Common Variation Neighbouring Micro-RNA 22 Is Associated with Increased Left Ventricular Mass

Aims

Previous genome-wide linkage analysis has suggested that chromosomal region 17p13.3 may harbour genes influencing left ventricular mass (LVM) in man. To date, the genetic factors accounting for LVM variability remain largely unknown but a non-coding RNA gene within this region, micro-RNA 22 (miR-22), has been implicated in cardiac hypertrophy and heart failure in animal models. We thus investigated the relationship between common genetic polymorphisms surrounding miR-22 and left ventricular mass in a family-based association study.

Methods and Results

We studied a cohort of 255 families comprising 1,425 individuals ascertained via a hypertensive proband. Ten single nucleotide polymorphisms which together tagged common genetic variation surrounding the miR-22 gene were genotyped. There was evidence of association between the rs7223247 polymorphism, which lies within the 3′UTR of a gene of unknown function, TLCD2, immediately downstream from miR-22, and left ventricular mass determined by Sokolow-Lyon voltage (Bonferroni corrected p-value = 0.038). The T allele at rs7223247 was associated with an 0.272 standard deviation higher Sokolow-Lyon voltage. Genotype was responsible for ∼1% of the population variability in LVM.

Conclusions

Genotype at the rs7223247 polymorphism affects left ventricular mass determined by Sokolow-Lyon voltage. The neighbouring genes miR-22 and TLCD2 are strong candidates to account for this observation.     

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r² = 0.83, r = 0.91, p < 0.01) better than NHP (r² = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.     

Sucrose nonfermenting AMPK-related kinase (SNARK) regulates exercise-stimulated and ischemia-stimulated glucose transport in the heart

The signaling mechanisms mediating myocardial glucose transport are not fully understood. Sucrose nonfermenting AMP-activated protein kinase (AMPK)-related kinase (SNARK) is an AMPK-related protein kinase that is expressed in the heart and has been implicated in contraction-stimulated glucose transport in mouse skeletal muscle. We first determined if SNARK is phosphorylated on Thr²⁰⁸, a site critical for SNARK activity. Mice were treated with exercise, ischemia, submaximal insulin, or maximal insulin. Treadmill exercise slightly, but significantly increased SNARK Thr²⁰⁸ phosphorylation. Ischemia also increased SNARK Thr²⁰⁸ phosphorylation, but there was no effect of submaximal or maximal insulin. HL1 cardiomyocytes were used to overexpress wild-type (WT) SNARK and to knockdown endogenous SNARK. Overexpression of WT SNARK had no effect on ischemia-stimulated glucose transport; however, SNARK knockdown significantly decreased ischemia-stimulated glucose transport. SNARK overexpression or knockdown did not alter insulin-stimulated glucose transport or glycogen concentrations. To study SNARK function in vivo, SNARK heterozygous knockout mice (SNARK^+/−) and WT littermates performed treadmill exercise. Exercise-stimulated glucose transport was decreased by ~50% in hearts from SNARK^+/− mice. In summary, exercise and ischemia increase SNARK Thr²⁰⁸ phosphorylation in the heart and SNARK regulates exercise-stimulated and ischemia-stimulated glucose transport. SNARK is a novel mediator of insulin-independent glucose transport in the heart.     

Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos

Pregnancy, parturition and calf survival following the transfer of embryos produced in vitro were monitored. A total of 44 blastocysts was transferred in pairs to 1 uterine horn ipsilateral to the corpus luteum (CL) of 22 synchronized heifers. At Day 42 of development 14 recipients (64%) were pregnant; the calving rate was also 64%. The twinning rate was 9/14 at Day 42 and 7/14 at birth, for an overall fetal mortality rate of 9%. The average gestation length was 281 and 275 d for single and twin pregnancies, respectively. Blood samples from recipients were collected for determination of bovine pregnancy associated glycoprotein (bPAG) from 2 wk after transfer and throughout the pregnancy. During the first trimester of pregnancy, the bPAG concentration was significantly higher in twin than in single bearing heifers, and the perinatal increase in bPAG was correlated positively with the total weight of the fetus(es). The percentage of male calves was 43%. The birth weight of twin individuals was 25 +/- 1 kg, which was 78% of the birthweight of the singletons (32 +/- 2 kg). One singleton calf was oversized, weighing 58 kg (80% more than the median weight of the other singletons). Stillbirths occurred in 21% of the twins, butin none of the singletons. Calf mortality during the first 14 d was higher for twins (4/11) than for singletons (1/7) due to infections and cerebellar hypoplasia. Karyotyping the calves detected no cytogenetically recognizable abnormalities. All calves were negative for BVD virus and IBR antibodies. The results of this study showed that although the incidence of fetal loss was low, there was an unacceptable high perinatal mortality of the calves. Thus it is likely that the blood supply through the placenta of animals pregnant with twins was impaired or it is possible that these fetuses and calves had increased stress susceptibility caused by the in vitro conditions. Furthermore, the birth of 1 oversized calf, 2 calves with cerebellar hypoplasia and 5 calves succumbing to infections seems to indicate that a proportion of in vitro produced calves may suffer from factors inherent in the in vitro production system.     

3-Alkylthio-1,2,4-triazine dimers with potent antimalarial activity

We report on the discovery of 3-alkylthio-1,2,4-triazine dimers that are potently toxic to Plasmodium falciparum, with single digit nanomolar activity, and up to several thousand-fold lower toxicity to mammalian cells. They are equipotent against chloroquine-resistant strains of P. falciparum.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

New mouse genetic models for human contraceptive development

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.