Incorporation of cytosol δ-aminolevulinate synthase of rat liver into the mitochondria in vitro

When ¹²⁵I-labeled cytosol δ-aminolevulinate synthase was incubated in suspensions of rat liver mitochondria, the enzyme was incorporated into the mitochondira at the rate that was linear with time and with the [¹²⁵I]enzyme added. Subfractionation of the mitochondria using a digitonin technique revealed that the [¹²⁵I]enzyme was incorporated into the innermembrane-matrix fraction where endogeneous δ-aminolevulinate synthase is located.     

Translational inhibition by heme of the synthesis of hepatic δ-aminolevulinate synthase in a cell-free system

Synthesis of δ-aminolevulinate synthase in a rabbit reticulocyte lysate system directed by total polysomes from the liver of allylisopropylacetamide-treated rats was studied with the combined use of [³H]leucine and a specific rabbit antibody. The protein synthesis observed in the cell-free system employed represented mainly the peptide chain elongation and its termination rather than the net synthesis involving initiation. Synthesis of δ-aminolevulinate synthase in this cell-free system was inhibited progressively with the increased addition of hemin; the synthesis was reduced to about 40% by about 30 μM hemin. Synthesis of total protein, however, was not significantly affected by the addition of hemin. The data obtained suggest that heme inhibits a peptide chain elongation step in the synthesis of δ-aminolevulinate synthase.     

The conversion of the precursor form of γ-glutamyltranspeptidase to its subunit form takes place in brush border membranes

The subcellular distributions of the precursor form and mature form of γ-glutamyltranspeptidase of rat kidney were studied by labeling the enzyme with [³H] fucose in vivo. In trans Golgi elements and basolateral membranes, γ-glutamyltranspeptidase was present as a precursor form with a single polypeptide chain. However, the brush border membranes contained the heavy and light subunits as well as precursor. These results suggest that the precursor is converted to the mature form after its transport to the brush border membranes.     

Quantum-chemical investigation of the structure and the antioxidant properties of α-lipoic acid and its metabolites

Quantum-chemical computations were used to investigate the structure-antioxidant parameter relationships of α-lipoic acid and its natural metabolites bisnorlipoic acid and tetranorlipoic acid in their oxidized and reduced forms. The enantiomers of lipoic and dihydrolipoic acid were optimized using the B3LYP/6-311+G(3df,2p), B3LYP/aug-cc-pVDZ and MP2(full)/6-31+G(d,p) levels of theory as isolated molecules and in the presence of water. The geometries of the metabolites and the values of their antioxidant parameters (proton affinity, bond dissociation enthalpy, adiabatic ionization potential, spin density, and the highest occupied molecular orbital energy) were calculated at the B3LYP/6-311+G(3df,2p) level of theory. The results obtained reveal similarities between these structures: a pentatomic, nonaromatic ring is present in the oxidized forms, while an unbranched aliphatic chain (as found in saturated fatty acids) is present in both the oxidized and the reduced forms. Analysis of the spin density and the highest occupied molecular orbital energy revealed that the SH groups exhibited the greatest electron-donating activities. The values obtained for the proton affinity, bond dissociation enthalpy and adiabatic ionization potential indicate that the preferred antioxidant mechanisms for α-lipoic acid and its metabolites are sequential proton loss electron transfer in polar media and hydrogen atom transfer in vacuum.     

Purification and general properties of δ-aminolaevulate dehydratase from cow liver

1. delta-Aminolaevulate dehydratase, the enzyme catalysing the condensation of delta-aminolaevulic acid to porphobilinogen, has been prepared from cow liver and its properties have been studied. The enzyme has been purified 310-fold. 2. The purified preparation behaves as a single protein under gel filtration on Sephadex and Bio-Gel columns; it migrates as a single band in disk and starch-gel electro-phoresis at different pH values and it sediments as a single symmetrical peak in the ultracentrifuge. 3. The pH optimum for the pure enzyme was 6.8, the K(m) at pH 6.8 and 38 degrees was 1.5x10(-4)m, the isoelectric point was about pH 4.9 and the molecular weight was 140000+/-14000 by the gel-filtration method. Maximal enzyme activity was observed at 65 degrees . 4. The presence of thiol groups in the enzyme system, essential for its activity, was indicated and the total number of thiol groups was determined. 5. After the first steps of purification the enzyme required cysteine or reduced glutathione for activity.     

Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

Relationship between kinetic properties of γ-glutamyl transpeptidase and the structure of its saccharide moiety

Gamma-glutamyl transpeptidase (EC 2.3.2.2; GGT) is a plasma-membrane bound glycoenzyme, the saccharide moiety of which is rather heterogeneous and organ specific. It has been stated that GGT catalyses three types of reactions, i.e., hydrolysis, transpeptidation and autotranspeptidation. The initial velocity equation, involving all these reactions, is shown in the present report. Mathematical analysis of the equation resulted in a. definition of the constant of half saturation (K_hs). The value of K_hs was used for characterization of kinetics of GGT from rat organs differing in the structure of GGT oligosaccharide chains. No significant organ differences were found, when the K_hs values of GGT from the brain, kidney and pancreas equalled 0.61 mM, 0.68 mM and 0.68 mM, respectively. On the contrary, when two different glycoforms of GGT from the pancreas were compared, distinct values of K_hs were obtained (l.43 mM and 0.67 mM, respectively). It is therefore being suggested that the saccharide chains of GGT are involved in its kinetic properties. However, this effect is masked when the enzyme, non-fractionated into glycoforms, is analysed, even though the saccharide moiety is specific for the organ studied.     

Influence of gestational diabetes on the activity of δ-aminolevulinate dehydratase and oxidative stress biomarkers

Objective: This study aimed to evaluate the activity of delta-aminolevulinate dehydratase (δ-ALA-D) and oxidative stress biomarkers in pregnant women with gestational diabetes mellitus (GDM), in order to demonstrate the involvement of oxidative stress in this condition, which presents pathophysiology still undetermined.

Methods: δ-ALA-D activity, lipid peroxidation estimated as the levels of thiobarbituric acid reactive substances (TBARS), protein (P-SH) and non-protein thiol (NP-SH) content, catalase (CAT) activity and concentration of vitamin C (VIT C) in samples of pregnant women with GDM (n?=?48) and in healthy pregnant women (n?=?30), who constituted the control group.

Results: The δ-ALA-D activity was significantly lower in pregnant women with GDM compared to controls, as well as levels of thiols, VIT C and CAT activity. Lipid peroxidation was higher in GDM group.

Discussion: The results suggest that the main factor for the increase in oxidative stress and reduced δ-ALA-D activity in diabetic pregnant women is gestational hyperglycemic environment, which changed the redox balance and interfered on mechanism of the δ-ALA-D activity in relation to normoglycemic pregnant women.     

Morphological structure and optical properties of the wings of Morphidae

The morphological structure and optical properties of the wings of 14 species of Morphidae have been investigated. Most of the scales of the iridescent species of Morphidae （Lepidoptera） present a very particular structure. The ground scales, responsible for the major part of the optical properties, are covered by a very regular set of longitudinal ridges. The ridges themselves are constituted by a superposition of lamellae that act locally as a multilayered structure. This very specific morphology leads to both interferences and diffraction effects. The first one is responsible of the brilliant blue coloration of the males, while the second one diffracts this colored light at a very large angle. These two phenomena give to the butterfly a very effective long-range communication system. The morphological characteristics of the scales of the various species are presented in detail. Two types of optical measurement were performed on the iridescent wings of 14 different species of Morphidae： spectroscopic measurements under various incidences and gonioscopic measurements for a given incidence angle and wavelength. The first allows a determination of the index of refraction of the cuticular material. The second leads to the drawing of spatial diffraction maps. It shows that most of the reflected light is diffracted laterally over a very large angle （90° 〈 0 〈 120°, according to the different species） and that this repartition depends of the polarization of incident light. As predicted by previous calculations, the dissymmetric structure of the ridge is responsible for the separation of the polarization modes in the various diffraction orders.     

