Bacteriophage T4-induced shut-off of host-specific translation.

To study the mechanism by which bacteriophage T4 inhibits the synthesis of inducible host enzymes we measured the formation of beta-galactosidase from preformed lac mRNA. Beta-Galactosidase was induced with isopropyl-beta-D-thiogalactopyranoside in the presence of 7-azatryptophan, a tryptophan analogue that is incorporated into proteins and renders the beta-galactosidase formed inactive. The accumulated las mRNA was measured by capacity to form active beta-galactosidase after a chase of the analogue with excess tryptophan. After T4 infection the ability to form beta-galactosidase from the preformed lac mRNA was rapidly lost even when T4 infection took place in the presence of rifampin. This restriction was dependent on the multiplicity of infection. At a multiplicity of infection of 8.6, 90% of the ability to express preformed lac mRNA was lost within 30 s. The kinetics of cessation of beta-galactosidase synthesis after T4 infection indicate that infection blocks initiation of lac mRNA translation.     

Transport of D-glucose and 3-O-methyl-D-glucose in the cyanobacteria Aphanocapsa 6714 and Nostoc strain Mac.

1. The cyanobacterium Aphanocapsa 6714 which grow in the dark on D-glucose, will take up D-glucose and the analogue 3-O-methyl-D-glucose; uptake of each of these compounds was inhibited competitively by the other and by 6-deoxy-D-glucose. 2. This cyanobacterium accumulated 3-O-methyl-D-glucose up to 100-fold relative to the medium but did not modify or metabolize it to a significant degree. 3. Intracellular 3-O-methyl-D-glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D-glucose and 3-O-methyl-D-glucose. 4. Although not characterized to the same extent, D-glucose and 3-O-methyl-D-glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D-glucose, was similar. 5. Other cyanobacteria that do not grow on D-glucose take up this compound at much lower rates which were unaffected by analogues of D-glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac. 6. It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D-glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D-glucose as a growth substrate.     

The mechanism of the synthesis of enzymes. II. Further observations with particular reference to the linear nature of the time course of enzyme formation

1. The pretreatment induction method of studying the formation of beta-galactosidase in E. coli B has been described. 2. It has been found that E. coli B cells have their maximum capacity to form beta-galactosidase, in response to a constant induction stimulus, when they are in the stationary phase of the growth cycle. 3. The concentration of inductor, the nature of the nitrogen source, the duration of the assimilatory phase, oxygen tension, and temperature are factors which affect, and may limit, the rate of beta-galactosidase formation. 4. When limitations imposed by these factors were removed, the time course of induced beta-galactosidase formation was strictly linear from the onset. 5. The implications of this finding were discussed and a new theory of the mechanism of enzyme formation has been proposed. 6. A very satisfactory method of synthesis of ortho-nitrophenol-alpha-D-galactoside has been described. This substance is a suitable chromogenic substrate for the specific determination of alpha-galactosidase activity. 7. Preliminary experiments using this substrate have confirmed the results of respiration studies and shown that in E. coli B alpha-galactosidase formation may be induced by beta- as well as by alpha-galactosides.     

Regulation of formation of the intracellular beta-galactosidase activity of Aspergillus nidulans

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.     

D-Glucose Isomerase: Constitutive and Catabolite Repression-Resistant Mutants of Streptomyces phaeochromogenes

As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression.     

New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.     

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.     

Investigations on isolated islets of langerhans in vitro. 16.Modification of the glucose-dependent inhibition of glucagon secretion.

Investigation of glucagon secretion in isolated Wistar rat islets was carried out to elucidate further the regulatory function of glucose and arginine on pancreatic A-cells. The suppressive effect of D-glucose could also be demonstrated with L-glucose, D-mannose, D-fructose, D-galactose, D-glyceraldehyde and DL-dihydroxyacetone, but not in the presence of 3-O-methylglucose or mannitol. Sugars other than D-glucose inhibited glucagon secretion only at much higher concentrations than those at which D-glucose was effective. Furthermore, although 7.5 mM D-glucose up to 80% inhibition, the effects of other sugars appeared to level off at only 50--60% inhibition. The inhibitory action of D-glucose or D-glyceraldedyde on glucagon secretion could not be overcome by L-arginine, but 3-O-methylglucose, mannoheptulose, 2-deoxy-D-glucose, iodoacetamide, theophylline, epinephrine and acetylcholine were effective. The insulin secretion in response to glucose was inhibited by the metabolic inhibitors used, whereas the B-cell response in the presence of glyceraldehyde was diminished by iodoacetamide only. Like D-glucose, a variety of other sugars markedly reduced the stimulatory effect of L-arginine in glucagon release. The results show that the suppression of glucagon secretion is not specific for D-glucose and not strongly connected on a stimulated insulin secretion.     

