DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Comment on: Wu R, et al. Clin Cancer Res 2011; 17:7359–72     

Enhanced skeletal muscle contraction with myosin light chain phosphorylation by a calmodulin-sensing kinase

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.     

Historical and Contemporary DNA Indicate Fisher Decline and Isolation Occurred Prior to the European Settlement of California

Establishing if species contractions were the result of natural phenomena or human induced landscape changes is essential for managing natural populations. Fishers (Martes pennanti) in California occur in two geographically and genetically isolated populations in the northwestern mountains and southern Sierra Nevada. Their isolation is hypothesized to have resulted from a decline in abundance and distribution associated with European settlement in the 1800s. However, there is little evidence to establish that fisher occupied the area between the two extant populations at that time. We analyzed 10 microsatellite loci from 275 contemporary and 21 historical fisher samples (1880–1920) to evaluate the demographic history of fisher in California. We did not find any evidence of a recent (post-European) bottleneck in the northwestern population. In the southern Sierra Nevada, genetic subdivision within the population strongly influenced bottleneck tests. After accounting for genetic subdivision, we found a bottleneck signal only in the northern and central portions of the southern Sierra Nevada, indicating that the southernmost tip of these mountains may have acted as a refugium for fisher during the anthropogenic changes of the late 19^th and early 20^th centuries. Using a coalescent-based Bayesian analysis, we detected a 90% decline in effective population size and dated the time of decline to over a thousand years ago. We hypothesize that fisher distribution in California contracted to the two current population areas pre-European settlement, and that portions of the southern Sierra Nevada subsequently experienced another more recent bottleneck post-European settlement.     

Impact of participant and interventionist race concordance on weight loss outcomes

Objective:

We have previously shown that racial composition of behavioral intervention groups does not affect achieved weight loss. However, it is unclear if the race of the interventionist affects intervention outcomes. The objective of this analysis is to estimate the impact of race concordance between participant and interventionist on weight change in the initial weight loss phase (phase I) of the Weight Loss Maintenance trial (WLM).

Design and Methods:

A total of 1,685 overweight or obese adults (BMI 25‐45 kg/m²) who were taking medication for hypertension and/or dyslipidemia participated in phase I of the WLM trial. All participants received a 6‐month intensive behavioral intervention in groups of 15‐20 facilitated by a trained interventionist. The main outcome is change in weight at 6 months.

Results:

Participants were on average 55 years of age, 67% female and 44% African American (AA). Three of seventeen interventionists were AA, 14 were non‐AA. Seventy‐three percent of participants shared race concordance with the interventionist. There was a small but statistically significant difference in weight change of participants who were the same race as the interventionist (?5.84 kg, s.e. 0.17) as compared with those who were not race concordant (?5.04 kg, s.e. 0.33), a difference of 0.8 kg, (P = 0.04). The impact of concordance on weight change differed by race (i.e., interaction of race and concordance was significant, P = 0.02).

Conclusions:

In a post hoc analysis of a group‐based behavioral intervention, race concordance for non‐AA participants was associated with slightly greater weight loss. Race concordance was not associated with weight loss for AA participants.

     

Alcohol preference in AXB/BXA recombinant inbred mice: gender differences and gender-specific quantitative trait loci

The purpose of the present study was to characterize the C57BL/6J, A/J, and AXB/BXA Recombinant Inbred (RI) strains of mice for voluntary alcohol consumption. Quantitative Trait Locus (QTL) analysis was used to provide provisional location of QTLs for alcohol consumption. The inbred strains were screened for levels of alcohol intake (calculated as alcohol preference and absolute alcohol consumption) by receiving 4 days of forced exposure to a 10% (wt/vol) solution of alcohol, followed by 3 weeks of free choice between water and 10% alcohol. A wide and continuous distribution of values for alcohol consumption and preference was obtained in the AXB/BXA RI strains, confirming polygenic influences on alcohol-related behaviors. Significant gender differences were found for both alcohol preference [F_28,651= 2.12, p < 0.001] and absolute alcohol consumption [F_28,647= 2.57, p < 0.001]. In males, putative QTLs were mapped to chromosomes (Chrs) 2, 5, 7, 10, 11, and 16. Multiple regression analysis indicated that approximately 75% of the genetic variance in alcohol preference in males could be accounted for by three of the QTL regions. Several of the putative QTLs appeared to be male-specific (Tyr on Chr 7; D10Mit126 on Chr 10; D11Mit61 on Chr 11). In females, seven putative QTLs were mapped to Chrs 2, 4, 5, 7, 11, 16, and 19. Approximately 90% of the genetic variance in alcohol preference in females could be accounted for by four QTL regions, as determined by multiple regression. The QTL on Chr 11 near D11Mit35 appeared to be female-specific. This site was close to a female-specific QTL (Alcp2) previously mapped in C57BL/6J × DBA/2J backcrosses by Melo and coworkers (Nat Genet 13, 147, 1996). The QTLs mapped for alcohol preference in the present study must be considered suggestive at the present time, since only D2Mit74 met very strict statistical criteria for significance. However, the concordance across several studies for the loci on Chrs 2, 4, 7, 9, and 11 suggest that some common QTLs influencing alcohol preference have been identified. Confirmation of QTLs mapped in the present study is currently being conducted in a new series of recombinant congenic (RC) strains developed from reciprocal backcrosses between the A/J and C57BL/6J progenitors. The concomitant use of both RI and RC strains developed from the same progenitors should provide a powerful means of detecting, confirming, and mapping QTLs for alcohol-related traits. Received: 25 August 1998 / Accepted: 8 October 1998     

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca²⁺‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca²⁺ efflux pump, PMCA4. This is the major Ca²⁺ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca²⁺] in capacitated and Ca²⁺ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca²⁺] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4‐nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co‐immunoprecipitated with eNOS in a Ca²⁺‐dependent manner. In Pmca4^?/? sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild‐type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co‐ordinates Ca²⁺ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4^?/? males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.     

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.     

Evaluation of the salivary proteome as a surrogate tissue for systems biology approaches to understanding appetite

Current measurement of appetite depends upon tools that are either subjective (visual analogue scales), or invasive (blood). Saliva is increasingly recognised as a valuable resource for biomarker analysis. Proteomics workflows may provide alternative means for the assessment of appetitive response. The study aimed to assess the potential value of the salivary proteome to detect novel biomarkers of appetite using an iTRAQ-based workflow. Diurnal variation of salivary protein concentrations was assessed. A randomised, controlled, crossover study examined the effects on the salivary proteome of isocaloric doses of various long chain fatty acid (LCFA) oil emulsions compared to no treatment (NT). Fasted males provided saliva samples before and following NT or dosing with LCFA emulsions. The oil component of the DHA emulsion contained predominantly docosahexaenoic acid and the oil component of OA contained predominantly oleic acid. Several proteins were present in significantly (p<0.05) different quantities in saliva samples taken following treatments compared to fasting samples. DHA caused alterations in thioredoxin and serpin B4 relative to OA and NT. A further study evaluated energy intake (EI) in response to LCFA in conjunction with subjective appetite scoring. DHA was associated with significantly lower EI relative to NT and OA (p=0.039). The collective data suggest investigation of salivary proteome may be of value in appetitive response. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance. Evidence that chronic fructose feeding in the hamster is accompanied by enhanced intestinal de novo lipogenesis and ApoB48-containing lipoprotein overproduction

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a) in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or in vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.