Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

The cyanogenic substrate for horseradish peroxidase is a conjugated enamine

We identify the cyanogenic substrate for horseradish peroxidase (HRP) as a conjugated enamine and explore this unusual reaction using alpha-aminocinnamate (RH) as follows. 1) HRP catalyzes the oxidation of RH by O2 (and its peroxidation by H2O2 to form R-R) to produce, simultaneously, CN- and benzaldehyde cyanohydrin. 2) RH is transient and must be generated in situ. The properties of the cyanogenic reaction of HRP are independent of the method of preparation of RH (whether this be condensation of NH3 with phenylpyruvate, enzymatic hydrolysis of glycyldehydrophenylalanine, or oxidation of L-phenylalanine by L-amino acid oxidase). 3) The oxidation of RH is a free radical chain reaction initiated by HRP Compounds I and II (I (or II) + RH----R. + II (or HRP], propagated by RO2. (R. + O2----RO2., RO2. + RH----R. + RO2H), and terminated by recombination reactions such as 2R.----R2 and RO2.----R' + HO2. followed by R. + HO2.----RH + O2. KMnO4 and K3Fe(CN)6 can substitute for HRP. 4) The proximal precursor of CN- and cyanohydrin is postulated to be RO2H (phi-CH(-O2H)-CCO2-(= NH]. These results explain why cyanide is generated from the synergistic action of HRP and L-amino acid oxidase on aromatic L-amino acids and O2 and suggest that the requirement for a beta-aryl substituent on the enamine originates in the reaction of RH with HRP, or of R with O2, rather than the imine/enamine tautomerization of the L-amino acid oxidase product.     

Phorbol esters and calcium ionophores inhibit internalization and accelerate recycling of receptors in macrophages

Exposure of macrophages to phorbol esters or the calcium ionophore A23187 increases the number of several surface receptors due to recruitment of receptors from internal pools (Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38, 569-576). We have examined the mechanism by which these agents increase surface receptor number. Cells which were preloaded with either fluid phase or receptor-mediated ligands did not lose ligand following exposure to ionophore or phorbol ester. The rate of movement of ligands to the lysosome was also unaffected. These results suggest that A23187 does not induce the fusion of ligand-containing compartments with the cell surface. Ionophore treatment did, however, produce a severalfold increase in the rate at which unoccupied receptors reappear on the cell surface. These results suggest that the compartment of receptors affected by the ionophore formed subsequent to the dissociation of ligand from receptor. The altered rate of receptor reappearance was transitory (90 s), and the increase in receptor number was subsequently maintained by a decrease in the rate of internalization. Changes in the rate of receptor internalization did not correlate with changes in the rate of fluid phase pinocytosis, suggesting that the effect on receptor internalization was selective.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.     

Autostimulation of dihydrostreptomycin uptake in Bacillus subtilis

In Bacillus subtilis it was shown that the membrane potential (delta psi) has to reach a threshold value of -180 to -190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of delta psi is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N'-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of delta psi. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing delta psi due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins.     

The use of biochemical markers, serotype and fimbriation in the detection of Escherichia coli clones

Biochemical reactions, O and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index less than 0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0.89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain O serotypes (O1, O4, O6, O7, O18 and O25) were more common among bacteraemic isolates than controls. K serotypes such as K1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.     

Uptake of galactose and lactose by Kluyveromyces lactis: biochemical characteristics and attempted genetical analysis

Study of the lactose and galactose transport systems in Kluyveromyces lactis has shown that lactose uptake is by active transport. The transport system is under monogenic control and is inducible. Galactose uptake is also by active transport but the system is controlled by two genes which, in the four strains we studied, are present only in K. lactis CBS 2359. Galactose uptake in the other K. lactis strains is by a simple diffusion process.     

Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.