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K Pratt

Publications and source records attributed to K Pratt.

At least 37 records · Page 2Linked to original sources
PubMed1990

Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.

Animals↗
PubMed1990

Structure of miniature swine class II DRB genes: conservation of hypervariable amino acid residues between distantly related mammalian species.

As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBc and the human DRB1-0101 allele. The percentage of differences in these residues between the pig DRBc allele and the human DRB1-0101 allele was significantly lower (29%) than that between the DRB1-0101 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBc and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in these widely disparate species. Consistent with this hypothesis, an examination of available murine and bovine class II sequences revealed a homology in hypervariable residues between the human DRB1-1401 allele and the mouse E beta s allele as well as a cow DRB allele. Consideration of these data along with intraspecies allelic sequence comparisons suggests that at least some of the interspecies similarities have emerged as the result of convergent evolution, possibly as the result of a need to react to common pathogens.

Amino Acid Sequence↗
PubMed1990

Kluyvera mediastinitis following open-heart surgery: a case report.

Reports of serious infections caused by Kluyvera spp. have been rare. A case of Kluyvera sp. mediastinitis and bacteremia in a man after open-heart surgery is described. The clinical significance of Kluyvera sp. isolates was debated in early descriptions of the organism. More recent reports, as well as the present case, suggest that the pathogenic potential of this organism is being increasingly recognized by clinicians and microbiologists.

Aged↗
PubMed1989

Endothelialization of vascular prosthetic surfaces after seeding or sodding with human microvascular endothelial cells.

The rapid establishment of an endothelial cell (EC) monolayer on the luminal surface of small-diameter vascular grafts may be necessary to prevent early thrombosis and failure. We have studied procedures used to promote EC coverage of vascular grafts and have compared preclotting prosthetic surfaces with ECs in platelet-rich plasma (seeding) with plating ECs onto a preestablished clot (sodding). We evaluated the rate of monolayer formation, the subsequent resistance to shear stress, and the effects of EC growth factors (ECGF and heparin) on these functions. Woven Dacron was seeded or sodded at a density of 2 x 10(5) cells/cm2 with human adult microvessel ECs derived from adipose tissue. In the presence of ECGF-heparin, the immediate establishment of an EC layer after sodding was observed, whereas seeded grafts required almost 48 hours for cells to reach the surface. In the absence of ECGF-heparin, sodded grafts still exhibited a complete monolayer of EC, whereas ECs were not observed at the surface of seeded grafts after 48 hours. After exposure to shear stress (up to 20 dynes/cm2) for 2 hours, most freshly sodded EC remained attached; however, the loss of loosely adherent cells did occur. EC seeded grafts remained covered with fibrin matrix after exposure to shear stress. We conclude that the use of a microvessel sodding technique as an alternative to previously reported seeding techniques is necessary for the immediate formation of an EC monolayer before implantation.

Blood Vessel Prosthesis↗
PubMed1988

Class II genes of miniature swine. I. Class II gene characterization by RFLP and by isolation from a genomic library.

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human alpha probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human beta probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique alpha genes were obtained which showed no evidence of cross-hybridization, while beta genes showed extensive cross-hybridization and were frequently detected in the library by more than one human beta gene probe. These data are consistent with early evolutionary divergence of alpha genes, prior to mammalian speciation, and with continuing evolution of beta genes, with possible shared usage of these genes by different alpha loci. The data also imply that alpha genes can readily be assigned to loci homologous to their human counterparts, but that beta genes will require further mapping and/or sequence analysis to confirm assignments.

Animals↗
PubMed1987

Rapid identification of Staphylococcus epidermidis.

