Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

MPD and DNA Bending in Crystals and in Solution

Bending of 15 to 24° is observed within crystal structures ofB-DNA duplexes, is strongly sequence-dependent, and exhibits no correlation with the concentration of MPD (2-methyl-2,4-pentanediol) in the crystallizing solution. Two types of bends are observed: facultative bends or flexible hinges at junctions between regions of G·C and A·T base-pairs, and a persistent and almost obligatory bend at the center of the sequence R-G-C-Y. Only A-tracts are characteristically straight and unbent in every crystal structure examined to date. A detailed examination of normal vector plots for individual strands of a double helix provides an explanation, in terms of the stacking properties of guanine and adenine bases. The effect of high MPD concentrations, in both solution and crystal, is to decrease local bending somewhat without removing it altogether. MPD gel retardation experiments provide no basis for choosing among the three models that seek to explain macroscopic curvature of DNA by means of microscopic bending: junction bending, bent A-tracts, or bent general- sequence DNA. Crystallographic data on the straightness of A-tracts, the bendability of non-A sequences, and the identity of inclination angles in A-tract and non-A-tractB-DNA support only the general-sequence bending model. The pre-melting transition observed in A-tract DNA probably represents a relaxation of stiff adenine stacks to a flexible conformation more typical of general-sequence DNA.     

Update in research and methods in proteomics and bioinformatics

The 3rd International Conference on Proteomics & Bioinformatics (Proteomics 2013)

Philadelphia, PA, USA, 15–17 July 2013

The Third International Conference on Proteomics & Bioinformatics (Proteomics 2013) was sponsored by the OMICS group and was organized in order to strengthen the future of proteomics science by bringing together professionals, researchers and scholars from leading universities across the globe. The main topics of this conference included the integration of novel platforms in data analysis, the use of a systems biology approach, different novel mass spectrometry platforms and biomarker discovery methods. The conference was divided into proteomic methods and research interests. Among these two categories, interactions between methods in proteomics and bioinformatics, as well as other research methodologies, were discussed. Exceptional topics from the keynote forum, oral presentations and the poster session have been highlighted. The topics range from new techniques for analyzing proteomics data, to new models designed to help better understand genetic variations to the differences in the salivary proteomes of HIV-infected patients.     

Deoxynivalenol and nivalenol in wheat and by-products in Argentina

The deoxynivalenol and nivalenol contamination in wheat and by-products obtained through milling was analized by Trucksess method slightly modified in the proportion of acetonitrile—water (3:1). Only one sample of wheat showed deoxynivalenol contamination, 1,200μg/kg. No samples obtained in different stages of the milling were contaminated with deoxynivalenol or nivalenol. In the commercial wheat flours the levels found ranged between 400 and 800μg/kg, as follows: 400μ/kg, 5 samples; 800jug/kg, 1 sample.     

Obestatin and ghrelin in obese and in pregnant women

We identified, through qPCR, receptor mRNA for a number of gut peptides in female human omental fat: the incretins, GIP and GLP-1, the orexigenic peptides PYY-Y1 and -Y2 and ghrelin, and the anorexigenic peptide obestatin. Four cohorts of women were examined: lean controls (BMI<23), obese (BMI>41), obese diabetic and term pregnant women. Human fat expressed receptor mRNAs for all six peptides. Pregnant women expressed roughly three times as much orphan GPR-39 receptor, a proposed obestatin receptor, than other women and less than half as much of the ghrelin receptor (GHSR-1a). An immunoblot probed with a GPR-39 selective antibody yielded a single band corresponding to the correct molecular weight (52 kDa) for the proposed obestatin receptor. Fluorescent immunohistochemistry of human fat employing the same antibody indicated the receptor protein was localized to the adipocyte cell membrane. The concentration of obestatin circulating in blood was measured in the same cohort of women and was significantly lower in obese and obese diabetic women compared to control.     

