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Biomedical subjects

J S Smith

Publications and source records attributed to J S Smith.

At least 19 recordsLinked to original sources
PubMed1992

Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3).

We have examined the specificity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase-associated RNase H in removing the tRNA(Lys3) (-)-strand primer in vitro using a model substrate. This substrate represents an intermediate in the reverse transcription process where the tRNA(Lys3) primer has not yet been removed after (+)-strand strong stop DNA synthesis. The substrate consists of an RNA oligonucleotide corresponding to the 3'-terminal 17 nucleotides of the tRNA(Lys3) linked to U5 DNA and annealed to single-stranded DNA containing the U5 and the primer-binding site. Upon incubation with HIV-1 reverse transcriptase p66/p51 heterodimer, the minus-strand DNA product resulting from RNase H cleavage retained the 3'-rA from the model tRNA primer. Changing the 3'-terminal AMP of the model tRNA primer from rA to dA did not alter the RNase H cleavage site. Further, the retention of AMP was not dependent on recognition of adjacent U5 sequences or the CCA terminus of the model tRNA(Lys3). The synthetic RNA primer was released as an intact species by a single endonucleolytic cleavage 5' of the rA. The cleavage patterns of Moloney murine leukemia virus and avian myoblastosis virus RNase H activities on the HIV-1 model substrate were more heterogeneous compared to HIV-1 RNase H. This specificity of HIV-1 RNase H would result in linear DNA molecules with a single rA at the U5 terminus and would provide two bases adjacent to the conserved CA dinucleotide to be cleaved away during the integration process.

Base Sequence↗
PubMed1992

Anion-proton cotransport through the human red blood cell band 3 protein. Role of glutamate 681.

The band 3 protein of the human red blood cell membrane contains a glutamate residue that must be protonated in order for divalent (SO4=) anion transport to take place at an appreciable rate. The carboxyl side chain on this glutamate residue can be converted to the primary alcohol by treatment of intact cells with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate) followed by reductive cleavage with BH4-. Edman degradation of CNBr fragments from band 3 labeled in intact cells with Woodward's reagent K and [3H]BH4- showed that Glu681 is heavily labeled under conditions in which Cl- exchange is inhibited, SO4= exchange is accelerated, and Cl- conductance is accelerated. No other glutamate residue in band 3 is detectably labeled under the conditions of these experiments, as demonstrated either by Edman degradation or by the lack of label in major known proteolytic fragments. It is concluded that Glu681 is the binding site for the H+ that is transported with SO4= during band 3-catalyzed H+/SO4= cotransport. This residue is conserved among all species of red cell band 3 (AE1) as well as the related proteins AE2 and AE3. Glu681 is the first amino acid residue in band 3 which has been identified as a binding site for a transported substrate (H+). The functional characteristics of this residue suggest that it lies within the transport pathway and can be alternately exposed to the intracellular and extracellular media.

Amino Acid Sequence↗
PubMed1992

Laser recanalization versus endoscopic intubation in the palliation of malignant dysphagia: a randomized prospective study.

Forty patients with histologically confirmed malignant dysphagia were randomized to either endoscopic intubation or laser recanalization. Age, sex, tumour histology and site were evenly distributed between the groups. Results were analysed on an 'intention to treat' basis. All patients treated by laser achieved patency; there was one failed intubation. The best swallowing grade achieved was significantly better with laser recanalization (median 4 (range 3-4)) than with intubation (median 3 (range 2-4)) (P < 0.001). The median survival was 21.5 (range 4-62) weeks in the group receiving laser treatment, compared with 14.5 (range 7-102) weeks in the intubated group (P = 0.09). The median inpatient stay as a proportion of survival time was 14 per cent in the group receiving laser treatment compared with 15 per cent in the intubated group (P > 0.05). The median weight loss was less in the laser-treated patients (2.0 (range 2-8) versus 3.0 (range 0-10) kg, P = 0.04). These results indicate that laser recanalization provides better palliation of dysphagia than does intubation, but this is not reflected in an improvement in survival time.

Adult↗
PubMed1992

The effect of environmental temperature and humidity on 24 h energy expenditure in men.