A panel of Minitek sugar disks, consisting of trehalose, mannitol, xylose, and sucrose, was evaluated for its ability to identify blood culture isolates of Staphylococcus epidermidis (SE). Using a heavy suspension of organism in Mueller-Hinton broth, 50 microL was pipetted onto each disk in wells of a flat-bottomed microtiter tray. The tray was covered, incubated in a moist chamber in non-CO2 at 35 degrees C, and examined after 5 and 24 hours. A color change of yellow or orange was positive; no color change (red) was negative. Expected reactions for SE were as follows: negative trehalose, mannitol, and xylose; positive, sucrose. On evaluation of 227 coagulase-negative staphylococci (CNS) at 5 and 24 hours, the panel had a sensitivity of 94 and 96%, specificity of 92 and 89%, predictive value of positive tests of 97 and 96%, and predictive value of negative tests of 84 and 87%. This panel offered an inexpensive and convenient method for differentiating SE from the other CNS in five hours.

Alkaline Phosphatase↗
PubMed1986

Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery.

Endothelial seeding of vascular graft surfaces may lead to a less thrombogenic surface. We examined the feasibility of using microvessel endothelial cells derived from human fat for seeding purposes. Human fat was treated with collagenase for 24 minutes, washed, and purified in a Percoll gradient separation. This yielded 1.25 +/- 0.45 X 10(6) cells/gm of fat. After a 1-hour incubation on plasma-coated Dacron, 2.8 +/- 1.5 X 10(4) cells remained firmly adherent to the surface. When exposed to flow for 2 hours at a shear stress of 0 to 80 dyne/cm2, between 50% and 100% of the initially adherent cells remained adherent. Statistical analysis of this data failed to demonstrate a strong relationship between the number of adherent cells and the shear rate. Scanning electron microscopy demonstrated endothelial cells in various stages of attachment to the plasma-coated Dacron. Although most cells were still round and only focally attached to the surface, some cells were maximally flattened, forming cell-to-cell contact. Because of the high cell yield and the firm adherence characteristics, we conclude that microvessel endothelial cells may offer the possibility for confluent endothelial cell seeding of a graft at the time of surgical implantation without the need for cell culture.

Adipose Tissue↗
PubMed1985

Conserved sequences in both coding and 5' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.

Animals↗
PubMed1985

A human opal suppressor tRNA gene and pseudogene.

A human DNA library, cloned in bacteriophage lambda, was screened with an opal suppressor tRNA probe. Two genes were isolated, subcloned into pBR322, and sequenced. One is a normal opal suppressor tRNA gene 87 nucleotides in length without intervening sequences. It has a TCA anticodon demonstrating that the mature tRNA reads the termination codon UGA. The 5' internal control region for transcription has two extra nucleotides compared to the consensus sequence for eucaryotic tRNA genes, while the 3' internal control region is normal. This gene differs from a previously sequenced chicken opal suppressor serine tRNA gene (Hatfield, D., Dudock, B., and Eden, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4940-4944) only at position 11. The second human gene appears to be a pseudogene truncated near the 3' end, since in the cloverleaf form of the mature tRNA there are three noncomplementary bases in the acceptor stem. The two human genes have a high degree of homology and, excluding the truncated 3' terminus of the pseudogene, differ in only two positions. The flanking sequences of the pseudogene are about 90% homologous to the consensus sequence of the human Alu family of repeated sequences. This gene appears to have been inserted between two adjacent Alu family members.

Animals↗
PubMed1984

Bovine estrogen receptor binds chromatin at pre-existing nuclease hypersensitive sites.

Partially purified estrogen receptor prepared from heifer uterine cytosol, and labeled in vitro with tritiated estradiol, was used to locate receptor binding sites in target and non-target nuclei from various bovine tissues. Nuclei were digested to various extents with bovine pancreatic deoxyribonuclease I, micrococcal nuclease or endogenous nuclease and then assessed for their ability to bind charged estrogen receptor. After very brief digestion with DNAase I, such that only hypersensitive sites were cleaved, calf uterus nuclei were no longer able to bind estrogen receptor. Brief digests with micrococcal nuclease or endogenous nuclease, such that most DNA was still of polynucleosomal length, eliminated the binding ability of both calf and heifer uterus nuclei. These results suggest that estrogen receptor binds to pre-existing nuclease hypersensitive sites. Interestingly, nuclei digested by HaeIII restriction endonuclease, which cleaves at specific sequences, demonstrated no loss of labeled estrogen receptor binding, even though digestion products were of similar size to those obtained from nuclei after treatment with the other nucleases. Since nuclease hypersensitive sites occur in regulatory regions of actively transcribed genes, including estrogen-inducible genes, binding of estrogen receptor at these sites, in vivo, may be part of the mechanism by which transcription is induced.

