Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.     

Analysis of the association of a heat shock protein70-1 gene promoter polymorphism with myocardial infarction and coronary risk traits

In 12 depressed inpatients referred for bilateral electroconvulsive therapy (ECT), each patient was titrated at the first treatment session by using an ascending method-of-limits procedure with a step-wise increase in pulse frequency (frequency titration) or train duration (duration titration). At the second treatment session, seizure threshold was redetermined by using the method (frequency or duration titration) not used at the first treatment. Frequency or duration was maintained at the lowest level when the other parameter was titrated. Seizure threshold was significantly lower with duration titration (mean, 90 mC; SD, 27.3) than frequency titration (mean, 114 mC; SD, 35.6; p = 0.03). On average, patients in the duration-titration group required 1.2 (SD, 0.6) subconvulsive stimulations before a seizure was elicited, and patients in the frequency-titration group required 1.7 (SD, 0.9) subconvulsive stimulations before a seizure was elicited, a nonsignificant difference. These findings suggest that to elicit a seizure during ECT, increasing train duration may be slightly more efficient than increasing frequency. Basic and other clinical research findings indicate that increasing pulse width may be an inefficient way to elicit a seizure. Therefore the following sequence in the determination of seizure threshold is worth considering when using dose-titration or related techniques: the train duration should be increased first before increasing pulse frequency, and the decision to increase pulse width should be reserved for patients who do not seize at the maximal duration and frequency settings. Further empiric research is needed to establish the utility of this approach.     

Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant

The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.     

Increased transversions in a novel mutator colon cancer cell line

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.     

Benign solitary fibrous tumour of the pre-sacral space: MRI findings

Benign solitary fibrous tumour, a rare mesenchymal tumour of adults, usually arises from the pleura. Only a few cases have been reported in the retroperitoneum and, to our knowledge, there has been no report of its imaging features. We describe the MRI features of benign solitary fibrous tumour arising from the pre-sacral space.     

Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.     

The role of enterocytes in gut dysfunction

Stem cells in the intestinal epithelium give rise to enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Each of these cell lines plays a role in cytoprotection of the intestinal mucosa. In particular, it has been demonstrated that mature enterocytes can act as antigen presenting cells. Parenteral and enteral nutrition are used to nourish critically ill patients. However, these regimens are unfortunately associated with gut atrophy. Glutamine, the preferred intestinal nutrient, reverses this gut atrophy and plays a key role in maintaining the barrier function of the gut. Specific nutrients (putrescine, spermidine, spermine) have been used to modulate intestinal adaption. In addition, ornithine has been shown to act as a regulator of intestinal adaption. In this review, we discuss the relationship between the biology of enterocytes and failure of the gut barrier.     

Comparison of wound culture and bronchial lavage in the severely burned child: implications for antimicrobial therapy

BACKGROUND: The relationship of the burn wound flora to microbial pathogens in the tracheobronchial tree has important implications for antimicrobial therapy in the severely burned patient. Management of septic complications is bolstered by surveillance quantitative wound cultures (QWC) and bronchial lavage fluid (BLF) cultures. OBJECTIVES: To compare the organisms present in BLF with those found in QWC and to determine if QWC can predict BLF results. DESIGN: Results of BLF cultures from all patients who underwent bronchial lavage from January 1, 1996, to December 31, 1996, at our institution were compared with QWC data from the same date. Criteria for a positive match included an identical antibiotic susceptibility pattern and biotype. Match rates were calculated qualitatively and quantitatively. RESULTS: In 30 (48%) of the 62 BLF cultures, there was a match between the organism identified in the BLF and the QWC. When strict quantitative criteria were applied, the match rate was only 9 (14%) of 62. Burn size and inhalation injury had no significant effect on match rate. CONCLUSIONS: Whereas the microbial pathogens were similar in the QWC and BLF, linear regression showed no value of QWC in predicting BLF culture results. The difference between qualitative and quantitative match rates suggests cross-colonization between the burn wound and tracheobronchial tree, but little to no cross-infection. The QWC and BLF cultures must be performed independently in determining antimicrobial specificity in the burned patient.