The effects of environmental temperature and humidity and their interaction on 24 h energy expenditure were measured using whole-body indirect calorimetry in eight normal-weight young men who wore standardized light clothing and followed a controlled activity regimen. A randomized-block experimental design was used, with temperature effects assessed by measurements at 20, 23, 26 and 30 degrees, while humidity was altered from ambient (50-65% relative humidity) to high (80-93% relative humidity) at 20 and 30 degrees only. There was no significant effect of humidity on 24 h energy expenditure at the two extreme temperatures in this range, though when periods of sleep and exercise were excluded the energy expenditure at high humidity was significantly higher than that ambient humidity (P < 0.02). The effect of temperature at ambient humidity levels showed lower values at 23 and 26 degrees than at 20 and 30 degrees (P < 0.02). The effect of temperature was not equally apparent in all components of the 24 h energy expenditure, as sleeping metabolic rate and the energy cost of walking and cycling showed no significant effect of temperature over this range. This raises the possibility that the effects of temperature are attributable to behavioural changes during the waking portion of the day rather than any non-shivering thermogenic mechanisms at tissue level.

Adult↗
PubMed1992

Effect of skim milk supplementation on blood cholesterol concentration, blood pressure, and triglycerides in a free-living human population.

In 82 subjects, aged 21-73, we studied the effect of skim milk supplementation on serum cholesterol concentration, blood pressure, and serum triglyceride level. The study involved a 1-week pretreatment baseline period followed by 8 weeks of milk supplementation. Sixty-four people were designated to a test group and 18 people were placed in a seasonal index group. The study was designed as a free-living trial, i.e., participants were requested to maintain their normal lifestyles, including dietary pattern, except for the supplementation of one quart of 2% solids-not-fat fortified skim milk to the daily diet in the test group. Supplemental milk treatment was associated with a 6.6% reduction (p = 0.0004) of serum cholesterol in the high cholesterol (greater than or equal to 190 mg/dl) test subgroup within the first 4 weeks. No change was noted in serum cholesterol in the low-cholesterol (less than 190 mg/dl) subgroup throughout the study. Body weight and seasonal variation of blood cholesterol did not significantly influence serum cholesterol levels. Reduction (p = 0.0140) in percentage of calories from fat in the high-cholesterol subgroup was not correlated with the decrease in serum cholesterol in this test subgroup. Reductions in systolic and diastolic blood pressure occurred in the test subgroups; the low-cholesterol subgroup had a greater reduction (p = 0.0002) in diastolic blood pressure than the high-cholesterol group (p = 0.0049). Milk supplementation was associated with reduction (p = 0.0370) in serum triglycerides in the high-cholesterol subgroup.

Adult↗
PubMed1992

Carba-prostacyclin inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation in sensitive murine epidermal JB6 cells.

The ability of carba-prostacyclin (cPGI2), a stable analog of prostacyclin (PGI2), to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 cells was investigated. JB6 cells sensitive (P+) and resistant (P-) to TPA-induced transformation to anchorage-independent growth were plated in soft agar in the presence or absence of cPGI2 for 14-21 days. Transformation frequencies were determined by recording colony numbers. cPGI2 was found to inhibit TPA-induced transformation of P+ cells in a dose-dependent fashion with 1 microM cPGI2 producing approximately 50% inhibition of colonies in soft agar. Our findings are consistent with the hypothesis that TPA-induced transformation in JB6 cell variants is mediated by PGI2 via regulation of adenylate cyclase activity and cAMP accumulation, with resultant inhibition of expression of the transformed phenotype, reflected in anchorage-independent growth.

Animals↗
PubMed1992

JB6 murine epidermal cell lines sensitive and resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation exhibit differential arachidonic acid metabolism in response to TPA and the calcium ionophore A23187.

