Antimicrobial activity of Citrus spp. and Anethum graveolens components against Candida metapsilosis in ranch sauce.

In this paper, a study was carried out to test the inhibitory effect of a natural food compound (NFC), based on flavonoids (naringenin, hesperetin, tangeritin, luteolin, apigenin and kaempferol) from citrus and dill, in ranch sauce. A strain of C. metapsilosis, isolated from a spoiled sample of ranch sauce, was used as target pathogen microorganism. The inhibitory effect of NFC was compared with a common mixture of chemical preservatives used in this type of sauces: potassium sorbate and sodium benzoate (S/B). An in vitro test was performed by the microtiter plate assay at 10, 25 and 37 °C for 24 h in modified Tryptic Soy Broth. An additive antimicrobial effect had been observed in the combination of acetic acid and NFC. The results of the microtiter assay were validated in a challenge test in ranch sauce at 5, 25 and 37 °C for 10 weeks. NFC showed partial fungicidal effect against C. metapsilosis, reducing two logarithmic units at 5 °C for 10 weeks. At 5 °C, the traditional doses of S/B used in ranch sauce decreased viable cells to non-detectable counts from the second week of the experiment. At 25 and 37 °C, the use of S/B mixture or the use of NFC showed the same fungicidal effect. The incorporation of NFC, alone or in combination with acetic acid, opens the possibility of formulating clean label sauces with good protection against the development of the acid resistant yeast C. metapsilosis.

A practical guide for the diagnosis of primary enteric nervous system disorders.

OBJECTIVE: Primary gastrointestinal neuropathies are a heterogeneous group of enteric nervous system (ENS) disorders that continue to cause difficulties in diagnosis and histological interpretation. Recently, an international working group published guidelines for histological techniques and reporting, along with a classification of gastrointestinal neuromuscular pathology. The aim of this article was to review and summarize the key issues for pediatric gastroenterologists on the diagnostic workup of congenital ENS disorders. In addition, we provide further commentary on the continuing controversies in the field. RESULTS: Although the diagnostic criteria for Hirschsprung disease are well established, those for other forms of dysganglionosis remain ill-defined. Appropriate tissue sampling, handling, and expert interpretation are crucial to maximize diagnostic accuracy and reduce interobserver variability. The absence of validated age-related normal values for neuronal density, along with the lack of correlation between clinical and histological findings, result in significant diagnostic uncertainties while diagnosing quantitative aberrations such as hypoganglionosis or ultrashort Hirschsprung disease. Intestinal neuronal dysplasia remains a histological description of unclear significance. CONCLUSIONS: The evaluation of cellular quantitative or qualitative abnormalities of the ENS for clinical diagnosis remains complex. Such analysis should be carried out in laboratories that have the necessary expertise and access to their own validated reference values.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Endoscopic transesophageal vs. thoracoscopic removal of mediastinal lymph nodes: a prospective randomized trial in a long term animal survival model.

BACKGROUND AND STUDY AIMS: In cases where biopsies remain inconclusive, removal of mediastinal lymph nodes for further analysis requires surgical means. Natural orifice transluminal endoscopic surgery (NOTES) procedures allow incision/closure of the gut wall, which might enable endoscopic excision of pre-marked nodes. The aims of the current study were to investigate the feasibility, safety, and reproducibility of lymph node generation in an animal model to enable endoscopic ultrasound-guided (EUS) lymph node removal (ELR) using transesophageal NOTES access/closure and to compare this procedure with thoracoscopic lymph node removal (TLR) in a randomized long term survival animal study. PATIENTS AND METHODS: Lymph node creation using graphite injection was performed in 12 pigs. After randomization into ELR and TLR groups, lymph nodes were marked with newly developed anchors under EUS guidance and removed using either ELR or TLR. ELR included incision of the esophageal wall and closure after lymph node removal. The main outcome measures were success in lymph node generation, technical success of lymph node removal, complications, and comparability of ELR and TLR. RESULTS: Generation of lymph nodes proved successful in all animals in 46/48 sites injected (96 %). Anchors were placed through the selected nodes in a mean of 9.4 minutes. TLR and ELR were successful in all cases. One bleeding occurred during esophageal incision in ELR, which was stopped endoscopically. After lymph node removal, endoscopic suturing of the incision took a mean of 18 minutes. Procedure time was longer for ELR than TLR (mean 48 vs. 42 minutes). All animals survived the procedures. Autopsy after 4 weeks showed two thoracic wall abscesses in the TLR group and none in the ELR group.  Microscopic analysis revealed well healed esophageal scars. CONCLUSION: ELR proved to be feasible in this limited sample size and complications were not observed more frequently in this group than in the TLR group.