The Occurence of δ-Tocopherylquinone in Higher Plants and Its Relation to Senescence

δ-Tocopherylquinone (δTQ) content was determined in tobacco and yellow maple leaves, green ivy leaves and cactus tissues. It was found that the concentration of δ-TQ was highest in mature or senescent tissues, such as white tobacco leaves (0.02 μmole/g dry wt) while its detection was uncertain in young, green leaves from the apex of tobacco plants. Fractionation by centrifugation of senescent tobacco leaves showed that the osmiophilic globule fraction was enriched in δ-TQ. Electron microscope studies of young, mature and senescent tobacco tissues showed progressive changes in the size and number of osmiophilic globules. After chloroplast breakdown in senescent tobacco leaves, these globules became the predominant constituents of the organelle. δ-TQ which is associated with osmiophilic globules may play a role in the development of plants, particularly during senescence.     

Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4–55 PHA synthase and an insight into its catalytic mechanism

Background  

Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4–55 PHA synthase 1 (PhaC1_{P.sp USM 4–55}).     

The Problem of Colliding Networks and its Relation to Cell Fusion and?Cancer

Cell fusion, a process that merges two or more cells into one, is required for normal development and has been explored as a tool for stem cell therapy. It has also been proposed that cell fusion causes cancer and contributes to its progression. These functions rely on a poorly understood ability of cell fusion to create new cell types. We suggest that this ability can be understood by considering cells as attractor networks whose basic property is to adopt a set of distinct, stable, self-maintaining states called attractors. According to this view, fusion of two cell types is a collision of two networks that have adopted distinct attractors. To learn how these networks reach a consensus, we model cell fusion computationally. To do so, we simulate patterns of gene activities using a formalism developed to simulate patterns of memory in neural networks. We find that the hybrid networks can assume attractors that are unrelated to parental attractors, implying that cell fusion can create new cell types by nearly instantaneously moving cells between attractors. We also show that hybrid networks are prone to assume spurious attractors, which are emergent and sporadic network states. This finding means that cell fusion can produce abnormal cell types, including cancerous types, by placing cells into normally inaccessible spurious states. Finally, we suggest that the problem of colliding networks has general significance in many processes represented by attractor networks, including biological, social, and political phenomena.     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.     

The precursor of the F1β subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F₁ subunit from Nicotiana plumbaginifolia (pF₁) into plant mitochondria. When pF₁ of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF₁. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF₁. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca²⁺-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF₁ proteins were strongly associated with the mitochondrial outer membrane.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Study on the influence of cranberry extract ?uravit S·O·S(?) on the properties of uropathogenic Escherichia coli strains, their ability to form biofilm and its antioxidant properties

Consumption of cranberries is known to exert positive health effects, especially against urinary tract infections. For this reason, presumably, they are widely used in folk medicine. Different aspects of cranberry phenolics activity were studied in individual papers but complex study in this matter is missing. The aim of the present study is to provide complex data concerning various aspects of cranberry extract activity. We studied the effects of subinhibitory concentrations of commercially available extract (?uravit S·O·S(?)) against two Escherichia coli strains isolated from urine of patients with pyelonephritis. Additionally the main extract anthocyanins were characterized. The activity of extract against lipid peroxidation and its radical scavenging ability were also assessed. ?uravit S·O·S(?) decreased the hydrophobicity of one of the studied E. coli strains, reduced swimming motility and adhesion to epithelial cells of both studied strains, it also limited the ability of bacteria to form biofilm. Expression of curli was not affected by cranberry extract, the assessment of P fimbriae expression was not reliable due to extract-induced agglutination of erythrocytes. Cranberry extract caused filamentation in both studied E. coli strains. It also showed pronounced antioxidant and radical scavenging properties. The properties of the studied cranberry extract show that it could be effectively used in prevention and/or elimination of urinary tract infections, specially the recurrent ones.