A chimeric tyrosine/tryptophan hydroxylase

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t_1/2 of 84 min at 37°C, TPH was much less stable (t _1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at _1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.     

Long-term survival in an infant with urethral atresia

Complete urethral atresia is an anomaly that previously was incompatible with life. We report on a surviving infant with this anomaly. As a fetus urinary decompression was accomplished with a vesicoamniotic shunt. Peritoneal dialysis was initiated shortly after birth and at 9 months supramembranous scrotal inlay urethroplasty was performed to provide for egress of urine from the bladder. A maternal renal allograft was performed when he was 12 months old. When the patient was 3 1/2 years old he had normal renal function and emptied the bladder to completion through the reconstructed urethra. Although mildly delayed, he continues to progress with all developmental milestones.     

Differentiation of Achromobacter-like strains from human blood by DNA restriction endonuclease digest and ribosomal RNA gene probe patterns

Variation amongst Achromobacter-like strains was examined by DNA restriction endonuclease digestion and rDNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 12 cultures representing Achromobacter groups B, E and F, all from human blood cultures. DNA fingerprinting using EcoRI, Hae III or HindIII sub-divided the strains in a similar manner to that obtained by their protein patterns. The HaeIII patterns, with their small number of bands, were the easiest to interpret. The EcoRI patterns included a species-species triplet of bands but minor band patterns allowed further differentiation. The Achromobacter group F strains comprised a separate taxon and were distinct from the group B and E strains by all techniques examined. The study demonstrates that, in addition to total DNA digest analysis, rDNA gene restriction patterns provide a simple but discriminatory electrophoretic method for distinguishing within Achromobacter groups B and E.     

Pre-clinical Cushing's syndrome: an unexpected frequent cause of poor glycaemic control in obese diabetic patients

OBJECTIVE Autonomous cortisol secretion without clinical stigmata of Cushing's syndrome (CS) has been recently recognized and termed pre-clinical or sub-clinical CS. The common assumption is that CS is an extremely rare cause of uncontrolled diabetes; however, the prevalence of this entity has not been studied. We assessed the prevalence of pre-clinical CS among obese patients with uncontrolled diabetes. PATIENTS AND DESIGN (1) In a retrospective analysis, the medical records of 63 patients with endogenous CS were reviewed. (2) In a cross-sectional study, 90 obese patients (BMI >25 kg/m²) followed in a University Hospital and the local Health Fund endocrine and diabetes clinics, with poorly controlled diabetes (glycosylated haemoglobin >9%), underwent an overnight 1 mg dexamethasone suppression. In patients with non-suppressible cortisol levels (>140 nmol/l), Liddle's 2 and 8 mg dexamethasone suppression tests and imaging studies were performed. MEASUREMENTS The prevalence of poorly controlled diabetes, the major presenting symptom of CS, was assessed in the retrospective analysis. The prevalence of ‘true’ CS and the false positive rate in the overnight dexamethasone suppression test were calculated. The endocrine evaluation of the patients with pre-clinical CS and the effects of surgical cure on glycaemic control are described. RESULTS In the retrospective analysis, 11 (17.5%) had diabetes and 2 (3.2%) lacked the classic physical characteristics of the syndrome. In the cross-sectional study, 4 patients failed to suppress plasma cortisol (<140 nmol/l). In one patient the diagnosis of CS was not confirmed by a standard Liddle’s test and was therefore considered false positive. In the other 3, the diagnosis of CS was confirmed (prevalence of 3.3%, 95% confidence interval 1–9%). In all other patients the overnight cortisol suppression test was normal (cortisol level 47.3 ± 2.5 nmol/l (mean ± SEM)). After surgical treatment of CS, glycaemic control was markedly improved in all 5 patients (2 from retrospective and 3 from cross-sectional studies). CONCLUSIONS The prevalence of pre-clinical Cushing's syndrome in obese patients with poorly controlled diabetes appears to be considerably higher than previously believed. The overnight dexamethasone suppression test proved to be a simple, sensitive and highly specific screening test for Cushing's syndrome despite the presence of obesity and hyperglycaemia.     