Loss of functional KATP channels in pancreatic beta-cells causes persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a disorder of childhood associated with inappropriate hypersecretion of insulin by the pancreas. The pathogenesis of the condition has hitherto remained controversial. We show here that insulin-secreting cells from a homogeneous group of five infants with PHHI lack ATP-sensitive K+ channel (KATP) activity. As a consequence, PHHI beta-cells are spontaneously electrically active with high basal cytosolic Ca2+ concentrations due to Ca2+ influx. Our findings define the pathogenesis of this disease as a novel K+ channel disorder.

The feasibility of wireless capsule endoscopy in detecting small intestinal pathology in children under the age of 8 years: a multicentre European study.

OBJECTIVE: To systematically evaluate the feasibility and methodology to carry out wireless capsule endoscopy (WCE) in children <8 years to define small intestinal pathology. DESIGN: Prospective European multicentre study with negative prior investigation. PATIENTS AND INTERVENTIONS: 83 children aged 1.5-7.9 years were recruited. Initially, all were offered "swallowing" (Group 1) for capsule introduction. If this failed endoscopic placement (Group 2) was used and the Roth net, Advance or custom-made introducers were compared. OUTCOME MEASURES: Primary endpoint: to determine pathology; secondary endpoint: comparison of capsule introduction methods. RESULTS: Capsule introduction: 20 (24%) children aged 4.0-7.9 years (mean, 6.9 years; 14 male) comprising Group 1 were older (p<0.025) than 63 (76%) aged 1.5-7.9 years (mean, 5.25 years; 30 male) forming Group 2. COMPLICATIONS: Roth net mucosal trauma in 50%; no others occurred. The available recording apparatus was inappropriate for those <3 years. INDICATIONS: gastrointestinal bleeding: n = 30 (16 positive findings: four ulcerative jejunitis, four polyps, two angiodysplasia, two blue rubber blebs, two Meckel's diverticula, one anastomotic ulcer, one reduplication); suspected Crohn's disease: n = 20 (11 had Crohn's disease); abdominal pain: n = 12 (six positive findings: three Crohn's disease, two lymphonodular hyperplasia, one blue rubber bleb); protein loss: n = 9 (four lymphangectasia); malabsorption: n = 12 (seven positive findings: six enteropathy, one ascaris). No abnormalities overall: 45%. CONCLUSION: WCE is feasible and safe down to the age of 1.5 years. 20 children >4 years swallowed the capsule. The Advance introducer proved superior for endoscopic placement. The pathologies encountered showed age specificity and, unlike in adolescents, obscure gastrointestinal bleeding was the commonest indication.

Sucrase-isomaltase deficiency in humans. Different mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme.

Eight cases of congenital sucrase-isomaltase deficiency were studied at the subcellular and protein level with monoclonal antibodies against sucrase-isomaltase. At least three phenotypes were revealed: one in which sucrase-isomaltase protein accumulated intracellularly probably in the endoplasmic reticulum, as a membrane-associated high-mannose precursor, one in which the intracellular transport of the enzyme was apparently blocked in the Golgi apparatus, and one in which catalytically altered enzyme was transported to the cell surface. All patients expressed electrophoretically normal or near normal high-mannose sucrase-isomaltase. The results suggest that different, probably small, mutations in the sucrase-isomaltase gene lead to the synthesis of transport-incompetent or functionally altered enzyme which results in congenital sucrose intolerance.

Therapy for persistent hyperinsulinemic hypoglycemia of infancy. Understanding the responsiveness of beta cells to diazoxide and somatostatin.

The neonatal disorder persistent hyperinsulinemic hypoglycemia of infancy (PHHI) arises as the result of mutations in the subunits that form the ATP-sensitive potassium (KATP) channel in pancreatic beta cells, leading to insulin hypersecretion. Diazoxide (a specific KATP channel agonist in normal beta cells) and somatostatin (octreotide) are the mainstay of medical treatment for the condition. To investigate the mechanism of action of these agents in PHHI beta cells that lack KATP currents, we applied patch clamp techniques to insulin-secreting cells isolated from seven patients with PHHI. Five patients showed favorable responses to medical therapy, and two were refractory. Our data reveal, in drug-responsive patients, that a novel ion channel is modulated by diazoxide and somatostatin, leading to termination of the spontaneous electrical events that underlie insulin hypersecretion. The drug-resistant patients, both of whom carried a mutation in one of the genes that encode KATP channel subunits, also lacked this novel K+ channel. There were no effects of diazoxide and somatostatin on beta cell function in vitro. These findings elucidate for the first time the mechanisms of action of diazoxide and somatostatin in infants with PHHI in whom KATP channels are absent, and provide a rationale for development of new therapeutic opportunities by K+ channel manipulation in PHHI treatment.

