, more recently, DNA chips. Subtractive hybridization has significantly improved after the polymerase chain reaction was incorporated into the original method and many new protocols have been established. Recently, the availability of the well-known coding sequences for some organisms has greatly facilitated gene expression analysis using high-density microarrays. Here, we describe some of these modifications and discuss the benefits and drawbacks of the various methods corresponding to the main advances in this field.

Enhanced mucosal re-epithelialization induced by short chain fatty acids in experimental colitis

The short chain fatty acids (SCFA) are the best nutrients for the colonocytes. Glucose is poorly used as a fuel but may be transformed into SCFA by colonic bacteria. The aim of this study was to investigate the effect of SCFA or glucose on experimental colitis. Colitis was induced in 30 Wistar rats by colonic instillation of 4% acetic acid. Five days later they were randomized to receive twice a day colonic lavage containing saline (controls, N = 10), 10% hypertonic glucose (N = 10) or SCFA (N = 10) until day 8 when they were killed. At autopsy, the colon was removed and weighed and the mucosa was evaluated macro- and microscopically and stripped out for DNA assay. Data are reported as mean ± SD or median [range] as appropriate. All animals lost weight but there was no difference between groups. Colon weight was significantly lower in the SCFA group (3.8 ± 0.5 g) than in the control (5.3 ± 2.1 g) and glucose (5.2 ± 1.3 g) groups (P<0.05). Macroscopically, the severity of inflammation was less in SCFA (grade 2 [1-5]) than in control (grade 9 [4-10]) and glucose-treated (grade 9 [2-10]) animals (P<0.01). Microscopically, ulceration of the mucosa was more severe in the glucose and control groups than in the SCFA group. The DNA content of the mucosa of SCFA-treated animals (8.2 [5.0-20.2] mg/g of tissue) was higher than in glucose-treated (5.1 [4.2-8.5] mg/g of tissue; P<0.01) and control (6.2 [4.5-8.9] mg/g of tissue; P<0.05) animals. We conclude that SCFA may enhance mucosal re-epithelialization in experimental colitis, whereas hypertonic glucose is of no benefit.

McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Polymerase chain reaction (PCR) has been widely investigated for the diagnosis of tuberculosis. However, before this technique is applied on clinical samples, it needs to be well standardized. We describe the use of McFarland nephelometer, a very simple approach to determine microorganism concentration in solution, for PCR standardization and DNA quantitation, using Mycobacterium tuberculosis as a model. Tuberculosis is an extremely important disease for the public health system in developing countries and, with the advent of AIDS, it has also become an important public health problem in developed countries. Using Mycobacterium tuberculosis as a research model, we were able to detect 3 M. tuberculosis genomes using the McFarland nephelometer to assess micobacterial concentration. We have shown here that McFarland nephelometer is an easy and reliable procedure to determine PCR sensitivity at lower costs.

Typing of Enterococcus faecium by polymerase chain reaction and pulsed field gel electrophoresis

Polymerase chain reaction (PCR) with JB1 or REP consensus oligonucleotides and pulsed field gel electrophoresis (PFGE) were used to study genomic DNA extracted from 31 strains of enterococci. Eleven ATCC strains, representative of 11 species of Enterococcus, were initially tested by JB1-PCR, revealing that Enterococcus malodoratus and Enterococcus hirae presented identical banding patterns. Eight Enterococcus faecium isolates from Stanford University and 12 from São Paulo Hospital were studied by JB1-PCR, REP-PCR 1/2R and PFGE. Among the isolates from Stanford University, 5 genotypes were defined by JB1-PCR, 7 by REP-PCR 1/2R and 4 by PFGE. Among the isolates from São Paulo Hospital, 9 genotypes were identified by JB1-PCR, 6 by REP-PCR and 5 by PFGE. The three methods identified identical genotypes, but there was not complete agreement among them.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

Long-chain n-3 fatty acids and inflammation: potential application in surgical and trauma patients

Lipids used in nutritional support of surgical or critically ill patients have been based on soybean oil, which is rich in the n-6 fatty acid linoleic acid (18:2n-6). Linoleic acid is the precursor of arachidonic acid (20:4n-6). In turn, arachidonic acid in cell membrane phospholipids is the substrate for the synthesis of a range of biologically active compounds (eicosanoids) including prostaglandins, thromboxanes, and leukotrienes. These compounds can act as mediators in their own right and can also act as regulators of other processes, such as platelet aggregation, blood clotting, smooth muscle contraction, leukocyte chemotaxis, inflammatory cytokine production, and immune function. There is a view that an excess of n-6 fatty acids should be avoided since this could contribute to a state where physiological processes become dysregulated. One alternative is the use of fish oil. The rationale of this latter approach is that fish oil contains long chain n-3 fatty acids, such as eicosapentaenoic acid. When fish oil is provided, eicosapentaenoic acid is incorporated into cell membrane phospholipids, partly at the expense of arachidonic acid. Thus, there is less arachidonic acid available for eicosanoid synthesis. Hence, fish oil decreases production of prostaglandins like PGE2 and of leukotrienes like LTB4. Thus, n-3 fatty acids can potentially reduce platelet aggregation, blood clotting, smooth muscle contraction, and leukocyte chemotaxis, and can modulate inflammatory cytokine production and immune function. These effects have been demonstrated in cell culture, animal feeding and healthy volunteer studies. Fish oil decreases the host metabolic response and improves survival to endotoxin in laboratory animals. Recently clinical studies performed in various patient groups have indicated benefit from this approach.

Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients

Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean ± SD, 9.6 ± 2.16 g/dl) than in untreated patients (8.07 ± 0.91 g/dl). Fetal hemoglobin levels were also higher in treated patients (14.16 ± 8.31%) than in untreated patients (8.8 ± 4.09%), as was the Ggamma/Agamma ratio (1.45 ± 0.78 vs 0.98 ± 0.4, P < 0.005). The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.