Relationship of "bleeding on probing" and "gingival index bleeding" as clinical parameters of gingival inflammation

Abstract Bleeding on probing (BOP) and the gingival index have been used to clinically characterize the degree of gingival inflammation. It is, however, unclear to what extent these parameters correlate to each other and to probing pocket depth (PD). The purpose of this clinical study was to evaluate the association between BOP and GI bleeding (scores of 2 and 3), as well as the relationship of these variables to PD, in a group of patients presenting with naturally-occurring gingivitis. Based on screening examinations of 125 subjects with at least 20 teeth, no more than 4 sites with PD over 6 mm, a BOP frequency of 30% or greater, and no systemic condition that would influence the inflammatory response, were selected. 2 weeks after screening they were examined at 6 sites per tooth for plaque index, GI, PD and BOP. A standardized pressure sensitive probe (Florida Probe) with 20 g probing force was used for BOP and PD measurements. In this population, means of 40.9% (S.E.= 1.36) BOP sites and 35.3% (S.E, = 1.81) GI bleeding sites per patient were found. A total of 20,008 sites ranging in PD up to 5.9 mm were evaluated; however, the majority of sites (19,723, 98.6%) presented with <4 mm PD. When sites were evaluated, BOP demonstrated a positive correlation with PD, whereas GI bleeding correlated with PH. For sites characterized by the absence of BOP as well as the absence of GI bleeding (scores 0 and 1), the highest % of agreement between the 2 indices (77.7%) was found in shallow sites (0.1–2 mm). In contrast, when sites presenting with both BOP and GI bleeding were analyzed, the highest % of agreement (85,4%) was found for sites with PD >4.0 mm. In this gingivitis population group, it appears that BOP and GI bleeding evaluate distinct inflammatory1 conditions of the gingival tissues, and the relationship between the 2 clinical parameters may vary according to PD at the individual site examined.     

Uninformed Decisionmaking The Case of Surrogate Research Consent

A New York court recently struck down state Office of Mental Health regulations governing research involving subjects with impaired decisionmaking capacity. The court held that neither incapacitated adults nor minors could participate in any research protocol that contained a nontherapeutic element, irrespective of possible benefits to the subject or the importance of the knowledge to be gained. Although the decision rested on a technical point of law and dealt only with psychiatric research, the court's holding has significantly broader implications.     

The induction of operational tolerance is not prevented by simultaneous administration of cyclosporin A1

In this study, the effect of combining anti-CD4 monoclonal antibody (mAb) and cyclosporin (CyA) therapy at the time of transplantation was examined. A mouse cardiac allograft model was used. Anti-CD4 mAb administered perioperatively induces long-term survival. The addition of a short course of CyA given subcutaneously in a regimen of either a high-dose treatment or a standard dose treatment to the anti-CD4 mAb treatment protocol did not have a detrimental effect on graft survival. Despite having no significant effect on graft survival, the addition of CyA to the treatment protocol did result in a significant decrease in the level of IL-2 present in the hearts 7 days after transplantation. The decrease in IL-2 production was directly related to the presence of CyA in vivo. When CyA treatment was continued throughout the period during which unresponsiveness to the graft is induced by anti-CD4 mAb therapy, 50 % of the grafted hearts were rejected once the CyA was discontinued. In conclusion, the combined use of anti-CD4 mAb therapy and CyA did not have a negative effect on graft survival in this model when the two agents were used concurrently at the time of transplantation. Received: 2 October 1996 Received after revision: 31 January 1997 Accepted: 5 February 1997     

The Role of the Clinical Research Coordinator in Multicenter Clinical Trials

The clinical research coordinator plays a crucial role in organizing a site's participation in the expanding arena of multicenter medical and pharmacologic clinical trials. This summary clarifies the role of the clinical research coordinator for inexperienced staff members assuming these responsibilities and outlines planning procedures leading to successful implementation. Emphasis is placed on establishing an interdependent relationship with the principal investigator, careful protocol assessment, team building, and staff feedback. Useful tools such as study manuals and physicians' study orders are described.     

Prospective comparative study of cefotetan with piperacillin for prophylaxis against infection in elective colorectal surgery

This study compared one dose of cefotetan with three doses of piperacillin as prophylaxis against wound infection in 153 patients undergoing elective colorectal surgery. The patients were randomized into two groups: the first received 2 g cefotetan intravenously with induction of anaesthesia (n = 75), and the second received three doses of 2 g piperacillin (n = 78). Wound infection was defined as the presence of an abscess or discharging pus from the wound. In the cefotetan group there were 14 (19%) wound infections and 13 (17%) in the piperacillin group. There were three septic deaths, one in the cefotetan group and two in the piperacillin group. Both groups were comparable with regard to age, sex, nature of pathology and pre- and perioperative risk factors. No significant haematological or biochemical abnormalities were detected. The only adverse reaction was one patient who had an allergic reaction (rash) to piperacillin. These data suggest that single-dose cefotetan is as effective as triple-dose piperacillin in prophylaxis against infection in elective colorectal surgery.