Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro

Mature uteroferrin (Uf; M = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a M of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man_6-4GlcNac₂ oligosaccharides found on mature Uf.Abbreviations Uf Uteroferrin - ConA Concanavalin A - WGA Wheat Germ Agglutinin - endoH endo--N-acetylglucosaminidase H - SDS Sodium Dodecyl Sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues

Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.     

Ploidy is an important determinant of fluoroquinolone persister survival

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Identification of a novel mechanism for the removal of glucose residues from high mannose-type oligosaccharides.

The role of glucosylated oligosaccharides in the biogenesis of the glycoprotein (G protein) of vesicular stomatitis virus was studied in PhaR2.7, a mouse lymphoma cell line deficient in glucosidase II activity. As expected, the great majority of cell-associated G protein remained glucosylated in PhaR2.7, and the G protein was rapidly deglucosylated in BW5147, the parental cell line. Despite these differences in glucosylation, the rates of G protein trimerization and transport to the cell surface were as rapid and efficient in the PhaR2.7 mutant as in BW5147. Surprisingly, greater than 73% of the oligosaccharides on G proteins recovered from released virions were complex-type units. The efficient processing of the G protein oligosaccharides coincided with the efficient removal of glucose residues from its oligosaccharides. After treatment with deoxynojirimycin, an inhibitor of endoplasmic reticulum (ER) glucosidases I and II, the total percentage of G protein-associated high mannose-type oligosaccharides increased more in the parental cells than in the mutant cells. Furthermore, when the G protein was retained in the ER of PhaR2.7 cells by depletion of the cellular ATP pools with carbonyl cyanide m-chlorophenylhydrazone, its oligosaccharides remained glucosylated. Under identical conditions, BW5147 cells removed the glucose residues from > 90% of the retained G protein's oligosaccharides. Thus, PhaR2.7 cells efficiently remove glucose residues from high mannose-type oligosaccharides of selected proteins using a deoxynojirimycin-insensitive enzyme located in a post-ER compartment. The existence of a second mechanism for the deglucosylation of N-linked oligosaccharides provides evidence for the important role of glucose removal in glycoprotein maturation.     

Differential and cell-type specific microheterogeneity of high mannose-type Asn-linked oligosaccharides of human transferrin receptor.

The structural analysis of high mannose-type Asn-linked (N-linked) oligosaccharides of the human transferrin receptor (hTR) from D-[2-3H]mannose metabolic-radiolabeled human cells--A431, K562, BeWo, and HL60--was investigated. The radiolabeled hTR glycopeptides were prepared and fractionated by a lectin chromatography of Concanavalin A-Sepharose. The composition analysis of hTR glycopeptides revealed that Con A-I contains both mannose and fucose, whereas Con A-III has mannose exclusively. The Con A-III glycopeptides were treated with Endo H. The released oligosaccharides were charge-fractionated by QAE-Sephadex. The neutral oligosaccharides were further size-fractionated by an amine absorption high performance liquid chromatography (HPLC). Our results demonstrate that the high mannose-type oligosaccharides of hTR ranged in size from Man5-R to Man9-R with cell-type specific patterns. A relative amount of each component was found to be differentially heterogeneous among the four different human cell lines.     

Biomass allocation is an important determinant of the tannin concentration in growing plants

