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Biomedical subjects

S C King

Publications and source records attributed to S C King.

At least 19 recordsLinked to original sources
PubMed1998

Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8-9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals.

The gab permease (GabP) catalyses transport of GABA (4-aminobutyrate) into Escherichia coli. Although GabP can recognize and transport many GABA analogues that exhibit activity at GABAergic synapses in the nervous system, the protein domains responsible for these transport and ligand recognition properties have not been studied. Here we report that an amphipathic domain extending through putative transmembrane helix 8 and into the adjoining cytoplasmic region (loop 8-9) contains a critical 20 residue zone within which mutagenesis of polar amino acids has a deleterious effect on [3H]GABA transport activity. This functionally important amphipathic domain is found to be highly conserved in the many APC family transporters that are homologous to GabP. And even though members of the GAT family of GABA transporters from the animal nervous system are not homologous to GabP, an analogous amphipathic structure is found in their loop 8-9 region. These results and observations suggest: (1) that the consensus amphipathic region (CAR) in the putative helix 8 and loop 8-9 region of GabP has functional significance, and (2) that nature has repeatedly used this CAR in transporters from bacteria to mammals.

Amino Acid Sequence↗
PubMed1996

High-dose, hyperfractionated, accelerated radiotherapy using a concurrent boost for the treatment of nonsmall cell lung cancer: unusual toxicity and promising early results.

PURPOSE: The treatment of nonsmall cell lung cancer (NSCLC) with conventional radiotherapy (RT) results in inadequate local tumor control and survival. We report results of a Phase II trial designed to treat patients with a significantly increased total dose administered in a reduced overall treatment time using a hyperfractionated, accelerated treatment schedule with a concurrent boost technique. METHODS AND MATERIALS: A total of 49 patients with unresectable Stage IIIA/IIIB (38 patients) or medically inoperable Stage I/II (11 patients) NSCLC were prospectively enrolled in this protocol. Radiation therapy was administered twice daily, 5 days/week with > 6 h between each treatment. The primary tumor and adjacent enlarged lymph nodes were treated to a total dose of 73.6 Gy in 46 fractions of 1.6 Gy each. Using a concurrent boost technique, electively irradiated nodal regions were simultaneously treated with a dose of 1.25 Gy/fraction for the first 36 fractions to a total dose of 45 Gy. RESULTS: Median survival for the entire group of 49 patients is 15.3 months. Actuarial survival at 2 years is 46%: 60% for 11 Stage I/II patients, 55% for 21 Stage IIIA patients, and 26% for 17 Stage IIIB patients. The actuarial rate of freedom from local progression at 2 years is 64% for the entire group of 49 patients: 62% for Stage I/II patients, 70% for Stage IIIA patients, and 55% for Stage IIIB patients. Patients who underwent serial bronchoscopic reevaluation (4 Stage I/II, 8 Stage IIIA, and 6 Stage IIIB) have an actuarial rate of local control of 71% at 2 years. The median total treatment time was 32 days. Nine of 49 patients (18%) experienced Grade III acute esophageal toxicity. The 2-year actuarial risk of Grade III or greater late toxicity is 30%. The 2-year actuarial rate of severe-late pulmonary and skin-subcutaneous toxicity is 20% and 15%, respectively. CONCLUSION: This treatment regimen administers a substantially higher biologically effective dose compared with conventional and pure hyperfractionation treatment schedules. The overall rate of acute and late toxicity was acceptable. Preliminary rates of overall survival and local control and freedom from local progression compare favorably to results reported with pure hyperfractionated radiotherapy and chemoradiotherapy.

Aged↗
PubMed1996

Substrate specificity of the Escherichia coli 4-aminobutyrate carrier encoded by gabP. Uptake and counterflow of structurally diverse molecules.

Transport of 4-aminobutyrate into Escherichia coli is catalyzed by gab permease (GabP). Although published studies show that GabP is relatively specific, recognizing the common alpha-amino acids with low affinity, recent work from this laboratory indicates that a number of synthetic compounds are high affinity transport inhibitors (50% inhibition at 5-100 microM). Here we present evidence that many of these structurally heterogeneous compounds not only inhibit transport but also function as alternative GabP substrates (i.e. a set of observations inconsistent with the idea that the core of the GabP transport channel exhibits rigid structural specificity for the native substrate, 4-aminobutyrate.

Aminobutyrates↗
PubMed1996

Cold blood and clinical research during World War I.

