Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from Rhodococcus erythropolis.

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli. Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding alpha-hydroxy amides and acids while retaining enantiopurity.

Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria.

The enzymatic hydrolysis of the nitrile group of different 2-acetoxynitriles was investigated in order to obtain catalysts that chemoselectively hydrolyse nitriles in the presence of ester groups. The biotransformation of four 2-acetoxynitriles [2-acetoxybutenenitrile (ABN), 2-acetoxyheptanenitrile (AHN), 2-acetoxy-2-(2-furyl)acetonitrile (AFN), and 2-acetoxy-2,3,3-trimethylbutanenitrile (ATMB)] by different bacterial strains that synthesise nitrilases or nitrile hydratases was studied. ABN, AHN and AFN were converted by various microorganisms belonging to different bacterial genera (e.g. Pseudomonas or Rhodococcus) expressing either nitrilase or nitrile hydratase activities. In contrast, no metabolism of the sterically hindered substrate ATMB was observed. All wild-type strains investigated formed considerable amounts of cyanide and aldehydes from the 2-acetoxynitriles. This indicated the presence of esterases converting the 2-acetoxynitriles to 2-hydroxynitriles, which then spontaneously decomposed to the corresponding aldehydes and cyanide. In order to suppress unwanted side-reactions, biotransformations were performed with recombinant Escherichia coli strains that heterologously expressed nitrilase activities originating from Pseudomonas, Rhodococcus, or Synechocystis strains. The attempted conversion of the 2-acetoxynitriles to almost stoichiometric amounts of the corresponding 2-acetoxycarboxylic acids was finally achieved by using either a recombinant E. coli strain that highly overexpressed the nitrilase gene from the pseudomonad or the purified enzyme derived from this strain.

Relation between depression and anxiety in dystonic patients: implications for clinical management.

Past clinical research has identified depression as the most common psychiatric disorder associated with cervical dystonia (CD). The purpose of our study is to document different patterns of psychopathology, the frequency of psychiatric disorders, and possible correlation with the neurological disorder in patients with CD. Forty patients with CD were investigated to assess levels of psychopathology on two self-rated scales: the Beck Depression Inventory (BDI) and Symptom Check List (SCL-90). To determine the presence of psychiatric disorders, the patients were evaluated using the standard instrument in the DSM-III-R (Structured Clinical Interview Schedule, SCID). A small group of dystonic patients (12%) had higher levels of psychopathology, with significant amounts of concomitant anxiety and depression on the BDI and SCL-90. SCID criteria for at least one psychiatric disorder were fulfilled in 22 patients (55%), including both the lifetime and current diagnoses. The most frequent diagnostic categories were anxiety (40%) and major depressive disorders (37.5%). In 17 patients (42.5%), criteria for at least one lifetime diagnosis were fulfilled prior to the onset of CD. Psychiatric evaluation does not indicate one specific disorder associated with CD. The presence of anxiety and depression symptoms before and during the course of dystonia, without a possible causal relationship, could mean that the alteration of a chain of physiological events in the central nervous system may not lead to a single clinical picture. The relatively high overall lifetime prevalence of anxiety and depressive disorders may indicate the need for a broader diagnostic and therapeutic approach to patients with focal dystonia.

Naltrexone as a treatment of self-injurious behavior--a case report.

OBJECTIVE: Self-injurious (SIB) is a phenomenon associated with different psychopathological conditions such as psychosis, mental retardation and especially severe personality disorders--mainly of the borderline subtype. It has been suggested that a blockade of the endogenous opoid system might lead to a reduction of the urge to SIB. METHOD: The case and successful treatment of SIB with naltrexone in a patient with borderline personality disorder and dysthymia is described. RESULTS: After treatment trials with neuroleptics, antidepressants and valproate the patient was treated with naltrexone in a dosis of 50 mg/day and did not show SIB over a follow-up period of 32 weeks. CONCLUSION: Naltrexone could be effective in reducing SIB in patients with psychiatric disorders by blocking the positive reinforcement of SIB, which is released by the release of endogenous opoides. Placebo-controlled studies of the efficacy of naltrexone in treating SIB should be undertaken.

The concept of docking and protecting groups in biohydroxylation.