Animals↗
PubMed1983

Nucleosome phasing in Tetrahymena macronuclei.

Core-protected DNA can drive only 60% of the Tetrahymena thermophila macronuclear genome into duplexes in hybridization experiments. This core-protected DNA therefore contains only a subset of the genome complexity. We interpret this to mean that a large fraction, if not all, of the genome is phased with respect to nucleosome placement. Among the sequences present in total DNA and absent from core-protected DNA are most of the sequences containing N6-methyladenine (MeAde) residues, consistent with our previous demonstration that most of these residues lie in linker DNA. We show that these results are not due to artifacts resulting from the small size of the DNA driver, nor are they due to any sequence preferences exhibited by staphylococcal (staph) nuclease. This is the first evidence that nucleosome phasing may be a bulk genome characteristic.

Adenine↗
PubMed1982

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.

Adenine↗
PubMed1981

Aminoglycoside resistance among isolates of nosocomial Enterobacteriaceae.

Fifty-seven gentamicin-resistant isolates of Enterobacteriaceae, obtained from patients attending hospital, were examined for the production of aminoglycoside-modifying enzymes. Of the 51 strains producing such enzymes, 34 were presumptively plasmid-mediated as indicated by conjugation experiments.

Aminoglycosides↗
PubMed1981

Evaluation of certain current chloramphenicol assay methods.

The rapid assay of chloramphenicol in serum and cerebrospinal fluid is an important service to the clinician, especially in the paediatric setting. More than 3 years' experience in the use of chloramphenicol transacetylase in conjunction with 14C-acetyl co-enzyme A has proved that this is an accurate method.

Chloramphenicol↗
PubMed1981

Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila.

Deoxyribonucleic acid (DNA) of the transcriptionally active macronucleus of Tetrahymena thermophila is methylated at the N6 position of adenine to produce methyladenine (MeAde); approximately 1 in every 125 adenine residues (0.8 mol%) is methylated. Transcriptionally inert micronuclear DNA is not methylated (< or = 0.01 mol% MeAde; M. A. Gorovsky, S. Hattman, and G. L. Pleger, J. Cell Biol. 56:697-701, 1973). There is no detectable cytosine methylation in macronuclei in Tetrahymena DNA (< or = 0.01 mol% 5-methylcytosine). MeAde-containing DNA sequences in macronuclei are preferentially digested by both staphylococcal nuclease and pancreatic deoxyribonuclease I. In contrast, there is no preferential release of MeAde during digestion of purified DNA. These results indicate that MeAde residues are predominantly located in "linker DNA" and perhaps have a function in transcription. Pulse-chase studies showed that labeled MeAde remains preferentially in linker DNA during subsequent rounds of DNA replication; i.e., there is little, if any, movement of nucleosomes during chromatin replication. This implies that nucleosomes may be phased with respect to DNA sequence.

Adenine↗
PubMed1980

Infectious drug resistance during an outbreak of samonellosis.

The sudden acquisition of aminoglycoside resistance among Salmonella group C1 isolates causing summer diarrhoea raised the possibility oif plasmid-mediated reistance. The demonstration of circular DNA species in the resistant, but not in the sensitive salmonellae and the transfer by conjugation of antibiotic resistance to a sensitive strain of Escherichic coli, was consistent with plasmid-mediated resistance.

Anti-Bacterial Agents↗
PubMed1978

Comparative study of DNA methylation in three unicellular eucaryotes.

We have analyzed the nature/content of methylated bases in the nuclear DNA of three unicellular eucaryotes. The pattern of methylation was different for each of the three organisms studied: Saccharomyces cerevisiae contained only 5-methylcytosine; Tetrahymena pyriformis contained only N6-methyladenine; and Chlamydomonas reinhardi contained both modified bases.

Adenine↗