Humoral immune responses of channel catfish (Ictalurus punctatus) fry and fingerlings exposed toEdwardsiella ictaluri

The ability of channel catfish to develop antibody responses to Edwardsiella ictaluri was evaluated. Fish were produced and reared under specific pathogen free (SPF) conditions. At the ages of 1, 2, 3 and 4 weeks and 2, 3, 4, 5, and 6 months post-hatch, a group of these naive fish were given a primary immersion exposure to E. ictaluri (mean doses of 6·4×10⁴ cfu ml⁻¹of tank water). Each group received a secondary immersion exposure 4 weeks after the primary exposure and were sampled 2 weeks after each exposure. Control groups were exposed to sterile culture medium. Specific antibody titres were first detected in fish exposed at 4 weeks post-hatch at an average weight of 85 mg. A secondary response was first demonstrated by fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. However, a true boosting effect was first demonstrated in fish that received their primary exposure at 2 months of age. Fish given a primary exposure before 4 weeks of age and a secondary exposure failed to produce a significant antibody response, even though they were the same age at secondary exposure as fish that produced strong antibody responses upon primary exposure. This phenomenon suggests that immunological tolerance was induced and indicates that channel catfish may not be capable of generating a humoral immune response before four weeks of age.     

Direct binding of the dynamin-like GTPase, Dnm1, to mitochondrial dynamics protein Fis1 is negatively regulated by the Fis1 N-terminal arm

Recruitment of a dynamin-like GTPase (Drp1/Dlp1/Dnm1) to membranes requires the mitochondrial dynamics protein Fis1. Mdv1 has been proposed to act as an adaptor between Fis1 and Dnm1 in Saccharomyces cerevisiae. We show that S. cerevisiae Fis1 binds directly to Dnm1 and to Mdv1. Two Fis1 regions have been previously implicated in Mdv1 recruitment: an N-terminal "arm" and a concave surface formed by evolutionarily conserved residues in the tetratricopeptide repeat domain. Perturbing either Fis1 region does not affect Mdv1 binding, but both regions influence Dnm1 binding. Fis1 lacking its N-terminal arm binds tightly to Dnm1, and binding is abolished by mutations to the Fis1 concave surface. The Fis1-Dnm1 interaction decreases more than 100-fold in the presence of the Fis1 arm, suggesting that the arm acts in an autoinhibitory manner to restrict access to the Dnm1 binding site on Fis1. Our data indicate that the concave surface of the Fis1 tetratricopeptide repeat-like domain is evolutionarily conserved to bind the dynamin-like GTPase Dnm1 and not Mdv1 as previously predicted.     

Activity of specific lipid-regulated ADP ribosylation factor-GTPase-activating proteins is required for Sec14p-dependent Golgi secretory function in yeast

Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.     

Serotonin 2C receptor agonists improve type 2 diabetes via melanocortin-4 receptor signaling pathways

The burden of type 2 diabetes and its associated premature morbidity and mortality is rapidly growing, and the need for novel efficacious treatments is pressing. We report here that serotonin 2C receptor (5-HT(2C)R) agonists, typically investigated for their anorectic properties, significantly improve glucose tolerance and reduce plasma insulin in murine models of obesity and type 2 diabetes. Importantly, 5-HT(2C)R agonist-induced improvements in glucose homeostasis occurred at concentrations of agonist that had no effect on ingestive behavior, energy expenditure, locomotor activity, body weight, or fat mass. We determined that this primary effect on glucose homeostasis requires downstream activation of melanocortin-4 receptors (MC4Rs), but not MC3Rs. These findings suggest that pharmacological targeting of 5-HT(2C)Rs may enhance glucose tolerance independently of alterations in body weight and that this may prove an effective and mechanistically novel strategy in the treatment of type 2 diabetes.     

Effect of Treatment of OSA With CPAP on Glycemic Control in Adults With Type 2 Diabetes: The Diabetes Sleep Treatment Trial (DSTT)

ObjectiveThe effect of obstructive sleep apnea (OSA) treatment with continuous positive airway pressure (CPAP) on glycemic measures in patients with type 2 diabetes (T2D) remains unclear. We aimed to determine whether CPAP treatment of OSA improves glycemic measures in patients with T2D.MethodsThis randomized controlled trial (N = 98) examined changes in glycemic measures following 12 weeks of active (n = 49) or sham (n = 49) CPAP and consideried participants’ adherence to CPAP therapy (percentage of days with ≥4 hours use and average hours/day of use).ResultsBaseline treatment groups were similar. Regarding the efficacy of active vs sham-CPAP over time, at 6 weeks, both groups had similar reductions in fructosamine (mean difference [MD], 95% confidence interval [CI]: CPAP ?13.10 [?25.49 to ?0.7] vs. sham ?7.26 [?20.2 to 5.69]; P = .519) but different in HbA1c (CPAP ?0.24 [?0.48 to ?0.003] vs sham 0.15 [?0.10 to 0.4]; P = .027). At 12 weeks, reductions in HbA1c values were similar by group (CPAP ?0.26 [?0.53 to 0.002] vs sham ?0.24 [?0.53 to 0.04]; P = .924). HbA1c reductions were associated with a greater percentage of cumulative days of CPAP usage ≥4 hours per day (b [SE] = 0.006 [0.002]; P = .013) and cumulative hours of CPAP use (b [SE] = 0.08 [0.08]; P = .012). CPAP use of ≥7 hours was associated with a significant reduction in HbA1c (b [SE] 0.54 [0.16]; P = .0012).ConclusionCPAP treatment of OSA did not result in sustained improved glycemic control compared to sham in the intent-to-treat analysis. CPAP adherence was associated with greater improvements in glycemic control.     