The hydroxylation of unactivated carbon atoms employing methods developed in the realms of classical organic chemistry is difficult to achieve and the processes available lack the degree of chemo-, regio- and enantioselectivity required for organic synthesis. To improve this situation, the concept of docking/protecting groups should enable the organic chemist to employ biohydroxylation as an easy tool for preparative work. Similar to the common practice of using protective groups in organic chemistry, a docking/protecting (d/p) group is introduced first, then the biotransformation is performed, and finally the d/p group is removed. The aim of this concept is not only to avoid time consuming microorganism screening methods, but also to improve hydroxylation position predictability, prevent undesired side reactions, aid substrate detection, and product recovery. This approach is successfully applied to carboxylic acids, ketones, aldehydes, and alcohols.

Psychological disorders in survivors of torture: exhaustion, impairment and depression.

Post-traumatic stress disorder (PTSD) has been described as the characteristic sequel to extreme events in life such as war and especially torture. This limitation to a single approach in regard to diagnosis and treatment has been criticised as being a too narrow concept to describe the effects following extreme events in life, especially as most studies so far were limited to PTSD and a small range of symptoms or disorders. The study presents data on psychiatric disorders in a group of exiled survivors of torture presenting to an out-patient department for psychiatry. A DSM-III-R-based psychiatric interview, including the general assessment of functioning scale (GAF), an open list of symptoms and the Vienna diagnostic criteria in regard to depression were used to evaluate a broader range of possible sequels. The most frequent present diagnosis in 44 patients seen over a period of 3 years was PTSD (n = 40), but criteria for a present diagnosis of other disorders were fulfilled in 34 patients, even years after torture, mainly major depression or dysthymia (n = 26). Criteria for functional psychosis were fulfilled in 4 patients. Many patients reported symptoms not assessed by DSM-III-R criteria, including feelings of shame and guilt, and ruminations on existential fears. The impairment as indicated by the GAF (mean 59.1) correlated best with the presence of the endogenomorphic-depressive axial syndrome, but not with duration of imprisonment, age or other factors. Research on sequels to extreme trauma should not be restricted to a simple diagnosis of PTSD, but should continue to look for a broader conceptualisation, including neglected categories like the axial syndrome, as PTSD is common, but might not be the only factor of importance for research and treatment. ICD-10 might offer a more adequate interpretation of sequels.

Psychiatric disorders in patients with blepharospasm - a reactive pattern?

OBJECTIVE: The purpose of this study was to assess the life-time prevalence of all major psychiatric disorders in patients suffering from blepharospasm. METHOD: A total of 31 consecutive patients with blepharospasm attending the Department of Neurology were interviewed at the Department of Psychiatry at the University of Vienna. Patients had been submitted to standard neurological diagnostic procedures, psychiatric diagnoses were made with the help of the SCID, functional impairment was assessed by the General Assessment of Functioning Scale (GAF). RESULTS: A current or life-time psychiatric diagnosis was made for 22 patients (71%). The most frequent disorders were depressive disorders, mainly major depression (five patients, 16.1%), secondary dysthymia (six patients, 19.3%), and recurrent major depression (five patients, 16.1%). A diagnosis of simple phobia was made for seven patients (22.5%), for obsessive-compulsive disorder in three patients (9.6%). The mean GAF score of our sample was 63.1%. CONCLUSION: In contrast to previously published results, we did not find a high rate of a single specific disorder or patterns in our study sample, though by the inclusion of life-time diagnostic criteria, the majority of patients fulfilled criteria for at least one diagnosis. This might indicate the considerable negative impact of blepharospasm on the patients' lives.

Delusional pregnancy in a patient with primary sterility.

The case of a patient suffering from primary sterility who developed a well-systemized delusion of pregnancy is described. The patient did not have a history of psychiatric disorder or organic cerebral pathology. No history of epilepsy was detected in this patient contrary to the reports in the literature, and these facts have pathogenic relevance in delusional pregnancy. The importance of psychological factors in the development of a delusion of pregnancy is discussed.

Dysphoria from a transnosological perspective.

