Biosynthesis of heparin

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[¹⁴C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.Abbreviations PAPS 3-phosphoadenylylsulfate - Hepes 4-(2-hydroxy-ethyl)piperazineethanesulfonic acid - GlcUA glucuronic acid This is Paper XIV of a series in which the preceeding reports are refs 10 and 12. A preliminary report has appeared [28].     

Capsomer vaccines protect mice from vaginal challenge with human papillomavirus

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18.     

Frequency of benign neutropenia among Black versus White individuals undergoing a bone marrow assessment

Healthy individuals in the United States identified as having Black race have lower neutrophil counts, on average, than individuals identified as having White race, which could result in more negative diagnostic evaluations for neutropenia. To test this hypothesis, the proportion of evaluations where the final diagnosis was clinically insignificant neutropenia for Black and White individuals who underwent an evaluation by a haematologist that included a bone marrow (BM) biopsy to investigate neutropenia was assessed. 172 individuals without prior haematological diagnoses who underwent a haematological evaluation to investigate neutropenia. Individuals diagnosed with clinically insignificant neutropenia between Black and White individuals were compared using a propensity‐score‐adjusted logistic regression. Of 172 individuals, 42 (24%) were classified as Black race, 86 (50%) were males, and the 79 (46%) were over 18 years old. A BM biopsy did not identify pathology in 95% (40 of 42) of Black individuals and 68% (89 of 130) of White Individuals. Black individuals (25 of 42 [60%]) received a final diagnosis of clinically insignificant neutropenia, compared to White individuals (12 of 130 [9%]) (adjusted odds ratio =7.9, 95% CI: 3.1 – 21.1). We conclude that black individuals were more likely to receive a diagnosis of clinically insignificant neutropenia after haematological assessment.     

Interaction of tSNARE syntaxin 18 with the papillomavirus minor capsid protein mediates infection

The papillomavirus capsid mediates binding to the cell surface and passage of the virion to the perinuclear region during infection. To better understand how the virus traffics across the cell, we sought to identify cellular proteins that bind to the minor capsid protein L2. We have identified syntaxin 18 as a protein that interacts with bovine papillomavirus type 1 (BPV1) L2. Syntaxin 18 is a target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (tSNARE) that resides in the endoplasmic reticulum (ER). The ectopic expression of FLAG-tagged syntaxin 18, which disrupts ER trafficking, blocked BPV1 pseudovirion infection. Furthermore, the expression of FLAG-syntaxin 18 prevented the passage of BPV1 pseudovirions to the perinuclear region that is consistent with the ER. Genetic studies identified a highly conserved L2 domain, DKILK, comprising residues 40 to 44 that mediated BPV1 trafficking through the ER during infection via an interaction with the tSNARE syntaxin 18. Mutations within the DKILK motif of L2 that did not significantly impact virion morphogenesis or binding at the cell surface prevented the L2 interaction with syntaxin 18 and disrupted BPV1 infection.     

Blocking fatty acids' mystery tour: a therapy for metabolic syndrome?

Elevated circulating fatty acids are associated with impaired insulin action and inflammation. During intracellular transit, fatty acids use fatty acid-binding proteins (FABPs) as shuttles. A recent study (Furuhashi et al., 2007) explores inhibiting FABP4/aP2 as a strategy for treating atherosclerosis and type 2 diabetes.     

Divergence across diet,time and populations rules out parallel evolution in the gut microbiomes of Trinidadian guppies

