Immunochemical studies of Rhizobium mutants

Summary It was shown in gel diffusion and in immunoelectrophoresis tests that somatic antigens of smooth cultures were more complex than those of rough mutants. Somatic antigens ofR. trifolii 24 contained glucuronic acid, glucosamine, galactose, glucose, mannose, xylose or fucose, ribose, and 2-keto-3-deoxyoctonate. Arabinose was identified in some preparations. The antigens of the smooth strain contained a large amount of rhamnose whereas those of the rough mutants were deficient in that sugar.     

Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene.

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.     

Pathways of Glucose Metabolism by Rough and Smooth Variants of Bacillus stearothermophilus

The radiorespirometric method was used to study the pathways of glucose metabolism in the rough and smooth variants of Bacillus stearothermophilus NCA 1518. The Embden-Meyerhof (EM) pathway was more active in the smooth variant than in the rough variant. The participation of the hexose monophosphate shunt (HMP) and EM pathways in the smooth variant was calculated as 4.2 and 95.8%, respectively. The rough variant utilized glucose via the EM pathway exclusively or in combination with a pathway other than the HMP pathway. The estimated efficiency of the tricarboxylic acid system in the rough and smooth variants was 81.3 and 4.9%, respectively.     

Variable Ammonia Production Among Smooth and Rough Strains of Pseudomonas pseudomallei: Resemblance to Bacteriocin Production

The colonial morphology of some strains of Pseudomonas pseudomallei was correlated with certain biochemical and physiological traits. After 3 days of growth on Wahba or heart infusion agars, smooth-colony strains generated toxic amounts of ammonia. Under the same conditions, the rough strains simultaneously produced oxalic acid which decreased the inhibitory concentration of ammonia. The ammonia-ammonium concentrations in smooth cultures exhibited certain bacteriocin-like characteristics. An unusually stable, smooth strain (strain 165) was chosen to compare and emphasize any differences with typical, rough strain 7815. Three-day-old smooth cultures grown on Wahba agar containing 3% (w/v) glycerol demonstrated ammonia toxicity. The substitution of glucose for glycerol completely obviated this toxicity. In highly aerated Wahba broth containing glucose, the amount of ammonia found in strain 165 smooth cultures and the amount of oxalic acid found in strain 7815 rough cultures were greatly reduced. In Difco nitrate broth smooth strain 165 did not form gas, and it reduced nitrate to nitrite only. Strain 7815 produced a gas and reduced both nitrate and nitrite.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO₂ fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O₂ evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected.The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient dl-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity.It is concluded that ribose 5-phosphate inhibits photosynthetic O₂ evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes — rough — smooth I — smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth microsomes. On the other hand, 5 mM MgCl₂ decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Fermentation characteristics of exopolysaccharide-producing lactic acid bacteria from sourdough and assessment of the isolates for industrial potential

Lactic acid bacteria (LAB) with antimicrobial activity and high exopolysaccharide (EPS) production ability isolated from sourdough were studied for their fermentation characteristics as potential new starter cultures. The values of pH, titratable acidity, and viable cell counts were 4.06+/-0.009-4.50+/- 0.015, 0.787+/-0.020%-1.172+/-0.018%, and 8.78+/-0.08-8.98+/- 0.06 log CFU/ml, respectively. In order to select probiotics with a high survival rate in the gut, isolates were tested to assess resistance against the artificial gastric acid and bile juice. Viable LAB counts were significantly (p<0.05) affected by the acidity. At pH 2.0, the total declines in the initial bacterial counts were 4.52+/-0.07 log for S. thermophilus St-Body-1, >7.98+/-0.03 log for E. flavescens DU-10, >7.95+/-0.05 log for E. faecium DU-12, and 3.15+/- 0.06 log for L. amylovorus DU-21. Among the strains, L. amylovorus DU-21 was the only strain that had bile tolerance under simulated gastrointestinal conditions. In order to improve EPS production by L. amylovorus DU- 21, the influence of carbon source was studied. When glucose was used as a carbon source, EPS production dramatically increased to 17.19+/-0.28 g/l (p<0.05). The maximum cell growth (10.012+/-0.012 log CFU/ml) and EPS production (18.71+/-0.19 g/l) were achieved when 15 g/ l of glucose was employed as the carbon source.     

Lactobacillus amylovorus as a phytase producer in submerged culture

Nineteen strains of lactic acid producing bacteria of the genera Lactobacillus and Streptococcus collected from different culture collections were screened for the production of extracellular phytase. A number of them exhibited the enzyme activity in the fermentation medium but Lactobacillus amylovorus B4552 produced the maximum amounts of phytase ranging from 125 to 146 units ml^-1 extracellularly under the submerged cultivation conditions. The optimum glucose and inorganic phosphate levels for highest phytase production were found to be 1% and 24% mg, respectively. Lactobacillus amylovorus has potential in improving nutritional qualities of cereal and pulse-based food fermentations.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

Properties of carbohydrate utilising variants of Chinese hamster cells

Variants of Chinese hamster ovary cells (CHO-K1) have been isolated which can grow on one of the following carbohydrates: lactose, sucrose, ribose or lactate. The ribose+ clones grow at the same rate on glucose as the parental cells whereas the others grow more slowly. With the exception of one ribose+ clone all excrete lactic acid while growing on glucose; none excrete significant amounts of lactic acid while growing on the alternative energy source. Wild-type cells and the variants accumulate radioactively labelled glucose and the corresponding radio-actively labelled alternative energy source to the same extent. The ribose+ variant that does not accumulate lactate while growing on glucose is also exceptional in its inability to utilise mannose.     

