Long-term adverse effects associated with isolated below-knee deep-vein thrombosis: a 10-year follow-up study.

AIM: To assess the effect of the presence and locality of symptomatic lower-limb deep vein thrombosis (DVT) on mortality and morbidity following contrast venography (CV), the reference standard for diagnosing below-knee DVT, with a view to determining the prevalence of recurrent episodes of DVT and post-thrombotic syndrome (PTS). MATERIALS AND METHODS: Patients with clinical DVT undergoing investigation using CV were prospectively recorded. By retrospective case note examination and mortality data evaluation, 347 patients with DVT were matched with negative controls for mortality follow-up. Long-term complications were recorded. RESULTS: Fifty-one (14.7%) of the DVT patients were diagnosed with PTS and 43 (12.4%) with possible PTS in the 10 years following presentation. The relative risk for developing definite PTS was 0.544 for below- versus above-knee DVT; 9.9% with below-knee DVT had PTS, and 9% had probable PTS. Recurrent DVT occurred in 23.3% of patients with proximal DVT as opposed to 12.6% of patients with isolated below-knee DVT. CONCLUSIONS: Morbidity is greater in patients with proximal DVT; however, a significant, albeit smaller, proportion of patients with isolated below-knee DVT develop recurrent DVT and PTS. Below-knee DVT carries sufficient morbidity and mortality to warrant vigilance in diagnosis and management of this condition.

Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys.

The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].

Membrane topology of the Escherichia coli gamma-aminobutyrate transporter: implications on the topography and mechanism of prokaryotic and eukaryotic transporters from the APC superfamily.

The Escherichia coli gamma-aminobutyric acid permease (GabP) is a plasma membrane protein from the amine-polyamine-choline (APC) superfamily. On the basis of hydropathy analysis, transporters from this family are thought to contain 12, 13 or 14 transmembrane domains. We have experimentally analysed the topography of GabP by using the cytoplasmically active LacZ (beta-galactosidase) and the periplasmically active PhoA (alkaline phosphatase) as complementary topological sensors. The enzymic activities of 32 GabP-LacZ hybrids and 43 GabP-PhoA hybrids provide mutually reinforcing lines of evidence that the E. coli GabP contains 12 transmembrane segments that traverse the membrane in a zig-zag fashion with both N- and C-termini facing the cytoplasm. Interestingly, the resulting model predicts that the functionally important 'consensus amphipathic region' (CAR) [Hu and King (1998) Biochem. J. 330, 771-776] is at least partly membrane-embedded in many amino acid transporters from bacteria and fungi, in contrast with the apparent situation in mouse cationic amino acid transporters (MCATs), in which this kinetically significant region is thought to be fully cytoplasmic [Sophianopoulou and Diallinas (1995) FEMS Microbiol. Rev. 16, 53-75]. To the extent that conserved domains serve similar functions, the resolution of this topological disparity stands to have family-wide implications on the mechanistic role of the CAR. The consensus transmembrane structure derived from this analysis of GabP provides a foundation for predicting the topological disposition of the CAR and other functionally important domains that are conserved throughout the APC transporter superfamily.

Functional significance of the "signature cysteine" in helix 8 of the Escherichia coli 4-aminobutyrate transporter from the amine-polyamine-choline superfamily. Restoration of Cys-300 to the Cys-less Gabp.

gab permease (GabP) is the exclusive mediator of 4-aminobutyrate (GABA) transport across the Escherichia coli plasma membrane. Helix 8 and a portion of the adjoining cytoplasmic region (loop 8-9) constitute the GabP "consensus amphipathic region" (CAR), a potential channel-forming domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily. Upon the polar surface of the CAR, all known gab permeases display a "signature cysteine" not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting specificity (kcat/Km) to the translocation reaction. Here we show that among the five cysteine residues in the E. coli GabP, only Cys-300, the signature cysteine, can restore wild type properties to the Cys-less GabP mutant. We conclude (i) from partial reaction studies (equilibrium exchange, counterflow) that rapid translocation of the GABA binding site from one side of the membrane to the other is greatly facilitated by Cys-300 and (ii) from pharmacological studies that loss of Cys-300 has little effect on the affinity that GabP exhibits for a structurally diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic acid, and cis-4-aminocrotonic acid) of competitive ligands. These results raise the possibility that other GABA transporters might rely analogously upon conserved cysteine residues positioned within the amphipathic helix 8 and loop 8-9 regions.

4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis.

A previous study [Ferson, Wray and Fisher (1996) Mol. Microbiol. 22, 693-701] has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis. Here it is demonstrated directly that the B. subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E. coli strain. Unexpectedly, the ligand-recognition profile of the B. subtilis GabP was found to differ substantially from that of the highly homologous E. coli GabP. Unlike the E. coli GabP, the B. subtilis GabP: (i) exhibits approx. equal preference for the 3-carbon (beta-alanine, Km=9.6 microM) and the 4-carbon (GABA, Km=37 microM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g. nipecotic acid, guvacine), which are active against GABA transporters from brain. The present study shows additionally that the B. subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against [3H]GABA. Thus, consistent with the idea that the ligand-recognition domain of the B. subtilis GabP is less spacious than that of the close homologue from E. coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation. These distinct functional characteristics of the E. coli and B. subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.

Suppressor scanning at positions 177 and 236 in the Escherichia coli lactose/H+ cotransporter and stereotypical effects of acidic substituents that suggest a favored orientation of transmembrane segments relative to the lipid bilayer.

Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents. Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.

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.

Standard vs conformal radiation therapy for adenocarcinoma of the prostate: no difference.

Objective: To compare results of treatment of adenocardinoma of the prostate using Standard (2D) vs Conformal (3D) treatment planning. Methods: The records of all patients with adenocarcinoma of the prostate treated curatively with radiation therapy alone from July 1991 to June 1994 were reviewed. Acute and late complications were scored by the RTOG criteria. Biochemical failure was defined as a rising PSA of at least 10% on two measurements separated >/=1 month or either a PSA nadir >4 ng/ml or >1 ng/ml. Disease free survival (DFS) was defined as no evidence of local, distant, or biochemical failure. 2D planning included standard simulation with target volume drawn from the treatment planning or diagnostic CT. 3D planning included a CT in the treatment position with computer simulation using beam's-eye-view for field design. Results: Two-hundred and seventeen 2D and 45 3D patients had similar median age and pre-treatment PSA, T-stage, and dose to the prostate. The median follow-up periods for the 2D and 3D groups were 32.0 and 21.5 months, respectively. The two-year actuarial survival, local or biochemical control, and DFS were not different. The 3D group had a significantly higher incidence of acute bladder side effects of all grades and acute grade 1/2 rectal complications. There were no differences in the incidence of late bladder or rectal complications. Conclusions: Careful 2D planning for the treatment of localized adenocarcinoma of the prostate is an acceptable means of treatment. Within the dose range of 64-70 Gy, this preliminary analysis demonstrated no reduction in complications nor improvement in local or biochemical control, or DFS was seen with the the use of 3D treatment planning.

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.

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.

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.

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.

Pyridine carboxylic acids as inhibitors and substrates of the Escherichia coli gab permease encoded by gabP.

Although considered selective for its natural substrate, 4-aminobutyrate, gab permease was inhibited by 1,2,3,6-tetrahydro-3-pyridinecarboxylate and 1,2,3,6-tetrahydro-4-pyridinecarboxylate. The former is a transported substrate, since its preloading into metabolically poisoned cells stimulated transient accumulation of 4-aminobutyrate via counterflow.

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.

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.

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).

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.

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.