Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production.

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

Molecular characterization of human and bovine endothelin converting enzyme (ECE-1).

A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE). The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture and oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.

Characterization of a membrane-bound metalloendoprotease of rat C6 glioblastoma cells.

C6 rat glioblastoma cells are able to attach to and to spread on culture dishes which are coated with purified central nervous system myelin, in contrast to normal astrocytes, fibroblasts or neurons which adhere poorly and are unable to spread on this substrate. The metalloprotease blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myelin, suggesting a crucial role for a metalloprotease. Here we characterize this metalloproteolytic activity of C6 cells using a peptide degradation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe-125I-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membranes cleaved the peptide between alanine and phenylalanine, an effect which is strongly inhibited by o-phenanthroline, but not by thiol-blocking agents or aspartic and serine protease inhibitors. The metalloendoprotease is highly sensitive to phosphoramidon but insensitive to thiorphan. The enzyme is tightly bound to the plasma membrane but not G protein-phosphatidylinositol linked. It can be solubilized in part by the detergents 3-(3-cholamidopropyldimethylamino)-1-propanesulfonate or Triton X-114. Gel filtration chromatography using the Triton X-114-solubilized proteins or the proteins removed by a short trypsin treatment revealed a molecular weight range for the C6 enzyme of 60,000-100,000. Polymerase chain reaction with primers corresponding to endopeptidase 24.11 or to the highly conserved motif of the "astacin family" showed that both enzymes were not detectable in the C6 glioblastoma cells.

HT7, Neurothelin, Basigin, gp42 and OX-47--many names for one developmentally regulated immuno-globulin-like surface glycoprotein on blood-brain barrier endothelium, epithelial tissue barriers and neurons.

The antigens HT7 and Neurothelin were recently described as inducible membrane glycoproteins on chick blood-brain barrier endothelium. Our results demonstrate that the Neurothelin antibody 1W5 does recognize the isolated HT7 protein as well as the HT7 protein expressed by COS-1 cells transfected with the HT7 cDNA. Therefore, the HT7 and Neurothelin epitopes can be found on an unique glycoprotein. By low stringency hybridization we have isolated the murine cDNA clone encoding the homologous murine protein. The nucleotide sequence of the open reading frame is identical to that of the cDNA clones encoding the gp42 protein and the Basigin antigen expressed in F9 cells and in the early mouse embryo. The murine amino acid sequence shows 94% identity with the rat OX-47 membrane glycoprotein which was shown to be expressed in blood-brain barrier endothelium and several epithelial cell layers. These results demonstrate that HT7, Neurothelin, gp42, Basigin and OX-47 are different names for probably the same glycoprotein in different species.

Correlation of blood-brain barrier function and HT7 protein distribution in chick brain circumventricular organs.

The HT7 protein defined by a monoclonal antibody is a specific marker for chick brain endothelial cells (EMBO J., 5 (1986) 3179-3183). In this study, we have investigated the expression of this protein in the brain circumventricular organs which lack a blood-brain barrier. Using immunohistochemical techniques we found that the protein was absent from the vascular system of the pituitary, median eminence, subfornical organ, pineal gland, the organum vasculosum lamina terminalis and the layer of sinusoid blood vessels of the area postrema. In some regions of the median eminence and, more strikingly, in the pineal gland, parenchymal cells expressed the HT7 antigen. Immunoblots of proteins from brain, pituitary, pineal gland and retina showed that the antigen is very abundant in the retina. Lower amounts were present in brain and pineal gland. The glucose transporter was found to be an independent reliable marker for blood-brain barrier endothelium. In the chick brain the distribution of the biochemically distinct proteins was very similar. Using a postembedding technique we have ultrastructurally localized the HT7 protein specifically in blood-brain barrier endothelial cells. Thus, the expression of the HT7 protein and glucose transporter correlated with blood-brain barrier function.

The inducible blood--brain barrier specific molecule HT7 is a novel immunoglobulin-like cell surface glycoprotein.

The unique properties of brain endothelial cells, which form the blood-brain barrier, are reflected by the expression of specific cell surface molecules. We report here the purification, cloning and expression of one such molecule which is recognized by HT7 monoclonal antibodies. The HT7 antigen is a highly glycosylated 45-52 kd protein localized in brain endothelial cells, kidney epithelial cells and erythroblasts. The protein was purified to homogeneity from plasma membrane proteins isolated from all three sources using immunoaffinity chromatography and reverse phase HPLC. The amino-terminal amino acid sequences of the proteins were found to be identical. Based on amino acid sequence information, specific primers were designed and the polymerase chain reaction was used to obtain a full length cDNA clone. The nucleotide sequence encoded a novel glycoprotein with two C2-like immunoglobulin related domains, one transmembrane domain and a cytoplasmic tail. Expression of the transfected cDNA in COS cells resulted in the appearance of the HT7 antigen on the surface of these cells. On the basis of our results we propose that the protein may be a receptor involved in cell surface recognition at the blood-brain barrier.