Immobilization of enzymes on alumina by means of pyridoxal 5′-phosphate

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum)

Summary Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 g/g body weight). Fish were sacrificed 30 min and 1,2 and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Increased 5-Hydroxytryptamine and Norepinephrine Release from Rat Brain Slices by the Red Sea Flatfish

The effect of the Red Sea flatfish toxin pardaxin was examined on K+-evoked and on basal release of either [3H]norepinephrine or [3H]5-hydroxytryptamine from preloaded rat cortical slices. The K+-induced release of the neurotransmitters was stimulated in a dose-related manner at concentrations ranging from 0.5 to 4 micrograms/ml. Basal release of the two transmitters was elevated to a lesser extent. Although the stimulation of evoked release was approximately equivalent for the two neurotransmitters, the response to 5-hydroxytryptamine was reversible whereas that of norepinephrine was not washed by 20 min of superfusion. The mechanisms involved in producing these actions of pardaxin are not known; however, they may be mediated by changes in electrolyte fluxes across the neuronal membranes.     

Cyclic AMP inhibits platelet activation independently of its effect on cytosolic free calcium

Stimulation of platelets with the ionophore A23187, thrombin, ADP or PAF-acether resulted in a rapid increase of cytosolic free Ca2+, as measured with Quin-2, and in aggregation, 5HT secretion and - in the case of the first two agonists - thromboxane generation. PGI2 and dibutyryl cyclic AMP inhibited all these responses, except in the case of A23187, in response to which the increase in Ca2+ was unaffected, although the other responses were inhibited. The inhibition of aggregation and secretion in response to the combination of thrombin and A23187 was indistinguishable from that in response to thrombin alone. It thus appears that cAMP inhibits these responses independently of its effect in lowering cytosolic free Ca2+.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.