Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

Insulin Resistance in Striated Muscle-specific Integrin Receptor ��1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1-5). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6, 7).Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS)² phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8-11). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12, 13). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14-18). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20, 21). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22-24).In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25, 26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27-33). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle.     

Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA

Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end.     

Comparative study of histological and kinetic variations of the digestive mucosa and pancreatic parenchyma after hypophysectomy in the rat. Light and electron microscopic study

Variations of the pancreatic parenchyma, the gastric mucosa and the intestinal mucosa were studied in adult male Wistar rats on day 8 and 15 after hypophysectomy. All results were compared with those obtained in pair-fed control rats. Hypophysectomy affected small intestine as well as gastric mucosa. Hypotrophy was observed on day 8 as most of the morphological parameters reached the maximal decrease. By contrast, hypoplasy occurred on day 15, when the labeling index (LI) decreased significantly. In the intestine, however, a decrease of the LI was observed only for the upper proliferative cells of the crypts. In the gastric mucosa, the LI was reduced only in the proliferative zone containing progenitor cells (isthmic region). Consequently, the cell differentiation is not similarly affected on all levels of the digestive tract.     

Integrative metabolic regulation of peripheral tissue fatty acid oxidation by the SRC kinase family member Fyn

Mice null for Fyn (a member of the Src family of nonreceptor tyrosine kinases) display a reduced percentage of adipose mass associated with decreased adipocyte cell size. In parallel, there is a substantial reduction in fasting plasma glucose, insulin, triglycerides, and free fatty acids concomitant with decreased intrahepatocellular and intramyocellular lipid accumulation. Importantly, the Fyn null mice exhibit improved glucose tolerance resulting from increased peripheral tissue (adipose and skeletal muscle) insulin sensitivity with a very small effect in the liver. Moreover, whole-body, adipose, and skeletal muscle fatty acid uptake and oxidation are increased along with AMP kinase activation and acetyl-CoA carboxylase inhibition. Together, these data demonstrate crosstalk between Src-family kinase activity and fatty acid oxidation and show that the loss of Fyn markedly improves peripheral tissue insulin sensitivity by relieving a selective negative modulation of AMP kinase activity in adipose tissue and skeletal muscle.