Association of polymorphic markers of the lipid metabolism genes with diabetic polyneuropathy in type 1 diabetes mellitus

A possible association of the polymorphic markers 2/3/4 of the apolipoprotein E gene (APOE) and I /D of the apolipoprotein B gene (APOB) with diabetic polyneuropathy (DPN) was analyzed in patients with type 1 diabetes mellitus (T1DM) with (N=86) or without (N=94) clinical signs of DPN. The two groups did not differ significantly in allele and genotype frequencies of the 2/3/4 polymorphic marker of the APOE gene. Analysis of the allele and genotype frequency distributions of the I/D polymorphic marker of the APOB gene showed that risk of DPN is higher in carriers of allele I or genotype I/I (OR=1.66 and 2.01, respectively) and lower in carriers of allele D (OR=0.60). The results implicate the APOB gene, which codes for one of the major components of the lipid metabolism system, in DPN development in patients with T1DM.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 230–234.Original Russian Text Copyright © 2005 by Voronko, Yakunina, Strokov, Lavrova, Nosikov.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Cellular fatty acid composition of six fastidious,gramnegative, xylem-limited bacteria from plants

Composition of cellular fatty acids was determined for strains of fastidious, Gramnegative, xylem-limited bacteria causing or associated with Pierce's disease of grapevine, phony disease of peach, plum leaf scorch, stunt of ragweed, elm leaf scorch, and periwinkle wilt. The most abundant fatty acids were straight-chain 150, 160, 170, and 180, unsaturated 161, 181, and unsaturated 17-and 19=carbon homologs. Minor fatty acids included straightchain 120, 140, 190, and 200; an unsaturated 15-carbon homolog; hydroxy-substituted 2-OH 120, 3-OH 120, and 3-OH 140; and branched chain iso-140 and iso-200. Cyclopropane acids were not detected. Physiological age had no effect on fatty acid composition. Class analysis of data indicated relative uniformity within the group. Saturated even-carbon chains comprised 31%–42%, unsaturated acids 41%–52%, saturated odd-carbon chains 10%–18%, hydroxysubstituted chains 2%–7%, and branched-chains 1%–4% of total fatty acids. The ratio of saturated-unsaturated acids ranged from 0.8 to 1.2.     

ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKC, PKC and PKC, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKC levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKC expression levels were also unaffected during the induction of LVH, but increased 3.2 ± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKC levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKC was not increased at any time point. In contrast, PKC autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKC autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKC, whereas total PKC levels were markedly increased (6.0 ± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKC levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKC may be involved in the induction of pressure overload LVH, whereas both PKC and PKC may be involved in the transition to heart failure.     

Comparative effects of sorbitol and sucrose as main carbon energy sources in micropropagation of apricot

In vitro proliferation and rooting capacity of San Castrese and Portici apricots (Prunus armeniaca L.) were tested on modified MS medium enriched with varying growth regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon energy sources. The interaction of proliferation and rooting media was also studied.Proliferation of both cultivars was proportional to benzyladenine (BA) concentration and enhanced with sorbitol media. However, 8.8 M BA was often associated with hyperhydricity, particularly when shoots were grown on sucrose media. Newly proliferated shoots elongated better on sorbitol media. The positive influence of sorbitol on proliferation and shoot growth was not due to osmotic effects. Moreover, sorbitol showed a positive carryover effect in hastening rooting of Portici. By contrast, when transferred to sorbitol rooting media, the shoots of both cultivars generally showed low rooting, with short, thick roots.Up to 70% of the plantlets that produced roots in sucrose media enriched with indolebutyric acid were successfully acclimatized when they were dipped in a benomyl (0.075% w/v) suspension before being transplanted with care being taken to prevent over-wetting of soil.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - GA₃ gibberellic acid - SEM standard error of mean     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

PKCε inhibits the hyperglycemia-induced apoptosis signal in adult rat ventricular myocytes

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC _I/_II, and ) or activate (PKC). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC _I/_II and blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKC (V1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKC translocation activator (RACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKC in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKC protects ARVM from the hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Effect of cell turgor on hydraulic conductivity and elastic modulus of Elodea leaf cells

Water relation parameters of leaf cells of the aquatic plant Elodea densa have been measured using the pressure probe. For cells in both the upper and lower epidermis it was found that the elastic modulus () and the hydraulic conductivity (Lp) were dependent on cell turgor (P). Lp was (7.8±5.5)·10^-7 cm s^-1 bar^-1 (mean±SD; n=22 cells) for P>4 bar in cells of the upper epidermis and was increasing by a factor of up to three for P0 bar. No polarity of water movement or concentration dependence of Lp was observed. For cells of the lower epidermis the Lp-values were similar and the hydraulic conductivity also showed a similar dependence on turgor. No wall ingrowth or wall labyrinths (as in transfer cells) could be found in the cells of the lower epidermis. The elastic modulus () of cells of the upper epidermis could be measured over the whole pressure range (P=0–7 bar) by changing the osmotic pressure of the medium. increased linearly with increasing turgor and ranged between 10 and 150 bar. For cells of the lower epidermis the dependence of on P was similar, although the pressure dependence could not be measured on single cells. The Lp-values are compared with literature data obtained for Elodea by a nuclear magnetic resonance (NMR)-technique. The dependence of Lp on P is discussed in terms of pressure dependent structural changes of the cell membranes and interactions between solute and water transport.Abbreviations P cell turgor pressure - Lp hydraulic conductivity - volumetric elastic modulus - T _1/2 half-time of water exchange of individual cell     

