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Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 145 records · Page 8Linked to original sources
PubMed1992

High speed automated DNA sequencing in ultrathin slab gels.

We have developed a high speed instrument for automated DNA sequence analysis. The apparatus employs laser excitation and a cooled CCD detector for the parallel detection of up to 18 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (50-100 microns) denaturing polyacrylamide gels. Four hundred and fifty bases of sequence information is obtained from 100 ng of M13 template DNA in less than one hour, corresponding to an overall instrument throughput of over 8000 bases/hr.

Autoanalysis↗
PubMed1992

Long-term measures of electrode impedance and auditory thresholds for the Ineraid cochlear implant.

Measures of electrode impedance and of detection thresholds for electrical stimuli were extracted from the records of patients implanted with the Ineraid cochlear prosthesis. An analysis of impedance measures, obtained at 1, 12, 24, and 36 months after surgery, demonstrated (a) a significant decrease in impedance over the first year for electrodes that carried current and (b) significant increases in impedance at 24 and 36 months for electrodes that did not carry current. An analysis of detection thresholds, obtained at the same times as the impedance measures, demonstrated that averaged thresholds for the current-carrying electrodes varied no more than 0.5 dB over the 3-year period. These results support the conclusion that stimulation with the Ineraid device does not produce deleterious changes in the electrodes or in the target neural tissue.

Acoustic Impedance Tests↗
PubMed1992

Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J.

The host ranges of the Rous sarcoma virus (RSV) pseudotype RSV(HPRS-103) of a novel avian leukosis virus (ALV), strain HPRS-103, and representative RSV pseudotypes of subgroups A to F, have been determined in embryo fibroblasts from 12 avian species. Domestic fowl, red jungle fowl, Sonnerat's jungle fowl and turkey were susceptible to infection by RSV(HPRS-103); ring-necked pheasant, Japanese green pheasant, golden pheasant, Japanese quail, guinea-fowl, Peking duck, Muscovy duck and goose were resistant. The host range pattern of RSV(HPRS-103) differs from those of viruses of subgroups A to G and I, and provides support for placing the HPRS-103 strain of ALV in a new envelope subgroup, designated J.

Animals↗
PubMed1992

Second cancers in long-term survivors of Ewing's sarcoma.

Previous reports suggest an increased risk of a second cancer, primarily osteosarcoma, in survivors of Ewing's sarcoma. In a retrospective review of 25 long-term irradiated survivors of Ewing's sarcoma, the incidence of second cancers was determined. The patients were free of disease for more than three years (except for one patient who developed a second cancer 2.5 years after diagnosis), with a median follow-up period of 7.6 years. All received megavoltage radiation to the primary tumor. Twenty-four of the 25 patients were treated with chemotherapy. Second cancers developed in two patients. Acute myelogenous leukemia (AML) developed in a seven-year-old 15 months after treatment. An osteosarcoma developed within an irradiated field in a 13-year-old three years after treatment. The actuarial risk of developing a second cancer at five years is 8% whereas the actuarial risk of developing a bone sarcoma is 4%. Genetic factors may play a role in the development of AML in patients with Ewing's sarcoma. Megavoltage radiation, particularly doses greater than 60 Gy, as well as alkylating agent chemotherapy may contribute to the risk for bone sarcoma. The risk of a second cancer after successful treatment of Ewing's sarcoma is similar to that expected for survivors of all childhood cancers.

Actuarial Analysis↗
PubMed1991

Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.

Base Sequence↗
PubMed1991

A universal method for the direct cloning of PCR amplified nucleic acid.

We have devised a simple, universal cloning strategy that permits the direct ligation of PCR amplified nucleic acid to a compatible vector preparation. The method does not require that special restriction sites or additional sequences be appended onto the amplification primers, nor the use of restriction endonucleases, modifying enzymes, or any purification procedures. This approach takes advantage of the single 3' deoxyadenylate extension that Thermus aquaticus, Thermus flavus, and Thermococcus litoralis DNA polymerases add to the termini of amplified nucleic acid. A new type of plasmid was constructed that allows the preparation of ends containing a single complementary 3' deoxythymidylate extension. Cloning PCR products by this method is approximately 50 times more efficient than blunt-ended ligation reactions. This direct PCR ligation technique has been engineered in-phase with a truncated lacZ gene to facilitate the discrimination of recombinants from non-recombinants. We have applied this method to directly clone amplified RNA and DNA from as little as 1 microliter of a PCR reaction, without prior modification or purification steps.

Base Sequence↗
PubMed1991

Allergic conjunctivitis: a survey of new antihistamines.

