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.     

Children's productions of the affix -ed in past tense and past participle contexts.

Children's productions of the affix -ed in past tense and past participle contexts (e.g., the boy kicked the ball vs. the ball was kicked) were examined in spontaneous conversations and elicited productions. The performances of 7 children with specific language impairment (SLI) were compared with those of 2 control groups of typically developing children (age matches, MLU matches). Children with SLI produced fewer obligatory contexts for both past tense and past participle forms than did the control children, and were more likely to omit past tense affixes. In contrast, few omissions of the past participle were observed across all 3 groups. Implications for theories regarding the morphological deficits associated with SLI are discussed.     

A Bayesian approach to aid in formulary decision making: incorporating institution-specific cost-effectiveness data with clinical trial results.

Pharmacy and therapeutics committees commonly cite a lack of generalizability as a reason for not incorporating cost-effectiveness information into decision making. To address this concern, many committees undertake site-specific economic evaluations, which are often limited by small sample sizes and nonrandomized designs. We show how 2 complementary approaches were used to minimize these limitations in an economic evaluation of abciximab at 1 institution. Using a propensity score methodology, we selected patients who did not receive abciximab for the comparison cohort. Then, we adopted a Bayesian, hierarchical, random-effects model to integrate site-specific and clinical trial data. We applied the posterior distributions of effectiveness with local cost data in a traditional decision-analytic model. In 74% of the simulations, abciximab was cost-effective at 1 institution at the $50,000 per life year saved threshold, assuming a 50:50 split of patients undergoing coronary stenting and angioplasty. Among patients undergoing coronary stenting, the cost-effectiveness ratio of the addition of abciximab was at or below the $50,000 per life year saved threshold in 66.0% of the simulations.     

Cerebral vascular effects of angiotensin II: new insights from genetic models.

Very little is known regarding the mechanisms of action of angiotensin II (Ang II) or the consequences of Ang II-dependent hypertension in the cerebral circulation. We tested the hypothesis that Ang II produces constriction of cerebral arteries that is mediated by activation of AT1A receptors and Rho-kinase. Basilar arteries (baseline diameter approximately 130 microm) from mice were isolated, cannulated and pressurized to measure the vessel diameter. Angiotensin II was a potent constrictor in arteries from male, but not female, mice. Vasoconstriction in response to Ang II was prevented by an inhibitor of Rho-kinase (Y-27632) in control mice, and was reduced by approximately 85% in mice deficient in expression of AT1A receptors. We also examined the chronic effects of Ang II using a model of Ang II-dependent hypertension, mice which overexpress human renin (R+) and angiotensinogen (A+). Responses to the endothelium-dependent agonist acetylcholine were markedly impaired in R+A+ mice (P<0.01) compared with controls, but were restored to normal by a superoxide scavenger (PEG-SOD). A-23187 (another endothelium-dependent agonist) produced vasodilation in control mice, but no response or vasoconstriction in R+A+ mice. In contrast, dilation of the basilar artery in response to a NO donor (NONOate) was similar in R+A+ mice and controls. Thus, Ang II produces potent constriction of cerebral arteries via activation of AT1A receptors and Rho-kinase. There are marked gender differences in cerebral vascular responses to Ang II. Endothelial function is greatly impaired in a genetic model of Ang II-dependent hypertension via a mechanism that involves superoxide.     

Variation in evoked potential measures over the menstrual cycle: A pilot study

Nancy Kluck, Scan J. O'Connor, Victor M. Hesselbrock, Allan Tasman and Donald Maier, Lance Bauer: Variation in Evoked Potential Measures Over the Menstrual Cycle: A Pilot Study. Prog. Neuro. Psychopharmacol. Biol. Psychiat. 1992. 16(6): 901–911.

1. 1. The P3 component of a visual event related potential (ERP) was studied for five consecutive weeks in six women with normal menstrual cycles. Serum concentrations of luteinizing hormone (LH), estradiol (E2) and progesterone were studied during the same period.

2. 2. Increases in P3 amplitude, although nonsignificant, were noted in the week preceding onset of menses.

3. 3. No significant changes in reaction times to target/nontarget stimuli were noted over the same time period.

Metathesis polymerization behavior of 2-cyanobicyclo[2.2.1]hept-5-ene

This paper describes the ring-opening methathesis polymerization of homopolymers and copolymers of exo- and endo-2-cyanobicyclo[2.2.1]hept-5-ene (norbornenenitrile) in solution. Polymerization kinetics were followed via ¹H NMR spectroscopy in real time while the final polymer microstructure was determined via ¹³C NMR. While pure endo monomer does not polymerize readily, it copolymerizes with exo monomer, rapidly and to high conversions. The structure of the copolymer appears to be quite similar to that of exo homopolymer. We conclude that exo monomer adds directly to either exo or endo, while endo-endo addition is sterically hindered.     

Don’t Let an Opportunity Go by: Validation of the EIGHT Gambling Screen

The EIGHT Screen is a brief problem gambling screen originally designed for use by family doctors. Its wider use indicated the need for further validation. A triangulated approach used a range of measures in different settings in both the current study and findings from a number of earlier projects, and reviewed current use. The EIGHT Screen had acceptable correlations with the SOGS (r = 74–90%) and with the NODS-12 months Screen (r = 62.4%). Measurements remained relatively constant amongst a range of cultures, settings, age and gender, while few false positives were produced by the screen. The EIGHT Screen appears to be a valid tool for untrained users to identify Level 2 and 3 problem gambling in a wide range of cultures and settings.     

D2-40 immunohistochemistry in the differential diagnosis of seminoma and embryonal carcinoma: a comparative immunohistochemical study with KIT (CD117) and CD30.

The distinction between seminoma and embryonal carcinoma based on morphology alone can sometimes be problematic, requiring the use of immunohistochemistry to facilitate diagnosis. D2-40 is a monoclonal antibody that reacts with an oncofetal antigen expressed by fetal germ cells and testicular germ cell tumors. The diagnostic value of D2-40 immunohistochemistry in distinguishing seminoma from embryonal carcinoma has not been determined. D2-40 immunoreactivity was evaluated in a series of testicular germ cell tumors and compared with that of KIT (CD117) and CD30, to assess the relative utility of this marker in discriminating between seminoma and embryonal carcinoma. Forty testicular germ cell neoplasms were examined, which included 19 seminomas, three embryonal carcinomas, three teratomas, one yolk sac tumor, and 14 mixed germ cell tumors. The 14 cases of mixed germ cell tumors contained components of seminoma (n=7), embryonal carcinoma (n=11), teratoma (n=10), yolk sac tumor (n=2), and choriocarcinoma (n=1). All cases of pure seminoma and the seminomatous components of mixed germ cell tumors exhibited positive immunoreactivity for D2-40. Focal positivity for D2-40 was also observed in 29% of the embryonal carcinoma samples. D2-40 immunoreactivity in seminomas was characterized by diffuse membrane staining, whereas for embryonal carcinomas, staining was focal and distributed along the apical surfaces of the neoplastic cells. Immunohistochemical staining for KIT was observed in 92% of the seminoma samples and in none of the embryonal carcinomas. Conversely, CD30 expression was identified in 93% of the embryonal carcinoma samples and in none of the seminomas. Other germ cell components showed no immunoreactivity for D2-40, KIT, or CD30. KIT and CD30 are effective immunohistochemical markers in separating seminoma from embryonal carcinoma. Although a highly sensitive marker for seminomas, D2-40 positivity was also observed in a subset of embryonal carcinomas, thus limiting the utility of this antibody for discriminating between these two malignancies.