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In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiester ases (PDEs), whic h modulate the intr acellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes to war ds a sessile lifestyle, the different c-di-GMP modulating enzymes are r esponsib le for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover tr anscriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus lowexpression of sigma factor encoding genes served the identification of their downstream regulons, we wer e una b le to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were r apidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon pr olonged cultivation of clinical P. aeruginosa isolates. [ABSTRACT FROM AUTHOR]

Ribonuclease H (RNase H) specifically hydrolyzes the 5'-phosphodiester bonds of the RNA of RNA/DNA hybrid. Both types 1 and 2 RNases H act on the RNA strand of the hybrid, while only type 2 acts on the single ribonucleotide embedded in DNA duplex. In this study, to explore the role of mammalian type 2 RNase H (RNase H2) in cells, we constructed the RNase H2 knockout NIH3T3 cells (KO cells) by CRISPR/Cas9 system. KO cells hydrolyzed RNA strands in RNA/DNA hybrid, but not single ribonucleotides in DNA duplex, while wild-type NIH3T3 cells (WT cells) hydrolyzed both. Genomic DNA in the KO cells was more heavily hydrolyzed than in the WT cells by the alkaline or RNase H2 treatment, suggesting that the KO cells contained more ribonucleotides in genomic DNA than the WT cells. The growth rate of the KO cells was 60% of that of the WT cells. Expression of interferon-stimulated genes (ISGs) in the KO cells was not markedly elevated compared with the WT cells. These results suggest that in NIH3T3 cells, RNase H2 is crucial for suppressing the accumulation of ribonucleotides in genomic DNA but not for the expression of ISGs. [ABSTRACT FROM AUTHOR]

Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling. [ABSTRACT FROM AUTHOR]

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ~60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 μM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters. [ABSTRACT FROM AUTHOR]

Endonuclease V (Endo V) is a DNA repair enzyme that recognizes deoxyinosine and cleaves the second phosphodiester bond on the 3′ side of the deaminated base lesion. A database search revealed the presence of homologous genes for Endo V in most archaeal species, but the absence in some methanogenic species. We cloned a gene encoding the sequence homologous to Escherichia coli Endo V from the genome of the hyperthermophilic euryarchaeon, Pyrococcus furiosus and purified gene product (PfuEndoV) to homogeneity. In vitro characterization showed that PfuEndoV possesses specific endonuclease activity for the deoxyinosine-containing DNA strand. The activity of the enzyme was maximal at 90°C. Stable complex formation between PfuEndoV and nicked DNA produced by the cleavage reaction was detected by gel mobility shift assays. The molecular mechanisms of the inosine repair pathway including Endo V in the archaeal cells are discussed. Interestingly, PfuEndoV cleaved inosine-containing RNA strands as well as DNA substrates. PfuEndoV may also be involved in RNA metabolism. [ABSTRACT FROM AUTHOR]

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation. [ABSTRACT FROM AUTHOR]

The disaccharided- N-acetylglucosamine-l-rhamnose plays an important role in the mycobacterial cell wall as a linker connecting arabinogalactan and peptidoglycan via a phosphodiester linkage. The first step of the disaccharide linker is the formation of decaprenyl phosphate-Glc NAc, which is catalyzed by Glc NAc-1-phosphate transferase. In Gram-negative bacteria, the wecA gene specifies the UDP-Glc NAc: undecaprenyl phosphate Glc NAc-1-phosphate transferase (WecA), which catalyzes the first step in the biosynthesis of lipopolysaccharide O-antigen. Mycobacterium tuberculosis Rv1302 and Mycobacterium smegmatis MSMEG_4947 show homology to Escherichia coli WecA protein. We cloned Rv1302 and MSMEG_4947 and introduced plasmids pYJ-1 (carrying Rv1302) and pYJ-2 (carrying MSMEG_4947) into a wecA-defective strain of E. coli MV501, respectively. Lipopolysaccharide analysis demonstrated that lipopolysaccharide synthesis in MV501 (pYJ-1) and MV501 (pYJ-2) was restored upon complementation with Rv1302 and MSMEG_4947, respectively. This provides the first evidence that Rv1302 and MSMEG_4947 have the same function as E. coli WecA. We also generated an M. smegmatis MSMEG_4947 knockout mutant using a homologous recombination strategy. The disruption of MSMEG_4947 in the M. smegmatis genome resulted in the loss of viability at a nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy results showed that the lack of the MSMEG_4947 protein causes drastic morphological changes in M. smegmatis. [ABSTRACT FROM AUTHOR]

The yeast Candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is directly mediated by cell wall components of the yeast, including a wide range of abundantly expressed glycoconjugates. Of particular interest in C. albicans are the β-mannosylated epitopes that show a complex expression pattern on N-glycan moiety of phosphopeptidomannans and are absent in the non-pathogenic species Saccharomyces cerevisiae. Being known as potent antigens for the adaptive immune response and elicitors of specific infection-protective antibodies, the exact delineation of β-mannosides regulation and expression pathways has lately become a major milestone toward the comprehension of host-pathogen interplay. Using the newly developed HR-MAS NMR methodology, we demonstrate the possibility of assessing the general profiles of cell-surface-exposed glycoconjugates from intact living yeast cells without any prior purification step. This technique permitted to directly observe structural modifications of surface expressed phosphodiester-linked β-mannosides on a series of deletion strains in β-mannosyltransferases and phospho-mannosyltransferases compared with their parental strains [ABSTRACT FROM PUBLISHER]

