Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution.     

Gα<Subscript>16</Subscript> interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Background  

G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα₁₆ as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).     

Conformational changes induced by binding UDP-2F-galactose to alpha-1,3 galactosyltransferase- implications for catalysis

Alpha-1,3 galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to beta-linked galactosides with retention of its alpha configuration. Although several complexes of alpha3GT with inhibitors and substrates have been reported, no structure has been determined of a complex containing intact UDP-galactose. We describe the structure of a complex containing an inhibitory analogue of UDP-galactose, UDP-2F-galactose, in a complex with the Arg365Lys mutant of alpha3GT. The inhibitor is bound in a distorted, bent configuration and comparison with the structure of the apo form of this mutant shows that the interaction induces structural changes in the enzyme, implying a role for ground state destabilization in catalysis. In addition to a general reduction in flexibility in the enzyme indicated by a large reduction in crystallographic B-factors, two loops, one centred around Trp195 and one encompassing the C-terminal 11 residues undergo large structural changes in complexes with UDP and UDP derivatives. The distorted configuration of the bound UDP-2F-galactose in its complex is stabilized, in part, by interactions with residues that are part of or near the flexible loops. Mutagenesis and truncation studies indicate that two highly conserved basic amino acid residues in the C-terminal region, Lys359 and Arg365 are important for catalysis, probably reflecting their roles in these ligand-mediated conformational changes. A second Mn(2+) cofactor has been identified in the catalytic site of a complex of the Arg365Lys with UDP, in a location that suggests it could play a role in facilitating UDP release, consistent with kinetic studies that show alpha3GT activity depends on the binding of two manganese ions. Conformational changes in the C-terminal 11 residues require an initial reorganization of the Trp195 loop and are linked to enzyme progress through the catalytic cycle, including donor substrate distortion, cleavage of the UDP-galactose bond, galactose transfer, and UDP release.     

Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines

A newly isolated fungus Penicillium verruculosum SG was evaluated for the production and characterization of bioactive colored secondary metabolites using solid-state fermentation along with their cytotoxic activities against normal and cancer cell lines. Logical fragmentation pattern following column chromatography, thin layer chromatography and liquid chromatography and mass spectrometry of crude culture filtrate of fungus revealed the presence of different polyketide pigments and other bioactive compounds. Cytotoxicity of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (μg/ml) values ranging from 5 to 100. It was significantly higher in case of orevactaene (5 + 0.44) and monascorubrine followed by pyripyropene (8 + 0.63) against cancer cell line KA3IT. Overall, these compounds considerably showed less toxicity toward normal cell lines NIH3T3, HSCT6, HEK293 and MDCK. XRD of a yellow crystalline compound (224.21 m/z) confirmed its 3-dimensional structure as phenazine 1 carboxylic acid (C13H8N2O2) (broad spectrum antibiotic), and it is first time reported in fungi.     

Meningoencephalitis Due to Listeria monocytogenes in a Pregnant Rhesus Macaque (Macaca mulatta)

We here report a spontaneous case of meningoencephalitis due to Listeria monocytogenes in an adult primiparous rhesus macaque (Macaca mulatta) during an outbreak of listeriosis in an outdoor enclosure. Clinical signs included tremors, abnormal posture, and altered mental status. Hematology and analyses of cerebrospinal fluid were consistent with bacterial infection. Pure cultures of L. monocytogenes were recovered from the placenta–abortus, cerebrospinal fluid, and brain tissue. The macaque did not respond to treatment and was euthanized. Histopathologic examination of the brain revealed acute meningoencephalitis. This case represents an unusual clinical and pathologic presentation of listeriosis in a nonhuman primate in which the dam and fetus both were affected.Listeria monocytogenes is a ubiquitous, facultative anaerobic, intracellular gram-positive coccobacillus. This bacterium is found in diverse environments including (but not limited to) soil, water, plant matter, food items, and the intestinal tract of mammalian hosts.^15,18 The organism is environmentally resistant, being able to survive in dried media for several months and in moist soil for up to a year.¹⁵ L. monocytogenes is the causative agent of listeriosis, a bacterial infection that has a worldwide distribution and affects a wide range of mammals and birds, including human beings.In people, L. monocytogenes is a relatively uncommon foodborne pathogen; its abilities to survive food processing and grow in cold conditions allow it to persist in appropriately stored or refrigerated foods.² In people, listeriosis occurs both sporadically and as large outbreaks,¹⁸ generally comprising 3 separate syndromes with clinical manifestations ranging from mild to life-threatening.³⁵ The most common form is seen in immunocompetent, nonpregnant adults as a febrile gastroenteritis.^2,18,21 The other 2 forms, which occur in fetuses and immunocompromised patients, are more severe.^19,21 In pregnant women, maternal listeriosis is asymptomatic or causes mild, flu-like symptoms, but the bacterium''s ability to cross the placenta and the blood–brain barrier of the fetus results in neonatal septicemia, meningitis, abortion, and stillbirth.¹⁶ In elderly and immunocompromised patients, septicemia and meningoencephalitis are life-threatening manifestations of literiosis.²⁶ The worldwide case fatality rate varies widely among countries, sometimes exceeding 50% despite what is considered to be appropriate antibiotic therapy.¹⁸ In 2009, the Centers for Disease Control reported 524 cases of listeriosis in the United States, which were associated with a 19% resulting in death.⁴In ruminants, listeriosis is also known as ‘circling disease’ and ‘silage disease.’^8,18,21 Foodborne infection with L. monocytogenes is well described, and many studies have shown that spoiled silage may be a source of listeria outbreaks.^8,18 Rhombencephalitis and diffuse meningoencephalitis are the most recognized forms of the infection in ruminants; sporadic abortion is reported also.²² Clinical signs of listeria encephalitis in cattle, sheep, and goats are characterized by unilateral or bilateral brainstem dysfunction and cranial nerve deficits. In sheep and goats, the course of the disease is acute, but the disease in cattle has a more chronic progression, with neurologic manifestations that can last 4 to 14 d.^1,22In rabbits, infection with L. monocytogenes is characterized by abortion in pregnant does or sudden death; neurologic signs are rare.¹ In poultry, an acute form with septicemia and sudden death occurs in adults, in contrast to a subacute–chronic form, with encephalitis, in the young.⁶The literature on L. monocytogenes in nonhuman primates is sparse^5,11,17,33 and more recently limited to experimental infection of pregnant animals. In pregnant rhesus macaques (Macaca mulatta), experimental infection during the last trimester of gestation can cause stillbirth with no other clinical signs.^23,24 In our colony, however, infection with L. monocytogenes is endemic. Every year, several spontaneous abortions or stillbirths in our outdoor colony are caused by infection of the dam with this organism. Culture of L. monocytogenes from both the abortus–fetus and placenta are well documented. As described in the literature,^23,24 the dams in our colony do not demonstrate any clinical signs prior to the abortion or stillbirth.During the winter to spring of 2011, one of our outdoor housing enclosures experienced an outbreak of listeriosis. This outside corral housed 100 rhesus macaques in a social group that included 42 reproductive females. Of these reproductive females, 37 (88%) were confirmed pregnant by abdominal palpation or ultrasonography or both. From January 2011 to May 2011, 19 (51%) stillbirths and neonatal deaths (in infants younger than 3 d) were reported in this enclosure; 13 (68%) of these tissues (placenta, 3; fetal lungs, 8; fetal peritoneum, 2) were culture-positive for L. monocytogenes. In all cases except the one presented here, the dam did not manifest any clinical signs prior to or after the delivery of a stillborn or premature birth with neonatal death.Here we describe an unusual case of listeriosis in a primiparous pregnant female rhesus macaque that manifested severe neurologic impairment and intrautero death of the fetus.     