The objective of this study was to determine the therapeutic value of a wide variety of H1 antihistamines for potential ophthalmic use by performing ocular toxicity and efficacy tests in rabbits and humans. Fourteen antihistamines were formulated into ophthalmic preparations and were screened in the rabbit model; of these, thirteen were preliminary evaluated for toxicity and efficacy in humans. Based on comfort and efficacy, four (pheniramine, chlorpheniramine, dexbrompheniramine and pyrilamine) were selected for more extensive dose response and efficacy testing. 0.3% chlorpheniramine, dexbrompheniramine, pyrilamine and pheniramine significantly reduced histamine-induced itching (p less than or equal to 0.01 for each drug) and conjunctival injection (p less than or equal to 0.02 for each drug), when compared to contralateral eyes receiving PBS. When compared to 0.3% pheniramine in the fellow eye (mean difference score = 0.79 +/- 0.21), 0.3% chlorpheniramine (1.5 +/- 0.22; p = 0.04) and 0.3% dexbrompheniramine (1.71 +/- 0.18; p = 0.01) were superior in decreasing histamine-induced itching. Dose-response curves demonstrated that 0.4% and 0.5% pheniramine were statistically superior to 0.3% in relieving itching and redness. Compared to 0.3% pheniramine, 0.1% and 0.2% chlorpheniramine and 0.2% pyrilamine were statistically superior in inhibiting redness and itching. The results of this study suggest that 0.1%, 0.2% and 0.3% chlorpheniramine, 0.3% dexbrompheniramine, 0.2% pyrilamine, and 0.4% and 0.5% pheniramine were effective in relieving the itching and conjunctival injection associated with topically applied histamine. These seven formulations should be considered as possible new preparations for use as ophthalmic antihistamines and may warrant further evaluation.

Animals↗
PubMed1991

Long-term scintigraphic appearance of extremities following bone tumor resection and allograft reconstruction.

The authors retrospectively reviewed the Tc-99m medronate scan findings in six patients who had no evidence of metastatic disease following en bloc resection of a primary osteosarcoma and subsequent limb reconstruction using allograft bone. Persistently increased radionuclide uptake was noted at the junction between the host bone and the graft, while the graft cortical bone showed persistently decreased activity. Radionuclide uptake at the periphery of the graft varied. Over time the youngest patient in the series had increasingly normal scan findings.

Adolescent↗
PubMed1991

Sodium-dependent magnesium uptake by ferret red cells.

1. Magnesium uptake can be measured in ferret red cells incubated in media containing more than 1 mM-magnesium. Uptake is substantially increased if the sodium concentration in the medium is reduced. 2. Magnesium uptake is half-maximally activated by 0.37 mM-external magnesium when the external sodium concentration is 5 mM. Increasing the external sodium concentration increases the magnesium concentration needed to activate the system. 3. Magnesium uptake is increased by reducing the external sodium concentration. Uptake is half-maximum at sodium concentrations of 17, 22 and 62 nM when the external magnesium concentrations are 2, 5 and 10 mM respectively. 4. Replacement of external sodium with choline does not affect the membrane potential of ferret red cells over a 45 min period. 5. Magnesium uptake from media containing 5 mM-sodium is inhibited by amiloride, quinidine and imipramine. It is not affected by ouabain or bumetanide. Vanadate stimulates magnesium uptake but has no effect on magnesium efflux. 6. When cell ATP content is reduced to 19 mumol (1 cell)-1 by incubating cells for 3 h with 2-deoxyglucose, magnesium uptake falls by 50% in the presence of 5 mM-sodium and is completely abolished in the presence of 145 mM-sodium. Some of the inhibition may be due to the increase in intracellular ionized magnesium concentration ([Mg2+]i) from 0.7 to 1.0 mM which occurs under these conditions. 7. Magnesium uptake can be driven against a substantial electrochemical gradient if the external sodium concentration is reduced sufficiently. 8. These findings are discussed in terms of several possible models for magnesium transport. It is concluded that the majority of magnesium uptake observed in low-sodium media is via sodium-magnesium antiport. A small portion of uptake is through a parallel leak pathway. It is believed that the antiport is responsible for maintaining [Mg2+]i below electrochemical equilibrium in these cells at physiological external sodium concentration. Thus in ferret red cells the direction of magnesium transport can be reversed by reversing the sodium gradient.

Adenosine Triphosphate↗
PubMed1991

Incidence and management of secondary malignancies in patients with retinoblastoma and Ewing's sarcoma.

Childhood cancer survivors at highest risk of developing a secondary malignancy are those with hereditary retinoblastoma. The majority of such secondary cancers will be sarcomas, most commonly of bone. One-third of these occur outside a typical radiation field, commonly in an extremity. Bone sarcoma is also the most commonly reported secondary cancer to develop among survivors of Ewing's sarcoma. In this group, radiation doses greater than 60 Gy as well as alkylating agent chemotherapy have been identified as contributors to the increased risk. The prognosis for patients with a secondary sarcoma has been poor, with few cures reported to date. However, an aggressive, combined modality approach, including radical resection, postoperative radiation, and adjuvant chemotherapy, may improve the survival rate.

Adolescent↗
PubMed1991

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template.

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.

Bacteriophages↗
PubMed1990

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.

Affinity Labels↗
PubMed1990

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma.

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.

Animals↗
PubMed1990

High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.

Base Sequence↗