BACKGROUND AND AIMS: Condensed tannins (CTs) in the diet affect consumers in a concentration-dependent manner. Because of their importance in plant defence against herbivores and pathogens as well as their potential application against gastrointestinal parasites of ruminants in agronomy, an understanding of the seasonal dynamics of CT concentrations during plant growth is essential. METHODS: Over a vegetation period, CT concentrations in leaves, stems and roots and the biomass proportions between these organs were investigated in Onobrychis viciifolia, Lotus corniculatus and Cichorium intybus. Based on the experimental data, a model has been suggested to predict CT concentrations in harvestable biomass of these species. KEY RESULTS: During the experiment, leaf mass fractions of plants decreased from 85, 64, 85 to 30, 18, 39 % d. wt in Onobrychis, Lotus and Cichorium, respectively, and proportions of stems and roots increased accordingly. While CT concentrations almost doubled in leaves in Onobrychis (from 52 to 86 mg g(-1) d. wt, P<0.001) and Lotus (from 25 to 54 mg g(-1) d. wt, P<0.001), they were stable at low levels in expanding leaves of Cichorium (5 mg g(-1) d. wt) and in stems and roots of all investigated species. Due to an inverse effect of the increasing CT concentrations in leaves and simultaneous dilution from increasing proportions of 'CT-poor' stems, CT concentrations in harvestable biomass were stable over time in all investigated species: 62, 26 and 5 mg g(-1) d. wt for Onobrychis, Lotus and Cichorium, respectively. CONCLUSIONS: As a consequence of the unequal distribution of tannins in different plant parts and due to the changing biomass proportions between them, various herbivores (e.g. a leaf-eating insect and a grazing ruminant) may find not only different concentrations of CT in their diets but also different CT dynamics during the season. For the prediction of seasonal variations of CT concentrations, biomass allocation and accumulation of none-CT plant material are likely to be as important predictors as the knowledge of CT synthesis and its regulation.     

Mannose 6-sulfate is present in the N-linked oligosaccharides of lysosomal enzymes of Dictyostelium

The major N-linked, anionic oligosaccharide found on several lysosomal enzymes of Dictyostelium discoideum contains five charges, composed of three sulfate esters and two residues of Man-6-P in phosphodiester linkage. Most of the SO4 was found as Man-6-SO4. This novel sulfated sugar was detected and quantitated by measuring the appearance of 3,6-anhydromannitol following acid hydrolysis and reduction of base-treated, reduced oligosaccharides. If SO4 is removed by solvolysis prior to the base treatment, the anhydrosugar is not formed, indicating that its presence is not an artifact of the procedure. That these oligosaccharides are derived from standard high-mannose-type oligosaccharides indicates that only one or, at most, two Man residues are unsubstituted at the 6-position.     

Genetic interaction with temperature is an important determinant of nematode longevity

As in other poikilotherms, longevity in C. elegans varies inversely with temperature; worms are longer‐lived at lower temperatures. While this observation may seem intuitive based on thermodynamics, the molecular and genetic basis for this phenomenon is not well understood. Several recent reports have argued that lifespan changes across temperatures are genetically controlled by temperature‐specific gene regulation. Here, we provide data that both corroborate those studies and suggest that temperature‐specific longevity is more the rule than the exception. By measuring the lifespans of worms with single modifications reported to be important for longevity at 15, 20, or 25 °C, we find that the effect of each modification on lifespan is highly dependent on temperature. Our results suggest that genetics play a major role in temperature‐associated longevity and are consistent with the hypothesis that while aging in C. elegans is slowed by decreasing temperature, the major cause(s) of death may also be modified, leading to different genes and pathways becoming more or less important at different temperatures. These differential mechanisms of age‐related death are not unlike what is observed in humans, where environmental conditions lead to development of different diseases of aging.     

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure

Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP‐dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

Norovirus RNA-dependent RNA polymerase is phosphorylated by an important survival kinase, Akt

Viruses commonly use host cell survival mechanisms to their own advantage. We show that Akt, an important signaling kinase involved in cell survival, phosphorylates the RNA-dependent RNA polymerase (RdRp) from norovirus, the major cause of gastroenteritis outbreaks worldwide. The Akt phosphorylation of RdRp appears to be a feature unique to the more prevalent norovirus genotypes such as GII.4 and GII.b. This phosphorylation event occurs at a residue (Thr33) located at the interface where the RdRp finger and thumb domains interact and decreases de novo activity of the polymerase. This finding provides fresh insights into virus-host cell interactions.     