Therapeutic transfusion was not a common procedure at the turn of the century. Although its safety was enhanced by the discovery of blood groups and preinfusion testing in the decade prior to World War I, techniques and indications remained cumbersome and clinically naive. By 1916, a stable Western Front, an efficient line of transport, and the operative requirements of a large number of wounded demonstrated the futility of pharmacotherapy or saline infusion for traumatic shock. In the same year, Rous and Turner at the Rockefeller Institute developed a preservative solution for whole blood. Rous' student, Dr. O.H. Robertson, arrived in France with Base Hospital 5 in June 1917 during a period of growing recognition by military surgeons that transfused blood was an effective therapy, although a practical delivery system was not available. Over the next 8 months, Robertson clinically tested a transfusion technique using preserved blood in glass jars carried to the front in specially designed cases. The method was accepted immediately, and by the Armistice transfusion was used frequently on the front line or during the perioperative period. The accessibility of preserved blood with an efficient transfusion system reinforced the introduction of "resuscitation teams" attached to Casualty Clearing Hospitals for the specialized management of traumatic shock. Robertson's success at technical innovation during World War I associated with a large clinical population resulted in the development of the indications and procedures for modern transfusion therapy.

Blood Transfusion↗
PubMed1995

Ligand recognition properties of the Escherichia coli 4-aminobutyrate transporter encoded by gabP. Specificity of Gab permease for heterocyclic inhibitors.

4-aminobutyrate metabolism in Escherichia coli begins with transport across the cytoplasmic membrane via the GabP, which is encoded by gabP. Although GabP is specific and exhibits poor affinity for many cellular constituents such as the alpha-amino acids, the range of compounds recognized with high affinity has yet to be investigated. In order to address this gap in knowledge, we developed a gabP-negative host strain, which permits evaluation of test compounds for inhibitory effects on cloned GabP (expression inducible by isopropyl-1-thio-beta-D-galactopyranoside). Using this inducible expression system, three structurally distinct categories of high affinity transport inhibitor were identified. The structural dissimilarity of these inhibitors significantly alters our view of ligand recognition by GabP. Any complete model must now account for the observation that inhibition of 4-aminobutyrate transport can be mediated either (i) by open chain analogs of 4-aminobutyrate, (ii) by cyclic amino acid analogs, or (iii) by planar heterocyclic compounds lacking a carboxyl group. Such results do not support a previously sustainable view of GabP that features a restrictive ligand recognition domain, unable to accommodate structures that differ very much from the native substrate, 4-aminobutyrate.

Base Sequence↗
PubMed1995

The efficacy of postprostatectomy radiotherapy in patients with an isolated elevation of serum prostate-specific antigen.

PURPOSE: To determine the efficacy of radiotherapy (RT) in patients with an isolated elevation of prostate specific antigen (PSA) after radical prostatectomy (RP). METHODS AND MATERIALS: Between November 1987 and May 1993, 53 patients with adenocarcinoma of the prostate were referred for pelvic RT for an elevated PSA after RP. No patient had clinically or radiographically apparent local or distant disease, nor had an undergone prior androgen ablation. Patients received a median dose of 61.2 Gy to the prostatic bed. An undetectable PSA was required to be considered disease free (NED). Univariate and multivariate analyses were performed to identify factors predictive of becoming disease free after RT. RESULTS: The median follow-up was 15 months. Of the 53 patients, 16 (30%) became NED after RT and 15 (28%) had a declining (n = 11) or stable (n = 4) PSA at last evaluation. The median time after RT to achieve an undetectable PSA was 9.3 months. At 12 and 24 months, the actuarial disease-free survival was 30 and 23%, respectively; actuarial progression-free survival was 71 and 26%, respectively. By univariate analysis, the last PSA level before RT (i.e., the pre-RT PSA) and an undetectable PSA after RP were significant predictors of becoming NED (p = 0.0001 and 0.04, respectively). However, on multivariate analysis, only the pre-RT PSA remained significant (p = 0.01). The mean pre-RT PSA differed significantly between patients who became NED after RT and those who did not (1.5 +/- 0.2 ng/ml vs. 7.6 +/- 1.6 ng/ml, respectively; p = 0.018). Fourteen of 27 (52%) patients with pre-RT PSA levels < or = 2.5 ng/ml became NED, vs. only 2 of 26 (8%) patients with higher levels. There were no severe acute or late sequelae of RT. CONCLUSION: Prostatic-bed RT for an elevated serum PSA after RP is most effective in patients with a pre-RT PSA < or = 2.5 ng/ml. Patients with significantly higher PSA values are unlikely to benefit from RT, possibly due to the presence of occult distant metastases. The optimal therapy for this latter group remains to be determined.