Specific antibodies to the N-termini of the interpeptide bridges of peptidoglycan

The synthetic peptides Gly₅--Ahx and l-Ala₃--Ahx, with structural similarity to the interpeptide bridge peptides of staphylococci or micrococci, respectively, were covalently linked to human serum albumin via their carboxylgroups. Antisera to these synthetic peptidyl-protein antigens contained fairly high amounts of antibodies with specificity to the N-terminal parts of the peptide chains attached to the carrier proteins. Antisera to (Gly₅--Ahx)₂₀-albumin gave, without exception, strong precipitin reactions in latex-agglutination with staphylococcal peptidoglycans. The antisera completely failed, however, in any reaction with peptidoglycans of micrococci or other bacteria which did not have these oligo-glycine peptides typical for staphylococci. On the contrary, antisera to (l-Ala₃--Ahx)₂₂-albumin strongly precipitated micrococcal peptidoglycans with oligo-l-alanine interpeptide bridges (e.g. Micrococcus varians, Micrococcus reseus), but showed no significant reaction with peptidoglycans of staphylococci or other bacteria lacking oligo-l-alanine interpeptide bridges.Abbreviations Use Ac acetyl- - -Ahx -amino caproic acid - ATCC American Type Culture Collection, Rockville, Md., U.S.A. - CCM Czechoslovak Collection of Microorganisms, Brno, CSSR - DSM Deutsche Sammlung für Mikroorganismen, München, FRG - IMRU Institute of Microbiology, Rutgers University, N.J., U.S.A. - Kiel Bundesanstalt für Milchforschung, Kiel, FRG - NPS o-nitrophenylsulphenyl- - -OMe methyl ester - -OSu succinimide ester - Z- benzyloxycarbonyl     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

Potency of Agonists and Competitive Antagonists on Adult- and Fetal-Type Nicotinic Acetylcholine Receptors

1. The potency of agonists and competitive antagonists on the two expressed forms of the nicotinic acetylcholine receptor (adult or junctional subtype, -AChR; fetal or extrajunctional subtype, -AChR) have not previously been compared systematically in homogeneous receptor preparations.2. Each subtype of the receptor was expressed separately in Xenopus oocytes by cytoplasmic injection of combinations of RNA transcribed in vitro. The presence of each type of receptor was confirmed by single-channel recordings. Expressing oocytes were assayed using discontinuous, single-electrode voltage clamp by measuring peak currents in response to test compounds.3. The extrajunctional subtype was more potently activated by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP) than was the junctional form. There was no statistically significant difference in potency between the two subtypes for other nicotinic agonists (nicotine, cytisine and succinylcholine). The rank order of potency for -AChR was succinylcholine>cytisine>DMPP>nicotine, and that for -AChR was DMPP>cytisine>succinylcholine>nicotine.4. Two agonists (cytisine and succinylcholine) displayed six- to eight-fold greater intrinsic activity in activating -AChR over -AChR. There was no difference between the two forms of receptor in efficacy for nicotine.5. The extrajunctional form was much more potently inhibited by the steroidal competitive antagonist pancuronium than was the junctional receptor. However, there was no significant difference in potency of inhibition by the curariform drug atracurium.6. Contrary to previous reports, there is no consistent relation between the effect of agonists and antagonists and the subtype of receptor. These data suggest that the resistance or sensitivity to these agents seen in various clinical settings are related to other cellular factors.     

Genetic Association between the Apolipoprotein E (APOE) Gene and Different Forms of Alzheimer's Disease

Allele 4of the apolipoprotein E (APOE) gene is associated with higher risk for family or sporadic Alzheimer's disease (AD) in many, though not all, ethnic groups. The APOEallele and genotype frequency distributions were evaluated in 207 AD patients without vascular disorders, 62 AD patients with vascular disorders (combined AD), and 206 control individuals (ethnic Russians from the Russian population). The frequency of allele 4in patients with early-onset and late-onset AD was three times higher than in controls (P< 0.000001). The increase in the frequency of 4in mixed dementia cases over controls was somewhat less but still significant (P= 0.0019). Relative risk of AD in carriers of allele 4was five times higher than in carriers of alleles 2and 3(P< 0.000001). Allele 2showed evidence of a protective effect in the early-onset AD group (P= 0.015). These results suggest that APOEallele 4is a universal factor of early-onset, late-onset, and combined AD in ethnic Russians from Russia.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. -carotene hydroxylases (-hydroxylases) add hydroxyl groups to the -rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts -ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on -ring structures. We have used library screening and genomics-based approaches to isolate a second -hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis -hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, -hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of -ring containing carotenes and are only weakly active towards -ring containing carotenes. Neither -hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for -ring hydroxylation but unrelated to -hydroxylases at the level of amino acid sequence.