Metabolic engineering Corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway

The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l^?1 h^?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum.     

Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats

To investigate the mechanism of apoptosis in myocardial cells of aging rats induced by d-galactose and to study the effect of the Polysaccharide isolated from the seeds of Cuscuta chinensis Lam (PCCL) on apoptosis of cardiomyocytes and its corresponding machinasim in aging rat model. Fifty male SD rats were randomly divided into 5 groups. Normal control group (NC). d-galactose (100 mg·kg^?1d^?1 for 56 day) indued aging group (MC), d-galactose plus 100 mg kg^?1 d^?1 PCCL group (ML), d-galactose plus 200 mg kg^?1 d^?1 PCCL group (MM), and d-galactose plus 400 mg kg^?1 d^?1 PCCL group (MH). Same volume of solution (water, or PCCL aqueous solution) was given by gavage for 56 days. Then the hearts were collected and apoptosis parameters were evaluated. Caspase-3 and Cyt c were determined by fluorescence spectrometer, the apoptosis rate was assessed by AnnexinV-FITC method by Flow-Cytometry, [Ca²⁺]i and [Ca²⁺]i overloaded by KCL were observed by laser scanning confocal microscopy (LSCM); Bcl-2 and Bax were examined by immunohistochemistry. The content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3, Bax expression level in d-galactose induced aging group were higher than NC (p < 0.05). The ratio of Bcl-2/Bax was decreased in d-galactose induced aging group compared to NC. On the other hand, the content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3 and apoptosis rate, as well as Bax expression level in all three PCCL groups were decreased compared to galactose induced group (p < 0.05). Bcl-2/Bax ratio was increased in all PCCL groups compared to galactose induced aging group. PCCL could decrease the apoptosis of cardiomyocytes by the mitochondria apoptosis pathway.     

New taxa and combinations inAlchemilla (Rosaceae) (2)

47 names and combinations are published as new, chiefly from the elaboration ofAlchemilla byA. Plocek inL. Bertová (ed.), Flóra Slovenska 4/3, Bratislava (in press). The populations recognized as varieties and species new to science (12 and 29, respectively) come from the summits of W. Beskids (9 taxa), Belianske Tatry (12), other parts of Tatra (8), Fatra (5), elsewhere in the Central Carpathians in Slovakia (4), the Low Beskids in E. Slovakia (1), W. and E. Carpathians (1), N. Moravia (1). In addition, three epithets are published in a new combination, and the names for two new subseries and a new forma are proposed.     

Different vasoactive effects of chronic endothelial and neuronal NO-synthase inhibition in young Wistar rats

While the unequivocal pattern of endothelial nitric oxide synthase (eNOS) inhibition in cardiovascular control is recognized, the role of NO produced by neuronal NOS (nNOS) remains unclear. The aim of this study was to compare the effects of chronic treatment with 7-nitroindazole (7-NI, nNOS inhibitor) and N^G-nitro-l-arginine methylester (l-NAME, general and predominantly eNOS inhibitor) on cardiovascular system of young normotensive rats. Wistar rats (4 weeks old) were used: controls and rats administered either 7-NI (10 mg/kg bw/day) or l-NAME (50 mg/kg bw/day) in drinking water for 6 weeks. The systolic blood pressure (sBP) was measured by plethysmographic method, and the vasoactivity of isolated arteries was recorded. 7-NI-treatment did not affect sBP; however, the sBP was increased after l-NAME-treatment. l-NAME inhibited acetylcholine-induced relaxation of thoracic aorta (TA), whereas it remained unchanged after 7-NI-treatment. The response of TA to sodium nitroprusside was increased in both experimental groups. The expression of eNOS and nNOS in TA was unchanged in both experimental groups, whereas the activity of NOS was decreased in l-NAME-treated group. Noradrenaline- and angiotensin II-induced contractions of TA were reduced in l-NAME-treated group; however, the contractions remained unchanged in 7-NI-treated group. In all groups, the endogenous angiotensin II participated in adrenergic contraction of TA; this contribution was significantly increased in l-NAME-treated group. Neurogenic contractions in mesenteric artery (MA) remained unchanged after 7-NI-treatment, but increased after l-NAME-treatment. Results show that NO deficiency induced by administration of 7-NI and l-NAME had different cardiovascular effects: eNOS and nNOS triggered distinct signaling pathways in young normotensive rats.     

Hydrolysis of the Leu-Gly bond of phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-D-Arg (a substrate of microbial collagenases) by treponemes isolated from the subgingival plaque of periodontitis patients

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl-l-prolyl-l-leucylglycyl-l-prolyl-d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US),Treponema denticola ATCC 35405, and 29 different clinical isolates ofT. denticola. TheTreponema US enzyme also hydrolyzed furylacryloyl-l-leucylglycyl-l-prolyl-l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators andp-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by “true” microbial collagenases.     

l-Arginine and its metabolites in kidney and cardiovascular disease

l-Arginine is a semi essential amino acid synthesised from glutamine, glutamate and proline via the intestinal-renal axis in humans and most mammals. l-Arginine degradation occurs via multiple pathways initiated by arginase, nitric-oxide synthase, Arg: glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine and agmatine with each having enormous biological importance. Several disease are associated to an l-arginine impaired levels and/or to its metabolites: in particular various l-arginine metabolites may participate in pathogenesis of kidney and cardiovascular disease. l-Arginine and its metabolites may constitute both a marker of pathology progression both the rationale for manipulating l-arginine metabolism as a strategy to ameliorate these disease. A large number of studies have been performed in experimental models of kidney disease with sometimes conflicting results, which underlie the complexity of Arg metabolism and our incomplete knowledge of all the mechanisms involved. Moreover several lines of evidence demonstrate the role of l-arg metabolites in cardiovascular disease and that l-arg administration role in reversing endothelial dysfunction, which is the leading cause of cardiovascular diseases, such as hypertension and atherosclerosis. This review will discuss the implication of the mains l-arginine metabolites and l-arginine-derived guanidine compounds in kidney and cardiovascular disease considering the more recent literature in the field.     

Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

The N ¹-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m¹A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.     

Annual nitrophilous pond and river bank communities in north part of Korean peninsula

The article deals with the herbal vegetation of flow and basin watersides in North Korea. Characterizations of associationsPolygonetum thunbergii Lohm. etMiyawaki 1962 andPolygonetum sieboldii-hydropiperis Okuda 1978 are given.Persicarietum posumbu, Aeschynomeno-Kummerowietum, Bidens bipinnata-community,Hemarthria sibirica-community,Persicaria dissitiflora-community andBidens tripartita-community are newly described. All phytosociological relevés were numerically classified by the computer program NUMCLAS. Brief notes on development, ecology and classification of waterside vegetation are mentioned.     

The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana

The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943–954, 2013). In the presence of Mg²⁺ ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous dl-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for dl-glycerol-3-phosphate.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

Bacidina genus novum familiaeLecideaceae s. lat. (Ascomycetes lichenisati)

The following eleven species currently classified in the generaBacidia s. lat. andCatillaria s. lat. are transferred to the new genusBacidina Vězda gen. n. (Lecideaceae s. lat.):Bacidina apiahica (Müll. Arg.) comb. n.,B. chloroticula (Nyl.)Vězda etPoelt comb. n.,B. egenula (Nyl.) comb. n.,B. inundata (Fr.) comb. n.,B. mirabilis (Vězda) comb. n.,B. neglecta (Vězda) comb.n.,B. pallidocarnea (Müll. Arg.) comb. n.,B. phacodes (Koerb.) comb.n.,B. scutellifera (Vězda) comb.n.,B. vasakii (Vězda) comb.n., andB. ziamensis (Vězda) comb.n.     

Low Energy Conformations for Gonadotropin-Releasing Hormone with d- and l-Amino Acid Substitutions for Gly 6: Possible Receptor-Bound Conformations

In the preceding paper, using ECEPP, including the effects of water, and the chain build-up procedure, we computed the low energy structures for GnRH and found that there were no distinct low energy structures or structures with high statistical weights. To attempt to deduce possible structures of GnRH that may bind to the GnRH receptor, we computed the low energy structures for GnRH peptides that have l- and d-amino acids substituting for Gly 6. The l-amino acid-substituted peptides (l-Ala and l-Val) have very low or no affinity for the receptor and on activity (release of FSH and LH) while the d-Ala-, d-Leu-, d-Trp- and d-Phe-substituted peptides have significantly higher relative affinities and activities than those for native GnRH; the d-Val-substituted peptide has about one-third of the affinity and activity as native GnRH. Unlike native GnRH, our computations suggest that both sets of peptides form well-defined structures in water: the l-amino acid-substituted peptides are predominantly α-helical while the d-amino acid-substituted peptides adopted E*A A A E D*(C*) A E C A(C*) and minor variants of these structures. By eliminating structures that lay in common to the d-Ala and l-Val peptides and further eliminating structures that differed between the d-Ala and d-Leu peptides, we reduced the number of possible distinct binding conformations to 254. Searching for structures among these 254 conformations that had relative statistical weights that paralleled their relative affinities, we found two candidate structures: D*E A A E C*A E C A and D*G A A E D*A E C G*, both of which have conformations for residues 3–9 that are similar to the computed most probable structures for the d-amino acid-substituted GnRH peptides in water.     

