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Biomedical subjects

S Kelly

Publications and source records attributed to S Kelly.

At least 163 records · Page 9Linked to original sources
PubMed1993

Wheat germ and yeast RNA polymerase II: photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound [32P]-containing transcript.

A stable ternary transcription complex was formed with either wheat germ or yeast RNA polymerase II using a ribotrinucleotide primer (GpCpG) to initiate transcription on a short synthetic single-strand DNA template. The template was designed to limit the incorporation of a photoprobe S4-UMP (4-thio-UMP) to a unique position at the 3' terminus of the transcript. The resulting stable ternary transcription complex was photolyzed to cross-link the bound transcript ([32P]-labeled by the incorporation of [alpha-32P]CMP) with the protein domain at or near the active site. Separation of the protein components by electrophoresis in polyacrylamide gel containing SDS and analysis by autoradiography and silver staining revealed that for either enzyme only the largest subunit was [32P] labeled.

Affinity Labels↗
PubMed1993

Novel splicing, missense, and deletion mutations in seven adenosine deaminase-deficient patients with late/delayed onset of combined immunodeficiency disease. Contribution of genotype to phenotype.

We examined the genetic basis for adenosine deaminase (ADA) deficiency in seven patients with late/delayed onset of immunodeficiency, an underdiagnosed and relatively unstudied condition. Deoxyadenosine-mediated metabolic abnormalities were less severe than in the usual, early-onset disorder. Six patients were compound heterozygotes; 7 of 10 mutations found were novel, including one deletion (delta 1019-1020), three missense (Arg156 > His, Arg101 > Leu, Val177 > Met), and three splicing defects (IVS 5, 5'ss T+6 > A; IVS 10, 5'ss G+1 > A; IVS 10, 3'ss G-34 > A). Four of the mutations generated stop signals at codons 131, 321, 334, and 348; transcripts of all but the last, due to delta 1019-1020, were severely reduced. delta 1019-1020 (like delta 955-959, found in one patient and apparently recurrent) is at a short deletional hot spot. Arg156 > His, the product of which had detectable activity, was found in three patients whose second alleles were unlikely to yield active ADA. The oldest patient diagnosed was homozygous for a single base change in intron 10, which activates a cryptic splice acceptor, resulting in a protein with 100 extra amino acids. We speculate that this "macro ADA," as well as the Arg156 > His, Arg101 > Leu, Ser291 > Leu, and delta 1019-1020 products, may contribute to mild phenotype. Tissue-specific variation in splicing efficiency may also ameliorate disease severity in patients with splicing mutations.

Adenosine Deaminase↗
PubMed1993

Suitable screening tests for cognitive impairment and depression in the terminally ill--a prospective prevalence study.

Although confusional states and depression are common accompaniments of advanced cancer, few objective data are available concerning the prevalence of these clinical states or what methods are most suitable for their accurate detection. We decided that a 10-question Abbreviated Mental Test Score (AMTS) and a semistructured application of modified DSMIII-R (Diagnostic and Statistical Manual of Mental Disorder, third edition-revised) criteria for a major depressive illness were the most suitable screening tests for a terminally ill population. Thirty of 87 patients (34%) displayed significant cognitive impairment. The AMTS rating declined with approaching death and also correlated negatively with age. Of 81 patients, 21 (26%) were depressed when screened using DSMIII-R criteria for depression. One-third of patients with impaired AMTS scores also satisfied DSMIII-R criteria for depression. Of cognitively impaired patients, 90% had at least two possible causes for their confused state. We have found that both the AMTS and semistructured interview using DSMIII-R criteria for depression are useful routine screening tests in the terminally ill.

Adult↗
PubMed1993

Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells.

C3H10T1/2 cells are an established mesenchymal stem cell line which can differentiate into muscle, fat and cartilage cells when treated with azacytidine. Bone morphogenetic protein-2 (BMP-2) caused a dose dependent differentiation of these cells into fat, cartilage and bone cells-low concentrations favoring adipocytes and high concentrations chondrocytes and osteoblasts. The differentiated phenotypes were stable in the absence of BMP-2. Furthermore, the addition of other growth factors during the differentiation process altered the frequency of the differentiated colony formation. Transfection of the C3H10T1/2 cells with a BMP-2 cDNA also induced a phenotypic change from the parental fibroblast to adipocytes and osteoblasts. Our results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.

3T3 Cells↗
PubMed1993

Fat necrosis. An unusual cause of polyarthritis.

Metastatic fat necrosis is a form of lobular panniculitis which together with synovitis is an unusual complication of pancreatic disease. It can precede an attack of pancreatitis. Pancreatic disease should therefore be remembered as a cause of panniculitis. Serum amylase and abdominal ultrasonography should be considered to exclude pancreatic disease in cases of panniculitis of uncertain etiology. We describe a case of metastatic fat necrosis and discuss the appearance on magnetic resonance imaging.

Arthritis↗
PubMed1993

Ultrasound guide wire-directed stent deployment.