Screening a limited structure-based library identifies UDP-GalNAc-specific mutants of alpha-1,3-galactosyltransferase

Complex glycans have important roles in biological recognition processes and considerable pharmaceutical potential. The synthesis of novel glycans can be facilitated by engineering glycosyltransferases to modify their substrate specificities. The choice of sites to modify requires the knowledge of the structures of enzyme-substrate complexes while the complexity of protein structures necessitates the exploration of a large array of multisite mutations. The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), which catalyzes the synthesis of the alpha-Gal epitope, has strict specificity for UDP-galactose as a donor substrate. Based on the structure of a complex of UDP-galactose with alpha3GT, the specificity for the galactose moiety can be partly attributed to residues that interact with the galactose 2-OH group, particularly His280 and Ala282. With the goal of engineering a variant of bovine alpha3GT with GalNAc transferase activity, we constructed a limited library of 456 alpha3GT mutants containing 19 alternative amino acids at position 280, two each at 281 and 282 and six at position 283. Clones (1500) were screened by assaying partially purified bacterially expressed variants for GalNAc transferase activity. Mutants with the highest levels of GalNAc transferase activity, AGGL or GGGL, had substitutions at all four sites. The AGGL mutant had slightly superior GalNAc transferase activity amounting to about 3% of the activity of the wild-type enzyme with UDP-Gal. This mutant had a low activity with UDP-Gal; its crystallographic structure suggests that the smaller side chains at residues 280-282 form a pocket to accommodate the larger acetamido group of GalNAc. Mutational studies indicate that Leu283 is important for stability in this mutant.     

Regional pattern of cell maturation and progesterone biosynthesis in the avian granulosa cell layer

The objective of the present study was to compare the structural and functional features of cells derived from histologically different regions of the granulosa cell layer of hen preovulatory follicles. Granulosa cells were isolated from a 0.8-1.5-cm diameter region of the granulosa layer overlying the germinal disc (GD) or from the remainder of the granulosa layer peripheral to the disc region (GP). In the first study, the isolated cells were prepared from each region of the five largest preovulatory follicles; fixed; stained with fluorescent dyes for DNA, total protein, and RNA; and analyzed by use of multiparameter flow cytometry. A greater percentage of cells from the GD region than from the GP region were in proliferative (S and G2/M) stages of the cell cycle in the four largest follicles. In addition, GD cells had lower relative protein content than GP cells in the two largest follicles. In the second study, progesterone biosynthesis in response to treatment with luteinizing hormone (LH) or forskolin was examined in granulosa cells from the GD and the GP regions of the largest preovulatory follicles. GP cells had greater responsiveness to the treatments than GD cells. In addition, conversion of 25-hydroxy-cholesterol to progesterone was greater in GP cells than in GD cells. There were no differences in cyclic adenosine 3',5'-monophosphate (cAMP) production by GD and GP cells in response to LH or forskolin or in the ability of cells from each region to convert pregnenolone substrate to progesterone via 3 beta-hydroxysteroid dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)