Aged↗
PubMed1995

Molecular characterization of further dystrophin gene microsatellites.

Microsatellites of the dystrophin gene have been used extensively in the genetic analysis of Duchenne and Becker muscular dystrophy families. The microsatellites that have been reported to date are clustered within disparate regions of the dystrophin gene, specifically at the 5'-end and in the central rod-domain. YACs encompassing the gene were screened for further microsatellites to improve the density of available genetic markers. Four microsatellites were localized to defined regions of the dystrophin gene by the analysis of patient DNA samples, somatic cell hybrids and YACs. In addition, varying combinations of microsatellite loci were amplified in multiplex PCRs, which complement those loci that have been studied to date.

Base Sequence↗
PubMed1995

Frequency of micronucleated-binucleated lymphocytes is not significantly affected by the harvest time following G0 exposure to X-radiation.

Whole blood from two male individuals was X-irradiated using a linear accelerator at 200 cGy/min to give a total exposure of 300 cGy. Lymphocytes were cultured using standard techniques with the addition of 3 micrograms/ml cytochalasin B at 26 h to produce binucleation through the inhibition of cytokinesis for the scoring of micronuclei after the first nuclear division. Replicate cultures from each individual were harvested at 48, 68, 70, 72, 74, 76, 92, 94, 96, 98 and 100 h postinitiation using a cytocentrifuge. Slides were stained with acridine orange, and binucleated cells were scored for the presence of micronuclei. There were no statistically significant differences in the frequency of micronucleated binucleates between replicate cultures, between individuals, or among cultures harvested from 48 to 100 h postinitiation. This indicates that the phytohaemagglutinin-stimulated lymphocytes are a relatively homogeneous population of cells with respect to X-radiation-induced chromosome damage. In addition, these data show that for determining the frequency of micronuclei in lymphocytes irradiated in G0, the harvest time (up to at least 100 h postinitiation) is not critical as long as analysis is confined to the first mitosis after irradiation (i.e. the binucleated cells).

Adult↗
PubMed1995

Oxygen desaturation on swallowing as a potential marker of aspiration in acute stroke.

We have assessed the measurement of oxygen saturation (SaO2) as a means of detecting aspiration in patients with stroke. For 10 weeks all acute stroke [AS] admissions were seen within 48 hours. Basal SaO2 was measured by pulse oximetry. Patients swallowed 10ml water while sitting up and SaO2 was noted for 2 minutes. Two control groups [young, fit (YF) and inpatient age- and sex-matched, non-neurological disease (IP)] underwent the same assessment. AS subjects underwent independent assessment of swallowing by a speech and language therapist (SLT). Exclusion criteria comprised impaired consciousness, other neurological disease and chest infection. Forty-nine AS subjects [20 men; aged 46-93 (mean 71) years], 55 YF [26 men; aged 18-55 (mean 32) years] and 65 IP [28 men; aged 53-96 (mean 71) years] were studied. Mean (SD) SaO2 fall in AS subjects [2.6 (2.9)%)] was significantly more than in YF [1.1 (0.8)%] or IP [1.1 (0.9)%]. The lower 95% confidence limit for variation in SaO2 did not differ between YF and IP (3.0% 'fall'); 19 (39%) AS subjects desaturated below this 95% lower confidence limit. Mean (SD) SaO2 fall was significantly more in SLT-graded 'aspirators' [4.6 (2.7)%] than 'nonaspirators' [1.4 (1.0)%]. We conclude that (1) a fall in SaO2 on swallowing fluid is common in patients with acute stroke; (2) the presence or absence of desaturation agrees statistically with SLT assessment of aspiration; (3) SaO2 measures may aid bedside assessment of swallowing.

Adolescent↗
PubMed1994

Clinical use of a concomitant boost technique using a gypsum compensator.

PURPOSE: To develop a clinical procedure to treat field within a field (concomitant boost) portals with a single compensated field. METHODS AND MATERIALS: An ordinary manual cerrobend block former was used to produce styrofoam molds from simulator film data. A special gypsum compound was poured into the molds. The compensator block is independently mounted to the treatment machine via a custom-made compensator holder. RESULTS: Measurements confirm that the inhomogeneous dose distribution has been reliably delivered via this technique. The accuracy of placement of the high dose region is sufficient for clinical use. CONCLUSION: The procedure enables the concomitant boost effect to be easily implemented in the clinic without increasing clinical setup time.

