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C L Smith

Publications and source records attributed to C L Smith.

At least 235 records · Page 13Linked to original sources
PubMed1990

Using estimated creatinine clearance for individualizing drug therapy: a reassessment.

Estimates of renal function are frequently used to design individual dosing regimens. The accuracy of these estimates naturally influences their ability to predict certain pharmacokinetic parameters and appropriate drug dosages. Creatinine clearance is the most widely used estimate of renal function. Many formulas have been developed to provide a quick, relatively accurate prediction of creatinine clearance and, supposedly, the glomerular filtration rate (GFR). However, an understanding of the limitations associated with creatinine clearance estimations raises questions concerning their reliability as an aid in individualizing drug therapy. Many factors such as disease states, age, diet, analytical variations, and drug interactions affect the relationship between estimates and measures of creatinine clearance and GFR. As a result, creatinine clearance estimates using these formulas are often poor reflections of measured creatinine clearance or GFR. Also, studies comparing measured creatinine clearance with more accurate methods of assessing renal function (i.e., inulin) reveal errors that are often exaggerated as renal function declines. Therefore, estimated creatinine clearance is twice removed from the associated pharmacokinetic parameter. Despite these limitations, no other clinically relevant and convenient assessment of renal function is available. The authors recommend that the appropriate caveats be considered when using these tools clinically. For drugs with narrow therapeutic indices, estimates of creatinine clearance should only be used to establish initial dosing regimens, with subsequent therapy based on parameters generated from concentration determinations.

Age Factors↗
PubMed1990

A role for corticosteroid-binding globulin in delivery of cortisol to activated neutrophils.

In human blood, cortisol is transported by a plasma protein known as corticosteroid-binding globulin (CBG). As anticipated from primary structure comparisons of CBG and alpha 1-proteinase inhibitor (A1-PI), CBG acts as a substrate for neutrophil elastase. However, unlike A1-PI, CBG does not alter the activity of this enzyme, but is cleaved by it at a single location close to its carboxy-terminus, and this reduces its molecular size by 5 kDa with the concomitant release of more than 80% of CBG-bound cortisol. Three small molecular size fragments are detected after elastase cleavage, and carbohydrate analysis of these fragments suggests that they represent the same polypeptide fragment which has been differentially glycosylated. To assess the biological significance of these observations, CBG was incubated with either mononuclear cells or granulocytes obtained from patients with acute inflammation (sepsis) and from a normal volunteer. Only granulocytes from septic patients reduced the mol wt of CBG by about 5 kDa and destroyed its steroid-binding activity. Preincubation with A1-PI prevented this, which demonstrates that neutrophil elastase plays a key role in this event. These results suggest a physiological role for CBG in the delivery of cortisol to sites of inflammation.

Adult↗
PubMed1990

Rabbit corticosteroid-binding globulin: primary structure and biosynthesis during pregnancy.

The cDNA-deduced primary structure of rabbit corticosteroid-binding globulin (CBG) contains 383 amino acids (mol wt, 42,326), including three cysteine residues and four sites for N-glycosylation. It is primarily the product of a 1.68-kilobase hepatic mRNA, but small amounts of CBG mRNA were also found in maternal lung, spleen, and ovary and fetal kidney. In the fetus, hepatic CBG mRNA concentrations increase markedly after day 11 and were 2- to 5-fold higher than those in maternal liver during days 17-23. They then declined to very low levels at term (31 days). By contrast, maternal hepatic CBG mRNA levels did not increase until day 23; reached a peak at about day 27, and then declined to prepregnancy values by 3 days after delivery. In general, fetal and maternal hepatic CBG mRNA concentrations reflect the corresponding serum CBG levels. Our data, therefore, indicate that the marked changes in fetal and maternal plasma CBG levels during pregnancy reflect changes in the biosynthesis of the protein rather than alterations in compartmentalization or clearance.

Amino Acid Sequence↗
PubMed1990

Swine as a model for cardiovascular research: improved cardiopulmonary bypass techniques.

A large-animal model is essential for the assessment of functional parameters in cardiovascular surgical research. To date the canine model has been used successfully because of its availability and tolerance to cardiopulmonary bypass. However, because of decreased availability and increased cost, an alternative animal model is now needed. The swine model has been used in experimental cardiac procedures, but complications during cardiopulmonary bypass have presented a formidable challenge. These complications include enormous fluid shifts from the vascular bed, increased metabolic acidosis, and marked hemoglobinuria. To eliminate these deleterious complications within the swine model, a number of technical alterations were achieved. The priming solution used for the extracorporeal circuit was altered to consist of 1000 mL lactated Ringer's solution. 500 mL 20% mannitol, 500 mL 6% dextran in 5% detrose solution. 50 mEq sodium bicarbonate, and 10,000 IU heparin. The extracorporeal circuit employed the use of membrane oxygenation. Three different blood flow rates (150, 175, and 200 mL/kg min-1) were studied. We conclude that the optimum blood flow rate for cardiopulmonary bypass in swine is in the range of 175-200 mL/kg min-1. Membrane oxygenation results in less damage to blood during cardiopulmonary bypass. The asanguinous hyperosmolar priming solution is beneficial for cardiopulmonary bypass in swine to greatly reduce fluid shifts, prevent metabolic acidosis, and eliminate hemoglobinuria.

Acidosis↗
PubMed1990

Prostaglandin catabolism and ulcerative colitis: effect of sulphasalazine, 5-aminosalicylic acid and other drugs.