Diverse microbial consortia profoundly influence animal biology, necessitating an understanding of microbiome variation in studies of animal adaptation. Yet, little is known about such variability among fish, in spite of their importance in aquatic ecosystems. The Trinidadian guppy, Poecilia reticulata, is an intriguing candidate to test microbiome-related hypotheses on the drivers and consequences of animal adaptation, given the recent parallel origins of a similar ecotype across streams. To assess the relationships between the microbiome and host adaptation, we used 16S rRNA amplicon sequencing to characterize gut bacteria of two guppy ecotypes with known divergence in diet, life history, physiology and morphology collected from low-predation (LP) and high-predation (HP) habitats in four Trinidadian streams. Guts were populated by several recurring, core bacteria that are related to other fish associates and rarely detected in the environment. Although gut communities of lab-reared guppies differed from those in the wild, microbiome divergence between ecotypes from the same stream was evident under identical rearing conditions, suggesting host genetic divergence can affect associations with gut bacteria. In the field, gut communities varied over time, across streams and between ecotypes in a stream-specific manner. This latter finding, along with PICRUSt predictions of metagenome function, argues against strong parallelism of the gut microbiome in association with LP ecotype evolution. Thus, bacteria cannot be invoked in facilitating the heightened reliance of LP guppies on lower-quality diets. We argue that the macroevolutionary microbiome convergence seen across animals with similar diets may be a signature of secondary microbial shifts arising some time after host-driven adaptation.     

Microbial Iron Redox Cycling in a Circumneutral-pH Groundwater Seep

The potential for microbially mediated redox cycling of iron (Fe) in a circumneutral-pH groundwater seep in north central Alabama was studied. Incubation of freshly collected seep material under anoxic conditions with acetate-lactate or H₂ as an electron donor revealed the potential for rapid Fe(III) oxide reduction (ca. 700 to 2,000 μmol liter⁻¹ day⁻¹). Fe(III) reduction at lower but significant rates took place in unamended controls (ca. 300 μmol liter⁻¹ day⁻¹). Culture-based enumerations (most probable numbers [MPNs]) revealed significant numbers (10² to 10⁶ cells ml⁻¹) of organic carbon- and H₂-oxidizing dissimilatory Fe(III)-reducing microorganisms. Three isolates with the ability to reduce Fe(III) oxides by dissimilatory or fermentative metabolism were obtained (Geobacter sp. strain IST-3, Shewanella sp. strain IST-21, and Bacillus sp. strain IST-38). MPN analysis also revealed the presence of microaerophilic Fe(II)-oxidizing microorganisms (10³ to 10⁵ cells ml⁻¹). A 16S rRNA gene library from the iron seep was dominated by representatives of the Betaproteobacteria including Gallionella, Leptothrix, and Comamonas species. Aerobic Fe(II)-oxidizing Comamonas sp. strain IST-3 was isolated. The 16S rRNA gene sequence of this organism is 100% similar to the type strain of the betaproteobacterium Comamonas testosteroni ({"type":"entrez-nucleotide","attrs":{"text":"M11224","term_id":"151536","term_text":"M11224"}}M11224). Testing of the type strain showed no Fe(II) oxidation. Collectively our results suggest that active microbial Fe redox cycling occurred within this habitat and support previous conceptual models for how microbial Fe oxidation and reduction can be coupled in surface and subsurface sedimentary environments.Changes in iron (Fe) redox state are linked to carbon and energy flow as well as the behavior of various inorganic compounds in modern soils and sediments. Microorganisms play a pivotal role in the Fe redox cycle in such environments (29, 35, 39). A growing body of literature indicates that aerobic lithotrophic Fe(II)-oxidizing bacteria (FeOB) can contribute significantly to circumneutral-pH Fe(II) oxidation (4, 9, 15, 23, 25, 34) and that microbial catalysis can dominate Fe(II) oxidation in diffusion-limited reaction systems (32, 34). Microbial catalysis is strictly required for anaerobic nitrate-dependent Fe(II) oxidation (36), since an abiotic reaction between Fe(II) and nitrate does not take place under typical near-surface conditions (40).Circumneutral-pH Fe(II) oxidation produces Fe(III) oxide mineral phases which can function as electron acceptors for anaerobic respiration by dissimilatory Fe(III)-reducing bacteria (FeRB) (8, 37). This metabolism is widespread among prokaryotic taxa (19) and plays a key role in oxidation of natural organic compounds and in the bioremediation of organic and metal contaminants in the subsurface (18). The coupling of Fe(III) oxide reduction to oxidation of organic carbon or H₂ leads to release of Fe(II) into the aqueous phase. When the oxidative and reductive parts of the Fe redox cycle come together with ongoing input of energy, a self-sustaining microbial community based on Fe redox cycling may develop. Sustained microbial Fe redox cycling has been proposed in various redox interfacial environments like groundwater Fe seeps (8), plant roots (10), the sediment-water interface in circumneutral-pH (29, 33) and acidic (24) aquatic ecosystems, and hot springs and hydrothermal vents (16a, 24a).Here we present data that support the existence of a sustained microbial Fe redox cycle in a circumneutral-pH groundwater Fe seep in north central Alabama. Potential microbial involvement in Fe redox cycling was assessed by most probable number (MPN) enumerations, in vitro Fe(III) reduction experiments, and isolation of representative Fe(III)-reducing and Fe(II)-oxidizing microorganisms. A simple kinetic model was used to explore the impact that decay of dead chemolithotrophic biomass coupled to Fe(III) reduction could have on rates of Fe turnover.     