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D is an important determinant of uterine anandamide levels during implantation

Implantation requires reciprocal interaction between blastocysts and a receptive uterus. In mice, one important player in this dialogue involves endocannabinoid signaling via cannabinoid receptor CB1. Anandamide is an endogenous cannabinoid ligand, and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing lower levels in the receptive uterus and at the implantation site. However, the mechanism by which differential uterine anandamide gradients are established under different pregnancy status is not clearly understood. Using multiple approaches, we show here that uterine anandamide levels conducive to implantation are primarily regulated by spatiotemporal expression of Nape-Pld, the gene encoding N-acylphosphatidylethanolamine-hydrolyzing phospholipase D that generates anandamide. The expression is well correlated with its activity and anandamide levels. This study is clinically relevant, since elevated anandamide levels in peripheral circulation are associated with spontaneous pregnancy failure in women.     

Morningness orientation is an important determinant to circadian misalignment and tolerance: an Asian perspective

ABSTRACT

Diurnal preferences refers to one’s preference of performing timely activities where one may prefer for late timings and the other for earlier, which account for their chronotype that is controlled partly by genetic factors and are also influenced by environmental factors. Individual’s circadian preference can be drawn using questionnaire tools as well as can be externally validated by studying various physiological parameters that has a rhythmicity. Determination of chronotype is well studied worldwide but mostly in European and American countries. Asia being the largest continent, comprising so many countries still did not have widespread studies over chronotype assessment and its implications. This review aims at jotting down the available literature regarding the chronotype evaluation and the various contributing factors in Asian perspectives such that there can be huge cross-cultural studies emphasizing environmental, geographical differences, lifestyle habits, work schedule, and other contributing factors regarding the understanding of circadian preference of humans, which is really hard to define. The dearth in studies regarding diurnal preferences and physiological altercations, which is in turn influenced by the work schedule and the resultant sleep-wake pattern disorientation, needs further investigation worldwide and this available literature may put some light in the path of future research scope.     

Gravity is an important but secondary determinant of regional pulmonary blood flow in upright primates

Original studies leading to the gravitationalmodel of pulmonary blood flow and contemporary studies showinggravity-independent perfusion differ in the recent use of laboratoryanimals instead of humans. We explored the distribution ofpulmonary blood flow in baboons because their anatomy, serialdistribution of vascular resistances, and hemodynamic responses tohypoxia are similar to those of humans. Four baboons wereanesthetized with ketamine, intubated, and mechanically ventilated.Different colors of fluorescent microspheres were given intravenouslywhile the animals were in the supine, prone, upright (repeated), andhead-down (repeated) postures. The animals were killed, and their lungswere excised, dried, and diced into~2-cm³ pieces with the spatialcoordinates recorded for each piece. Regional blood flow was determinedfor each posture from the fluorescent signals of each piece. Perfusionheterogeneity was greatest in the upright posture and least when prone.Using multiple-stepwise regression, we estimate that 7, 5, and 25% ofperfusion heterogeneity is due to gravity in the supine, prone, andupright postures, respectively. Although important, gravity is not thepredominant determinant of pulmonary perfusion heterogeneity in uprightprimates. Because of anatomic similarities, the same may be true for humans.     

Age is an important determinant of the growth hormone response to sprint exercise in non-obese young men

BACKGROUND: The factors that regulate the growth hormone (GH) response to physiological stimuli, such as exercise, are not fully understood. The aim of the present study is to determine whether age, body composition, measures of sprint performance or the metabolic response to a sprint are predictors of the GH response to sprint exercise in non-obese young men. METHODS: Twenty-seven healthy, non-obese males aged 18-32 years performed an all-out 30-second sprint on a cycle ergometer. Univariate linear regression analysis was employed to evaluate age-, BMI-, performance- and metabolic-dependent changes from pre-exercise to peak GH and integrated GH for 60 min after the sprint. RESULTS: GH was elevated following the sprint (change in GH: 17.0 +/- 14.2 microg l(-1); integrated GH: 662 +/- 582 min microg l(-1)). Performance characteristics, the metabolic response to exercise and BMI were not significant predictors of the GH response to exercise. However, age emerged as a significant predictor of both integrated GH (beta = -0.547, p = 0.003) and change in GH (beta = -0.448, p = 0.019) after the sprint. CONCLUSION: In non-obese young men, age is a more important predictor of GH following sprint exercise than BMI, sprint performance or the metabolic response to sprint exercise.     

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.