Differential responses of certain brain phosphoesterases to aluminium in dietary protein adequacy and inadequacy.

The aluminium-induced neurotoxic consequences include, among other factors, dephosphorylation, phosphorylation as well as hyperphosphorylation of specific macromolecules. Accordingly, activities of phosphoesterases were measured in different regions of rat brain, maintained with either adequate or inadequate protein diet, following aluminium exposure. Male Wistar rats weighing 80-100 g were treated with aluminium chloride at a dose of 15% of the LD50 for 4 weeks. In different regions of the brain of aluminium-exposed rats, significant variation in both phosphomonoesterase and phosphodiesterase activities have been recorded. These alterations were found to be varied when the rats were subjected to dietary protein insufficiency. These findings demonstrate the specificity of aluminium on different phosphoesterases. These regional variations may be attributed to the accumulated level of aluminium or may be due to cellular localization of these enzymes and linked to whether the enzymes are compartmentalized with different aluminium hydration species.

Impact of protein malnutrition on subcellular nucleic acid and protein status of brain of aluminum-exposed rats.

Nucleic acid and protein content in various cellular fractions of different regions of the brain were investigated in male albino rats following aluminum (Al) exposure (at the dose of 15% of LD50 i.p. for 28 days) on either an adequate or inadequate protein diet. It was observed that there was a decrease in homogenate DNA content in the thalamic area (Th), midbrain-hippocampal region (MH) and cerebellum (CL), but not in the cerebrum (CC) of the protein-restricted group of animals. Increased RNA content was recorded in the ribosomal and soluble fractions of CL of the adequately protein-fed animals compared to pair-fed controls. In the low-protein-fed animals, on the other hand, a decrease in RNA content was observed in the whole homogenate and nuclear fractions of CC, MH and CL, the ribosomal and soluble fractions of MH and CL, and in the mitochondrial fraction of TH. Ribonucleolytic activity was found to be increased only in the Th and CL of the adequately protein-fed group. Protein contents in the subcellular fractions of these four regions remain almost unaltered with the present dose and duration of Al-exposure; only the soluble fraction of CC and microsomal fraction of Th of the low-protein-fed group showed a significant decrease. The results of the present investigation confirm that Al has generally depressive effects on the nucleic acid metabolism of the brain and suggest that these effects are region-specific as well as dependent on dietary protein level. It is further suggested that alterations in the cellular microenvironment, caused by protein malnutrition, may play a significant role in the modification of the effects of Al in the brain.

Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

The soft-rotting bacterium, Erwinia carotovora subsp. carotovora 71, produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh), and protease (Prt). While the extracellular levels of these enzymes are extremely low when the bacterium is grown in salts-yeast extract-glycerol (SYG) medium, the enzymatic activities are highly induced in SYG medium supplemented with celery extract. By transposon (mini-Tn5) mutagenesis, we isolated a RsmA- mutant, AC5070, which overproduces extracellular enzymes; the basal levels of Pel, Peh, and Cel in AC5070 are higher than the induced levels in the RsmA+ parent, AC5047. While Peh production is mostly constitutive in AC5070, Pel, Cel, and Prt production is still inducible with celery extract. The high basal levels of pel-1, pel-3, and peh-1 mRNAs in AC5070 demonstrate that overproduction of the pectolytic enzymes is due to the stimulation of transcription. Using chromosomal DNA flanking mini-Tn5 as a probe, we cloned the wild-type rsmA+ allele, which suppresses Pel, Peh, Cel, and Prt production in both RsmA+ and RsmA- strains. The RsmA- mutant, like its parent, produces N-(3-oxohexanoyl)-L-homoserine lactone (HSL), a starvation/cell density-sensing signal required for extracellular enzyme production. To examine the role of HSL, we constructed HSL-deficient strains by replacing hslI, a locus required for HSL production, with hslI::Tn3HoHo1-Spc. While the basal levels of Pel, Peh, Cel, and Prt are comparable in the RsmA- mutant and its HSL- derivative, these enzymes are barely detectable in the Hsl- derivative of the RsmA+ parent strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone mediated changes of brain glutamic acid system in amino acid imbalance.

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.

Impact of amino acid imbalance on brain amines.

The brain levels of serotonin (5-HT), norepinephrine (NE) and dopamine (DM) were studied in amino acid imbalance, induced in rats by feeding them an 8% casein diet supplemented with 0.3% L-threonine. In addition, tryptophan hydroxylase (TPH) and monoamine oxidase (MAO) activities in the brain were measured. The studies revealed that induction of amino acid imbalance decreased the brain levels of 5-HT and NE, and increased the DM level significantly. These changes in the amine levels were found to be accompanied by increases in the activities of TPH and MAO of brain. It has been suggested that the enhanced activity of brain TPH in threonine imbalance was probably brought about by a conformational change of the enzyme protein and might be an adaptive response to the low level of 5-HT in the brain due to increased MAO activity. The increased brain DM level in threonine imbalance could be ascribed to enhanced entry of tyrosine in the catecholamine anabolic pathway, while the diminished brain NE level under the same condition might result from its increased degradation or diminished conversion of DM to NE due to supposedly inhibition of DM-beta-hydroxylase.

