Dihydromyricetin enhances glucose uptake by inhibition of MEK/ERK pathway and consequent down‐regulation of phosphorylation of PPARγ in 3T3‐L1 cells

Accumulating evidence suggests that inhibition of mitogen‐activated protein kinase signalling can reduce phosphorylation of peroxisome proliferator‐activated receptor γ (PPARγ) at serine 273, which mitigates obesity‐associated insulin resistance and might be a promising treatment for type 2 diabetes. Dihydromyricetin (DHM) is a flavonoid that has many beneficial pharmacological properties. In this study, mouse fibroblast 3T3‐L1 cells were used to investigate whether DHM alleviates insulin resistance by inhibiting PPARγ phosphorylation at serine 273 via the MEK/ERK pathway. 3T3‐L1 pre‐adipocytes were differentiated, and the effects of DHM on adipogenesis and glucose uptake in the resulting adipocytes were examined. DHM was found to dose dependently increase glucose uptake and decrease adipogenesis. Insulin resistance was then induced in adipocytes using dexamethasone, and DHM was shown to dose and time dependently promote glucose uptake in the dexamethasone‐treated adipocytes. DHM also inhibited phosphorylation of PPARγ and ERK. Inhibition of PPARγ activity with GW9662 potently blocked DHM‐induced glucose uptake and adiponectin secretion. Interestingly, DHM showed similar effects to PD98059, an inhibitor of the MEK/ERK pathway. DHM acted synergistically with PD98059 to improve glucose uptake and adiponectin secretion in dexamethasone‐treated adipocytes. In conclusion, our findings indicate that DHM improves glucose uptake in adipocytes by inhibiting ERK‐induced phosphorylation of PPARγ at serine 273.     

Genetic analysis of the gas vesicle gene cluster in haloarchaea

Gas vesicles are buoyant intracellular organelles composed of a rigid proteinaceous membrane surrounding a gas-filled space. Many prokaryotic microorganisms including photosynthetic and heterotrophic bacteria and halophilic and methanogenic archaea produce gas vesicles. In the majority of cases gas vesicles function in providing vertical motility to cells in aquatic environments. Recent genetic analysis of several halophilic archaeal (haloarchaeal) species has shown that a large cluster of genes [gvpMLKJIHGFEDACN(O)] is necessary for gas vesicle formation.     

Prokaryotes in the WAIS Divide ice core reflect source and transport changes between Last Glacial Maximum and the early Holocene

We present the first long‐term, highly resolved prokaryotic cell concentration record obtained from a polar ice core. This record, obtained from the West Antarctic Ice Sheet (WAIS) Divide (WD) ice core, spanned from the Last Glacial Maximum (LGM) to the early Holocene (EH) and showed distinct fluctuations in prokaryotic cell concentration coincident with major climatic states. The time series also revealed a ~1,500‐year periodicity with greater amplitude during the Last Deglaciation (LDG). Higher prokaryotic cell concentration and lower variability occurred during the LGM and EH than during the LDG. A sevenfold decrease in prokaryotic cell concentration coincided with the LGM/LDG transition and the global 19 ka meltwater pulse. Statistical models revealed significant relationships between the prokaryotic cell record and tracers of both marine (sea‐salt sodium [ssNa]) and burning emissions (black carbon [BC]). Collectively, these models, together with visual observations and methanosulfidic acid (MSA) measurements, indicated that the temporal variability in concentration of airborne prokaryotic cells reflected changes in marine/sea‐ice regional environments of the WAIS. Our data revealed that variations in source and transport were the most likely processes producing the significant temporal variations in WD prokaryotic cell concentrations. This record provided strong evidence that airborne prokaryotic cell deposition differed during the LGM, LDG, and EH, and that these changes in cell densities could be explained by different environmental conditions during each of these climatic periods. Our observations provide the first ice‐core time series evidence for a prokaryotic response to long‐term climatic and environmental processes.     

Isolation and identification of siderophores produced by cyanobacteria

Cyanobacteria are one of the most successful and oldest forms of life that are present on Earth. They are prokaryotic photoautotrophic microorganisms that colonize so diverse environments as soil, seawater, and freshwater, but also stones, plants, or extreme habitats such as snow and ice as well as hot springs. This diversity in the type of environment they live in requires a successful adaptation to completely different conditions. For this reason, cyanobacteria form a wide range of different secondary metabolites. In particular, the cyanobacteria living in both freshwater and sea produce many metabolites that have biological activity. In this review, we focus on metabolites called siderophores, which are low molecular weight chemical compounds specifically binding iron ions. They have a relatively low molecular weight and are produced by bacteria and also by fungi. The main role of siderophores is to obtain iron from the environment and to create a soluble complex available to microbial cells. Siderophores play an important role in microbial ecology; for example, in agriculture they support the growth of many plants and increase their production by increasing the availability of Fe in plants. The aim of this review is to demonstrate the modern use of physico-chemical methods for the detection of siderophores in cyanobacteria and the use of these methods for the detection and characterization of the siderophore-producing microorganisms. Using high-performance liquid chromatography-mass spectrometry (LC-MS), it is possible not only to discover new chemical structures but also to identify potential interactions between microorganisms. Based on tandem mass spectrometry (MS/MS) analyses, previous siderophore knowledge can be used to interpret MS/MS data to examine both known and new siderophores.     