Which parameters might predict complications after natural orifice endoluminal surgery (NOTES)? Results from a randomized comparison with open surgical access in pigs.

BACKGROUND AND STUDY AIMS: Natural orifice transluminal endoscopic surgery (NOTES) is currently developed and assessed mainly in pig experiments. The vast majority of studies show a good outcome in short-term follow-up. The current study aims at comparing various parameters of postinterventional assessment and surveillance in relation to clinical behavior and autopsy results to find suitable control parameters and also to assess the pig as suitable model for NOTES compared with open surgery. METHODS: Within the framework of a randomized prospective study of 20 pigs with iatrogenic colonic perforation comparing endoscopic with open surgical closure, clinical examination, including observation of behavior, food intake, and body temperature, was carried out daily. Laboratory parameters (white blood cells [WBC], granulocytes) were measured in 14 animals. Weight was measured preoperatively and on days 2 and 7 postoperatively. Results were matched with complications found during/after 2 weeks' survival. Pre-autopsy sterile cultures were taken from the peritoneal cavities to determine possible bacterial contamination. RESULTS: Three animals from the surgical group were sacrificed on days 4, 8, and 12 because they became severely ill, with autopsy revealing intussusception from adhesions, peritoneal abscess, and peritonitis, in one pig each; another animal had culture positive for ESCHERICHIA COLI. Three minor complications (2 cough, 1 continuing fever with adhesions to the bladder found on autopsy) occurred in the endoscopic group without compromised recovery. WBC were measured in 14 animals, and found to be elevated (8 - 36 x 10 (9)/l) in six on day 2 including the two animals with severe complications. Between pre- and post-procedure, WBC increased about twofold in the uneventful cases but fourfold in the two animals with severe complications. Cultures from the abdominal cavity before autopsy were negative in all but one animal. CONCLUSION: Animal behavior was a reliable indicator of severe complications. Fever, body weight, and the results of in vitro cultures of the peritoneal fluid did not indicate complications. WBC proved not to be specific but showed a larger increase in pigs with severe complications.

Pharmacokinetic evaluation of gemcitabine and 2',2'-difluorodeoxycytidine-5'-triphosphate after prolonged infusion in patients affected by different solid tumors.

BACKGROUND: The study determined pharmacokinetic parameters, toxicity profile and preliminary clinical activity of gemcitabine administered i.v. at different infusion rates in patients with a range of solid tumors. PATIENTS AND METHODS: Twenty patients were enrolled for both pharmacokinetic and clinical studies. Gemcitabine 300 mg/m(2) was administered during 1 h, 2 h or 3 h, and as a conventional dose of 1000 mg/m(2) during 30 min infusion. Administration was on days 1, 8 and 15 every 4 weeks. RESULTS: Patients were randomly assigned to one of the four arms. After 30 min infusion of 1000 mg/m(2) gemcitabine the plasma concentration remained above the saturation level of 10-20 microM, whereas after 1, 2 or 3 h infusion 300 mg/m(2) gemcitabine it remained below the saturation level for most of the time (being in the range 2.5-10 microM). Gemcitabine triphosphate was determined in the four arms in white blood cells; for infusion times from 0.5 to 3 h there was a progressive enhancement of gemcitabine triphosphate levels. In all evaluable patients the toxicity was mild, myelosuppression being the main toxicity. No grade 3 or 4 toxicities occurred. Clinical response was similar in patients receiving 300 mg/m(2) gemcitabine in 2 and 3 h and in the 1000 mg/m(2) arm. CONCLUSIONS: 300 mg/m(2) gemcitabine during 3 h infusion produced the highest accumulation of gemcitabine triphosphate. Thus, to achieve the highest possible gemcitabine triphosphate level, prolonged infusion time would appear to be more important than a high dose administered as a short infusion. However, there was no substantial difference in toxicity or antitumoral activity in the all different patient groups.

Successful resection of a congenital superior mesenteric arteriovenous malformation.

