Biomedical subjects

R Reeves

Publications and source records attributed to R Reeves.

At least 127 records · Page 7Linked to original sources

PubMed1988

A rapid method for the large scale preparation of bovine leukemia virus antigen.

Bovine leukemia virus (BLV) is the etiologic agent responsible for enzootic bovine leukosis. Detection of cattle seropositive for BLV and laboratory studies of BLV require the preparation of large quantities of BLV antigen. A convenient and economical method for concentrating virus from large volumes of cell culture medium involves the precipitation of the virus in the cold by polyethylene glycol (PEG). The present work demonstrates the feasibility of rapidly precipitating BLV with 10% PEG and subsequently separating the resuspended virus from the residual PEG on a disposable desalting column.

Antigens, Viral↗

PubMed1988

Comparison of quality of life on nitrendipine and propranolol.

A multicenter, randomized, double-blind, comparative study was conducted in 274 patients with mild to moderate hypertension to assess the impact of nitrendipine and propranolol on quality of life. After placebo baseline, 136 patients were given nitrendipine (5-20 mg b.i.d.) and 138 were given propranolol (40-120 mg b.i.d.). Quality of life was evaluated at baseline, weeks 6-10, and weeks 14-18 of the maintenance period. At weeks 6-10, the nitrendipine group became significantly more vigorous (p less than 0.01) and less fatigued (p less than 0.05) than the propranolol group. Propranolol subjects noted decreased problems of trembling hands (p less than 0.01) and alcohol use (p less than 0.05) than the nitrendipine subjects. No other significant differences between groups in mood states, troublesome conditions (insomnia, headaches, and loss of appetite), or sexual satisfaction were noted at this visit, and patient willingness to continue study medication was marginally significantly higher (p less than 0.1) in the nitrendipine group than in the propranolol group. At weeks 14-18, the propranolol subjects perceived significantly decreased problems with the "felt worried, tense, and drank alcohol to cope" factor (p less than 0.05); however, there were no differences between groups at this visit for Profile of Mood States (POMS) scores, sex life variables, or medication preference. Based on within-group analysis, the propranolol group perceived a reduction in partner sexual satisfaction (p less than 0.05). Overall, nitrendipine seemed to be better tolerated than propranolol.

Adult↗

PubMed1988

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.

Amino Acid Sequence↗

PubMed1988

A conformational study of the sequence specific binding of HMG-I (Y) with the bovine interleukin-2 cDNA.

The DNA sequence specific interaction of the high mobility group non-histone protein HMG-I (Y) with the 3' untranslated region of the bovine interleukin-2 cDNA has been studied. Circular dichroism and thermal denaturation studies suggest that HMG-I (Y) alters the conformational state and increases the thermal stability of the DNA. Additionally, amino acid sequence analysis suggests that the previously identified non-histone protein HMG-Y is an isoform of HMG-I.

Amino Acid Sequence↗

PubMed1987

Non-histone chromatin proteins that recognize specific sequences of DNA.

Numerous chromatographic procedures have recently been developed to purify to near homogeneity various eukaryotic non-histone chromatin proteins that recognize and bind to specific sequences of DNA. In this brief review we have discussed a number of these different methods that we feel are important and probably represent the starting points for much of the future research work in this area. We view this coverage as being only an introduction, however, and strongly recommend that the reader consult the original papers for details of methods and protocols. We, nevertheless, hope that the information presented here will be of some assistance to those researchers and students who wish to become acquainted with the latest developments in this rapidly advancing field of chromatography. Although it is evident from what has been presented that the purification of each DNA sequence-specific non-histone chromatin protein initially starts with common or classical isolation and fractionation procedures, the final (and often crucial) steps of enrichment and purification often involve distinctive or unique procedures for each individual protein of interest. In many cases these final steps involve new techniques such as DNA sequence-specific bioaffinity and photoaffinity chromatography which not only ensure the isolation of specific protein species from complex mixtures but also result in a tremendous enrichment for nuclear proteins that are often present in the nucleus in extremely low concentrations. Furthermore, the entire process of protein purification has been remarkedly facilitated with the advent of high-performance liquid chromatographic and fast protein liquid chromatographic techniques which now allow for the very rapid separation and purification of proteins in a matter of minutes from mixtures that in the recent past would have required hours or days to purify. Thus, separation and purification techniques are now available that set the stage for the rapid isolation of rare, DNA sequence-specific, NHC nuclear proteins from almost any cellular source. It is therefore reasonable to anticipate that in the near future there will be major advances made in our understanding of the specific nuclear proteins that regulate gene expression in eukaryotic cells.

Animals↗

PubMed1987

Specific A . T DNA sequence binding of RP-HPLC purified HMG-I.

