CAMK2γ in intestinal epithelial cells modulates colitis-associated colorectal carcinogenesis via enhancing STAT3 activation.

Inflammation is one of the major risk factors for cancer. Here, we show that calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) in intestinal epithelial cells (IECs) modulates inflammatory signals and promotes colitis-associated cancer (CAC) in mice. We have identified CAMK2γ as a downstream target of colitis-induced WNT5A signaling. Furthermore, we have shown that CAMK2γ protects against intestine tissue injury by increasing IEC survival and proliferation. Calcium/calmodulin-dependent protein kinase II gamma knockout mice displayed reduced CAC. Furthermore, we used bone marrow transplantation to reveal that CAMK2γ in IECs, but not immune cells, was crucial for its effect on CAC. Consistently, transgenic over-expression of CAMK2γ in IECs accelerated CAC development. Mechanistically, CAMK2γ in IECs enhanced epithelial signal transducer and activator of transcription 3 (STAT3) activation to promote survival and proliferation of colonic epithelial cells during CAC development. These results thus identify a new molecular mechanism mediated by CAMK2γ in IECs during CAC development, thereby providing a potential new therapeutic target for CAC.

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension.

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.

Metabolic responses of the turkey hen (Meleagris gallopavo) to an intravenous injection of chicken or porcine glucagon.

The purpose of this study was to compare the metabolism of chicken and porcine glucagon in the turkey hen. Six hens each were infused with either porcine or chicken glucagon (16.6 micrograms/kg body weight). Blood samples were obtained at intervals pre- and post-infusion and analyzed for glucagon, glucose, insulin, free fatty acids, thyroxine (T4) and triiodothyronine (T3). The half-life (T 1/2) of porcine (4.5 min) and chicken glucagon (5.5 min) were similar. A 13% increase in glucose concentrations occurred within 10 min and remained elevated (22%) for 2 hr. A concomitant increase in insulin was noted, though not significant from pre-injection levels. A 50% increase in free fatty acids occurred in 2 min, reaching a zenith at 10-15 min post-treatment, and returned to baseline by 2 hr. Significant (P < 0.05) decreases in the circulating concentrations of both T3 and T4 hormones were observed within 5-10 min post-glucagon treatment and remained suppressed for the duration of sampling. These data indicate that the hormonal and metabolic responses of female turkeys to mammalian and avian glucagon are similar.

Relaxin gene expression in the porcine follicle during preovulatory development induced by gonadotrophins.

Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.

Relaxin gene expression in the sow corpus luteum during the cycle, pregnancy, and lactation.

Relaxin (RLX) mRNA was sought in the corpus luteum of the pig during the cycle, pregnancy, and lactation using Northern analysis and in situ hybridization. Three oligonucleotide probes to regions of the preprorelaxin molecule were used for hybridization to detect RLX message and gave similar results. Northern analysis showed a single 1-kilobase RLX transcript at all three stages of the reproductive cycle studied. The intensity of the RLX hybridization signal was greatest in pregnancy and varied during the cycle. The signal in ovaries from day 3 cyclic animals increased by day 13 and declined on day 19. However, the hybridization signal at midcycle was only 2% of that during pregnancy. After parturition on day 2 of lactation, RLX message was still detected in the ovary, although at reduced levels compared with those during the cycle and pregnancy. In situ hybridization results showed hybridization to RLX mRNA in luteal tissue on day 13 of the cycle, but not on day 3 or 19. An increased hybridization signal was observed on days 40, 60, and 90 of pregnancy, with a decline on day 2 of lactation. Control sections incubated with labeled heterologous probe or preincubated with excess unlabeled probe did not hybridize. These results indicate a good correlation between the relative concentrations of RLX transcript and immunohistochemical results previously reported in the corpus luteum of the sow. In addition, they demonstrate that the RLX gene is expressed in luteal tissue, not only in pregnancy, but also in the cycle and early lactation.