Heterodimeric GTPase core of the SRP targeting complex

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.     

Proteome half-life dynamics in living human cells

Cells remove proteins by two processes: degradation and dilution due to cell growth. The balance between these basic processes is poorly understood. We addressed this by developing an accurate and noninvasive method for measuring protein half-lives, called "bleach-chase," that is applicable to fluorescently tagged proteins. Assaying 100 proteins in living human cancer cells showed half-lives that ranged between 45 minutes and 22.5 hours. A variety of stresses that stop cell division showed the same general effect: Long-lived proteins became longer-lived, whereas short-lived proteins remained largely unaffected. This effect is due to the relative strengths of degradation and dilution and suggests a mechanism for differential killing of rapidly growing cells by growth-arresting drugs. This approach opens a way to understand proteome half-life dynamics in living cells.     

Nucleus accumbens D2/3 receptors predict trait impulsivity and cocaine reinforcement

Stimulant addiction is often linked to excessive risk taking, sensation seeking, and impulsivity, but in ways that are poorly understood. We report here that a form of impulsivity in rats predicts high rates of intravenous cocaine self-administration and is associated with changes in dopamine (DA) function before drug exposure. Using positron emission tomography, we demonstrated that D2/3 receptor availability is significantly reduced in the nucleus accumbens of impulsive rats that were never exposed to cocaine and that such effects are independent of DA release. These data demonstrate that trait impulsivity predicts cocaine reinforcement and that D2 receptor dysfunction in abstinent cocaine addicts may, in part, be determined by premorbid influences.     

Evaluation of reproductive traits and ovarian and uterine morphology of sows with different genotypes for the estrogen receptor,prolactin receptor,and follicle-stimulating hormone subunit beta genes

The purpose of this study was to analyze the morpho-functional features of the ovaries and uterus of sows with different genotypes for the estrogen receptor (ESR), prolactin receptor (PRLR), and follicle-stimulating hormone subunit beta (FSHβ) genes associated with reproductive traits. Healthy Large White sows were studied. The genotypic status of the ESR, PRLR, and FSHβ genes was detected by polymerase chain reaction-restriction fragment length polymorphism. The structure of the ovaries and uterus was studied using quantitative assessment of organs and histological research.Sows with the ESR^BB genotype significantly exceeded animals with the ESR^AA genotype in milk yield (by 0.3 kg) and in the number of piglets at birth (by 0.9 animals) and at weaning (by 0.7 animals). Sows with the ESR^AB genotype were midway between those with ESR^BB and ESR^AA genotypes in terms of these reproductive traits. Animals with the PRLR^AA genotype significantly exceeded those with the PRLR^BB genotype in the number of piglets born (P < 0.05); the differences in litter weight at birth were not significant. Compared to other genotypes, sows with genotypes ESR^BB (P < 0.05) and PRLR^{AA (AB)} (P < 0.05) had larger uteruses and more yellow bodies, tertiary follicles, and primordial follicles in their ovaries. Animals with the FSHβ^BB genotype significantly exceeded animals with the FSHβ^AB genotype in the length of uterus by 21 cm (P < 0.05).     

Detection of nonesterified (free) fatty acids in bovine serum: comparative evaluation of two methods.

A method comparison study for the determination and quantitation of nonesterified fatty acids (NEFAs) in serum, using the commercial "NEFA C" enzymatic test kit, was performed using the spectrophotometric method recommended by the manufacturer and a modified procedure optimized for the use of a microplate reader, a 96-well microtiter plate, and small sample volumes (10 microl). Linearity, sensitivity, and precision using the test kit were determined for each method of detection. The assay was linear from 0 to 1.97 mEq/liter for both procedures, and the limits of detection were determined to be 0.22 (+/- 0.074) and 0.05 (+/- 0.002) mEq/liter for the spectrophotometer and microplate reader, respectively. Pairs of measurements for bovine serum samples were compared and evaluated by a mean difference plot method and not regression analysis, a method that has been shown to be inappropriate for method comparison studies. The difference plot was used to evaluate the systematic bias between the 2 methods. Random error is reported on the basis of SD differences, and "limits of agreement" are used to describe the maximum differences likely to occur between the 2 methods. Results suggest that the microplate reader method can be used reliably in place of the recommended spectrophotometric method. The microplate reader method is preferred because of its high throughput capabilities, simultaneous analysis of all the standards and samples, use of small sample and reagent volumes, and reduction in labor requirements and costs.     

Root System Distribution of Highbush Blueberry Crops of Various Ages in Medium-Textured Soils

The paper deals with the spatial distribution of root density in highbush blueberry plantations of various ages for a proper planting distance recommendation and a better irrigation and fertilization management. The environmental conditions consist of sandy-loamy soils and a continental temperate climate. Two highbush blueberry cultivars were studied, ‘Bluecrop’ and ‘Blueray’. Root density (RD) and density of root cross-sectional area (RCSA) were investigated for various root diameters. It was found that the prevalent type of roots in highbush blueberry crops is the hair type, with root diameters less than 0.1?mm. Both RD and RCSA decreased with distance from plant and with soil depth; the 38-year-old crop showed a more developed root system versus the 8?year-old crop. In both cases the roots did not grow more than 0.6?m laterally and more than 0.7?m deep. According to these results, the planting distances in highbush blueberry could be reduced to substantially increase the number of plants/ha and fruit yield. Irrigation application should be carried out to wet a proper soil volume, about 0.6–0.7?m deep for older crops, and about 0.5?m deep for younger crops.     

Sperm cryopreservation in the Far‐Eastern wildcat (Prionailurus bengalensis euptilurus)

The Far‐Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far‐Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far‐Eastern wildcat captive‐born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen–thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far‐Eastern wildcat. The motility of frozen–thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far‐Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far‐Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far‐Eastern wildcat, respectively). Domestic cat epididymal and Far‐Eastern ejaculatory spermatozoa fertilized in vitro‐matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far‐Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.     

Atypical Reactive Center Kunitz-Type Inhibitor from the Sea Anemone Heteractis crispa

The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the K(i) of InhVJ to trypsin and α-chymotrypsin (7.38 × 10(-8) M and 9.93 × 10(-7) M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.     

An outbreak of tuberculosis by Mycobacterium bovis in coatis (Nasua nasua)

Mycobacterium tuberculosis complex, which includes Mycobacterium bovis, infrequently causes severe or lethal disease in captive wildlife populations. A dead coati from a wildlife triage center showing pulmonary lesions compatible with tuberculosis had raised suspicion of a potential disease caused by mycobacteria species and was further investigated. Four native coatis (Nasua nasua) with suspected mycobacterial infection were sedated, and bronchoalveolar lavages and tuberculin skin tests (TSTs) were performed. All animals tested positive upon TST. Mycobacterial culturing, Ziehl-Neelsen staining, and genetic testing were performed on postmortem samples and the etiologic agent was identified as M. bovis. Molecular genetic identification using a polymerase chain reaction panel was crucial to achieving a definitive diagnosis.