DCAF14 promotes stalled fork stability to maintain genome integrity

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The DCC Gene: Structural Analysis and Mutations in Colorectal Carcinomas

DCC is a candidate tumor-suppressor gene encoding a protein with sequence similarity to cell adhesion molecules such as N-CAM. A set of overlapping YAC clones that contains the entire DCC coding region was isolated. Studies of this YAC contig showed that the DCC gene spans approximately 1.4 Mb. For elucidation of exon-intron structure, lambda phage clones containing all known coding sequences were isolated from a genomic library. These clones were used to demonstrate the existence of 29 DCC exons, and the sequences of the exon-intron boundaries were determined for each. Twenty-three polymorphic markers from chromosome 18 were then studied in a panel of primary colorectal tumors that had lost some, but not all, of chromosome 18. In most of these tumors, the region that was lost included DCC. Finally, Southern blot and PCR-based approaches were used to search for subtle mutations in several DCC exons. One tumor that had a point mutation in exon 28 was found, resulting in a proline to histidine substitution. A second tumor with a point mutation in intron 13 was also found. The regional map and genomic structure of DCC should provide the means to more extensively study DCC gene alterations and protein function in normal and neoplastic cells.     

Binding of an analog of the simian virus 40 T antigen to wild-type and mutant viral replication origins

DNAase footprint analyses of purified AD2 + D2 (D2) T protein binding to simian virus 40 origin region fragments revealed a series of four specific interactions with contiguous sequences constituting a 120 base-pair block, in keeping with previous DNAase protection results reported by others. Protection was observed to extend from a 30 base-pair strong affinity site located on the early side of the replication origin (site 1) to two adjacent lower affinity sites, including the origin of replication (site 2) and a 11 to 13 base-pair site between site 1 and the beginning of the T/t coding sequence (site 1′). A fourth site (site 3) was noted abutting the late border of site 2. Binding to site 1 was associated with enhanced D2T binding to sites 2 and 1′. Thus, binding to these sites is co-operative and/or the sequences which constitute site 1 affect the conformation of the sites 1′ and 2 sequences such that they now serve as sites of more efficient D2T binding. In addition, while deletion of all of site 2 and its substitution by late viral sequences ablated processive T binding to sequences abutting site 1 on its late side, various site 2 deletions comprising up to approximately 40% of that sequence did not affect binding to that site to a major degree. Therefore, binding to the replication origin is a sequence-specific event, but there may be multiple strong protein contact sites within that sequence.     

Antisense glutaminase inhibition decreases glutathione antioxidant capacity and increases apoptosis in Ehrlich ascitic tumour cells.

Glutamine is an essential amino acid in cancer cells and is required for the growth of many other cell types. Glutaminase activity is positively correlated with malignancy in tumours and with growth rate in normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen peroxide. It is shown that Ehrlich ascites tumour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathione than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apoptotic cells and are more sensitive to both methotrexate and hydrogen peroxide toxicity than normal cells. Taken together, these results provide insights into the role of glutaminase in apoptosis by demonstrating that the expression of antisense mRNA for glutaminase alters apoptosis and glutathione antioxidant capacity.     

Cholera Toxins: Immunogenicity of the Rabbit Ileal Loop Toxin and Related Antigens

A method of assay of immunogenic potency of the cholera gut toxin is described; it is based on the relation of dose of antigen to neutralizing antibody titer produced in the rabbit under defined conditions and allows quantification of immunogenicity as immunogenic units per milligram of protein. Evidence, based on immunogenicity and rabbit ileal loop toxicity, is presented which indicates that the positively charged fraction of liquid-culture supernatant fluid eluted in deionized water from diethylaminoethyl Sephadex, or in electrolyte from carboxymethyl Sephadex, is a complex made up of a nonantigenic toxic moiety, a nontoxic protein component which elicits the formation of toxin-neutralizing antibody, and an inactive fraction. The complex may also be dissociated in high-salt concentrations with apparent recombination of the toxic moiety with a nondialyzable constituent of peptone to give a negatively charged complex. The immunogenic component is found in nontoxic supernatant fluids of cultures grown at pH 6.5 or in media deficient in peptone. It is also present in the nontoxic fraction eluted from diethylaminoethyl Sephadex in electrolyte or in deionized water from carboxymethyl Sephadex. When separated from the positively charged toxic moiety, the net charge of the antigen is reduced as shown by immunoelectrophoresis. On primary fractionation, the antigen may be associated with a minor antigenic component of the negatively charged complex containing a major antigen eliciting vibriocidal antibody formation, but antisera to the toxin antigen preparations, either in this form or freed of antigenic contamination by recycling, do not contain vibriocidal antibody. It is suggested that this antigen be designated the T (toxin) antigen, and the antigen producing vibriocidal antibody the V antigen. These two antigens would appear to represent the major antigenic specificities associated with the antitoxic and antibacterial elements of the immune response to infection.