HMG-I (alpha-protein) is a high mobility group protein which recognizes and binds specifically to A . T rich double stranded DNA. We have investigated, by electrophoretic shift assays and DNase I footprinting, the ability of reverse-phase high performance liquid chromatography purified HMG-I to bind to specific A . T rich duplex DNA sequences. We show here that when HMG-I is isolated and purified under denaturing conditions it retains its specific A . T DNA binding activity. These results suggest that reverse-phase high performance liquid chromatography to be the method of choice for the preparation of HMG-I.

Adenine↗

PubMed1987

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Amino Acid Sequence↗

PubMed1987

Posttranscriptional gene regulation and specific binding of the nonhistone protein HMG-I by the 3' untranslated region of bovine interleukin 2 cDNA.

The 3' untranslated tail region (3'-UTR) of the cDNA of bovine interleukin 2 (bIL-2) acts as a lymphoid cell-specific gene regulatory element in vivo when ligated to the 3' end of the "marker" bacterial gene coding for chloramphenicol acetyltransferase (CAT) and the hybrid fusion gene is introduced into bovine lymphoid cells by transfection. Evidence is also presented that the 3'-UTR with its conserved (TATT)n motif probably has multiple functions in lymphoid cells operating both at the chromosomal level, where the sequence may be involved in the specific binding of the nonhistone chromatin high mobility group protein HMG-I, and at the RNA level, where the conserved sequence is involved in selective posttranscriptional mRNA degradation by a lymphocyte-specific nuclease(s). These results suggest a complex in vivo role for the 3'-UTR of bIL-2 cDNA and the conserved (TATT)n sequences found within it. They also offer a plausible explanation for the high degree of conservation of similar A + T-rich sequences in the 3'-UTRs of many of the other immune-response and growth-regulatory genes of mammals.

Acetyltransferases↗

PubMed1986

Xrep, a plasmid-stimulating X chromosomal sequence bearing similarities to the BK virus replication origin and viral enhancers.

The human X chromosome-linked fragment, "Xrep," was sequenced because it exerts a positive effect on plasmid growth in both E. coli and Saccharomyces cerevisiae. The sequence revealed three features similar to the human BK virus replication origin: Xrep has a true palindrome, CCTCC(T)3CCTCC, which is similar to "true" palindrome-like sequences found at the replication origins of polyoma [CCTC(T/C)10CTCC], BK [CCTC(A/G)8CCTCC] and SV40 [CCTCC(A)6GCCTCC] viruses. Twenty nucleotides away from the true palindrome, Xrep has the sequence GAATCCTATTCACTTTT while BK virus, the human analogue of SV40, has GAAATCCCTATTCTTTT in exactly the same position relative to the true palindrome. These two 17-mers differ only in the positions of two nucleotides comparing Xrep and BK virus. Also similar to the replication origins of DNA viruses, Xrep appears to have a cluster of enhancers adjacent to the origin-like sequences. Potent enhancer-like activity was detected in pSV1 X CAT/Xrep constructs. Xrep may originate from an endogenous virus, or from an X chromosomal replication origin.

Acetyltransferases↗

PubMed1986

Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I.

We have previously detected and purified a Friend erythroleukemic mouse cell nonhistone chromatin protein having extraction and acid-solubility properties like the low molecular weight "high mobility group" (HMG) nuclear proteins. We show here that the electrophoretic properties and the amino acid composition of this mouse cell "HMG-like" protein is comparable to those of the HMG-I proteins isolated from human HeLa S3 cells, African green monkey cells, Ehrlich ascites mouse cells, and rat fibroblast cells. Therefore, we have also designated the Friend erythroleukemic mouse cell protein as HMG-I. In common with the other HMG proteins the Friend cell HMG-I protein can undergo a variety of post-translational biochemical modifications including acetylation, ADP-ribosylation, glycosylation, and phosphorylation. Surprisingly, in the course of these studies we found that in vivo radiolabeling experiments revealed that only two minor HMG-14 subspecies (and/or possibly a minor HMG-I subspecies) are phosphorylated whereas HMG-1, -2, -17, and the major HMG-14 are not heavily phosphorylated.

Animals↗

PubMed1986

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.

Amino Acid Sequence↗

PubMed1986

Conserved chromosomal positions of dual domains of the ets protooncogene in cats, mice, and humans.