The objective of our psychopathological analyses is to shed light on the position of irritable mood (dysphoria) in psychiatric diagnostics and nosology. In today's most commonly applied classification systems, the ICD-10 and the DSM-IV, dysphoria is mentioned mostly in the context of diagnostic criteria of personality and affective disorders. Other authors have emphasized the importance of dysphoric states in organic psychoses and delusional disorders. Summarizing the various publications on the nosological position, dysphoria is a nosological nonspecific syndrome which may occur in the course of all psychiatric disorders and illnesses. According to the results of our psychopathological analyses, the pathogenesis of dysphoria has to be considered as a multidimensional circular process in which various mental, physical and social factors act as predisposing, triggering and disorder-maintaining factors. Stressors induced by particular experiences and perceptions and by impaired health may lead to a dysphoric state if adequate coping mechanisms are missing. Dysphoria itself usually leads to a deterioration in the mental and physical state of the patient, and shows a clear impact on the patient's social network. The reactions of people close to the patient combined with the impaired mental and physical conditions of the patient cause the circle to restart. As contemporary diagnostic entities do not refer to pathogenesis, classical categorical diagnostics cannot provide the basis for effective pathogenesis-oriented therapy. A change of paradigm in diagnostics from a categorical to a dimensional approach thus becomes necessary. Following a dimensional diagnostic approach based on a dynamic model of vulnerability, a precise differential diagnosis of the complex constellation of conditions and their interactions becomes necessary in order to develop effective treatment strategies. Disorder-maintaining factors determine the treatment of the acute symptomatology, whereas predisposing and triggering factors serve as the basis for the prophylactic treatment.

The synthesis of chiral cyanohydrins by oxynitrilases.

Enantiomerically pure cyanohydrins are important synthetic intermediates for pharmaceuticals and agrochemicals. They are produced by enzyme-catalysed synthesis using oxynitrilases. Sufficient quantities of enzyme are available via cheap natural sources and there have been recent advances in overexpression production of cyanohydrins on an industrial scale.

Optimization of the hydroxylation of 2-cyclopentylbenzoxazole with Cunninghamella blakesleeana DSMZ 1906.

Biohydroxylation of 2-cyclopentyl-1,3-benzoxazole with the filamentous fungus Cunninghamella blakesleeana DSMZ 1906 was studied in a 15-l stirred tank reactor. The aim of the work was to avoid substrate limitation through sub-optimal mixing by formation of pellets with a uniform pellet size distribution of 250-500 microm, obtained at an inoculum concentration of 10(7) spores ml(-1) and an agitation rate of 390 rpm. Due to the high toxicity of the educt, 2-cyclopentyl-1,3-benzoxazole, on the fungus, the medium composition, the time of educt addition, and the educt starting concentration were optimized to reach high educt tolerance and hydroxylation activity. A good maintenance of biotransformation capacity was obtained without excessive loss of activity of the biocatalyst by addition of 30 mg 2-cyclopentyl-1,3-benzoxazole/g biomass (cell dry mass) during the stationary phase in a medium which was optimized in batch fermentations with experimental designs. An increase in product yield and quality (enantiomeric excess) was achieved by developing feeding strategies combining the educt and medium components. The resulting fermentation broth contained 450 mg l(-1) of the product (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol with an enantiomeric excess of 95%, which represents a 48% increase over former reported results.

Oxynitrilases for asymmetric C-C bond formation.

Oxynitrilases for the preparation of (R)- or (S)-cyanohydrins are now readily available. The research efforts of a number of groups have established these enzymes as catalysts with significant potential for application to asymmetric synthesis. Advances made in molecular cloning and genetics have delivered information on the oxynitrilase mechanism of action and sufficient quantities of enzyme to satisfy industrial requirements.

The Concept of Docking/Protecting Groups in Biohydroxylation.

A general principle for biohydroxylation, in which time-consuming screening and enrichment techniques are avoided, is demonstrated by the introduction of a docking/protecting group into the substrate. This facilitates acceptance by the microorganism and allows the use of a narrow range of microorganisms, for example Beauveria bassiana ATTC 7159 (B. b.), for the hydroxylation of compounds with diverse structures. After the biohydroxylation, the docking/protecting group is removed (see scheme).

Proton-nuclear magnetic resonance analyses of the substrate specificity of a beta-ketolase from Pseudomonas putida, acetopyruvate hydrolase.

A revised purification of acetopyruvate hydrolase from orcinol-grown Pseudomonas putida ORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of approximately 38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by (1)H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three (1)H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A (1)H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized pi-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg(2+) bound to the enzyme was made. (1)H-(2)H exchange reactions showed the complete, fast equilibration of (2)H into the C-3 of acetopyruvate chemically; this accounts for the appearance of (2)H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional (1)H-NMR in normal (1)H(2)O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.

Dimensions of quality of life and self-monitoring therapy with oral anticoagulants--still research or everyday practice?

