Detection of cystathionine and lanthionine ketimines in human urine

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.     

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).     

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

N-dealkylation of chlorimipramine and chlorpromazine in patients undergoing chronic treatment with both drugs: preliminary results

Data obtained in this preliminary study show that in patients chronically treated either with Chlorimipramine (n = 6) or with Chlorpromazine (n = 2), significant amounts of the corresponding Nor1- and Nor2-metabolites were detected in plasma. The role of these metabolites in the overall therapeutic effect is under investigation.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.