Haem ligands of the ferricytochrome c of Ustilago sphaerogena

The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.     

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.     

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N-bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu-

-Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.     

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.     

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.     

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine     

Overexpression of ornithine-δ-aminotransferase increases proline biosynthesis and confers osmotolerance in transgenic plants

Accumulationof proline is a way to increase tolerance to water stress in plants. Therefore,considerable attention has been devoted to optimise proline biosynthesis intransgenic plants. Glutamate and ornithine are both precursors of proline butwhile genes of the glutamate pathway were overexpressed in transgenic plants,no gene encoding an enzyme of the ornithine pathway was considered until now. Thepresent study aims to establish if the overexpression ofornithine--aminotransferase (-OAT) represents an additional wayto increase proline content. To achieve this goal, anArabidopsis -OAT cDNA was fused to the CaMV35Spromoter and introduced via Agrobacterium transformationinto Nicotiana plumbaginifolia. Overexpression of the-OAT cDNA in the analysed transgenic lines was linked to an increase in-OAT enzyme activity. The transgenic lines presenting high enzymaticactivity synthesized more proline than the control plants and showed a higherbiomass and a higher germination rate under osmotic stress conditions. Thesestudies reveal a new and efficient way to increase proline content in plantsand to enhance crop tolerance.     

Safety and efficacy of office-based surgery with monitored anesthesia care/sedation in 4778 consecutive plastic surgery procedures

Office-based surgery has several potential benefits over hospital-based surgery, including cost containment, ease of scheduling, and convenience to both patients and surgeons. Scrutiny of office-based surgery by regulators and state-licensing agencies has increased and must be addressed by improved documentation of safety and efficacy. To evaluate the safety and efficacy of the authors' office-based plastic surgery, a review was undertaken of 3615 consecutive patients undergoing 4778 outpatient plastic surgery procedures under monitored anesthesia care/sedation in a single office. The charts of 3615 consecutive patients who had undergone office-based surgery with monitored anesthesia care/sedation between May of 1995 and May of 2000 were reviewed. In all cases, the anesthesia protocol used included sedation with midazolam, propofol, and a narcotic administered by a board-certified registered nurse anesthetist with local anesthesia provided by the surgeon. Charts were reviewed for patient profile, types of procedures, multiple procedures, duration of anesthesia, American Society of Anesthesiologists class, and complications related to anesthesia. Outcomes measured included death, airway compromise, dyspnea, hypotension, venous thrombosis, pulmonary emboli, protracted nausea and vomiting lasting more than 24 hours, and unplanned hospital admissions. Statistical analyses were performed using the Microsoft Excel program and the SAS package. Results were as follows: 92.3 percent of the patients were female and 7.7 percent were male, with a mean age of 42.7 years (range, 3 to 83 years). Patients underwent aesthetic (95.6 percent) and reconstructive (4.4 percent) plastic surgery procedures. Same-session multiple procedures occurred in 24.8 percent of patients. The vast majority of patients were healthy: 84.3 percent of patients were American Society of Anesthesiologists class I, 15.6 percent were class II, and 0.1 percent were class III. The operations required a mean of 111 minutes. There were no deaths, ventilator requirements, deep venous thromboses, or pulmonary emboli. Complications were as follows: 0.05 percent (n = 2) of patients had dyspnea that resolved, 0.2 percent (n = 6) of patients had protracted nausea and vomiting, and 0.05 percent (n = 2) of patients had unplanned hospital admissions (<24 hours). One patient had an emergent intubation. No prolonged adverse effects were noted. There was a 30-day follow-up minimum. Outpatient surgery is an important aspect of plastic surgery. It was shown that office-based surgery with intravenous sedation, performed by board-certified plastic surgeons and nurse anesthetists, is safe. Appropriate accreditation, safe anesthesia protocols, and proper patient selection constitute the basis for safe and efficacious office-based outpatient plastic surgery.