In correlation with increased life expectancy of patients, quality of life (QOL) has become a factor of increasing interest by the patient himself and also of importance in health-care planning and recruitment of financial resources. In this context, self-monitoring of long-term anticoagulant treatment might be a strategy that could mean a step forward in health-related as well as general life satisfaction for patients participating in self-monitoring programs. Also, the new strategy of increased home-control of anticoagulant treatment illustrates the complexity of multiple factors that can lead to changes in the subjective feeling and objective aspects of QOL. Our intention in a pilot study was to probe the feasibility of QOL research and relevant factors of influence by retrospectively evaluating data from two groups of outpatients seen in a large treatment center. The high frequency (n = 8 in sample 2) of disturbed sleep as a simple screening indicator stresses the probable importance of undetected depression, which might require treatment and could confound research as to QOL. Instruments to measure QOL in oral anticoagulation self-monitoring should therefore be adapted to the heterogeneous structure of factors in the target population, and include psychological parameters, especially in regard to health-related locus of control and mood.

Kinetic studies on the enzyme (S)-hydroxynitrile lyase from hevea brasiliensis using initial rate methods and progress curve analysis

(S)-Hydroxynitrile lyase (EC 4.1.2.39) from Hevea brasiliensis(rubber tree) catalyzes the reversible cleavage of cyanohydrins to aldehydes or ketones and prussic acid (HCN). Enzyme kinetics in both directions was studied on a model system with mandelonitrile, benzaldehyde, and HCN using two different methods-initial rate measurements and progress curve analysis. To discriminate between possible mechanisms with the initial rate method, product inhibition was studied. Benzaldehyde acts as a linear competitive inhibitor against mandelonitrile whereas HCN shows S-linear I-parabolic mixed-type inhibition. These results indicate an Ordered Uni Bi mechanism with the formation of a dead-end complex of enzyme, (S)-mandelonitrile and HCN. Prussic acid is the first product released from the enzyme followed by benzaldehyde. For progress curve analysis, a kinetic model of an Ordered Uni Bi mechanism including a dead-end complex, enzyme inactivation, and the chemical parallel reaction was set up, which described the experimental values very well. From the reaction rates obtained the kinetic constants were calculated and compared with the ones obtained from the initial rate method. Good agreement could be achieved between the two methods supporting the suggested mechanism. Copyright 1999 John Wiley & Sons, Inc.

Psychiatric wills of mental health professionals: a survey of opinions regarding advance directives in psychiatry.

Psychiatric wills are advance directives for an eventual involuntary treatment in psychiatry. We attempted to determine psychiatric professionals' knowledge and opinion about this legal option and obtain their formulations of advance directives for themselves. A total of 101 psychiatric nurses and psychiatrists at the Department of Psychiatry of the University of Vienna responded to a questionnaire about psychiatric wills and anonymously drafted advance directives for themselves concerning psychiatric treatment in case of an acute psychosis. Fifty-four percent knew about this legal option, 55% considered it an appropriate legal possibility, and 29% considered it inappropriate. The study also found that 75% of respondents reject certain methods of therapy, e.g. 30% want to exclude the use of neuroleptic medications, and 46% reject ECT. We conclude that although there is little experience so far with advance directives for psychiatric patients, there is an interest and predominance of positive attitudes towards this legal option among mental health professionals. Concerning their preferences, professionals felt inclined to make very specific statements as to which available treatment strategies they would reject and which they would request for their treatment. This bodes well for the widespread use of advance directives in mental health settings.

Panic disorder and cigarette smoking behavior.

Smoking has been discussed both as a risk factor for panic disorder and as a contributing factor to elevated cardiovascular risk in panic disorder patients. Smoking habits and their association with panic disorder were studied in a sample of 102 panic disorder patients. Both for female and for male patients, rates of smokers and of exsmokers were substantially higher than in the general population. However, a surprisingly high number of patients had succeeded in reducing or quitting cigarette smoking because of their panic disorder, although they experienced little benefit in regard to panic symptoms from doing so. We conclude that the motivation for changing smoking habits is high in this population with elevated smoking prevalence and should be taken into consideration by therapists.

["Because it concerns me"--patient-directed discharge letters]. / "Weil es mich betrifft"--direkt an Patientlnnen gerichtete Entlassungsbriefe.

PURPOSE: Evaluation of practicability and acceptance of discharge summaries addressed directly to patients after psychiatric hospitalisation. METHODS: Over a period of 3 months 65 patients got discharge summaries addressed directly to them. Doctors and patients--4 months after discharge--were asked to evaluate this procedure. RESULTS: Both doctors' and patients' acceptance and evaluation was very positive. Argumentations for this view were the need to give and get information and the impression that this procedure can enhance confidence and trust.