Nicking by transesterification: the reaction catalysed by a relaxase

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site- and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3'-OH and the relaxase covalently bound to the 5' end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.     

Mandibular movement and muscle activity during mastication in the guinea pig (Cavia porcellus)

Optoelectronic analysis of mandibular movement and electromyography (EMG) of masticatory muscles in Cavia porcellus indicate bilateral, unilateral, and gnawing cycles. During bilateral and unilateral cycles, the mandibular tip moves forward, lateral, and down during the lingual phase of the power stroke to bring the teeth into occlusion. EMG activity is generally asymmetric, with the exception of activity of the temporalis muscle during bilateral cycles. During gnawing cycles, the mandible moves in an anteroposterior direction that is opposite that during bilateral and unilateral chew cycles. Bilateral and unilateral cycles of pellets were significantly longer than carrot. With the exception of the width of bilateral cycles, the magnitude of cycle width, length, and height during the mastication of carrots was greater than that during the mastication of pellets. Significant differences exist between EMG durations during mastication of pellets and carrots. The lateral pterygoid displays continuous activity during gnawing cycles. Significant differences also exist in the durations of EMG activity between the working and balancing side during all three cycle types. High level activity of balancing side temporalis and anterior belly of digastric (ABD) during bilateral cycles occurs during rotation and depression of the mandible during the power stroke. The temporalis apparently provides a ?braking”? or compensatory role during closing and power strokes. Differences between Cavia masticatory patterns and those shown by Rattus and Mesocricetus are apparently due to differences in dental morphology, occlusal relationships, and, possibly, the poorly developed temporalis in Cavia. The large number and wide diversity of rodent groups afford students of mammalian mastication an opportunity to investigate and compare different masticatory specializations.     

Osteopathology in Rhinocerotidae from 50 Million Years to the Present

Individual elements of many extinct and extant North American rhinocerotids display osteopathologies, particularly exostoses, abnormal textures, and joint margin porosity, that are commonly associated with localized bone trauma. When we evaluated six extinct rhinocerotid species spanning 50 million years (Ma), we found the incidence of osteopathology increases from 28% of all elements of Eocene Hyrachyus eximius to 65–80% of all elements in more derived species. The only extant species in this study, Diceros bicornis, displayed less osteopathologies (50%) than the more derived extinct taxa. To get a finer-grained picture, we scored each fossil for seven pathological indicators on a scale of 1–4. We estimated the average mass of each taxon using M1-3 length and compared mass to average pathological score for each category. We found that with increasing mass, osteopathology also significantly increases. We then ran a phylogenetically-controlled regression analysis using a time-calibrated phylogeny of our study taxa. Mass estimates were found to significantly covary with abnormal foramen shape and abnormal bone textures. This pattern in osteopathological expression may reflect a part of the complex system of adaptations in the Rhinocerotidae over millions of years, where increased mass, cursoriality, and/or increased life span are selected for, to the detriment of long-term bone health. This work has important implications for the future health of hoofed animals and humans alike.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.     

Randomized Control Trial to Improve Adiposity and Insulin Resistance in Overweight Latino Adolescents

Few randomized trials attempt to improve insulin sensitivity and associated metabolic risks in overweight Latino youth. The purpose of this study is to examine the effects of a modified carbohydrate nutrition program combined with strength training on insulin sensitivity, adiposity, and other type 2 diabetes risk factors in overweight Latino adolescents. In a 16‐week randomized trial, 54 overweight Latino adolescents (15.5 ± 1.0 years) were randomly assigned to: (i) Control (C; n = 16), (ii) Nutrition (N; n = 21), or (iii) Nutrition + Strength training (N+ST; n = 17). The N group received modified carbohydrate nutrition classes (once per week), while the N+ST received the same nutrition classes plus strength training (twice per week). The following were measured at pre‐ and postintervention: strength by 1‐repetition maximum, dietary intake by 3‐day records, body composition by dual‐energy X‐ray absorptiometry, glucose/insulin indices by oral glucose tolerance test (OGTT) and intravenous glucose tolerance test with minimal modeling. Across intervention group effects were tested using analysis of covariance with post hoc pairwise comparisons. A significant overall intervention effect was found for improvement in bench press (P < 0.001) and reductions in energy (P = 0.05), carbohydrate (P = 0.04) and fat intake (P = 0.03). There were no significant intervention effects on insulin sensitivity, body composition, or most glucose/insulin indices with the exception of glucose incremental area under the curve (IAUC) (P = 0.05), which decreased in the N and N+ST group by 18 and 6.3% compared to a 32% increase in the C group. In conclusion, this intense, culturally tailored intervention resulted in no significant intervention effects on measured risk factors with the exception of a beneficial effect on glycemic response to oral glucose.