Studies on heterogeneous lipopolysaccharide fractions of Vibrio cholerae 569B

By SDS-PAGE or gel filtration on Sephadex G-25, lipopolysaccharide (LPS) isolated from Vibrio cholerae 569B (Inaba) can be separated into two distinct fractions, one corresponding to smooth LPS and the other to rough LPS. Pulse-labelling of LPS with [14C]glucose showed that the rough form is synthesized first followed by the biosynthesis of the smooth form. A preferential release of the smooth LPS of V. cholerae 569B was also detected during normal growth of cells.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

Human erythrocyte phosphoglucomutase: comparison of the kinetic properties of PGM1 and PGM2 isoenzymes

Kinetic properties of PGM1 and PGM2 phosphoglucomutase "primary" isoenzymes from human erythrocytes were studied. The two enzyme forms share a "ping-pong" kinetic mechanism and show similar Km for substrate (glucose 1-P) and cofactor (glucose 1,6-P2). Micromolar concentrations of fructose 1,6-P2 and glycerate 2,3-P2 inhibit both PGM1 and PGM2 isoenzymes to a similar extent. The sole PGM2 form is affected by ribose monophosphates (ribose 1-P and ribose 5-P) that act as mutase inhibitors vs. glucose 1,6-P2 and as apparent activators vs. glucose 1-P. The interaction between PGM2 isoenzyme and ribose monophosphates is discussed in the light of the ability of this form to also display phosphoribomutase activity.     

Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH₄Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH₄Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH₄Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH₄Cl. 7. NH₄Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH₄Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.     

The chemical composition of the lipopolysaccharide from Pseudomonas aeruginosa strain PAO and a spontaneously derived rough mutant.

The chemical composition of the lipopolysaccharide (LPS) of the smooth strain Pseudomonas aeruginosa PAO 307 and a spontaneously derived rough mutant, obtained by selection for resistance to the LPS-specific phage E79, are compared. The rough LPS was shown to contain lipid A, heptose, 2-keto 3-deoxyoctonic acid, galactosamine, alanine and phosphate but lacked glucose, rhamnose and fucosamine which were important constituents, on a weight basis, of the smooth LPS. These results, and chromatographic analysis of the polysaccharide fraction indicate that the rough strain lacked side chain material and was defective in its inner core region. The chemical date obtained were consistent with a core in the PAO strain similar to that of strain NCTC 1999, enhancing the evidence for a common core polysaccharide in the LPS of P. aeruginosa strains.     

Comparison of bovine serum albumin glycation by ribose and fructose in vitro and in vivo

Advanced glycation end products (AGEs) play a critical pathogenic role in the development of diabetic complications. Recent studies have shown that diabetes is associated with not only abnormal glucose metabolism but also abnormal ribose and fructose metabolism, although glucose is present at the highest concentration in humans. The glycation ability and contribution of ribose and fructose to diabetic complications remain unclear. Here, the glycation ability of ribose, fructose and glucose under a mimic physiological condition, in which the concentration of ribose or fructose was one-fiftieth that of glucose, was compared. Bovine serum albumin (BSA) was used as the working protein in our experiments. Ribose generated more AGEs and was markedly more cytotoxic to SH-SY5Y cells than fructose. The first-order rate constant of ribose glycation was found to be significantly greater than that of fructose glycation. LC-MS/MS analysis revealed 41 ribose-glycated Lys residues and 12 fructose-glycated residues. Except for the shared Lys residues, ribose reacted selectively with 17 Lys, while no selective Lys was found in fructose-glycated BSA. Protein conformational changes suggested that ribose glycation may induce BSA into amyloid-like monomers compared with fructose glycation. The levels of serum ribose were correlated positively with glycated serum protein (GSP) and diabetic duration in type 2 diabetes mellitus (T2DM), respectively. These results indicate that ribose has a greater glycation ability than fructose, while ribose largely contributes to the production of AGEs and provides a new insight to understand in the occurrence and development of diabetes complications.     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

Translocation of sugars into rat liver lysosomes. Evidence against a common carrier for D-glucose and D-ribose.

A conflict exists in the literature concerning the mode of translocation of D-glucose and D-ribose across the lysosome membrane. The more rapid net uptake of ribose, when measured by the osmotic-protection technique, has been attributed either to its smaller size and lower hydrogen-bonding capacity, or to a lower affinity for a transport system shared by both sugars. The latency of acid beta-hexosaminidase in isolated rat liver lysosomes was measured after preincubation for periods up to 1 h in various solutions containing glucose and/or ribose, and in some cases sucrose. After confirmation of the superior osmotic protection afforded by glucose (than by ribose), it was shown that a solution 0.125 M in both glucose and ribose provided protection intermediate between that given by 0.25 M-glucose and that given by 0.25 M-ribose. This result is inconsistent with the common-carrier hypothesis.