1. Evidence is presented for the occurrence of type 1 prostaglandin 15-hydroxydehydrogenase in human rectal mucosa. No evidence of the presence of type 2 enzyme was found. 2. A 15-keto-prostaglandin reductase, responsible for the breakdown of 13,14-dihydro-15-keto prostaglandins to 13,14-dihydro prostaglandins, was also present in human rectal mucosa. 3. Ulcerative colitis patients catabolized prostaglandins to the same extent as the control group. PGE1 was catabolized significantly better than PGF2 alpha. 4. 5-Aminosalicylic acid and sulphapyridine did not affect prostaglandin catabolism. Sulphasalazine, methylsulphasalazine, indomethacin, flurbiprofen, disodium cromoglycate, sodium salicylate and carbenoxolone sodium inhibited prostaglandin catabolism to the same extent in both groups. Salicylazosulphadimidine was a more potent inhibitor of PGE1 catabolism than of PGF2 alpha. 5. The increased prostaglandin synthesis reported for ulcerative colitis patients was not paralleled by increased catabolism, a fact possibly contributing to the accumulation of such compounds in the diseased state.

Adult↗
PubMed1990

HydroLink gel electrophoresis: rapid electroblotting of dsDNA.

Blotting and probing of DNA, RNA and proteins after electrophoresis is a powerful technique for the study of the structure and function of biomolecules. Key factors in successful blotting experiments are efficiency of transfer, maintenance of the resolution obtained during gel electrophoresis, accuracy of the probes used and sensitivity of the detection method. We have recently developed a system for the high performance resolution of DNA with 10-fold greater capacity for sample loads than agarose or polyacrylamide. In the present study, we describe conditions for the rapid (less than one hour) and quantitative electrotransfer of DNA in the 100-23,000-base pair range, with subsequent conditions for probing of transfer membranes using radioactive or biotinylated probes. Our results suggest complete maintenance of the high-resolution characteristic of HydroLink gel electrophoresis and potentially increased sensitivity due to the high loading capacity in HydroLink gel electrophoresis.

Autoradiography↗
PubMed1989

Nucleoside transport and proliferative rate in human thymocytes and lymphocytes.

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Isolation and characterization of a human telomere.

A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.

Animals↗
PubMed1989

Tracking bacterial DNA replication forks in vivo by pulsed field gel electrophoresis.

The location of chromosomal DNA replication forks was identified in synchronously replicating E. coli cultures by pulse labeling DNA at specific times with 14C-thymidine and following incorporation of radionucleotide into genomic Not I restriction fragments. This technique could be used to characterize chromosomal DNA replication, to characterize mutations which affect this process, to identify the location of DNA replication origins and termini as well as aid in the construction of macrorestriction maps. Here, we further characterize the DNA replication mutations divE and dnaK and preliminary characterize the genomic organization of E. coli isolate 15.

Chromosome Mapping↗
PubMed1989

Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.

Animals↗
PubMed1989

Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome.

Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.

Blotting, Southern↗
PubMed1989

The role of methylation in regulating the expression of the alpha-fetoprotein gene in developing rat liver and hepatoma cell lines.

We have examined four possible sites of methylation in the 5' flanking region of the alpha-fetoprotein (AFP) gene during liver development in the rat, paying particular attention to the neonatal period, in which AFP gene transcription changes rapidly. These sites are found in MspI/HpaII sites located at -4197, -3038, -2431, and +3 bp relative to the transcription start site. Three of these sites are associated with sequence regions important for the regulation of AFP gene transcription. We found that, in general, the 5' flanking region of the gene was methylated more in the adult liver than in the livers of fetal and neonatal rats. In addition, the degree of methylation of all four sites examined was increased in the adult liver. One of these sites showed increased methylation as AFP gene activity decreased, whereas the other became more methylated only after transcriptional activity of the gene had ceased. In particular, the site (+3 bp) just adjacent to the transcriptional initiation site of the gene was fully methylated in the adult liver. In various rat hepatoma and liver cell lines methylation of this same site showed a particularly close correlation with the amount of transcriptional activity of the AFP promoter in these cell lines. Treatment of the hepatoma and liver cell lines with dexamethasone, which influences AFP gene expression, did not result in any changes in methylation of these sites in the 5' flanking region.

Animals↗
PubMed1989

HydroLink gel electrophoresis (HLGE). II. Applications of a new polymer matrix to dsDNA analysis.

HydroLink materials represent a novel family of gels composed of unique polymer matrices. The applications of HydroLink to molecular biology and, specifically, to DNA technology have been carefully investigated. Our results indicate that the HydroLink matrix developed for double-stranded DNA (dsDNA) is an excellent tool for electrophoretic separations in fixed electric fields. Excellent linear resolution from 100 to 5000 base pairs is easily achieved with good resolution albeit non-linear from 6000 to 23000 base pairs. The broad range of separation in addition to increased mechanical strength of dsDNA HydroLink represents a distinct advantage over other matrices currently used in DNA electrophoretic analysis.

Base Composition↗
PubMed1989

HydroLink gel electrophoresis (HLGE). III. High DNA loading capacity and recovery of dsDNA.

Novel polymers have been prepared for high performance electrophoretic separations of double-stranded DNA (dsDNA). These materials are part of a family of HydroLink high performance electrophoresis polymers. A comparison of the resolving capabilities of dsDNA HydroLink gels to agarose and polyacrylamide separations has been described in an accompanying paper. In this study, we demonstrate that dsDNA HydroLink gels possess ten times the loading capacity of comparable polyacrylamide or agarose gels without compromise to resolution or biological integrity of the separated DNA. A simplified procedure for recovery of separated components is also described.

Base Composition↗