Determination and stereochemistry of proteinogenic and non-proteinogenic amino acids in Saudi Arabian date fruits

Whereas an abundance of literature is available on the occurrence of common proteinogenic amino acids (AAs) in edible fruits of the date palm (Phoenix dactylifera L.), recent reports on non-proteinogenic (non-coded) AAs and amino components are scarce. With emphasis on these components we have analyzed total hydrolysates of twelve cultivars of date fruits using automated ion-exchange chromatography, HPLC employing a fluorescent aminoquinolyl label, and GC–MS of total hydrolysates using the chiral stationary phases Chirasil^®-L-Val and Lipodex^® E. Besides common proteinogenic AAs, relatively large amounts of the following non-proteinogenic amino acids were detected: (2S,5R)-5-hydroxypipecolic acid (1.4–4.0 g/kg dry matter, DM), 1-aminocyclopropane-1-carboxylic acid (1.3–2.6 g/kg DM), γ-amino-n-butyric acid (0.5–1.2 g/kg DM), (2S,4R)-4-hydroxyproline (130–230 mg/kg DM), l-pipecolic acid (40–140 mg/kg DM), and 2-aminoethanol (40–160 mg/kg DM) as well as low or trace amounts (<70 mg/kg DM) of l-ornithine, 5-hydroxylysine, β-alanine, and in some samples (<20 mg/kg DM) of (S)-β-aminoisobutyric acid and (<10 mg/kg DM) l-allo-isoleucine. In one date fruit, traces of α-aminoadipic acid could be determined. Enantiomeric analysis of 6 M DCl/D₂O hydrolysates of AAs using chiral capillary gas chromatography–mass spectrometry revealed the presence of very low amounts of d-Ala, d-Asp, d-Glu, d-Ser and d-Phe (1.2–0.4 %, relative to the corresponding l-enantiomers), besides traces (0.2–1 %) of other d-AAs. The possible relevance of non-proteinogenic amino acids in date fruits is briefly addressed.     

Roles of glycosaminoglycans and glycanmimetics in tumor progression and metastasis

Various tumor cells exhibit structural alterations in the sulfated modifications to glycosaminoglycans (GAGs). The altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) on the surfaces of tumor cells is known to play a key role in malignant transformation and tumor metastasis. The receptor molecule for the CS chains containing E-disaccharide units (CS-E) expressed on Lewis lung carcinoma (LLC) cells was recently revealed to be Receptor for Advanced Glycation End-products (RAGE). RAGE is also involved in the development of various pathological conditions including aging, infection, pulmonary fibrosis, diabetes, and Alzheimer’s disease, by binding to a wide range of ligands. RAGE binds strongly not only to CS-E, but also to HS-expressing LLC cells. Recombinant RAGE bound CS-E and HS with high affinity. Furthermore, in a mouse model, the colonization of the lungs by LLC cells was inhibited by intravenously injected CS-E, an anti-CS-E antibody, or an anti-RAGE antibody. These findings demonstrated that RAGE is at least one of the critical receptors for CS and HS chains expressed on the tumor cell surface and is involved in experimental lung metastasis, and also that CS/HS and RAGE are potential molecular targets for the treatment of pulmonary metastasis. We, hence, reviewed these findings and also several chemically synthesized small GAGmimetics that exhibit potent anti-metastatic and/or anti-tumor activities.     

Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.     

Observation of algal colonization on Acropora aspera killed by Acanthaster planci

Algae started colonizing branches of the coral Acropora aspera (Dana) killed by the sea star Acanthaster planci (Linnaeus) within less than 24 hours. Two blue-greens ((Microcoleus lyngbyaceus (Crouan) Ag. and Hormothamnion solutum B. & F.)) dominated the early community but became less abundant than a brown ((Giffordia indica (Sonder) Papenfuss & Chihara)) after 26 days.     

The neurobiological link between OCD and ADHD

Obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) are two of the most common neuropsychiatric diseases in paediatric populations. The high comorbidity of ADHD and OCD with each other, especially of ADHD in paediatric OCD, is well described. OCD and ADHD often follow a chronic course with persistent rates of at least 40–50 %. Family studies showed high heritability in ADHD and OCD, and some genetic findings showed similar variants for both disorders of the same pathogenetic mechanisms, whereas other genetic findings may differentiate between ADHD and OCD. Neuropsychological and neuroimaging studies suggest that partly similar executive functions are affected in both disorders. The deficits in the corresponding brain networks may be responsible for the perseverative, compulsive symptoms in OCD but also for the disinhibited and impulsive symptoms characterizing ADHD. This article reviews the current literature of neuroimaging, neurochemical circuitry, neuropsychological and genetic findings considering similarities as well as differences between OCD and ADHD.