Liver cell necrosis during administration of clozapine

A 38-year-old woman was treated for mutism with clozapine. After a week liver function disturbances developed, which disappeared when the treatment was discontinued. Histopathological investigation of a liver biopsy specimen revealed extensive liver cell necrosis. So far two patients have been described with cholestatic jaundice induced by clozapine, and one patient with toxic hepatitis due to clozapine.     

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection

The title compounds (6-9) were prepared and evaluated for serotonin 5-HT4 agonistic activity in in vitro tests. Introducing a propyl or allyl group at the 3-position of benzamide caused only a slight enhancement of agonistic activity. Construction of the benzo[b]furan skeleton and 2,3-dihydrobenzo[b]furan skeleton caused a significant enhancement of the activity. 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2-methylbenzo[b]furan-7-carboxamide (7b) hemifumarate was as potent as cisapride. 4-Amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide (8a) hemifumarate, 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dihydro-2-ethylbenzo[b]furan-7-carboxamide (8c) hemifumarate, and 4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro- 2,3-dimethylbenzo[b]furan-7-carboxamide (8d) hemifumarate were more potent than cisapride. Furthermore, 8a hemifumarate was free from dopamine D1, D2, serotonin 5-HT1, 5-HT2 and muscarine M1, M2 receptor binding activity in the in in vitro tests. On the other hand, construction of the indole skeleton caused a remarkable decrease in activity.     

Using Mutability Landscapes To Guide Enzyme Thermostabilization

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature T_m (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in T_m value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.     

Detection of bone marrow metastases in small cell lung cancer. Comparison of magnetic resonance imaging with standard methods

In small cell lung cancer (SCLC), bone marrow metastases are frequently detected by bone scintigraphy (BS) and/or unilateral bone marrow biopsy and aspiration (BMBA). In this study, the value of magnetic resonance imaging (MRI) of thoracic spine and pelvis was compared with BS and BMBA and its clinical implication was evaluated in 42 patients with SCLC. Patients were staged (including BS, BMBA, CT thorax, Liver ECHO) as limited (LD) or extensive disease (ED) before and after MRI. MRI was positive in 12 BS negative (P = 0.003) and in 14 BMBA negative patients (P < 0.001), while in 8 patients, MRI was the only sign of ED, which resulted in a decrease of patients categorised with LD from 52 to 33%. However, in this small group of LD patients, there was no significant survival difference between LD (MRI pos) and LD (MRI neg) patients. It is concluded that MRI can be of value in the staging of LD patients, but it has no influence on survival.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Alpha and beta glucocorticoid receptor mRNA expression in skeletal muscle

The aim of the present study was to investigate the occurrence and autoregulation of both glucocorticoid receptor mRNAs in rat gastrocnemius muscle. The expression of both receptor forms was studied 1, 4 or 12 hours after intra-tracheal instillation of a high dose (100 micrograms) of budesonide; muscular expression was compared with glucocorticoid receptor expression in lung tissue. After Northern blot analysis, hybridization was performed with glucocorticoid receptor, glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase probes. In the gastrocnemius muscle, both the alpha and beta glucocorticoid receptor mRNA forms were detected and found to be downregulated four hours after the budesonide instillation. alpha/beta glucocorticoid receptor ratios were lower in the gastrocnemius (1.1 +/- 0.2) than in the lungs (2.6 +/- 0.6). In the lungs, at all time points, the average alpha glucocorticoid receptor mRNA levels did not differ from controls, although glutamine synthetase mRNA levels were upregulated. The beta glucocorticoid receptor mRNA was slightly reduced at 1 and 4 hours. In conclusion, after intra-tracheal instillation of budesonide, both alpha and beta glucocorticoid receptor forms were downregulated in muscle tissue. The difference in alpha/beta glucocorticoid receptor mRNA ratios and concentrations between lung and gastrocnemius muscle supports the hypothesis of differential gene regulation by glucocorticoids in different cell types.     

Preserved catalytic activity in an engineered ribonucleotide reductase R2 protein with a nonphysiological radical transfer pathway. The importance of hydrogen bond connections between the participating residues

A hydrogen-bonded catalytic radical transfer pathway in Escherichia coli ribonucleotide reductase (RNR) is evident from the three-dimensional structures of the R1 and R2 proteins, phylogenetic studies, and site-directed mutagenesis experiments. Current knowledge of electron transfer processes is difficult to apply to the very long radical transfer pathway in RNR. To explore the importance of the hydrogen bonds between the participating residues, we converted the protein R2 residue Asp237, one of the conserved residues along the radical transfer route, to an asparagine and a glutamate residue in two separate mutant proteins. In this study, we show that the D237E mutant is catalytically active and has hydrogen bond connections similar to that of the wild type protein. This is the first reported mutant protein that affects the radical transfer pathway while catalytic activity is preserved. The D237N mutant is catalytically inactive, and its tyrosyl radical is unstable, although the mutant can form a diferric-oxo iron center and a R1-R2 complex. The data strongly support our hypothesis that an absolute requirement for radical transfer during catalysis in ribonucleotide reductase is an intact hydrogen-bonded pathway between the radical site in protein R2 and the substrate binding site in R1. Our data thus strongly favor the idea that the electron transfer mechanism in RNR is coupled with proton transfer, i.e. a radical transfer mechanism.     

The prognostic significance of accumulation of p53 protein in stage III non-small cell lung cancer treated by radiotherapy

In the present study the prognostic significance of accumulation of nuclear p53 protein on survival and freedom from local progression was investigated. Formalin-fixed, paraffin-embedded sections obtained by bronchoscopy or mediastinoscopy were used to examine the expression of nuclear p53 protein using immunohistochemistry. In 37 cases (57%), overexpression of the p53 protein was detected. No relation was found between p53 expression and other pretreatment variables. Response to radiotherapy was found in 11 p53-negative cases (65%) versus 10 p53-positive cases (42%). Freedom from local progression was significantly better in the p53-negative cases as compared with the p53-positive cases. The p53-negative cases who responded to radiotherapy showed an excellent freedom from local progression rate after 2 years of 100%, whereas all p53-positive cases without response to radiotherapy showed local progression within 24 months. Overall survival between p53-negative and -positive cases did not differ, however the disease-specific survival was found to be worse in the p53-positive cases as compared to the negative cases (median survival 8.4 vs. 14.4 months (P < 0.05)). No correlation was found between p53 expression and the frequency of distant metastases. In conclusion, the results of this study suggest that p53 protein expression may be of prognostic value on freedom from local progression in non-small cell lung carcinoma.     

Validation of the Point-EXACCT method in non-small cell lung carcinomas

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42-52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.     

Evidence for the Formation of an Enamine Species during Aldol and Michael‐type Addition Reactions Promiscuously Catalyzed by 4‐Oxalocrotonate Tautomerase

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which has a catalytic N‐terminal proline residue (Pro1), can promiscuously catalyze various carbon–carbon bond‐forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael‐type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ‐nitroaldehydes. To gain insight into how 4‐OT catalyzes these unnatural reactions, we carried out exchange studies in D₂O, and X‐ray crystallography studies. The former established that H–D exchange within acetaldehyde is catalyzed by 4‐OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4‐OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4‐OT‐catalyzed aldol and Michael‐type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1‐dependent formation of an enamine intermediate.