In a study of arachidonic acid metabolism in murine epidermal JB6 cells, promoter-sensitive (P+) and promoter-resistant (P-) variants, labeled with [3H]arachidonic acid, were treated successively with 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore A23187. Released radiolabel was separated by HPLC and identified by coelution of standards. Prostacyclin release was then quantified by radioimmunoassay for 6-keto prostaglandin (PG)F1 alpha. A23187 alone resulted in a small but significant enhanced release of radiolabel from both cell variants (0.7 +/- 0.2% for P- and 0.6 +/- 0.3% for P+ cells; mean +/- SD). Treatment with TPA and subsequent treatment with A23187 resulted in a synergistically enhanced release of radiolabel from both cell variants (4.1 +/- 0.8% for P- and 3.4 +/- 0.9% for P+ cells) relative to that with either agent alone. Although the predominant product for each treatment regimen was prostaglandin E2 (PGE2), the TPA-resistant cells (P-) released significantly more 6-keto PGF1 alpha, a stable breakdown product of PGI2, than did the TPA-sensitive (P+) cells. These results indicate differential arachidonic acid metabolism between JB6 cell variants resistant and sensitive to TPA-induced transformation.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis.

Nucleotide sequence analysis of a 200-bp region of the nucleoprotein (N) gene of rabies virus differentiated unique genetic groups of rabies virus from samples collected in areas where dog rabies is enzootic in Asia, Africa, Europe, and the Americas. Patterns of nucleotide sequence identified for an outbreak area were conserved in samples collected over three decades. Epidemiologic relationships among isolates were determined by patterns of conserved nucleotide sequence, and the degree of sequence divergence between samples from separate outbreak areas were measured. This approach suggested that a historical reconstruction of events leading to the introduction of rabies into an area would be possible. In this broader view of rabies epidemiology, the cultural legacy of European exploration and colonization may have also included zoonotic disease.

Amino Acid Sequence↗
PubMed1992

Prospective validation of criteria, including age, for safe, nonsurgical management of the ruptured spleen.

One hundred twelve cases of blunt splenic rupture were prospectively entered (October 1987-October 1991) into surgical or nonsurgical management groups using these criteria for the nonsurgical group: hemodynamic stability+age less than 55 years+CT scan appearance of grade I, II, or III injury+absence of concomitant injuries precluding abdominal assessment+absence of other documented abdominal injuries. All ages were included and AAST injury scaling was used. Patients were grouped from the trauma room. The surgical treatment group included 66 patients (49 splenectomies, 17 splenorraphies). These patients were generally older and more severely injured, required more transfused blood, and a longer ICU stay. The nonsurgical group included 46 patients with 33 older than 14 years. There were 3 patients over the age of 55 years inappropriately included in this group, and nonsurgical therapy failed in all three. Statistical analysis (chi 2) showed that more splenic injuries were observed and more spleens were saved with these criteria applied prospectively compared with a previous retrospective series in the same institution. Our series had a success rate of 93%, and validates the criteria used for safe, nonsurgical management of the ruptured spleen and adds a new criterion: a maximum age of 55 years.

Abbreviated Injury Scale↗
PubMed1992

Amygdalo-hippocampectomy for pathological aggression.

Two patients are reported, one with severe brain damage and epilepsy, and the other with limbic epilepsy, who were treated with unilateral microsurgical amygdalo-hippocampectomy for the control of rage and aggression. Both had significant improvement in their aggressiveness, and the second patient also improved in the frequency of his seizures and psychotic episodes. The significance of these observations for our understanding of the morphophysiological basis of rage and aggression is discussed.

Adult↗
PubMed1991

N-acylation of Aplysia egg-laying hormone with biotin. Characterization of bioactive and inactive derivatives.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

Acylation↗
PubMed1991

Shaw-like rat brain potassium channel cDNA's with divergent 3' ends.

The complete amino acid sequence of a potassium channel protein of rat brain, Kv3.2b, plus a partial sequence of a related channel, Kv3.2c, are deduced from molecular cloning of the respective cDNA's. Kv3.2b and Kv3.2c share extensive amino acid sequence identity with a previously identified channel, RKShIIIA[1], before diverging to unique carboxy termini. Probes specific for Kv3.2b and RKShIIIA detect similarly sized mRNA's on Northern blots. These two proteins are encoded by a single gene based on genomic Southern blotting, and therefore arise by alternative splicing. In vitro transcribed mRNA for Kv3.2b induces the expression of outward K+ currents in Xenopus oocytes under voltage-clamp conditions.

Amino Acid Sequence↗
PubMed1991

Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization.

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

Amino Acids↗
PubMed1991

Biotinylated peptides/proteins. II. Identification of biotinylated lysyl phenylthiohydantoins.

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.

Amino Acid Sequence↗
PubMed1991

Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.

Amino Acid Sequence↗