IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes

Diabetic cardiomyopathy is characterized by impaired ventricular contraction and altered function of insulin-like growth factor I (IGF-I), a key factor for cardiac growth and function. Endogenous IGF-I has been shown to alleviate diabetic cardiomyopathy. This study was designed to evaluate exogenous IGF-I treatment on the development of diabetic cardiomyopathy. Adult rats were divided into four groups: control, control + IGF-I, diabetic, and diabetic + IGF-I. Streptozotocin (STZ; 55 mg/kg) was used to induce experimental diabetes immediately followed by a 7-wk IGF-I (3 mg. kg(-1). day(-1) ip) treatment. Mechanical properties were assessed in ventricular myocytes including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were evaluated as Ca(2+)-induced Ca(2+) release and Ca(2+) clearing constant. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and glucose transporter (GLUT4) were assessed by Western blot. STZ caused significant weight loss and elevated blood glucose, demonstrating the diabetic status. The diabetic state is associated with reduced serum IGF-I levels, which were restored by IGF-I treatment. Diabetic myocytes showed reduced PS and +/-dL/dt as well as prolonged TPS, TR(90), and intracellular Ca(2+) clearing compared with control. IGF-I treatment prevented the diabetes-induced abnormalities in PS, +/-dL/dt, TR(90), and Ca(2+) clearing but not TPS. The levels of SERCA and GLUT4, but not PLB, were significantly reduced in diabetic hearts compared with controls. IGF-I treatment restored the diabetes-induced decline in SERCA, whereas it had no effect on GLUT4 and PLB levels. These results suggest that exogenous IGF-I treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.     

Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila

Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.     

Metagenomic Analysis from the Interior of a Speleothem in Tjuv-Ante's Cave,Northern Sweden

Speleothems are secondary mineral deposits normally formed by water supersaturated with calcium carbonate percolating into underground caves, and are often associated with low-nutrient and mostly non-phototrophic conditions. Tjuv-Ante’s cave is a shallow-depth cave formed by the action of waves, with granite and dolerite as major components, and opal-A and calcite as part of the speleothems, making it a rare kind of cave. We generated two DNA shotgun sequencing metagenomic datasets from the interior of a speleothem from Tjuv-Ante’s cave representing areas of old and relatively recent speleothem formation. We used these datasets to perform i) an evaluation of the use of these speleothems as past biodiversity archives, ii) functional and taxonomic profiling of the speleothem’s different formation periods, and iii) taxonomic comparison of the metagenomic results to previous microscopic analyses from a nearby speleothem of the same cave. Our analyses confirm the abundance of Actinobacteria and fungi as previously reported by microscopic analyses on this cave, however we also discovered a larger biodiversity. Interestingly, we identified photosynthetic genes, as well as genes related to iron and sulphur metabolism, suggesting the presence of chemoautotrophs. Furthermore, we identified taxa and functions related to biomineralization. However, we could not confidently establish the use of this type of speleothems as biological paleoarchives due to the potential leaching from the outside of the cave and the DNA damage that we propose has been caused by the fungal chemical etching.     

The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.     

The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER

Proteins are co-translationally transferred into the endo-plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells ( Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.     

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

Laboratory evaluation of the toxicity of systemic insecticides for Control of Anoplophora glabripennis and Plectrodera scalator (Coleoptera: Cerambycidae)

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) is one of the most serious nonnative invasive forest insects discovered in North America in recent years. A. glabripennis is regulated by federal quarantines in the United States and Canada and is the subject of eradication programs that involve locating, cutting, and chipping all infested trees. Other control methods are needed to aid in eradication and to form an integrated management program in the event eradication fails. We conducted laboratory bioassays to determine the toxicity of two systemic insecticides, azadirachtin and imidacloprid, for potential control of A. glabripennis and the cottonwood borer, Plectrodera scalator (F.) (Coleoptera: Cerambycidae), a closely related native cerambycid. Larvae of both cerambycid species were fed artificial diet with dilutions of azadirachtin or imidacloprid for 14 wk. Both insecticides exhibited strong antifeedant effects and some toxicity against A. glabripennis and P. scalator larvae. For A. glabripennis, the highest larval mortality at the end of the bioassay was 60% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (1.6 ppm). For P. scalator, the highest larval mortality at the end of the bioassay was 100% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (160 ppm). At 14 wk, the LC50 values for P. scalator were 1.58 and 1.78 ppm for azadirachtin and imidacloprid, respectively. Larvae of both species gained weight when fed diet treated with formulation blanks (inert ingredients) or the water control but lost weight when fed diet treated with increasing concentrations of either azadirachtin or imidacloprid. In a separate experiment, A. glabripennis adults were fed maple twigs treated with high and low concentrations of imidacloprid. A. glabripennis adult mortality reached 100% after 13 d on twigs treated with 150 ppm imidacloprid and after 20 d on twigs treated with 15 ppm imidacloprid. There was no visible feeding by A. glabripennis adults on twigs treated at the higher imidacloprid rate, and feeding was significantly reduced for adults placed on twigs treated at the low imidacloprid rate compared with adults on untreated twigs. In summary, imidacloprid and azadirachtin had both antifeedant and toxic effects against A. glabripennis and P. scalator and have potential for use in management programs. Based on our results, the delivery of high and sustained insecticide concentrations will be needed to overcome the antifeedant effects and lengthy lethal time for both larvae and adults exposed to these insecticides.     

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.     

Common antigenic determinants in the glycoproteins of plants,molluscs and insects

An antiserum raised against -fructosidase isolated from the cell walls of suspension-cultured carrot cells cross-reacts with many plant proteins and hemocyanin ofHelix pomatia. The shared epitope appears to be a small complex glycan with a (1–2)-linked xylose residue attached to the -linked mannose residue of the core of an asparagine-linked oligosaccharide. There is strong cross-reactivity with the proteins of many seed plants, molluscs and insects, and no cross-reactivity with the proteins of fungi, algae, mosses, ferns, or any of the vertebrates tested. Xylose-containing glycans appear to increase the immunogenicity of the proteins to which they are attached, and we suggest that they may be responsible for some allergic responses of people that are repeatedly exposed to plant or insect proteins.