Effect of glucose on endothelin-1-induced calcium transients in cultured bovine retinal pericytes.

Published work has shown that endothelin-1-induced contractility of bovine retinal pericytes is reduced after culture in high concentrations of glucose. The purpose of the present study was to establish the profile of endothelin-1-induced calcium transients in pericytes and to identify changes occurring after culture in high concentrations of glucose. Glucose had no effect on basal levels of cytosolic calcium or on endothelin-1-induced calcium release from intracellular stores. However, influx of calcium from the extracellular medium after endothelin-1 stimulation was reduced in pericytes that had been cultured in 25 mM D-glucose. L-type Ca(2+) currents were identified by patch clamping. The L-type Ca(2+) channel agonist, (-)-Bay K8644, caused less influx of calcium from the extracellular medium in pericytes that had been cultured in 25 mM D-glucose than in those cultured with 5 mM D-glucose. However, 3-O-methylglucose, a nonmetabolizable analogue of glucose which can cause glycation, had similar effects to those of high concentrations of glucose. The results suggest that reduced function of the L-type Ca(2+) channel that occurs in bovine retinal pericytes after culture in high concentrations of D-glucose is probably due to glycation of a channel protein.     

Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana.

Both genomic and full length cDNA clones of an Arabidopsis thaliana sugar carrier, STP1, have been obtained using a cDNA clone of the H+/hexose cotransporter from the green alga Chlorella kessleri as hybridization probe. The peptide predicted from these sequences in 522 amino acids long and has a molecular weight of 57,518 kd. This higher plant sugar carrier contains 12 putative transmembrane segments and is highly homologous to the H+/hexose cotransporter from Chlorella, with an overall identity in the amino acid sequence of 47.1%. It is also homologous to the human HepG2 glucose transporter (28.4%), and other sugar carriers from man, rat, yeast and Escherichia coli. The definite proof for the function of the STP1 protein as a hexose transporter and data on its substrate specificity were obtained by heterologous expression in the fission yeast Schizosaccharomyces pombe. Transformed yeast cells transport D-glucose with a 100-fold lower KM value than control cells. Moreover only the transformed cells were able to accumulate the non-metabolizable D-glucose analogue 3-O-methyl-D-glucose, indicating that the Arabidopsis carrier catalyses an energy dependent, active uptake of hexoses. Expression of STP1 mRNA is low in heterotrophic tissues like roots or flowers. High levels of expression are found in leaves.     

Physiological and histopathological changes in the intestines of mice challenged with S. typhi

Transport of Na+, Cl-, Ca2+ and 3-0-methyl-D-glucose (a non-metabolizable analogue of D-glucose) was worked out in control, infected and immunized-infected animals along with histopathological studies of the intestines in these groups. There was significant decrease (p less than 0.01) in net absorption of Na+ and Cl- in the infected group as compared to that in the control group. There was also a significant decrease (p less than 0.01) in 3-0-methyl-D-glucose uptake in the infected group. However, no change was found in the immunized group in any of the electrolytes and non-metabolizable analogue of D-glucose. These findings correlated well with the histopathological studies as the infected intestines showed complete disorganization of mucosal tissues with loss of epithelial cells from the villi whereas the mucosal lining and epithelial cells from the intestines of immunized mice were normal looking.     

Oxidation of L-glucose by a Pseudomonad.

A new enzyme, D-threo-aldolse dehydrogenase (2S,3R-aldose dehydrogenase), found in Pseudomonas caryophylli, was capable of oxidizing L-glucose L-xylose, D-arabinose, and L-fucose in the presence of NAD+. The enzyme was synthesized constitutively and purified about 120-fold from D-glucose-grown cells. The Km values for L-glucose, L-xylose, D-arabinose, and L-fucose were 1.5 . 10(-2), 4.5 . 10(-3), 2.8 . 10(-3), and 2.1 . 10(-3), respectively. D-glucose and other aldoses inhibited the enzyme reaction; this inhibition was competitive with L-glucose as substrate and D-glucose as inhibitor. The optimum pH for the enzyme reaction was 10; the molecular weight of the enzyme was determined by gel filtration to be 7 . 10(4).     

Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.     

Cloning the gene of beta-galactosidase from the industrial strain of Streptococcus lactis 111 in E. coli cells and conjugated transfer of this gene to Streptococcus thermophilus cells

The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.     

The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization

The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.     

Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12.

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 x 10(6) Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: [formula: see text] Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: [formula: see text] This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker.

Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.