NOVEL ICE‐BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE,CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.     

How the Motility Pattern of Bacteria Affects Their Dispersal and Chemotaxis

Most bacteria at certain stages of their life cycle are able to move actively; they can swim in a liquid or crawl on various surfaces. A typical path of the moving cell often resembles the trajectory of a random walk. However, bacteria are capable of modifying their apparently random motion in response to changing environmental conditions. As a result, bacteria can migrate towards the source of nutrients or away from harmful chemicals. Surprisingly, many bacterial species that were studied have several distinct motility patterns, which can be theoretically modeled by a unifying random walk approach. We use this approach to quantify the process of cell dispersal in a homogeneous environment and show how the bacterial drift velocity towards the source of attracting chemicals is affected by the motility pattern of the bacteria. Our results open up the possibility of accessing additional information about the intrinsic response of the cells using macroscopic observations of bacteria moving in inhomogeneous environments.     

Macromolecular synthesis by yeasts under frozen conditions

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8°C min⁻¹), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at −5°C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [³H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15°C and −15°C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at −5°C under frozen and liquid conditions revealed 2–3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of ≈ 1.4 mol l⁻¹ of NaCl at −5°C). The mesophile Saccharomyces cerevisae also incorporated [³H]leucine at −5°C and −15°C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.     

Ca(2+)-dependent regulation of the motor activity of myosin V

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.     

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA content. This study represents an attempt to improve detection of marine prokaryotes by increasing cellular rRNA content without changing the species composition. Cells from three California coastal sites were treated with chloramphenicol, an inhibitor of protein synthesis and rRNA degradation, at 100 (mu)g/ml and then were probed with a "universal" 16S rRNA fluorescent probe and viewed by image-intensified video microscopy. Counts of fluorescent cells increased from ca. 75% for untreated samples to ca. 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4(prm1),6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages include autofluorescent cells, which averaged 6%). This suggests that most cells in these samples were active. We hypothesize that the low fluorescent-cell counts previously reported were probably often due to the fluorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments. This method may aid in the characterization of bacterioplankton with fluorescent probes.     

Lateral flagella are required for increased cell adherence,invasion and biofilm formation by Aeromonas spp

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.     

Synthesis of NF-kappaB activation inhibitors derived from epoxyquinomicin C

In order to develop new inhibitors of NF-kappaB activation, we designed and synthesized dehydroxymethyl derivatives of epoxyquinomicin C, namely, DHM2EQ and its regioisomer DHM3EQ. These derivatives were synthesized from 2,5-dimethoxyaniline in 5 steps. Since DHM2EQ was more active and less toxic than DHM3EQ, its stereochemical configuration was determined by X-ray crystallographic analysis. Each enantiomer of the protected DHM2EQ was separated by a chiral column and deprotected. DHM2EQ inhibited TNF-alpha-induced activation of NF-kappaB in human T cell leukemia cells, and also inhibited collagen-induced arthritis in a rheumatoid model in mice.     

Unmasking imaging forces on soft biological samples in liquids when using dynamic atomic force microscopy: a case study on viral capsids

Dynamic atomic force microscopy is widely used for the imaging of soft biological materials in liquid environments; yet very little is known about the peak forces exerted by the oscillating probe tapping on the sample in liquid environments. In this article, we combine theory and experiments in liquid on virus capsids to propose scaling laws for peak interaction forces exerted on soft samples in liquid environments. We demonstrate how these laws can be used to choose probes and operating conditions to minimize imaging forces and thereby robustly image fragile biological samples.     

Influence of dissolved humic materials on carbon assimilation and alkaline phosphatase activity in natural algal-bacterial assemblages

