Rec dependence of mu transposition from P22-transduced fragments.

Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics. Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22. When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome. Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion. First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome. Second, transposition requires the host recA, B, and C functions. Transposition of Mu following injection by a Mu virion is rec independent. While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22. We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.     

A proteomic approach for studying insect phylogeny: CAPA peptides of ancient insect taxa (Dictyoptera, Blattoptera) as a test case

Background  

Neuropeptide ligands have to fit exactly into their respective receptors and thus the evolution of the coding regions of their genes is constrained and may be strongly conserved. As such, they may be suitable for the reconstruction of phylogenetic relationships within higher taxa. CAPA peptides of major lineages of cockroaches (Blaberidae, Blattellidae, Blattidae, Polyphagidae, Cryptocercidae) and of the termite Mastotermes darwiniensis were chosen to test the above hypothesis. The phylogenetic relationships within various groups of the taxon Dictyoptera (praying mantids, termites and cockroaches) are still highly disputed.     

Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.     

Auto-Antibodies and Their Association with Clinical Findings in Women Diagnosed with Microscopic Colitis

Background

Microscopic colitis (MC) is a disease manifested by diarrhoea and is divided into collagenous and lymphocytic colitis. The aetiology is unknown, but auto-immunity is suggested. Auto-antibodies have been only rarely examined in this entity. The aim of the study was to examine the prevalence of auto-antibodies, and to examine associations between the presence of antibodies and clinical findings.

Methods and Findings

Women with MC verified by biopsy and younger than 73 years, at any Department of Gastroenterology, in the district of Skåne, between 2002 and 2010 were invited to participate in this study. The patients were asked to complete both a questionnaire describing their medical history and the Gastrointestinal Symptom Rating Scale (GSRS). Blood samples were collected. Anti-nuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisiae antibodies (ASCA), and antibodies against glutamic acid decarboxylase (anti-GAD), islet antigens-like insulin 2 (anti-IA2), thyroid peroxidase (anti-TPO), and thyrotropin receptor (TRAK) were analysed. Of 240 women identified, 133 were finally included in the study, median age 63 (59–67) years. Apart from the MC diagnosis, 52% also suffered from irritable bowel syndrome, 31% from hypertension and 31% from allergy. The prevalence of ANA (14%), ASCA IgG (13%), and anti-TPO antibodies (14%) for these patients was slightly higher than for the general population, and were found together with other concomitant diseases. Patients had more of all gastrointestinal symptoms compared with norm values, irrespective of antibody expression.

Conclusions

Women with MC have a slightly increased prevalence of some auto-antibodies. These antibodies are not associated with symptoms, but are expressed in patients with concomitant diseases, obscuring the pathophysiology and clinical picture of MC.     

Control of reproduction in female cockroaches with special reference to Nauphoeta cinerea—I. First pre-oviposition period

Prior to the first oviposition, a receptivity centre, perhaps neurosecretory cells in the brain, controls the female's acceptance of courting males. In L. maderae this centre is affected by starvation. A brief exposure to food can induce mating but is inadequate for oöcyte development. Before the first ovulation starvation has no effect on receptivity in N. cinerea.

In N. cinerea mechanical stimulation caused by the firm insertion of the spermatophore in the bursa copulatrix releases stimuli via the nerve cord to the brain which render the female unreceptive and, at the same time, increases the activity of the corpora allata resulting in rapid development of the oöcytes.

The mechanical presence of the oötheca in the uterus also has two principal effects. Like spermatophore insertion, it inhibits mating. But its effect on the corpora allata is inhibitory, rather than stimulatory, and, consequently, the oöcytes remain underveloped for almost the entire gestation period. The effectiveness of inhibitory stimulation from the stretched uterus depends upon the period in the reproductive cycle in which it occurs-i.e. on the physiological state of the female. In N. cinerea uterine stretching inhibits mating and oöcyte development after oviposition or during gestation but is not effective when exerted during the first pre-oviposition period. In P. surinamensis, uterine stretching does not inhibit the corpora allata prior to the first ovulation but does prevent oöcyte development during gestation.

In fed L. maderae and N. cinerea there appears to be a synergistic action of nutrition and mating in controlling the rate of oöcyte development. Mating (mechanical) and feeding (chemical) stimuli are both usually required for activating the corpora allata to their fullest extent so that the oöcytes mature at their maximum rate. There is some indication that mating stimuli in N. cinerea and L. maderae are effective in further stimulating the corpora allata only if the corpora allata have reached a certain level of activity, if activating stimuli have begun to occur in the brain, or if the mating stimulus occurs in combination with nutritional factors. Thus, the corpora allata in starved virgin females of N. cinerea become sufficiently active so that some yolk is deposited in the oöcytes but these oöcytes do not mature; mating is effective in further stimulating the endocrine glands in these starved females and oviposition occurs in about the normal period. In starved virgins of L. maderae the corpora allata are virtually inactive and yolk is not deposited in the oöcytes; mating has no effect on oöcyte development in starved females. D. punctata differs from both the above species in that the corpora allata in the virgin female usually remain inactive whether she feeds or starves. Mating stimuli alone can activate the corpora allata, in fed or starved females, and consequently the oöcytes mature.