Hydroxycinnamates and their in vitro and in vivo antioxidant activities

Hydroxycinnamates are among the most widely distributed plant phenylpropanoids present in the free, conjugated-soluble and insoluble-bound forms. This review will focus on the occurrence, in vitro and in vivo antioxidant activities of ferulic, coumaric, caffeic and sinapic acids and their derivatives. Hydroxycinnamates are found in almost all food groups though they are abundant in cereals, legumes, oilseeds, fruits, vegetables and beverages and render antioxidant activity by scavenging hydroxyl radical, superoxide radical anion, several organic radicals, peroxyl radical, peroxinitrite and singlet oxygen, among others. Further, their antioxidant activity as chain breaking antioxidants and reducing agents is also notable. Ferulic acid and its derivatives such as ferulic acid ethyl ester, ferulic acid dehydrodimers, feruloyl glycosides and curcumin have demonstrated potent antioxidant activity in both in vitro and in vivo systems. Similarly, caffeic acid and some of its derivatives such as caffeic acid phenethyl ester, rosmarinic acid, and chlorogenic acid exhibit antioxidant activity. The highest antioxidant activity was observed for caffeic acid whereas p-coumaric acid had the least effect among major hydroxycinnamic acids. The importance of structural effects on the potency of antioxidant activity of hydroxycinnamates is discussed. While this review also shows the existence of substantial body of evidences for in vitro antioxidant activity of hydroxycinnamates, there is a clear gap for in vivo information, particularly for sinapic and p-coumaric acids and their derivatives. The role of grains, fruits, vegetables and red wine in disease risk reduction and health promotion could partly be attributed to their constituent hydroxycinnamates.     

Variation in infectivity and aggressiveness in space and time in wild host-pathogen systems: causes and consequences

Variation in host resistance and in the ability of pathogens to infect and grow (i.e. pathogenicity) is important as it provides the raw material for antagonistic (co)evolution and therefore underlies risks of disease spread, disease evolution and host shifts. Moreover, the distribution of this variation in space and time may inform us about the mode of coevolutionary selection (arms race vs. fluctuating selection dynamics) and the relative roles of G × G interactions, gene flow, selection and genetic drift in shaping coevolutionary processes. Although variation in host resistance has recently been reviewed, little is known about overall patterns in the frequency and scale of variation in pathogenicity, particularly in natural systems. Using 48 studies from 30 distinct host–pathogen systems, this review demonstrates that variation in pathogenicity is ubiquitous across multiple spatial and temporal scales. Quantitative analysis of a subset of extensively studied plant–pathogen systems shows that the magnitude of within‐population variation in pathogenicity is large relative to among‐population variation and that the distribution of pathogenicity partly mirrors the distribution of host resistance. At least part of the variation in pathogenicity found at a given spatial scale is adaptive, as evidenced by studies that have examined local adaptation at scales ranging from single hosts through metapopulations to entire continents and – to a lesser extent – by comparisons of pathogenicity with neutral genetic variation. Together, these results support coevolutionary selection through fluctuating selection dynamics. We end by outlining several promising directions for future research.     

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.     

Production of arsine and methylarsines in soil and in culture

Arsenate, arsenite, monomethylarsonate, and dimethylarsinate were added to different soils, and evolution of gaseous arsenical products was determined over 3 weeks. Arsine was produced in all three soils from all substrates, whereas methylarsine and dimethylarsine were produced only from methylarsonate and dimethylarsinate, respectively. At least three times more arsine than dimethylarsine was produced in soil incubated with dimethylarsinate. Resting cell suspensions of Pseudomonas and Alcaligenes produced arsine as the sole product when incubated anaerobically in the presence of arsenate or arsenite. In all instances, no trimethylarsine was observed, nor could any evidence be shown for the methylation of any arsenical substrate in soil or in culture. It was concluded that reduction to arsine, not methylation to trimethylarsine, was the primary mechanism for gaseous loss of arsenicals from soil.     

Exercise- and cold-induced changes in plasma beta-endorphin and beta-lipotropin in men and women

The plasma beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) response of men, eumenorrheic women, and amenorrheic women (n = 6) to 1 h of rest or to a bicycle ergometer test [20 min at 30% maximum O2 uptake (VO2max), 20 min at 60% VO2max, and at 90% VO2max to exhaustion] was studied in both normal (22 degrees C) and cold (5 degrees C) environments. beta-EP and beta-LPH was measured by radioimmunoassay in venous samples collected every 20 min during rest or after each exercise bout. Exhaustive exercise at ambient temperature (Ta) 22 degrees C induced significant increases in plasma beta-EP and beta-LPH in all subjects as did work at 60% VO2max in amenorrheic and eumenorrheic women. During work at Ta 5 degrees C, the relative increase in beta-EP and beta-LPH was suppressed in eumenorrheic women and completely prevented in amenorrheic women. Although significant lowering of beta-EP and beta-LPH was observed in men and eumenorrheic women during rest at 5 degrees C, amenorrheic women maintained precold exposure levels. These findings suggest that plasma beta-EP and beta-LPH may reflect a thermoregulatory response to heat load. There appears to be a sexual dimorphism in exercise- and cold-induced release of beta-EP and beta-LPH and amenorrhea may be accompanied by alterations in these responses.     

Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo

Guanine (Gua) modification by nitrating and hydroxylating systems was investigated in DNA. In isolated calf thymus DNA, 8-NO(2)-Gua and 8-oxo-Gua were dose-dependently formed with peroxynitrite, and 8-NO(2)-Gua was released in substantial amounts. Myeloperoxidase (MPO) with H(2)O(2) and NO(2)(-) reacted with calf thymus DNA to form 8-NO(2)-Gua dose dependently without release of 8-NO(2)-Gua. The frequency of strand breaks was higher than the sum of 8-NO(2)-Gua and 8-oxo-Gua, particularly in the MPO-treated DNA, indicating the importance of other types of damage. The activation of human neutrophils and lymphocytes with phorbol ester did not induce 8-NO(2)-Gua and 8-oxo-Gua in their nuclear DNA. However, 8-NO(2)-Gua was found in calf thymus DNA co-incubated with activated neutrophils in the presence of NO(2)(-). No significant formation of 8-NO(2)-Gua was found in liver DNA from mice treated with Escherichia coli lipopolysaccharide. The incubation of peroxynitrite or MPO-H(2)O(2)-NO(2)(-)-treated DNA with formamidopyrimidine glycosylase (Fpg) released 8-oxo-Gua, but not 8-NO(2)-Gua, indicating that 8-NO(2)-Gua is not a substrate for Fpg. Although 8-NO(2)-Gua was generated in isolated DNA by different nitrating systems, other types of damage were formed in abundance, and the lesion could not be found reliably in nuclear DNA, suggesting that the biological importance is limited.     

Actinomycetes and Fungi in Surface Waters and in Potable Water

In Finnish lakes and rivers used as water supplies, mesophilic fungi and actinomycetes were common, whereas thermophilic fungi and actinomycetes were present only in low concentrations. Fungi and actinomycetes were more abundant in eutrophic and mesotrophic lakes than in oligotrophic lakes. River water contained more thermophilic actinomycetes and fungi and mesophilic actinomycetes than did lake water. Runoff from soil seemed to be an important factor contributing to the incidence of these microbes in water. Chemical coagulation removed actinomycetes and fungi efficiently, but sand filtration allowed their passage. Disinfection could not prevent actinomycetes and fungi from reaching the distribution system. During infiltration in the production of recharged groundwater, mesophilic actinomycetes could even multiply appreciably.     

Seasonal and Diel Variability in Dissolved DNA and in Microbial Biomass and Activity in a Subtropical Estuary

Dissolved DNA and microbial biomass and activity parameters were measured over a 15-month period at three stations along a salinity gradient in Tampa Bay, Fla. Dissolved DNA showed seasonal variation, with minimal values in December and January and maximal values in summer months (July and August). This pattern of seasonal variation followed that of particulate DNA and water temperature and did not correlate with bacterioplankton (direct counts and [³H]thymidine incorporation) or phytoplankton (chlorophyll a and ¹⁴CO₂ fixation) biomass and activity. Microautotrophic populations showed maxima in the spring and fall, whereas microheterotrophic activity was greatest in late summer (September). Both autotrophic and heterotrophic microbial activity was greatest at the high estuarine (low salinity) station and lowest at the mouth of the bay (high salinity station), irrespective of season. Dissolved DNA carbon and phosphorus constituted 0.11 ± 0.05% of the dissolved organic carbon and 6.6 ± 6.5% of the dissolved organic phosphorus, respectively. Strong diel periodicity was noted in dissolved DNA and in microbial activity in Bayboro Harbor during the dry season. A noon maximum in primary productivity was followed by an 8 p.m. maximum in heterotrophic activity and a midnight maximum in dissolved DNA. This diel periodicity was less pronounced in the wet season, when microbial parameters were strongly influenced by episodic inputs of freshwater. These results suggest that seasonal and diel production of dissolved DNA is driven by primary production, either through direct DNA release by phytoplankton, or more likely, through growth of bacterioplankton on phytoplankton exudates, followed by excretion and lysis.     