A new mechanically rotated 20 MHz intravascular ultrasound guide wire (0.032 inch) with a transducer core was placed through the central lumen of a peripheral arterial balloon-expandable stent. Using an anesthetized canine model, 11 stents were then deployed into the iliac or femoral arteries. Eight stents were successfully deployed with proper position and full stent expansion documented by ultrasound imaging. Four of the stents were overlapping and the double row of stent struts at the region of overlap was easily seen. Three stents were unsuccessfully deployed because of undersizing, as clearly documented by ultrasound imaging showing stent strut recoil. As a result, the procedure was modified by performing ultrasound measurements of arterial dimensions before stent selection. There were no complications attributed to the ultrasound guide wire. This study demonstrates an effective combination of diagnostic and therapeutic devices that may allow more precise placement of intravascular stents.

Animals↗
PubMed1992

Red cell filterability determined using the cell transit time analyzer (CTTA): effects of ATP depletion and changes in calcium concentration.

Cell transit time analysis (CTTA) is a new filtrometric technique for assessing red blood cell deformability by measuring the conductivity change caused by passage of erythrocytes through a polycarbonate filter. Most reported studies to date using CTTA have focused on the transit time (TT), the duration of passage of an individual red cell through a micropore. Bulk flow rate has not been previously measured via CTTA. The use of new enzyme based cleaning solutions make it possible to reduce clogging in micropore filters. Therefore, valid measures of the number of red cell transits per unit time (counts/s: C/S) can now be obtained. We evaluated both parameters, TT and C/S, as indicators of red cell filterability. Our goal was to evaluate the effect of metabolic changes shown by alternative techniques to affect red cell deformability. The two best established factors are changes in intracellular [ATP] and [Ca2+]. ATP depletion produces a very small increase in TT but a very marked decrease in C/S. In contrast, the addition of low concentrations of calcium produces an increase in TT with minimal decrease in C/S. The effects of calcium appear to be complex. The substantial changes in intracellular calcium induced by the ionophore A23187 result in a curvilinear pattern of increase in transit times and reduction in counts per s. Lanthanum, which inhibits egress of intracellular calcium, causes an increase in TT with a drop in C/S. We conclude that CTTA demonstrates the same changes in red cell deformability measurable by alternative filtrometric techniques; however, CTTA furnishes two separate and independent parameters which may be used to evaluate red cell deformability.

Adenosine Triphosphate↗
PubMed1992

HLA-identical sibling donor bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: influence of GVHD prophylaxis on outcome.

We have analysed the results of treating 140 consecutive patients with chronic myeloid leukaemia (CML) in chronic phase by bone marrow transplantation (BMT) using marrow from HLA-identical siblings performed between February 1981 and July 1991. Three different regimens were used sequentially to prevent graft-versus-host disease (GVHD): cyclosporin A (CsA) alone (n = 39), T-cell depletion of donor marrow (n = 51) and CsA with methotrexate (MTX) (n = 50). Eighty-four patients (61%) survive at a median of 49 months from BMT (range 3-120). The actuarial overall and leukaemia-free survivals at 5 years were 52% and 41% respectively. The actuarial probabilities of leukaemia-free survival and haematological relapse at 2 years for the CsA only group were 65% and 4%, for the T-cell depletion group 40% and 41% and for the CsA/MTX group 68% and 6% respectively. For the T-cell depletion group the probability of leukaemia-free survival was significantly lower (P less than 0.001) and the probability of relapse significantly higher (P less than 0.001) than for other methods of GVHD prophylaxis; differences between the other two groups were not significant. Previous reports that T-cell depletion with Campath-1M results in a high rate of relapse are confirmed. Patients in the CsA/MTX group have been monitored with cytogenetic and polymerase chain reaction studies for residual BCR/ABL transcripts. We conclude that the combination of CsA/MTX is currently the best available approach to prevention of GVHD after BMT for CML and in our hands it is not associated with a major risk of relapse.

Adolescent↗
PubMed1992

Medical cover for 'The Open' golf championship.

A review of 10 years' provision of medical cover at a major golf championship is presented. The average consultation rate was approximately 1 in 200 of those attending of whom 3% required transfer to hospital, the majority to orthopaedic or general medical departments. Familiarity with acute resuscitation and musculoskeletal complaints is required of the medical attendants. The provision of physiotherapy and chiropody services is essential, especially for the participants' complaints, two-thirds of which affected the axial skeleton. Good communications and close liaison with the Chief Marshal, Police and Ambulance Services are vital.

Emergency Medical Service Communication Systems↗
PubMed1992

Liver transplantation: living-related donations.

The LRLT procedure appears to be a promising alternative and adjunct to conventional liver transplantation. The advantages to this technique are a donor for each recipient, an improved quality of graft, and a reduction in pretransplant mortality in children. The procedure is not without risks, but the overall benefits seem to outweigh these risks, and only future experience will tell otherwise. Will there continue to be a need for such a procedure? We believe there will be, because the number of candidates needing transplantation will continue to grow and the demand for cadaveric organs will never be met. However, the procedure is extremely labor and resource intensive, and it is our belief that only a handful of large major medical centers will be able to adopt a live-donor program.

Adult↗
PubMed1991

Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion.

A high proportion of patients with myelodysplasia show characteristic karyotypic abnormalities in bone marrow cells. The most distinctive of the myelodysplastic syndromes is the 5q- syndrome characterized by refractory anemia, poorly lobulated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 (5q deletion) as the sole karyotypic abnormality. Recently, several genes encoding hemopoietic growth factors and receptors, comprising the interleukins 3, 4, and 5, macrophage colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, and the receptor for macrophage-colony-stimulating factor [the CSF1R (formerly FMS) gene product], have been localized to the long arm of chromosome 5, and there has been much speculation that deletion of one or more of these genes may be critical to the pathogenesis of the associated myeloid disorders. One candidate gene is CSF1R, which is required for normal proliferation and differentiation of hemopoietic cells of the myeloid lineage. We have carried out a molecular examination of the CSF1R, both on the 5q- chromosome and on the apparently normal homologous chromosome 5, in 10 patients with myelodysplasia and a 5q deletion. We have found, using restriction fragment length polymorphism analysis and gene dosage experiments, that all 10 patients showed deletion of CSF1R; 6 of 10 were hemizygous and 4 of 10 homozygous for CSF1R loss. The homozygous CSF1R loss has been confirmed in 2 patients by an in situ hybridization technique comparing the signal in affected cells to that in control sex-mismatched cells on the same slides. In those patients considered to have homozygous CSF1R loss by DNA experiments the gene was deleted from the 5q chromosome in all cells and from the apparently normal chromosome 5 in a subset of cells. This loss of one CSF1R allele, together with loss in some cells of the remaining allele on the homologous chromosome 5, in patients with myelodysplasia indicates that this is a region of critical gene loss on 5q. The loss of the hemopoietic growth factor receptor gene CSF1R may be important in the pathogenesis of human myeloid leukemia.

Adult↗
PubMed1991

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand.

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.

Calorimetry, Differential Scanning↗
PubMed1991

Translocation 1;7 in hematologic disorders--a report of three further cases. Absence of amplification of the gene for the epidermal growth factor receptor.

We report three new cases with a hematologic disorder and the unbalanced translocation der(1)t(1;7)(p11;p11). It has been speculated that the gene for the epidermal growth factor receptor, localized to the short arm of chromosome 7, might be amplified in cases with this translocation. We have demonstrated that there is no amplification of this gene in these three cases.

Aged↗
PubMed1991

Clonality of cell populations in refractory anaemia using combined approach of gene loss and X-linked restriction fragment length polymorphism-methylation analyses.

We have used X-linked restriction fragment length polymorphism (RFLP)-methylation and gene deletion analyses to investigate the nature of the progenitor cell of origin in the myelodysplastic syndromes (MDS). Gene deletion studies were performed on the granulocyte and T-lymphocyte fractions of six women with refractory anaemia (RA) and either a partial deletion of the long arm of chromosome 5 (5q-) or monosomy 7. All six showed gene loss in the granulocyte but not the T-lymphocyte fractions, indicating monoclonality of the granulocytes but not the T-lymphocytes. In order to further investigate this finding, we subsequently performed X-RFLP-methylation studies using the probe M27 beta, and also a probe for the phosphoglycerate kinase (PGK) gene. These studies have confirmed the monoclonality of the granulocytes and the polyclonality of the T-lymphocytes in these cases. Our findings suggest that in this group of patients with MDS the T-lymphocytes were not involved in the disorder, and furthermore, in the one case where B-lymphocytes were also available, that the progenitor cell of origin was restricted to the myeloid lineage.

Anemia, Refractory↗
PubMed1990

Yeast RNA polymerase I. Derivatization of the 190 and 135 subunits by 4-thiouridine monophosphate positioned uniquely at the 3' terminus of an enzyme-bound 32P-containing transcript initiated by a triribonucleotide primer on synthetic single-stranded DNA.

Specific transcription complexes were formed with yeast RNA polymerase I using a cognate oligoribotri-nucleotide primer (GCG) to initiate transcription on short synthetic single-stranded DNA templates. The templates were designed to limit the incorporation of a photoprobe, 4-thiouridine triphosphate, to a single unique position at the 3' terminus of the product RNA (position 12, 13, 14, or 15). The resulting transcription complexes were photolyzed to cross-link the bound transcript (radiolabeled with [alpha-32P]CTP) to the protein with the probe located at the catalytic site. Separation of the protein subunit components by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by autoradiography and silver staining revealed that the two largest subunits (A190 and A135) were radiolabeled. The ratio of subunit labeling (A190/A135) decreased as the RNA transcript increased from 12 to 15 nucleotides in length. This decrease in ratio resulted from a progressive reduction of A190 subunit labeling while the A135 subunit derivatization remained essentially constant. It was also observed that the DNA template was radiolabeled.

Affinity Labels↗