Search

Cellulose is the most abundant biological compound on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in many diverse bacterial species. While bacterial cellulose was first described in the 19th century, it was not until this last decade that a string of structural works provided insights into how the cellulose synthase BcsA, assisted by its inner-membrane partner BcsB, senses c-di-GMP to simultaneously polymerize its substrate and extrude the nascent polysaccharide across the inner bacterial membrane. It is now established that bacterial cellulose can be produced by several distinct types of cellulose secretion systems and that in addition to BcsAB, they can feature multiple accessory subunits, often indispensable for polysaccharide production. Importantly, the last years mark significant progress in our understanding not only of cellulose polymerization per se but also of the bigger picture of bacterial signaling, secretion system assembly, biofilm formation and host tissue colonization, as well as of structural and functional parallels of this dominant biosynthetic process between the bacterial and eukaryotic domains of life. Here, we review current mechanistic knowledge on bacterial cellulose secretion with focus on the structure, assembly and cooperativity of Bcs secretion system components.

Taxol is currently a valuable anticancer drug; however, the accumulated mixture of taxoids can vary greatly among Taxus species. So far, there is very little genomic information for the genus Taxus, except for Taxus baccata. Transcriptome analysis is a powerful approach to explore the different regulatory mechanisms underlying the taxoid biosynthesis pathway in Taxus species. First, we quantified the variation in the taxoid contents between Taxus media and Taxus mairei. The contents of paclitaxel and 10-deacetylpaclitaxel in T. media are higher than that in T. mairei. Then, the transcriptome profiles of T. media and T. mairei were analyzed to investigate the altered expressions. A total of 20,704 significantly differentially expressed genes (DEGs), including 9865 unigenes predominantly expressed in T. media and 10,839 unigenes predominantly expressed in T. mairei, were identified. In total, 120 jasmonic acid-related DEGs were analyzed, suggesting variations in ‘response to JA stimulus’ and ‘JA biosynthetic process’ pathways between T. media and T. mairei. Furthermore, a number of genes related to the precursor supply, taxane skeleton formation and hydroxylation, and C13-side chain assembly were also identified. The differential expression of the candidate genes involved in taxoid biosynthetic pathways may explain the variation in the taxoid contents between T. media and T. mairei.

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

Phoslactomycins (PLMs) represent an unusual structural class of natural products secreted by various streptomycetes, containing an α,β-unsaturated δ-lactone, an amino group, phosphate ester, conjugated diene and a cyclohexane ring. Phosphazomycins, phospholines and leustroducsins contain the same structural moieties, varying only in the acyl substituent at the C-18 hydroxyl position. These compounds possess either antifungal or antitumor activities or both. The antitumor activity of the PLM class of compounds has been attributed to a potent and selective inhibition of protein phosphatase 2A (PP2A). The cysteine-269 residue of PP2Ac-subunit has been shown to be the site of covalent modification by PLMs. In this article, we review previous work on the isolation, structure elucidation and biological activities of PLMs and related compounds and current status of our work on both PLM stability and genetic manipulation of the biosynthetic process. Our work has shown that PLM B is surprisingly stable in solution, with a pH optimum of 6. Preliminary biosynthetic studies utilizing isotopically labeled shikimic acid and cyclohexanecarboxylic acid (CHC) suggested PLM B to be a polyketide-type antibiotic synthesized using CHC as a starter unit. Using a gene (chcA) from a set of CHC-CoA biosynthesis genes from Streptomyces collinus as a probe, a 75 kb region of 29 ORFs encoding PLM biosynthesis was located in the genome of Streptomyces sp. strain HK803. Analysis and subsequent manipulation of plmS 2 and plmR 2 in the gene cluster has allowed for rational engineering of a strain that produces only one PLM analog, PLM B, at ninefold higher titers than the wild type strain. A strain producing PLM G (the penultimate intermediate in PLMs biosynthesis) has also been generated. Current work is aimed at selective in vitro acylation of PLM G with various carboxylic acids and a precursor-directed biosynthesis in a chcA deletion mutant with the aim of generating novel PLM analogs.

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.