Acetylcholine and oxidative metabolism in septum and hippocampus in vitro

Regulation of acetylcholine metabolism varied in brain slices from hippocampus and septum which have different proportions of cholinergic nerve cell bodies and nerve endings. Anoxia (0% oxygen) inhibited acetylcholine synthesis (-77%) and its calcium-dependent release (-87%) from hippocampal slices but had no effect on synthesis or release by septal slices. [1,5-14C]Citrate incorporation into acetylcholine was higher in septum than in hippocampus, which suggested that citrate metabolism differs regionally. (-)Hydroxycitrate, a specific inhibitor of ATP citrate (pro3S)-lyase (EC 4.1.3.8), reduced [U-14C]glucose incorporation into acetylcholine more in septal than in hippocampal slices. 14CO2 production from glucose or citrate was similar in control and experimental conditions in the two regions. These findings indicate that acetylcholine metabolism varies regionally, which may partially explain the selective vulnerability of certain brain areas to anoxia and other metabolic insults.     

Quantitative changes in in vitro and in vivo protein synthesis in aging and rejuvenated soybean cotyledons

Cotyledons of light-grown soybean (Glycine max L. var Wayne) seedlings were used as a model system to study the possibility that aging requires qualitative changes in protein synthesis. Cotyledons reached a final stage of senescence and then abscised about 22 days after imbibition. Cotyledon senescence was reversed at 20 days after germination by epicotyl removal. Thereafter, the cotyledons regained much of the chlorophyll, RNA, protein, and polyribosomes lost during aging.

Total poly(A)mRNA was extracted from 4-, 12-, 20-day-old, and rejuvenated cotyledons and translated in a wheat germ system. Comparison of translation products on two-dimensional O'Farrell gels showed that many translation products increased in quantity during aging, while roughly half as many decreased. Rejuvenation returned the translation products to approximately 4-day-old levels in roughly half of those products which were diminished with age. Conversely, almost one-third of the products which had increased with age decreased with rejuvenation. None of the translation products were totally lost nor were newly synthesized products detected during aging. Therefore, aging in this system probably does not involve complete gene repression or depression. The observation that epicotyl removal causes a reversal in the levels of various proteins synthesized in vitro was corroborated by similar observations following in vivo labeling of cotyledon sections and analysis by SDS-polyacrylamide gel electrophoresis and fluorography. Densitometric scans of fluorograms revealed a gradual shift in profiles of both in vitro and in vivo translation products during aging. Rejuvenated cotyledon proteins had a profile resembling that of 4-day-old cotyledons. The overall level of [³⁵S]methionine incorporation into protein in vivo declined gradually during aging but was restored to 4-day-old levels within 2 days after epicotyl removal.

     

DNA stress and strain, in silico, in vitro and in vivo

A vast literature has explored the genetic interactions among the cellular components regulating gene expression in many organisms. Early on, in the absence of any biochemical definition, regulatory modules were conceived using the strict formalism of genetics to designate the modifiers of phenotype as either cis- or trans-acting depending on whether the relevant genes were embedded in the same or separate DNA molecules. This formalism distilled gene regulation down to its essence in much the same way that consideration of an ideal gas reveals essential thermodynamic and kinetic principles. Yet just as the anomalous behavior of materials may thwart an engineer who ignores their non-ideal properties, schemes to control and manipulate the genetic and epigenetic programs of cells may falter without a fuller and more quantitative elucidation of the physical and chemical characteristics of DNA and chromatin in vivo.     

Advances in purine and pyrimidine metabolism in health and diseases

ABSTRACT

In June, 2015, the Purine and Pyrimidine Society organized the 16th biennial symposium on Purine and Pyrimidine metabolism at the Faculty House of Columbia University, New York City. This exciting meeting focused on these important molecules, new developments in inborn errors of metabolism; therapeutic analogs. In addition, the biochemistry of mammalian and non-mammalian systems were discussed. Due to significant advances in molecular medicine, the boundaries between clinical and basic sciences have merged into exciting translational research, of which a small portion was highlighted in the presymposium.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics