d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.     

Remnants of trees from archaeological finds in Bohemia,I

The present paper gives an account of the archaeobotanical results of charcoal investigations from the lower valley of the river Oh?e (Bohemia). According to the analysis the primal vegetation may be the allianceCarpinion betuli.     

A method for estimating the number of invariant amino acid coding positions in a gene using cytochrome c as a model case

This paper shows, within the limitations of the assumption stated below, that approximately 27–29 of the unmutated codons which determine the amino acids of cytochrome c are invariant because of biological requirements. A mutation is defined here as the change of a single base in the sequence of a trinucleotide codon, which change alters the amino acid coded for. Codons, if any, in which mutations would be vigorously selected against are termed invariant codons. We assume that, subject to one adjustment, those mutations in the cytochrome c gene which survived in the descent of today's species are randomly distributed among the variable codons. The one adjustment arises from the possibility that a very few codon positions may exhibit frequencies of mutation sufficiently great to justify the exclusion of these codons from the overall distribution on the grounds that the frequency of mutation occurring in these few positions is clearly inconsistent with the assumption of randomness. There are 5 out of the total 110 codons in the cytochrome c structural gene which have clearly sustained an abnormally large number of mutations.This project received support from grants to W.M.F. from the National Institutes of Health (NB-04565) and the National Science Foundation (GB-4017).     

Assignment of the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22

The chromosomal location of the human intestinal Na+/glucose cotransporter gene (SGLT1) was determined using human cDNA and genomic probes for this transporter gene. Southern blot analysis of genomic DNA from 15 mouse-human somatic cell hybrids showed that the human gene for this transporter resides on chromosome 22. Analysis of hamster-human hybrids selectively retaining chromosome 22 or a portion of it allowed specific assignment of the locus to the q11.2----qter region of chromosome 22. A restriction fragment length polymorphism was identified with EcoRI.     

Capillary GC quantification of cholesterol oxidation products in plasma lipoproteins of fasted humans

Cholesterol oxidation products have been hypothesized to be important factors in atherosclerosis, a process which can culminate in myocardial infarction. The relative importance of exogenous or in vivo sources of cholesterol oxidation products has not been determined. However, methodology used for cholesterol oxidation products analysis of foods is applicable to the determination of cholesterol oxidation products in human plasma lipoproteins. Such methodology, outlined in this report, permits numerous critical experiments to be conducted on the possible role of cholesterol oxidation products in coronary heart disease.     

Characterization of Cellular Transport, Subcellular Distribution, and Secretion of the Neurotoxicant 1-Methyl-4-Phenylpyridinium in Bovine Adrenomedullary Cell Cultures

Cultures of bovine adrenomedullary chromaffin cells accumulated 1-[methyl-3H]methyl-4-phenylpyridinium ([3H]MPP+) in a time- and concentration-dependent manner with an apparent Km of 0.7 microM and a Vmax of 3 pmol/min/10(6) cells. The uptake was sodium dependent and sensitive to inhibitors of the cell-surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine-containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 microM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time-dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 microM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+ -dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium-induced release of both species was independent of external Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)