The mammalian protooncogene homologue of the avian v-ets sequence from the E26 retrovirus consists of two sequentially distinct domains located on different chromosomes. Using somatic cell hybrid panels, we have mapped the mammalian homologue of the 5' v-ets-domain to chromosome 11 (ETS1) in man, to chromosome 9 (Ets-1) in mouse, and to chromosome D1 (ETS1) in the domestic cat. The mammalian homologue of the 3' v-ets domain was similarly mapped to human chromosome 21 (ETS2), to mouse chromosome 16 (Ets-2), and to feline chromosome C2 (ETS2). Both protooncogenes fell in syntenic groups of homologous linked loci that were conserved among the three species. The occurrence of two distinct functional protooncogenes and their conservation of linkage positions in the three mammalian orders indicate that these two genes have been separate since before the evolutionary divergence of mammals.

Animals↗

PubMed1985

Collateral flow in patients with acute myocardial infarction.

To assess the change in angiographically visualized collaterals in evolving acute myocardial infarction (AMI), coronary arteriograms from 53 patients obtained 6.2 +/- 0.2 hours after onset of AMI symptoms were compared with follow-up angiograms obtained 14 +/- 1 days later. Collaterals were graded according to intensity score and percent of distal infarct-related artery visualized. Collateral intensity score and the percent of distal infarct vessel visualized by collaterals at baseline were low, and there was a significant increase in both values at follow-up angiography. The group of 20 patients with occluded infarct vessels at follow-up study accounted for these increases. In 33 patients with patent infarct vessels at repeat angiography, collateral intensity score and percent of segment visualized were unchanged. Among the patients with occluded infarct vessels at baseline and subsequent improvement in left ventricular (LV) ejection fraction (EF), baseline collateral score and percent of segment visualized were significantly greater than in patients in whom LVEF did not improve. Thus, in patients with evolving AMI, (1) angiographically visible collaterals are not extensive within the early hours of AMI, (2) the extent of collaterals on follow-up angiography may not be representative of that on the day of AMI, (3) collaterals are considerably more common 2 weeks after AMI, especially in patients with occluded infarct arteries during follow-up, and (4) collaterals present at the time of AMI are associated with improved LVEF at 2 weeks.

Aged↗

PubMed1985

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.

Acetylation↗

PubMed1985

Microheterogeneity of the mammalian high-mobility group proteins 14 and 17 investigated by reverse-phase high-performance liquid chromatography.

Microheterogeneity within the HMG-14 and HMG-17 group of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid as a weak ion-pairing agent) which separate proteins primarily on the basis of differences in their overall hydrophobicities. Ion-pair RP-HPLC proves to be a fast and efficient means for separating multiple subspecies of both the HMG-14 and the -17 proteins from both crude nuclear extracts and from ion-exchange column-purified protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least two different HMG-14 protein species (one major and one minor) and three different HMG-17 species (two major and one minor) can be resolved by ion-pair RP-HPLC. The identity and purity of these HMG-14 and -17 protein species were assayed by polyacrylamide gel electrophoresis and amino acid analysis. The amount of HMG protein microheterogeneity observed by RP-HPLC equals or exceeds that found for these proteins by other analytical techniques and the results suggest that this heterogeneity may be due to factors other than protein size or overall net charge variability.

Amino Acids↗

PubMed1985

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography.

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.

Amino Acids↗

PubMed1985

The effects of oxidation on the reverse-phase high-performance liquid chromatography characteristics of the high mobility groups 1 and 2 proteins.

Ion-pair reverse-phase high-performance liquid chromatography is a quick and convenient method for obtaining essentially pure preparations of the HMG (high mobility group)-1 and HMG-2 proteins if dithiothreitol is added to the eluted HMG protein fractions to prevent oxidation and their subsequent altered migration on acid-urea polyacrylamide gels. Unexpectedly, we found that this chromatographic separation technique can resolve the oxidized and reduced forms of both HMG-1 and HMG-2 proteins. We show that oxidized HMG-1 and -2 protein subfractions are responsible for some, but by no means all, of the HMG-1 and -2 protein heterogeneity previously reported by Elton and Reeves (2). At least two different HMG-1 protein species (one major and one minor) and at least four different HMG-2 protein species (two major and two minor) are consistently found in fully reduced "enriched" HMG-1 and -2 pig thymus protein preparations.

Animals↗

PubMed1985

A selection system for unequal crossing-over: plasmid construction and initial studies in Escherichia coli.

Unequal crossing-over is involved in genetic duplication and deletion in such diverse genetic systems as Drosophila, bacteria, and animal viruses. It is proposed to be involved in the form of unequal sister chromatid exchange in gene amplification in cultured animal cells and during carcinogenesis. Studies of the process of unequal crossing-over have been hampered by the lack of genetic systems allowing specific selection for cells that have undergone such unequal crossing-over. We report here on the construction of plasmids designed to provide specific selection of unequal crossing-over. One such plasmid was studied in Escherichia coli. We show that kanamycin resistance is generated, as predicted, by the expected unequal crossover event.

Crossing Over, Genetic↗