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R Boom

Publications and source records attributed to R Boom.

35 records · Page 2Linked to original sources
PubMed1990

Rapid and simple method for purification of nucleic acids.

We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

DNA↗
PubMed1988

Heat-shock induction of the human immunodeficiency virus long terminal repeat.

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.

Animals↗
PubMed1988

Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent.

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Acetyltransferases↗
PubMed1987

Resistance to methylation de novo of the human cytomegalovirus immediate early enhancer in a model for virus latency and reactivation in vitro.

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.

Animals↗
PubMed1987

Transcriptional activation of the major immediate early transcription unit of human cytomegalovirus by heat-shock, arsenite and protein synthesis inhibitors.

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.

Animals↗
PubMed1986

Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition.

Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.

Animals↗
PubMedDate not supplied

Looking for "indicants" in the differential diagnosis of jaundice.

The differential diagnosis between intra- and extrahepatic causes of jaundice was studied. At the "20 Noviembre" ISSSTE Hospital in Mexico City, between January 1977 and May 1984, data were collected in 1,263 jaundiced patients. To the clinical data for 1,000 of these 1,263 patients and a new set of 105 jaundiced patients, the COMIC study group algorithm was applied. The differential diagnosis between medical and surgical causes of jaundice was correct in 90% of the cases. In 85% the algorithm could also differentiate between acute and chronic disease, or between benign and malignant causes of jaundice. The COMIC algorithm was then modified and applied to the same 1,000 cases examined previously, with 96% accuracy in distinguishing medical and surgical causes of jaundice and 94% accuracy in discriminating between acute and chronic, or benign and malignant, disease. In a new set of 105 cases of jaundiced patients the modified COMIC algorithm made the correct diagnosis between intra- and extrahepatic causes for 98% of the patients, and for 93% it was also capable of distinguishing benign from malignant and acute from chronic causes of jaundice.

Adult↗
PubMedDate not supplied

Physicians' diagnoses compared with algorithmic differentiation of causes of jaundice.

Clinical data were collected in 194 cases of jaundiced patients treated at the "Adolfo Lopez Mateos" ISSSTE Hospital in Mexico City from July 1985 to July 1986. A copy of the clinical history of each patient was given to each of four physicians--one recently graduated from medical school, another in his first year of gastroenterology, and two others who were experienced gastroenterologists. The same clinical data were processed by a computer set up to use a modified Danish COMIC algorithm. All physicians and the computer technician were blinded to the "gold standard" pathologic diagnoses, with which their diagnoses were compared. Accuracy rates of the physicians in distinguishing intrahepatic (medical) from extrahepatic (surgical) jaundice were 78%, 86%, 86%, and 91%, and the accuracy of computer-assisted diagnoses was 96%. Chi-squared analysis of the diagnoses of three of the physicians and those of the computer showed significant differences (p between 0.1 and 0.01). For the diagnoses of the remaining physician, however, no significant difference was found after chi-squared continuity correction.

Adult↗
PubMedDate not supplied

[A 10-year prolective study on cancer of the digestive system].

OBJECTIVE: A ten year prolective and multicentre study of cancer of the Digestive Tract aims to evaluate the variations on frequency and to identify risk factors. METHODS: Ten hospitals are participating five in Mexico City and five in other states. Data were obtained through the hospitals files and specially designed questionnaires, the latter mainly diet and habits oriented. RESULTS: Through this ten years in the 4,715 cases studied we can appreciate a decreasing tendency of the carcinoma of the stomach (GC) that has been the most frequent until 1984 in the general group General Hospital and Instituto Nacional de la Nutrición. Since then and up to date, first place in frequency in shared between the stomach and colorectal carcinoma (CRC). CRC has been increasing even in the General Hospital in which GC was very high. Eighth years ago we found that people in those hospitals in which GC was high the diet was poor in proteins, fat A and C vitamin. People in those hospitals with high frequency of CRC had diet high in protein, fat and low in fiber. A new study on diet is ongoing. Gallbladder and biliary tract carcinoma has been increasing in all the hospitals. A high O blood group was found in the Juarez Hospital in which gallbladder carcinoma was the highest. O group is highest in the indian people. Cancer of the gallbladder is very high in Pimas and Navajos indians. A plan to study litogenic index and culture of salmonella is going to be practiced in bile from normal and gallbladder carcinoma. Concerning habits, tobacco and alcohol consumption are most frequent in cancer of the esophagus and pancreas. Also coffee consumption is high in the latter. CONCLUSIONS: Along this 10-year study, the frequency of gastric cancer has been decreasing. Instead, the frequencies of colo rectal and biliary tract cancers have been increasing. Variations in frequencies correlated with the composition of the diet, fiber consumption habits and racial characteristics of the different groups.

Adult↗