Phosphoenolpyruvate Cycling via Mitochondrial Phosphoenolpyruvate Carboxykinase Links Anaplerosis and Mitochondrial GTP with Insulin Secretion

Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K_ATP-dependent glucose-stimulated insulin secretion (GSIS), there are important K_ATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel ¹³C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.β-Cells in pancreatic islets of Langerhans make and release insulin in response to changes in blood glucose levels. The mechanisms by which high concentrations of glucose stimulate insulin release from islets remain unclear. The canonical explanation for GSIS² is that glucose metabolism increases mitochondrial ATP production, thereby raising the cytosolic ATP:ADP ratio that triggers the closure of ATP-sensitive K⁺ channels. This, in turn, depolarizes the membrane and stimulates the opening of voltage-dependent Ca²⁺ channels with increased Ca²⁺ influx promoting the exocytosis of insulin. Although K_ATP channels certainly have an important role in β-cells, K_ATP-independent signals are implicated to play a fundamental role in GSIS. In particular, β-cells are known to have notably elevated rates of anaplerotic flux of the carbon from glucose into the mitochondria and back out to pyruvate (pyruvate cycling) that is tightly correlated with insulin secretion (1–4).Recently, mtGTP synthesis was identified as a novel K_ATP-independent mitochondrial signal for insulin secretion (5). mtGTP is synthesized as a product of glucose metabolism by the GTP-specific isoform of the matrix enzyme SCS. mtGTP synthetic rates are determined by the rate of TCA cycle flux as well as by the ratio of activities of the ATP-specific and GTP-specific isoforms of SCS. The mtGTP signal is trapped within the matrix of the mitochondria, suggesting that another GTPase in the matrix transmits the mtGTP signal to the cytosol. Because both mtGTP synthesis and anaplerotic flux correlate with insulin secretion, we investigated whether the GTP-dependent mitochondrial isoform of PEPCK, an enzyme that lies at the intersection of anaplerosis and mtGTP metabolism (see Fig. 1A), is important for GSIS.Open in a separate windowFIGURE 1.PEP cycle. PEP is produced during glycolysis and is further metabolized to pyruvate by PK. Pyruvate that enters the TCA cycle by pyruvate dehydrogenase will generate GTP via direct synthesis by SCS-GTP. Anaplerotic pyruvate entry by PC will generate oxaloacetate. PEPCK-M will then consume oxaloacetate and GTP to produce PEP, GDP, and CO₂. PEP is then transported out of the mitochondrial matrix by an anion transporter (Ex) in exchange for another metabolite depending on the transporter. Mitochondrial PEP, thus, contributes to the PEP pool that is determined by the rate of appearance (ν_PEPCK-M+1+ ν_Glyc+1) of PEP minus the rate of disappearance (ν_PK+1). One turn of the PEP cycle will result in the net exchange of one ion into the mitochondrial matrix. Unidirectional fluxes are indicated by ν followed by the enzyme with the forward direction being +1 and the reverse −1. GDP in turn can be reused by SCS-GTP. PDH, pyruvate dehydrogenase.     

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen''s type III secretion system. Xcv ΔxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, α-helical repeats, suggesting multiple protein–protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv ΔxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv–tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.