SUMMARY. Mixed natural assemblages of algae and bacteria exhibited lower rates of ¹⁴C assimilation and high rates of dissimilation of recent photosynthate when amended with low concentrations (7.2 mg 1^-1) of unfractioned dissolved humic materials (DHM). The extent of the inhibition or stimulation was greatest in the smaller (1–5 μm) assemblage particles. In different algal-bacterial assemblages, additions of DHM markedly enhanced community alkaline phosphatase activity (APA), particularly under low light regimes, DHM of low apparent molecular weight was much more stimulatory to both ¹⁴C assimilation and APA than DHM of high apparent molecular weight, supporting the belief that DHM molecular weight is an important determinant of DHM interactive capacity. Higher concentrations of D HM (either unfractionated, or molecular weight fractionated) produced greater APA responses. Addition of phosphate enhanced the disparity in rates of ¹⁴C assimilation of samples incubated under low and high light regimes, increased the rates of ¹⁴C assimilation, and depressed APA. There were indications of interactions between DHM and phosphorus in several experiments. Two hypotheses were invoked to explain increases in APA in response to DHM: (1) increased competition between algae and bacteria for phosphate following bacterial release from substrate limitation, or (2) DHM may have acted as a sequestering agent for organophosphorus compounds, and in so doing, gradually depleted available phosphate. In either case, it is clear that DHM alters phosphorus cycling. This DHM characteristic may be ecologically as important as its ability to complex trace metals.     

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.     

Bacterial Activity at -2 to -20 degrees C in Arctic wintertime sea ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and ARCHAEA: Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks.

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences. In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.     

Flow and image cytometry for quality assessment of fresh and frozen human sperm samples

A previous study reported that sperm mitochondrial activity and sperm motility can be evaluated by combined flow and image cytometry, suggesting their potential interest in fertility clinical applications and for studying the effect of physical and chemical agents that modify sperm motility and/or metabolism. This paper focuses on the effect of freezing sperm in liquid nitrogen, extensively used in artificial insemination (AI), combined with different sperm manipulations (washing, swim-up in capacitating medium, CM) using flow and image cytometry on sperm samples from three fertile donors. Rhodamine 123 (Rh123) uptake profiles were bimodal both for fresh and frozen/thawed samples. The mean value of fluorescence of active cells (m+) remained nearly similar after freezing while the percentage of active cells (%C+) was significantly decreased and the percentage of dead cells (%dc, revealed by propidium iodide uptake) significantly increased. In all experiments, the decrease of MOT (percentage of motile sperm) due to freezing was concomitant, to a smaller extent, with a drop in %C+ and an increase in %dc; there was a good relationship between velocity (VCL, VSL) or trajectory characteristics (ALH) and the mean fluorescence values of active cells (m+). Sperm immobilization was neither found related to a major decrease of Rh123 fluorescence nor to an increase in dead cells.     

Culturable Bacteria in Subglacial Sediments and Ice from Two Southern Hemisphere Glaciers

Viable prokaryotes have been detected in basal sediments beneath the few Northern Hemisphere glaciers that have been sampled for microbial communities. However, parallel studies have not previously been conducted in the Southern Hemisphere, and subglacial environments in general are a new and underexplored niche for microbes. Unfrozen subglacial sediments and overlying glacier ice samples collected aseptically from the Fox Glacier and Franz Josef Glacier in the Southern Alps of New Zealand now have been shown to harbor viable microbial populations. Total direct counts of 2–7 × 10⁶ cells g^–1 dry weight sediment were observed, whereas culturable aerobic heterotrophs ranged from 6–9 × 10⁵ colony-forming units g^–1 dry weight. Viable counts in the glacier ice typically were 3–4 orders of magnitude smaller than in sediment. Nitrate-reducing and ferric iron–reducing bacteria were detected in sediment samples from both glaciers, but were few or below detection limits in the ice samples. Nitrogen-fixing bacteria were detected only in the Fox Glacier sediment. Restriction fragment analysis of 16S rDNA amplified from 37 pure cultures of aerobic heterotrophs capable of growth at 4°C yielded 23 distinct groups, of which 11 were identified as -Proteobacteria. 16S rDNA sequences from representatives of these 11 groups were analyzed phylogenetically and shown to cluster with bacteria such as Polaromonas vacuolata and Rhodoferax antarcticus, or with clones obtained from permanently cold environments. Chemical analysis of sediment and ice samples revealed a dilute environment for microbial life. Nevertheless, both the sediment samples and one ice sample demonstrated substantial aerobic mineralization of ¹⁴C-acetate at 8°C, indicating that sufficient nutrients and viable psychrotolerant microbes were present to support metabolism. Unfrozen subglacial sediments may represent a significant global reservoir of biological activity with the potential to influence glacier meltwater chemistry.     

Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.     

Thermostable dextranases: screening, detection and preliminary characterization

Screening methods have been developed for detection of micro-organisms producing thermostable dextranases. They utilize the incorporation of Blue Dextran into agar or liquid culture media for isolation of active dextranase producers growing at temperatures above 55°C. A variety of high temperature environments in sugar factories and naturally occurring thermal water samples were excellent sources of dextranase producers. A number of aerobic and anaerobic thermophilic bacteria, isolated from these sources, were found to produce thermostable dextranases. Dextranases with the greatest thermostability were found in cultures of anaerobic bacteria grown above 65°C. Temperature optima were determined for several crude enzyme preparations, four of which exhibited temperature optima in the range 65–85°C.