The pathogenic yeast Candida albicans produces large N-glycans with outer regions containing only mannose residues. The outer region comprises a primary branch with multiple secondary and tertiary branches. Tertiary branches are linked to secondary branches by phosphodiester bridges. In the current model of outer chain elongation in the genetically related yeast Saccharomyces cerevisiae, synthesis of the branches occurs sequentially, primary to tertiary. Thus, disruption of mannosylphosphorylation, the initial step in tertiary branch formation, should not affect primary or secondary branch production. Compared to its wild-type parent, a C. albicans mutant defective in tertiary branch mannosylphosphorylation (mnn4Δ/mnn4Δ) made outer regions with reduced susceptibility to low acid acetolysis treatment, suggesting that the secondary or primary region had been modified. Higher acid acetolysis conditions were required to release the secondary branches from the primary branches. The released secondary branches constitute the subset of the wild-type secondary branches that lack a phosphate group. In contrast, the acid-stable region of both wild-type and mnn4Δ S. cerevisiae strains required high acid acetolysis conditions to release the secondary branches, despite having smaller and less complex secondary and tertiary branches. These results suggest that the complex and longer secondary and tertiary branches of C. albicans affect the conformation of the acid-stable region to render it more susceptible to acetolysis which implies secondary and tertiary branch formation in C. albicans are interdependent events and occur concurrently, rather than sequentially. [ABSTRACT FROM PUBLISHER]

Ribonuclease MCI (RNase MCI) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5′-side of uridine. The crystal structures of RNase MCI in complex with 2′-UMP or 3′-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al (2000) Biochem. Biophys. Res. Commun. 275, 572–576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3′,5′-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased Km and 27.6-fold decreased kcat while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the kcat value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (kcat/Km) with a minimum Km value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2′,3′-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner. [ABSTRACT FROM AUTHOR]

Previously, lipoteichoic acid (LTA) of Enterococcus hirae was found to exhibit definite cytokine-inducing activity but synthetic specimens which share the fundamental structural principles proposed for LTA had no corresponding activity. We also showed recently that several minor components totally less than 5% of the LTA fraction from E. hirae ATCC 9790 possessed the activity, whereas the major component (over 90%) did not [Suda, Y., Tochio, H., Kawano, K., Takada, H., Yoshida, T., Kotani, S., and Kusumoto, S. (1995) FEMS Immun. Med. Microbiol. 12, 97–112]. In the present study, the structure of the major component of LTA was studied in an attempt to elucidate the reason for the lack of the activity in the synthetic compounds. The major component of the LTA was first digested by hydrofluoric acid hydrolysis to cleave phosphodiester linkages present. The hydrolysis products were separated and characterized by means of NMR and MS. The linkage positions of the original phosphodiesters were determined from the NMR spectra of an alkali-treated product without hydrofluoric acid degradation. The compound was proved to consist of 1,3-linked poly(glycerophosphate) and a lipid anchor, Glc(αl-2)Glc(αl-3)acyl2Gro, the former being linked to the 6-position of the distal glucose of the latter. The 2-position of the glycerol residues in the glycerophosphate part were substituted by oligoglucose esterified partially with alanine. The gross structure elucidated here thus coincides with the previous conclusion described by Fischer [Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M., ed.) pp. 123–234, Plenum Press, New York]. Thus, the molecular species with this so-called “LTA structure” is not responsible for the cytokine-inducing activity. [ABSTRACT FROM AUTHOR]

The article focuses on inhibition of gene expression by anti-sense oligodeoxynucleotides (ODN). Important requirements of ODN are, high specificity and high efficiency. Most modifications to anti-sense oligonucleotides are aimed at increasing their efficacy. The site of nucleolytic attack is the phosphodiester link, and modifications to this bond have been introduced to reduce degradation. ODN are likely to be taken up by pinocytosis and endocytosis. The intracellular concentration is reportedly higher than that of the surrounding medium, and ODN are found in both the cytoplasmic and nuclear fraction.

A dinuclear La3+ complex hydrolyzes the phosphodiester bond in diribonucleotides efficiently under mild conditions. The rate of hydrolysis is remarkably accelerated on the complex formation between and TPHP (TPHP=N,N,N',N'-tetrakis [(2-pyridyl)meth-yl]-2-hydroxy-1,3-dianinopropane), and the highest activity is attained when the dinu-clear structure is formed. The half-life of ApA hydrolysis by the dinuclear La3+ complex (1 mM) is 350 s, whereas that by the free La3+ ion (1 mM) is 23,000 s at pH 7.2 and 50°C. The dinuclear La3+ complex is promising as a catalytic center of artificial nucleases. [ABSTRACT FROM AUTHOR]

Focuses on the topical application of mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid, in treating plaque-type psoriasis. Brief information on MMF; Effectiveness of MMF as an immunosuppressive therapy.