Thyroid hormone production is regulated via pituitary thyrotropin (TSH) modulation of thyroxine (T4) prohormone secretion by the thyroid gland and regulation of active triiodothyronine (T3) production in peripheral tissues via metabolic events influencing activities of the iodothyronine monodeiodinase enzyme systems. Control at both levels is developmentally regulated and modified in serious nonthyroidal illness (trauma, infection, cancer, metabolic diseases). Racial and gender differences are of little significance except for the effects of placental estrogens and chorionic gonadotropin during pregnancy. There is a circadian rhythm of TSH secretion, with peak values at the onset of sleep and nadir concentrations during the afternoon hours. Peak and nadir concentrations differ by approximately +/- 50%. The effect on circulating T4 and T3 concentrations is not significant because of the large size of the extrathyroidal T4 pool. In healthy subjects there is no significant impact of body weight, physical training, body habitus, posture, immobilization, exercise, or ambulatory status on thyroid function, and no significant geographic environmental variation. Nutrition also has a minimal impact except for variation in iodine intake. Subthreshold concentrations of iodine intake are associated with increased TSH secretion, goiter, increased thyroid iodine uptake, decreased T4 production, an increased T3/T4 secretion ratio, and an increased ratio of circulating T3/T4 concentrations. Excessive iodine intake can block thyroid hormone biosynthesis by inhibiting the enzymes involved in the biosynthetic process, resulting in reduced T4 secretion, increased TSH concentrations, goiter, and hypothyroidism if the iodine excess is chronic.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, is very heterogeneous and has been classified into several subtypes. Missense mutations have been found to be responsible for the dominant type II vWD, characterized by qualitative abnormalities affecting von Willebrand factor (vWF) function. The breakpoints of a heterozygous vWF gene deletion (31 Kb), occurring 'de novo' in a patient with a variant of type II vWD, were localized to introns 25 and 34 and sequenced. An Alu repeat in intron 25 was interrupted between the transcriptional boxes A and B. The new junction present in the abnormal von Willebrand factor mRNA was sequenced after reverse transcription of platelet RNA. The codon 1104 (Cys) is followed in frame by the mutated codon 1926 (Cys to Arg), thus removing the complete A domains, found in a wide variety of genes and characterized by independent assembly 'in vitro'. We propose that the abnormal vWF, which carries intact protein domains responsible for vWF dimer and multimer formation, makes ineffective interactions with the normal molecules in the biosynthetic process, causing the dominant type II phenotype through a novel mechanism.

A correlation existed between the productivity of sclerin (SCL) and formation of sclerotia or dark pigmentation of old hyphae in Sclerotinia libertiana Fuckel and S. sclerotiorum de Bary. SCL stimulated the activities of phenoloxidase and peroxidase in a mycelial extract of S. libertiana, which appeared to be involved in the maturation and pigmentation of sclerotia. On the other hand, p-aminobenzoic acid (PABA), an inhibitor of phenoloxidase, inhibited the production of both SCL and sclerotia, where assimilation of PABA probably interrupted the biosynthetic process of SCL. When SCL was added at a high concentration, 4×10−3m, to Czapek’s agar medium a strain of S. libertiana which originally formed sclerotia and a strain of S. sclerotiorum which did not form sclerotia but produced melanin-like pigments at the edge of colony formed numerous sclerotium initials, which did not develop normal sclerotia. It was suggested that SCL played a role on induction of sclerotial formation and melanogenesis in these stra...

Changes in the contents and compositions of lipid classes and water-soluble components were examined in maturing soybean seed. At the first stage of seed development, triglyceride formation was very slow, and considerably large quantity of free fatty acids, phosphatidic acid, monoglyceride, and diglyceride were accumulated. At the second stage triglycerides were produced rapidly and the other components decreased. Glycerophosphate also appeared in the maturing seed. These results suggest that Kennedy’s pathway to triglycerides and other side pathway occur in seed. Positional distribution of fatty acids in mono-, di-, and triglycerides was also determined, and synthetic process of glyceride structure was discussed.

A cell-free system for the biosynthesis of l-threo-neopterin, a growth factor for protozoan, Crithidia fasciculata from guanosine-5′-triphosphate (GTP) was obtained from extracts of Serratia indica IFO 3759. This preparation catalyzed the production of a specific pteridine from GTP, which was isolated and characterized as a cyclic phosphate of neopterin (cNP). Among the other products, l-threo-neopterin, as the Crithidia factor, 6-hydroxymethylpterin, and erythro-neopterin were tentatively identified. Requirements for the synthesis of these products include GTP, Mg2+, and disodium phosphate. Fluorescence formation was inhibited by purine nucleotides.When a disodium phosphate was included in the reaction system, cNP and erythro-neopterin were effectively synthesized from GTP. On the other hand, when the phosphate was omitted 6-hydroxymethylpterin was formed.The possible biosynthetic process of l-threo-neopterin was discussed.