A 15-month-old male presented with severe gastrointestinal bleeding and heart failure. Imaging revealed a superior mesenteric artery arteriovenous malformation, associated with a congenital portosystemic shunt. The heart failure was cured by resection of the arteriovenous malformation.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity.

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.

Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.

Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study.

Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site".

Cloning of HOXD1 from unfertilised human oocytes and expression analyses during murine oogenesis and embryogenesis.

We describe the cloning of HOXD1 in human unfertilised oocytes and detailed expression analyses during mouse oogenesis and embryogenesis. The cDNA of 1991bp has an open reading frame of 987bp encoding a protein of 329 amino acids. A comparison of the amino acid sequence with the mouse homologue revealed an overall homology of 85.5% with 99% identity within the homeodomain. Expression was detected in unfertilised human oocytes and 2-, 4-, 8-cell and blastocyst stage embryos. Expression analyses in mature mouse ovaries, early embryos and isolated gut revealed expression in the oocytes of the primary and secondary ovarian follicles, and in embryonal mesodermal derivatives such as dermatomes, urogenital tubercle, tail bud, kidney, ovaries, testes and enteric mesoderm adjacent to the caecum where expression was up-regulated in vitro in response to increasing doses of retinoic acid. Our observations indicate a possible role for HOXD1/Hoxd1 in the ovarian oocytes and the establishment of mesodermal derivatives during embryogenesis.

[SIXTH JESUS CULEBRAS' LECTURE: GLUTAMINE AND THE CRITICAL PATIENT: THE END OF AN AGE?]. / SEXTA LECCIÓN JESÚS CULEBRAS: GLUTAMINA Y PACIENTE CRÍTICO ¿EL FIN DE UNA ERA?

In the last few years, glutamine has changed its status from a "non-essential" amino acid to "almost essential or indispensable" in the critical patient. This has occurred thanks to a series of studies and meta-analysis highlighting the beneficial effects on nosocomial infection, stay in ICU and hospital stay and mortality. After two multicentre studies (REDOXS and MetaPlus) which reviewed the effects of glutamine on critically ill patients, comments changed to: "we do strongly recommend that glutamine is not used in critically ill patients in shock or multiple organ failure" and: "there is an important questioning about the safety of this approach (combination of high- dose enteral and parenteral glutamine) which should not be ignored" and, therefore: "the committee decides to decrease the degree of recommendation for endovenous glutamine"; it currently states that glutamine "should be considered". According to another multicentre study with severe trauma patients our group (a group which in theory was much benefitted from glutamine actions), and 143 patients, did not experience any observable benefit at the usual parenteral doses. We do agree with previous studies on the prognostic value of low levels of glutamine at admittance, which was confirmed if those levels were not back to normal after its administration, although there are no readily available analytic trials for this. This divergence about the usefulness of glutamine grows up as more and more multicentre studies in critical patients show there should be a change of attitude, and probably the clinical guidelines that welcomed its use should now be amended.

La glutamina es un aminoácido que en pocos años ha pasado de "no esencial" a "casi imprescindible en el enfermo crítico", gracias a una serie de estudios y metaanálisis en los que destacaban sus beneficiosos efectos sobre la infección nosocomial, estancias en UCI y hospitalarias y mortalidad, sobre todo tras dos estudios multicéntricos (REDOXS y MetaPlus) que revisaban los efectos de la glutamina en pacientes críticos, los comentarios pasaban a: "recomendamos fuertemente que la glutamina no sea utilizada en pacientes críticos en shock o fallo multiorgánico" a través de un "importante cuestionamiento sobre la seguridad de esta estrategia (combinación de altas dosis de glutamina enteral y parenteral) que no debe ser ignorada" y, por tanto, "el comité decide disminuir el grado de recomendación para la glutamina endovenosa"; y actualmente destaca que la misma "debería ser considerada". Nuestro grupo, también según otro estudio multicéntrico en enfermos traumáticos graves, un grupo teóricamente más beneficiado de la acción de la glutamina, y en 143 pacientes, a las dosis parenterales habituales, no observamos ningún beneficio. Sí que coincidimos con anteriores estudios en el valor pronóstico de valores bajos de glutaminemia al ingreso, que se veía confirmado si no se normalizaban tras su administración, aunque su determinación no es una prueba analítica asequible. Esta divergencia sobre la utilidad de la glutamina aparece con la proliferación de estudios multicéntricos en pacientes críticos que obligan a un cambio de actitud y, probablemente también, en las guías clínicas que tan favorablemente acogieron su uso.

Differential inhibition of fungal oxidosqualene cyclase by 6E and 6Z isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene.

Inhibitory properties of 6E (compound 1) and 6Z (compound 2) isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene against oxidosqualene-lanosterol cyclase were assayed on microsomes and whole cells of Saccharomyces cerevisiae and Candida albicans. Only the 6E isomer (compound 1), bearing a correct substrate-like configuration, strongly inhibited the enzyme both in microsomes and cell cultures. The difference between compounds 1 and 2 (which had an unfavorable geometry) was especially evident when measuring [14C]acetate incorporation into non-saponifiable lipids extracted from treated cells. While isomer Z was totally ineffective at up to 30 microM, in cells treated with 5 microM isomer E, labelled oxidosqualene, the level of which was negligible in the control, rose to over 60% of the non-saponifiable lipids.

Active site titration of bovine beta-trypsin by N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis.

Kinetics of bovine beta-trypsin (trypsin) with the N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0 degrees C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin.N alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl.enzyme adduct has been solved by X-ray crystallography at 2.0 A resolution (R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue.

29-Methylidene-2,3-oxidosqualene derivatives as stereospecific mechanism-based inhibitors of liver and yeast oxidosqualene cyclase.

Two pairs of isomers (18Z)- (8), (18E)-29-methylidene-2,3-oxidohexanorsqualene (21), and (18Z)- (31), (18E)-29-methylidene-2,3-oxidosqualene (34), have been obtained in a fully stereospecific manner, as inhibitors of rat and yeast oxidosqualene cyclase. A new method for the synthesis of C22 squalene aldehyde 2,3-epoxide is reported, as well as that of other 19-modified 2,3-oxidosqualene analogues. We found that the activity is the opposite in the two series: the (E)-hexanormethylidene 21 and the (Z)-methylidene 31 are potent and irreversible inhibitors of oxidosqualene cyclase, while (Z)-hexanormethylidene 8 and (E)-methylidene 34 are almost completely inactive. Reduction of the 18,19-double bond, such as in 39, eliminates the activity, while removal of both of the 19-linked groups such as in heptanor derivative 40 greatly reduces inhibition of the enzyme. (E)-Hexanormethylidene 21 results the first irreversible inhibitor of the series toward the yeast enzyme.

Effects of chronic vitamin E deficiency on vascular function--a study of sympathetic nerves, smooth muscle and endothelium of the mesenteric arterial bed of the rat.

1. Male rats were deprived as weanlings of dietary vitamin E for 2, 4, 6, 10 and 12 months. Mesenteric arterial beds from these rats and from age-matched controls were isolated and perfused with Krebs solution at a constant flow rate (5 ml min-1). The function of perivascular sympathetic nerves, smooth muscle and endothelium was assessed. 2. At 12 months vitamin E deficient rats exhibited the characteristic symptoms of vitamin E deficiency, namely poor coat condition, muscle wasting, kyphoscoliosis and impaired gait. In the isolated mesenteric arterial bed electrical field stimulation (EFS) of perivascular nerves (4-32 Hz, 90 V, 1 ms, for 30 s) elicited frequency-dependent vasoconstrictor responses which were unaffected by vitamin E deficiency except at 12 months, at which age responses were significantly greater than those of the controls at 24 and 32 Hz (P < 0.01). 3. Exogenous noradrenaline (NA; 0.15-500 nmol) elicited dose-dependent vasoconstriction which was similar in vitamin E-deficient and control preparations at all ages. Potassium chloride (0.15 mmol) also produced similar vasoconstrictor responses in vitamin E-deficient and control preparations at each age. 4. Tone of the preparations was raised by continuous perfusion with methoxamine (4-70 microM), producing similar increases in perfusion pressure in vitamin E-deficient and control preparations at each age. Endothelium-dependent dose-dependent vasodilatation to adenosine 5'-triphosphate was significantly impaired in mesenteric arterial beds from 12 month-old vitamin E-deficient rats compared with the controls (P < 0.05). Relaxation to acetylcholine was not significantly different at any age. 5. Endothelium-independent vasodilatation to sodium nitroprusside was similar in vitamin E-deficient rats and age-matched controls. 6. These results suggest that long term (12 months) deprivation of dietary vitamin E may impair endothelial function in mesenteric arteries of the rat. Sympathetic perivascular nerve constrictor function was increased at 12 months. There were no functionally expressed changes in the vascular smooth muscle, which appears to be more resilient to the effects of oxidative stress in vitamin E deficiency.