Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey,Lampetra japonica

Summary Distribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH₂ (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Synthesis of an affinity ligand ('UPHIT') for in vivo acylation of the kappa-opioid receptor

The isothiocyanate analog (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective kappa-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of kappa-opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)-(+)-trans-2-pyrrolidinyl-N-methyl-cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to kappa receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 +/- 0.36 nM, whereas 827.42 +/- 5.88 and 115.10 +/- 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (+/-)-3 respectively. Intracerebroventricular (ICV) injection of 100 micrograms of (+/-)-3 into guinea-pig brain followed by analysis of remaining kappa-binding sites 24 h later revealed that (+/-)-3 depleted 98% of the kappa receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the kappa-opioid receptor.     

Repetitive recycling of guanosine triphosphate cyclohydrolase I for synthesis of dihydroneopterin triphosphate

A procedure for enzymatic production of dihydroneopterin triphosphate is described that allows GTP cyclohydrolase I to be reused repetitively. The reaction takes place in an ultrafiltration cell, and the product is collected in the filtrate, whereas the enzyme remains in the cell to be reused with additional substrate. This is repeated until the enzyme activity drops below a desirable level. The purity of the dihydroneopterin triphosphate is satisfactory for utilization of this compound for studies on enzymes involved in the synthesis of tetrahydrobiopterin and drosopterin. A procedure for purification of dihydroneopterin triphosphate is described that uses C18-silica and silica cartridges.     

Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.     

Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer

The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.