Presence Of Spermidine Research Articles

Spermidine-induced improvement of reconsolidation of memory involves calcium-dependent protein kinase in rats.

In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus. Twenty-four hours after training, animals were re-exposed to the apparatus in the absence of shock (reactivation session). Immediately after the reactivation session, spermidine (2–200 pmol/site), the PKC inhibitor 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl) maleimide hydrochloride (GF 109203X, 0.3–30 pg/site), the antagonist of the polyamine-binding site at the NMDA receptor, arcaine (0.2–200 pmol/site), or the PKC activator phorbol 12-myristate 13-acetate (PMA, 0.02–2 nmol/site) was injected. While the post-reactivation administration of spermidine (20 and 200 pmol/site) and PMA (2 nmol/site) improved memory reconsolidation, GF 109203X (1, 10, and 30 pg/site) and arcaine (200 pmol/site) impaired it. GF 109203X (0.3 pg/site) impaired memory reconsolidation in the presence of spermidine (200 pmol/site). PMA (0.2 nmol/site) prevented the arcaine (200 pmol/site)-induced impairment of memory reconsolidation. Anisomycin (2 µg/site) also impaired memory reconsolidation in the presence of spermidine (200 pmol/site). Drugs had no effect when they were administered in the absence of reactivation. These results suggest that the spermidine-induced enhancement of memory reconsolidation involves PKC activation.

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40±15) mM in freshly prepared solution and (0.28±0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43±0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4°C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.

Cuminaldehyde as the Major Component of Cuminum cyminum, a Natural Aldehyde with Inhibitory Effect on Alpha-Synuclein Fibrillation and Cytotoxicity.

Fibrillation of alpha-synuclein (α-SN) is a critical process in the pathophysiology of several neurodegenerative diseases, especially Parkinson's disease. Application of bioactive inhibitory compounds from herbal extracts is a potential therapeutic approach for this cytotoxic process. Here, we investigated the inhibitory effects of the Iranian Cuminum cyminum essential oil on the fibrillation of α-SN. Analysis of different fractions from the total extract identified cuminaldehyde as the active compound involved in the antifibrillation activity. In comparison with baicalein, a well-known inhibitor of α-SN fibrillation, cuminaldehyde showed the same activity in some aspects and a different activity on other parameters influencing α-SN fibrillation. The presence of spermidine, an α-SN fibrillation inducer, dominantly enforced the inhibitory effects of cuminaldehyde even more intensively than baicalein. Furthermore, the results from experiments using preformed fibrils and monobromobimane-labeled monomeric protein also suggest that cuminaldehyde prevents α-SN fibrillation even in the presence of seeds, having no disaggregating impact on the preformed fibrils. Structural studies showed that cuminaldehyde stalls protein assembly into β-structural fibrils, which might be achieved by the interaction with amine groups through its aldehyde group as a Schiff base reaction. This assumption was supported by FITC labeling efficiency assay. In addition, cytotoxicity assays on PC12 cells showed that cuminaldehyde is a nontoxic compound, treatment with cuminaldehyde throughout α-SN fibrillation showed no toxic effects on the cells. Taken together, these results show for the first time that the small abundant natural compound, cuminaldehyde, can modulate α-SN fibrillation. Hence, suggesting that such natural active aldehyde could have potential therapeutic applications.

Local de-condensation of double-stranded DNA in oppositely charged polyelectrolyte as induced by spermidine.

How polyamines such as spermidine cooperate with histone to condense and de-condense DNA during transcription has not been clarified. In this work, using the complex of DNA and poly(L-lysine) (PLL) at +/- ratio of 0.5 as a model of nucleosome, we monitored the de-condensation of DNA in the presence of spermidine. As revealed by the results from atomic force microscopy and time-resolved laser light scattering, spermidine was able to transform the spherical complex into a core-shelled structure, with the hard core being the DNA-PLL complex and the soft shell being DNA and spermidine. The soft shell evolved into a coiled DNA conformation with time. Such a local de-condensation process should be helpful in understanding the DNA transcription and cell division process in vivo.

Effects of benzyladenine purine and its interaction with polyamines on growth of Spathoglottis plicata PLBs

Spathoglottis plicata is highly sought after by florists and orchid growers due to its unique structure and colors. However, this orchid is a slow-growing type and thus the planting materials are acutely limited. Therefore, there is a need to improve its multiplication efficiency and speed up its growth in order to meet the market demand. Benzyladenine purine (BAP) has been widely used to improve plantlet multiplication in plant cultures. Polyamines have also been reported to improve the growth and development of in vitro cultures. The objective of this study was to investigate the effects of BAP and different polyamines on the physical and biochemical changes of protocorm-like bodies (PLBs) of S. plicata. Different concentrations of BAP and polyamines (putrescine, spermine, and spermidine) were used. BAP and polyamines were supplemented singly in half-strength MS medium and PLBs were cultured for 2 weeks. It was observed that 5 µM BAP and 25 µM spermidine resulted in the highest fresh weights of 0.38 g and 0.31 g, respectively. The total soluble protein and carbohydrate content for PLBs treated with 25 µM spermidine was 4.85 mg/g FW and 6.36 mg/g FW. A separate experiment was carried out to investigate the interactive effects of 25 µM spermidine with 5 µM BAP. The presence of spermidine in the media reduced the peroxidase and catalase activities by increasing the nitrate reductase activity.

Experimental and theoretical study of the silica particle interactions in the presence of multivalent rod-like ions.

The silica particle interactions in the presence of spermidine were systematically investigated both from experimental and theoretical points of view. The hydrodynamic radii and the corresponding polydispersity indices of the colloidal silica particles were determined by dynamic light scattering (DLS) as a function of spermidine concentration. Whereas the effective size of the silica particles increases with increasing spermidine concentration (pointing to the particle aggregation), the polydispersity index first increases reaches a maximum and then further decreases with the increasing spermidine concentration. From the mobility measurements it was concluded that the increase in spermidine concentration causes less negative values of zeta potential, meaning that the adsorption of spermidine leads to the less negative silica surface. Moreover, Monte Carlo (MC) simulations also confirmed that the addition of spermidine reduces the repulsion between silica particles. The MC concentration profiles of spermidine close to the charged silica particle are in a very good agreement with the results obtained by theory. An important motivation for our study is the effectiveness of multivalent ions to coagulate colloidal suspensions; e.g., the multivalent ions are exploited in the water purification process.

Isolation, Purification and Characterization of Nucleoids from <i>Synechococcus elongatus</i> PCC 7942

The genomic DNA of bacteria is highly compacted in one or a few bodies known as nucleoids. In order to understand the overall configuration and physiological activities of the cyanobacterial nucleoid under various growth conditions and the role(s) of each nucleoid protein in clock function, thylakoid membrane-associated nucleoids from the Synechococcus elongatus (se) PCC 7942 strain were isolated and purified in presence of spermidine at low salt concentrations by sucrose density gradient centrifugation. The sedimentation rates, protein/DNA composition and microscopic appearances as well as variation in structural components of clock proteins from the isolated nucleoids were compared under identical conditions. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The nucleoid structure in the wild type was more tightly compacted than that in the KaiABC mutant strain. Western immunoblot analyses revealed that the KaiC was associated with the nucleoid fraction whereas maximum KaiA was localized in the cytosolic fraction, supposedly in association with the translation machinery.

Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems

This study was undertaken to investigate the possible involvement of the antioxidant defense and glyoxalase systems in protecting rice seedlings from heat-induced damage in the presence of spermidine (Spd). Hydroponically grown 14-day-old seedlings were subjected to foliar spray with Spd (1 mM, 24 h) prior to heat stress (42 °C, 48 h) followed by subsequent recovery (27 °C, 48 h). Lipoxygenase activity, malondialdehyde (MDA), hydrogen peroxide (H2O2) and proline (Pro) content increased significantly whereas fresh weight (FW) and chlorophyll (Chl) content decreased during heat stress and after recovery, indicating unrecoverable damage to rice seedlings. Heat-induced damage was also evident in decreased levels of ascorbate (AsA), glutathione (GSH), and AsA and GSH redox ratios. Superoxide dismutase (SOD) and catalase (CAT) activities increased during heat stress but declined after recovery. Activities of glutathione peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR) and glutathione reductase (GR) decreased during heat stress but an opposite trend for most of these enzymes was observed after recovery. Heat stress also resulted in significant increases in the activities of glyoxalase enzymes (Gly I and Gly II). In contrast, exogenous Spd protected rice seedlings from heat-induced damage as marked by lower levels of MDA, H2O2, and Pro content coupled with increased levels of AsA, GSH, FW, Chl, and AsA and GSH redox status. After recovery, Spd-pretreated heat-exposed seedlings displayed higher activities of SOD, CAT, GPX, GST APX, DHAR and GR as well as of Gly I and Gly II. In addition, polyamine analysis revealed that exogenously applied Spd significantly elevated the levels of free and soluble conjugated Spd. Therefore, we conclude from our results that heat exposure provoked an oxidative burden while enhancement of the antioxidative and glyoxalase systems by Spd rendered rice seedlings more tolerant to heat stress. Further, co-induction of the antioxidative and glyoxalase systems was closely associated with Spd mediated enhanced level of GSH.

Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps

The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30–60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.

Sphingobium fontiphilum sp. nov., isolated from a freshwater spring

To investigate the biodiversity of bacteria in the spring water of the Chengcing Lake Park in Taiwan, a Gram-stain-negative, rod-shaped, non-motile, non-spore-forming and aerobic bacterial strain, designated strain Chen16-4(T), was isolated and characterized in a taxonomic study using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest relatives of strain Chen16-4(T) were Sphingobium amiense YT(T), Sphingobium yanoikuyae GIFU 9882(T) and Sphingobium scionense WP01(T), with sequence similarities of 97.6, 97.1 and 97.0 %, respectively. A phylogenetic tree obtained with 16S rRNA gene sequences indicated that strain Chen16-4(T) and these three closest relatives formed an independent phylogenetic clade within the genus Sphingobium. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of C18 : 1ω7c in the cellular fatty acid profile and the DNA G+C content also supported affiliation of the isolate to the genus Sphingobium. The DNA-DNA relatedness of strain Chen16-4(T) with respect to recognized species of the genus Sphingobium was less than 70 %. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Chen16-4(T) represents a novel species in the genus Sphingobium, for which the name Sphingobium fontiphilum sp. nov. is proposed. The type strain is Chen16-4(T) ( = BCRC 80308(T) = LMG 26342(T) = KCTC 23559(T)).

Spermidine inhibits MMP-2 via modulation of histone acetyltransferase and histone deacetylase in HDFs

In recent years, aging has been reported to be regulated by HAT. In this study, the inhibitory effects of spermidine on the matrix metalloproteinase-2 (MMP-2) activity and expression were investigated in human dermal fibroblasts (HDFs). It was observed that spermidine inhibits MMP-2 activity and expression. In addition, the expression levels of histone acetyltransferase (HAT), phospho-extracellular-signal related kinase (p-ERK), phospho-c-jun N-terminal kinase (p-JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are decreased in the presence of spermidine. In contrast, the expression levels of histone deacetylase 1 (HDAC1), sirtuin 1 (SIRT1), phospho-p38 (p-p38) are increased by spermidine. In conclusion, our results suggest that spermidine could have a therapeutic potential in inhibition of metastasis through the inhibitory effects on activity and expression of MMP-2 via regulation of HAT and HDAC.

Sphingobium limneticum sp. nov. and Sphingobium boeckii sp. nov., two freshwater planktonic members of the family Sphingomonadaceae , and reclassification of Sphingomonas suberifaciens as Sphingobium suberifaciens comb. nov.

Two novel chemo-organoheterotrophic members of the Sphingomonadaceae were isolated from alpine and pre-alpine lakes. Cells stained gram-negative, were motile and rod-shaped, and formed yellow, circular, convex colonies on different agar media. Strains 301(T) and 469(T) were strictly aerobic, catalase- and oxidase-positive, and grew at temperatures between 10 and 40 °C (optimum, 28 °C), and at pH values between 5 and 10 (optimum, pH 7). Both strains contained Q-10 as the dominant quinone, sphingoglycolipids and 2-hydroxymyristic acid, whereas 3-hydroxy fatty acids were absent. Major fatty acids of strain 301(T) were C(18 : 1)ω7c (53.3 %) and C(16 : 1)ω7c (22.9 %), with C(14 : 0) 2-OH (10.8 %) as the major 2-hydroxy fatty acid. Fatty acids of strain 469(T) were dominated by C(18 : 1)ω7c (34.4 %), C(16 : 1)ω7c (32.0 %) and C(14 : 0) 2-OH (15.2 %) as the major 2-hydroxy fatty acid. The genomic DNA G+C contents of strains 301(T) and 469(T) were 63.4 and 64.6 mol%, respectively. 16S rRNA gene sequence comparison indicated that both strains belonged to the genus Sphingobium. This classification was supported by the presence of spermidine as the major polyamine. The phylogenetically closest relatives of strain 301(T) were Sphingobium amiense DSM 16289(T), Sphingobium vermicomposti DSM 21299(T), Sphingobium yanoikuyae DSM 7462(T) and Sphingobium scionense DSM 19371(T) (98.8, 98.0, 97.9 and 97.4 % sequence similarity, respectively). DNA-DNA hybridization of genomic DNA yielded similarities in the range 43.2-12.1 % between strain 301(T) and the type strains of these four Sphingobium species. Closest relatives of strain 469(T) were Sphingomonas suberifaciens DSM 7465(T) and Sphingobium scionense DSM 19371(T) (97.1 and 96.5 % 16S rRNA gene sequence similarity, respectively). The degree of DNA-DNA hybridization between strain 469(T) and Sphingomonas suberifaciens DSM 7465(T) was 17.9 %. Based on the results of the molecular analyses and their phenotypic characteristics, strains 301(T) and 469(T) represent two novel species of the genus Sphingobium. The name Sphingobium limneticum sp. nov. is proposed for strain 301(T)( = DSM25076(T) = LMG 26659(T)). The name Sphingobium boeckii sp. nov. is proposed for strain 469(T) ( = DSM 25079(T) = LMG 26901(T)). The polyphasic analysis also suggests that Sphingomonas suberifaciens should be reclassified as Sphingobium suberifaciens comb. nov. with Ca1(T) ( = EY 2404(T) = ATCC 49355(T) = CIP 105429(T) = DSM 7465(T) = ICMP 12535(T) = NBRC 15211(T) = JCM 8521(T) = LMG 17323(T) = NCPPB 3629(T)) as the type strain.

Sphingobium baderi sp. nov., isolated from a hexachlorocyclohexane dump site

A Gram-stain-negative, rod-shaped and white-coloured bacterial strain, designated LL03(T), was isolated from hexachlorocyclohexane-contaminated soil at Spolana Neratovice, Czech Republic, where lindane was formerly produced. Strain LL03(T) was found to be a degrader of α-, γ- and δ-isomers of hexachlorocyclohexane, although no significant degradation activity was observed for the β-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL03(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest similarity with Sphingobium wenxiniae JZ-1(T) (99.2 %). The DNA G+C content of strain LL03(T) was 67.0 mol%. DNA-DNA relatedness values of strain LL03(T) with its close phylogenetic neighbours were below the threshold level of 70 %, supporting its identification as a representative of a novel species of the genus Sphingobium. The predominant respiratory quinone was ubiquinone Q-10. The polar lipid profile of strain LL03(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid), supporting its identification as a member of the genus Sphingobium. Spermidine was identified as the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1ω7c and/or 16 : 1ω6c), summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c) and 14 : 0 2-OH. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH and the G+C content of the genomic DNA supported the affiliation of the strain to the genus Sphingobium. The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished it from closely related species of the genus Sphingobium. Therefore, strain LL03(T) represents a novel species of the genus Sphingobium for which the name Sphingobium baderi LL03(T) sp. nov. is proposed; the type strain is LL03(T) ( = CCM 7981(T) = DSM 25433(T)).

Sphingobium jiangsuense sp. nov., a 3-phenoxybenzoic acid-degrading bacterium isolated from a wastewater treatment system

A non-sporulating, non-motile, catalase- and oxidase-positive, Gram-negative, rod-shaped bacterial strain, designated BA-3T, was isolated from activated sludge of a wastewater treatment facility. The strain was able to degrade about 95 % of 100 mg 3-phenoxybenzoic acid l(-1) within 2 days of incubation. Growth occurred in the presence of 0-2 % (w/v) NaCl [optimum, 0.5 % (w/v) NaCl], at pH 5.5-9.0 (optimum, pH 7.0) and at 10-37 °C (optimum, 28 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain BA-3T was a member of the genus Sphingobium; it showed highest gene sequence similarity to Sphingobium qiguonii X23T (98.2 %), and similarities of <97.0 % with strains of other Sphingobium species. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of summed feature 8 (C18:1ω6c and/or C18:1ω7c) in the cellular fatty acid profile and the DNA G+C content also supported affiliation of the isolate to the genus Sphingobium. Strain BA-3T showed low DNA-DNA relatedness values (21.3±0.8 %) with Sphingobium qiguonii X23(T). Based on phenotypic, genotypic and phylogenetic data, strain BA-3T represents a novel species of the genus Sphingobium, for which the name Sphingobium jiangsuense sp. nov. is proposed; the type strain is BA-3T (=CCTCC AB 2010217T= KCTC 23196T=KACC 16433T).

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Sphingobium wenxiniae sp. nov., a synthetic pyrethroid (SP)-degrading bacterium isolated from activated sludge in an SP-manufacturing wastewater treatment facility

A synthetic pyrethroid (SP)-degrading bacterial strain, designated JZ-1(T), was isolated from activated sludge of a SP-manufacturing wastewater treatment facility and studied using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1(T) belonged to the genus Sphingobium, showing highest sequence similarities to Sphingobium faniae DSM 21829(T) (98.6 %), Sphingobium cloacae JCM 10874(T) (98.5 %), Sphingobium vermicomposti DSM 21299(T) (97.4 %) and Sphingobium ummariense CCM 7431(T) (96.9 %). The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(19 : 0) cyclo ω8c, 11 methyl C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH, and the G+C content of the genomic DNA also supported the affiliation of the strain with the genus Sphingobium. Strain JZ-1(T) showed low DNA-DNA relatedness values with S. faniae DSM 21829(T) (30.2 %), S. cloacae JCM 10874(T) (23.3 %), S. vermicomposti DSM 21299(T) (10.9 %) and S. ummariense CCM 7431(T) (7.9 %). Based on its phylogenetic position and its phenotypic and genotypic properties, strain JZ-1(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1(T) ( = CGMCC 1.7748(T) = DSM 21828(T)).

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act in vitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca 2+. Identification of ε-(γ-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of ε-(γ-glutamyl)lysine formation. Indeed, in the presence of 1 mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N 1- mono(γ-glutamyl)SPD and N 8- mono(γ-glutamyl)SPD. Negligible amount of N 1,N 8- bis(γ-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.

Sphingobium qiguonii sp. nov., a carbaryl-degrading bacterium isolated from a wastewater treatment system

A Gram-staining-negative, catalase-positive, carbaryl-degrading, non-spore-forming, non-motile, rod-shaped bacterium, designated strain X23(T), was isolated from a wastewater treatment system. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain belongs to the genus Sphingobium. The highest 16S rRNA gene sequence similarity observed for the isolate was 96.6 % with the type strain of Sphingobium amiense. Chemotaxonomic data [major ubiquinone: Q-10; major polar lipids: diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine and unknown aminolipids and phospholipids; major fatty acids: summed feature 7 (C(18 : 1)ω7c, C(18 : 1)ω9t and/or C(18 : 1)ω12t), C(16 : 1)ω5c, C(14 : 0) 2-OH and C(16 : 0) 2-OH] as well as the inability to reduce nitrate and the presence of spermidine as the major polyamine supported the affiliation of the strain to the genus Sphingobium. Based on the phylogenetic analysis, whole-cell fatty acid composition and biochemical characteristics, the strain could be separated from all recognized species of the genus Sphingobium. Strain X23(T) should be classified as a novel species of the genus Sphingobium, for which the name Sphingobium qiguonii sp. nov. is proposed, with strain X23(T) (=CCTCC AB 208221(T) =DSM 21541(T)) as the type strain.

Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl- d-asparate-type glutamate receptors

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl- d-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [ 125I]MK-801 binding) and spermidine (100 μM)-potentiated [ 125I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1–10 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [ 125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (∼350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or dl-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [ 125I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

Sphingobium vermicomposti sp. nov., isolated from vermicompost

Strain VC-230(T) was isolated from homemade vermicompost produced from kitchen waste. The isolate was a Gram-negative-staining, catalase- and oxidase-positive, motile rod-shaped bacterium able to grow at 15-37 degrees C and pH 6-8. On the basis of 16S rRNA gene sequence analysis, strain VC-230(T) was determined to belong to the family Sphingomonadaceae by its clustering with type strains of the genus Sphingobium, with Sphingobium chlorophenolicum ATCC 33790(T) (97.7 %) and Sphingobium herbicidovorans DSM 11019(T) (97.4 %) as its closest neighbours. The polar lipid pattern, the presence of spermidine and ubiquinone 10, the predominance of the cellular fatty acids C(18 : 1)omega7c/9t/12t, C(16 : 1)omega7c and C(16 : 0) and the G+C content of the genomic DNA supported the affiliation of this organism to the genus Sphingobium. The phylogenetic, chemotaxonomic, phenotypic and DNA-DNA hybridization analyses verify that strain VC-230(T) represents a novel species, for which the name Sphingobium vermicomposti sp. nov. is proposed. The type strain is VC-230(T) (=CCUG 55809(T) =DSM 21299(T)).