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Studies on certain drug-metabolizing enzymes in deoxypyridoxine-treated rats.

The effect of deoxypyridoxine on the activities of drug-metabolizing enzymes was investigated in male rats. Phenylbutazone oxidase and aminopyrine N-demethylase decreased in both liver and kidney of deoxypyridoxine-treated rats that received either an 18% or 8% casein diet. However, the magnitude of decrease in activities was more when the rats received an 8% casein diet. The NADPH oxidase activity remained unchanged following deoxypyridoxine treatment. The diminished activities of phenylbutazone oxidase and aminopyrine N-demethylase noted after deoxypyridoxine treatment were restored by pyridoxine supplementation. The decreased activities of drug-metabolizing enzymes in deoxypyridoxine treated rats were not reversed by thyroxine supplementation. It is suggested that pyridoxine in the form of pyridoxal phosphate might be involved in the regulation of drug-metabolizing activities.

Studies on the possible mechanism of hydrazine action on ascorbic acid-metabolising enzymes.

The activities of enzymes associated with the synthesis and degradation of L-ascorbic acid were studied in hydrazine-treated rats supplemented with pyridoxine. The effects of hydrazine in vitro were also examined on these enzymes. The activity of liver D-glucuronoreductase was reduced in hydrazine-treated rats even after receiving pyridoxine in excess. But, the decreased activities of liver and kidney dehydroascorbatases in hydrazine-treated rats were reversed by pyridoxine supplementation. Hydrazine in vitro could not inhibit the activity of liver D-glucuronoreductase or L-gulonooxidase. The drug was also unable to inhibit in vitro the activity of liver and kidney dehydroascorbatases. Studies with dialyzed liver homogenates showed that the diminished activity of D-glucuronoreductase in the liver of hydrazine-treated rats was maintained even after dialyzing the tissue preparations. In contrast, the reduced activity of dehydroascorbatase in the liver of hydrazine-treated rats was abolished following dialysis of the liver preparations. It has been suggested that the diminished activity of liver D-glucuronoreductase after hydrazine treatment might arise from the reduced synthesis of enzyme protein, while the reduction in the activity of dehydroascorbatase following hydrazine treatment could be ascribed to depletion in vivo of pyridoxal phosphate and/or to the involvement of some dialyzable factors.

Possible influence of gonadotrophins on formation in vivo of pyridoxal phosphate.

FSH administered to normal rats increased the activity of pyridoxine phosphate oxidase of both liver and kidney and, consequently, pyridoxal phosphate levels in these tissues were elevated. LH administration, on the other hand, decreased the activity of pyridoxine phosphate oxidase, resulting in diminished pyridoxal phosphate level in the tissues. The stimulatory effect of FSH on the activity of liver and kidney pyridoxine phosphate oxidase was not observed in castrated-adrenalectomised rats unless supplemented with cortisone and testosterone, respectively. Puromycin treatment prevented the FSH-induced rise in the activity of liver and kidney pyridoxine phosphate oxidases. It is suggested that FSH stimulates the activity of liver and kidney pyridoxine phosphate oxidase by increasing the synthesis of apoproteins of the enzyme, and the effect of FSH on liver is dependent on the presence of adrenal corticoids while the presence of testosterone is a prerequisite for the FSH to have its effect on kidney pyridoxine phosphate oxidase.

Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity.

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.

Enzymatic degradation of polygalacturonic acid by Yersinia and Klebsiella species in relation to clinical laboratory procedures.

As scored by several specified plating procedures, clinical and environmental strains of Yersinia enterocolitica, Yersinia pseudotuberculosis, and Klebsiella pneumoniae "Oxytocum" showed detectable, albeit generally weak, ability to digest polygalacturonic (pectic) acid. None of these bacterial strains had the vigorous and rapid pectolytic activity on these polygalacturonic acid-containing media that is typical of soft-rot Erwinia species, although some of the Oxytocum strains came fairly close. Analyses of the pectolytic enzyme contents of the cells and culture supernatants of the Yersinia and Klebsiella species revealed that readily detectable quantities of cell-bound polygalacturonic acid trans-eliminase and hydrolytic polygalacturonase were formed by the Yersinia and Klebsiella species; however, the total units of enzyme activity produced by these bacteria were, in general, lower than were produced by soft-rot Erwinia species. Furthermore, unlike the situation in soft-rot Erwinia cultures, these pectolytic enzymes of Yersinia and Klebsiella species were not excreted rapidly and massively into the growth medium. Cultures of other enterobacteria (Citrobacter species, Enterobacter species, Erwinia amylovora, Erwinia herbicola, Escherichia coli, Proteus species, Salmonella typhimurium, and Serratia marcescens) showed no pectolytic ability whatsoever by any of the plating procedures used and (to the extent they were so examined) produced no pectolytic enzymes detectable either in their cells or culture supernatants. This slow or weak release of pectolytic enzymes by Yersinia and Klebsiella species has a bearing on clinical laboratory procedures suitable for detecting their pectolytic activity; methods adequate for this purpose are detailed.

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.