The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux

During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na⁺, or the ionophore X537A. Accompanying the sperm reaction induced by low Na⁺ are H⁺ efflux and Ca²⁺ influx in a ratio of near 100:1 as determined by ⁴⁵Ca²⁺ and atomic absorption analysis. Simultaneous pH and Ca²⁺ electrode measurements suggest that the movement of H⁺ begins 10–13 sec before the movement of Ca²⁺. Ca²⁺ uptake can be inhibited by verapamil without affecting H⁺ efflux or the sperm reaction. Acid release and Ca²⁺ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na⁺ is proportional to Ca²⁺ concentrations above 2 mM. H⁺ and Ca²⁺ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H⁺ is probably not exchanged for Ca²⁺. Instead we propose that loss of H⁺ triggers the uptake of Ca²⁺, which initiates the sperm reaction.     

Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise.

We examined the effects of menstrual cycle phase and oral contraceptive (OC) use on triglyceride mobilization during 90 min of rest and 60 min of leg ergometry exercise at 45 and 65% peak O(2) uptake (Vo(2 peak)) in eight moderately physically active, eumenorrheic women (24.8 +/- 1.2 yr). Subjects were tested during the follicular phase (FP) and the luteal phase (LP) before OC use and during the inactive phase (IP) and high-dose phase (HP) after 4 complete mo of OC use. Glycerol rate of appearance (R(a)), a measure of triglyceride mobilization, was determined in a 3-h postabsorptive state using a primed constant infusion of [1,1,2,3,3-(2)H]glycerol. Before OC use (BOC), there were no significant differences between FP and LP in any of the variables studied. Dietary composition, exercise patterns, plasma glycerol concentrations, growth hormone concentrations, and exercise respiratory exchange ratio did not change with OC use. However, 4 mo of OC use significantly (P < 0.05) increased glycerol R(a) in HP during exercise at 45% Vo(2 peak) (6.2 +/- 0.2, 6.5 +/- 0.4, and 7.7 +/- 1.1 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively) and in IP and HP at 65% Vo(2 peak) (6.6 +/- 0.1, 8.2 +/- 0.6, and 8.1 +/- 0.7 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively). Plasma cortisol concentrations were significantly higher with OC use at rest and during exercise at 45 and 65% Vo(2 peak). In summary, although fluctuations of endogenous ovarian steroids have little effect on triglyceride mobilization, the synthetic ovarian steroids found in OCs increase triglyceride mobilization and plasma cortisol concentrations in exercising women. We conclude that the hierarchy of effects of ovarian steroids and their analogs on triglyceride mobilization in exercising women is as follows: energy flux > OC use > recent carbohydrate nutrition, menstrual cycle effects.     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

The Use of Positive Reinforcement in Training Zebra Sharks (Stegostoma fasciatum)

Few studies have examined how personality traits may be related to the amounts and types of attachments humans have toward companion animals (pets). In this study, 1,098 companion animal guardians (owners) completed a survey that included the Big Five Inventory, the Lexington Attachment to Pets Scale, and the Pet Attachment Questionnaire. Each participant chose whether he or she identified as a Cat Person, Dog Person, Both, or Neither. Results indicated that neuroticism, conscientiousness, choosing a dog as a favorite pet, and identifying as a Cat Person, Dog Person, or Both predicted affection for a pet. Conscientiousness, extraversion, and openness decreased avoidant attachment to pets, and neuroticism increased anxious attachment to pets. Both dogs and cats could benefit from pet owners who are conscientious, and there may be some benefits of neuroticism in pet owners. The findings of this study will advance understanding of the human–animal bond. As this understanding increases, measurements of human attachment and personality may be useful for the development of tools that could assist shelter employees and veterinarians in counseling people about pet ownership.     

A Copy Number Variant at the KITLG Locus Likely Confers Risk for Canine Squamous Cell Carcinoma of the Digit

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (P_raw = 1.60×10⁻⁷; P_genome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10⁻⁸). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.     

Physical maturity in common bottlenose dolphins (Tursiops truncatus) from Sarasota Bay,Florida

Cetacean physical maturity is defined by growth cessation and complete fusion of epiphyses to vertebral bodies indicated by invisible sutures. Many studies have shown epiphyseal fusion is highly variable among individuals. In-depth examinations into fusion variability are lacking. We analyzed vertebrae of 37 (n = 21 female, n = 16 male) stranded common bottlenose dolphins (Tursiops truncatus) from the well-studied Gulf of Mexico, Sarasota Bay community. For each specimen, vertebrae were examined by vertebral region for degree of fusion anteriorly and posteriorly of each centrum and categorized from unfused to fused in five degrees. An ordinal logistic regression was used to estimate degree of fusion probability for each epiphysis. The model had fixed effects for age, number of offspring, sex, sexual maturity, and a random effect for epiphysis. Results show that age/reproductive status significantly explains an individual's degree of fusion. Adult females with fewer calves had more fusion than those with more reproductive experience across multiple ages. Access to long-term observational and sample data on the dolphins residing in the area served by Mote Marine Laboratory's Stranding Investigations Program offers a unique opportunity to examine the relationship between energetic demands of reproduction (calcium production/reproductive output) versus preconceived definitions of physical maturity (skeletal fusion) more closely.     

Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.