Calcium Sulfate↗
PubMed1994

Prognostic importance of restaging gallium scans following induction chemotherapy for advanced Hodgkin's disease.

PURPOSE: This study was intended to assess the ability of restaging gallium scanning to distinguish between patients with residual radiographic abnormalities who still have active Hodgkin's disease (HD) and those who are truly complete responders. Early identification of the former patients might increase the success of secondary salvage therapy. MATERIALS AND METHODS: The charts of all patients with advanced HD treated at Duke University Medical Center during the years 1983 to 1991 who underwent gallium scanning were reviewed. Thirty-three patients were identified who had gallium scans performed as part of restaging following induction combination chemotherapy; no patient had other signs or symptoms of active or progressive HD. RESULTS: Thirteen of 33 patients had positive restaging gallium scans; 20 patients had negative scans. The 4-year actuarial relapse-free survival (RFS) rate was 75% for patients with negative restaging gallium scans compared with 8% for those with positive restaging scans (P < .001). The 4-year actuarial overall survival (OS) rate was 100% for those with negative scans compared with 51% for gallium-positive patients (P = .001). Twenty-four patients had residual chest x-ray or computed tomographic scan abnormalities. Calculated negative and positive predictive values for gallium scanning are 92% and 90%, respectively, compared with values of 48% and 83% for computed tomographic scanning. CONCLUSION: Restaging gallium scans separate complete responders from induction failures with a high degree of accuracy. Gallium scanning is clearly superior to computed tomography in this regard. Patients with advanced HD who have positive restaging gallium scans after induction chemotherapy should be classified as induction failures and are highly unlikely to be cured with involved-field low-dose radiotherapy.

Actuarial Analysis↗
PubMed1993

Molecular cloning and structure of the human (GABATHG) GABA transporter gene.

A cDNA molecule encoding the human GABA transporter was synthesized by means of polymerase chain reaction (PCR) technique and used as probe for selecting a human genomic DNA fragment encoding GABA transporter. A positive clone harboring the whole gene was obtained from a human lymphocyte genomic library through utilizing the genomic 'walking' technique. The clone, designated as pHGAT, harbours a DNA fragment of about 39 kb in length inserted into the BamHI site in cosmid pWE15. The gene covers about 25 kb in length and is constituted by four EcoRI restricted fragments which are 13.7 kb, 3.1 kb, 4.2 kb and 7.2 kb long, respectively. The genomic clone contains 15 introns, including two introns prior to the initiator methionine (i.e., the translation start site is in exon 3). Eleven exons encode the twelve transmembrane regions in the transporter protein. Thus as in the case for a number of other membrane proteins, there appears to be a strong tendency for the putative transmembrane domains to be encoded by separate exons. It is noted that the structure of the human GABA transporter gene reported here differs from the mouse gene which is contains 12 introns.

Base Sequence↗
PubMed1992

Differential regulation of the imipramine-sensitive serotonin transporter by cAMP in human JAr choriocarcinoma cells, rat PC12 pheochromocytoma cells, and C33-14-B1 transgenic mouse fibroblast cells.

The imipramine-sensitive serotonin transporter appears to be the receptor for clinically important antidepressants. Some studies suggest that this protein may fall under the influence of abnormal and as yet uncharacterized regulatory effects during depressive illness. Despite these putative disease-related effects, regulation has never been demonstrated in either the platelet or synaptosome model systems. Here we demonstrate for the first time that the imipramine-sensitive serotonin transport activity in either JAr choriocarcinoma or PC12 pheochromocytoma cell lines is subject to regulation by cAMP. Unexpectedly, the regulatory effect is opposite in the two cases, causing stimulation in JAr (increased Vmax) and inhibition in PC12 (kinetically complex). Appearance of these kinetic effects lagged 15 to 20 hours behind peak cAMP levels. The results are consistent with the interesting possibility that different tissues may express isoforms of the recently cloned serotonin transporter cDNAs. We suggest, therefore, that JAr and PC12 are attractive models in which to pursue detailed analysis of serotonin transport and its manner of regulation by cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Amino acid substitution in the lactose carrier protein with the use of amber suppressors.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.

Amino Acid Sequence↗
PubMed1991

The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli.

The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N. These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively. The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype. Eleven melibiose-positive revertants of p020-K358T were isolated. All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr). Twelve melibiose-positive revertants of pAE43-D237N were isolated. Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237. We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge.

Amino Acid Sequence↗