Platelet-derived Growth Factor Differentially Regulates the Expression and Post-translational Modification of Versican by Arterial Smooth Muscle Cells through Distinct Protein Kinase C and Extracellular Signal-regulated Kinase Pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.     

Structure and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide 10F by Carbohydrate Engineering in Streptococcus oralis

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfβ1–6GalNAcβ1–3Galα) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfβ1–6GalNAcβ1–3Galα rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a β1–3 linkage between Galf and GalNAcβ1–3Galα. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of β1–6-linked Galf in recognition of adjacent GalNAcβ1–3Galα in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.     

Crystal Structures of Trypanosoma brucei Sterol 14��-Demethylase and Implications for Selective Treatment of Human Infections

Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.     

Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-β-l-threo-pentopyranosyl-4″-ulose in the presence of NAD⁺. The second activity converts UDP-β-l-threo-pentopyranosyl-4″-ulose and NADH to UDP-xylose and NAD⁺, albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.     

Analysis of myxobacterial secondary metabolism goes molecular

During the last 20 years myxobacteria have made their way from highly exotic organisms to one of the major sources of microbial secondary metabolites besides actinomycetes and fungi. The pharmaceutical interest in these peculiar prokaryotes lies in their ability to produce a variety of structurally unique compounds and/or metabolites with rare biological activities. This review deals with the recent progress toward a better understanding of the biology, the genetics, the biochemistry and the regulation of secondary metabolite biosynthesis in myxobacteria. These research efforts paved the way to sophisticated in vitro studies and to the heterologous expression of complete biosynthetic pathways in conjunction with their targeted manipulation. The progress made is a prerequisite for using the vast resource of myxobacterial diversity regarding secondary metabolism more efficiently in the future.     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.     

Studies on the late steps of (+) pisatin biosynthesis: evidence for (-) enantiomeric intermediates

Pisatin, a 6a-hydroxyl-pterocarpan phytoalexin from pea (Pisum sativum L.), is relatively unique among naturally occurring pterocarpans by virtue of the (+) stereochemistry of its 6a-11a C-C bond. However, pisatin synthesizing pea tissue has an isoflavone reductase, first identified in alfalfa, which acts on the (-) antipode. In order to establish the natural biosynthetic pathway to (+) pisatin, and to evaluate the possible involvement of intermediates with a (-) chirality in its biosynthesis, we administered chiral, tritium-labeled, isoflavanones and pterocarpans to pisatin-synthesizing pea cotyledons and compared the efficiency of their incorporation. Pea incorporated the isoflavanone, (-) sophorol, more efficiently than either its (+) antipode, or the pterocarpans (+) or (-) maackiain. (-) Sophorol was also metabolized by protein extracts from pisatin-synthesizing pea seedlings in a NADPH-dependent manner. Three products were produced. One was the isoflavene (7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene), and another had properties consistent with the isoflavanol (7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol), the expected product for an isoflavanone reductase. A cDNA encoding sophorol reductase was also isolated from a cDNA library made from pisatin-synthesizing pea. The cloned recombinant sophorol reductase preferred (-) sophorol over (+) sophorol as a substrate and produced 7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol. Although no other intermediates in (+) pisatin biosynthesis were identified, the results lend additional support to the involvement of intermediates of (-) chirality in (+) pisatin synthesis.     

Strigol: biogenesis and physiological activity

The role played by molecules of the strigolactone family in stimulating the germination of seeds of parasitic weeds of the genera Striga, Orobanche and Alectra has never been clearly elucidated. The biogenesis of these unusual terpenoid lactones, originally identified in minute quantities in the root exudates of a small number of host plants and two or three "false hosts", also remains obscure. These lactones, as the chemical signals which initiate the life cycle of Striga, are consequently at the forefront of the Striga research effort. This paper reviews recent key discoveries relating to the biosynthesis and mode of action of strigolactones, and summarises the evidence suggesting that these molecules may be far more widely distributed and have a greater physiological significance than has hitherto been appreciated.     

Hinokiresinol is not a precursor of agatharesinol in the norlignan biosynthetic pathway in Japanese cedar

The biosynthetic relationship between the two norlignans agatharesinol and trans-hinokiresinol was investigated. Fresh sapwood sticks of Cryptomeria japonica were fed with stable isotope-labeled compounds, namely p-coumaryl alcohol-[9,9-²H], p-coumaryl alcohol-[9-¹⁸O] and trans-hinokiresinol-[1-²H], and then incubated under high-humidity for approximately 20 days, during which the two norlignans were produced simultaneously. While trans-hinokiresinol was strongly deuterium-labeled after feeding with p-coumaryl alcohol-[9,9-²H], agatharesinol was only lightly labeled after feeding with either p-coumaryl alcohol-[9,9-²H] or -[9-¹⁸O]. These results suggest that p-coumaryl alcohol, which is a precursor of hinokiresinol, is not involved in the biosynthesis of agatharesinol. Therefore, the norlignan carbon skeleton of agatharesinol must be framed from different types of phenylpropanoid monomers compared to those utilized by the trans-hinokiresinol pathway. The biosynthesis of these two norlignans seems to branch at an early stage, i.e., before the framing of the norlignan carbon skeleton. Furthermore, agatharesinol was not labeled with deuterium after feeding with ²H-labeled trans-hinokiresinol, which has the simplest norlignan structure. This result strongly supports the suggestion that the conversion of trans-hinokiresinol to agatharesinol is not part of the biosynthesis of norlignans and that early branching occurs instead.