On the origin of vertical line bisection errors

Four experiments examined the biases of individual subjects when attempting to bisect vertical lines, and tested various hypotheses concerning the origin of vertical bisection errors (VBEs). In each experiment, individual differences on the vertical line bisection task were compared to individual differences on another task to evaluate whether the tasks were systematically related. In the first experiment, VBEs were found not to correlate with horizontal line bisection errors (HBEs). In the second experiment, VBEs were found not to correlate with the size of the horizontal-vertical illusion (HVI). In the third experiment, VBEs were found not to correlate with differences in perceived length of vertical lines presented in the upper and lower visual fields that were displaced horizontally. In the fourth experiment, VBEs were found to correlate with bisection errors of an open vertical interval. The results of the first three studies are counter to various hypotheses of the origins of vertical bisection errors. The last experiment suggests that lines, per se, are unnecessary for VBEs. Rather, it is important that the upper and lower segments of the stimulus that are judged to be equal on vertical bisection tasks are co-extensive.     

Uterine leucocyte infiltration after artificial insemination in bitches

In the present study, polymorphonuclear neutrophils (PMN) were enumerated to evaluate acute uterine inflammation after artificial insemination in the bitch. It was concluded that the canine seminal plasma possessed an immunomodulating action. However, the most commonly used extender for freezing canine semen (Tris glucose with egg yolk and glycerol) was a potential inducer of uterine inflammation.     

Mitochondria and membrane cryoinjury in micropatterned cells: Effects of cell-cell interactions

The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell “viability”. Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell-cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell-cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell-cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 °C/min to −40 °C after extracellular ice nucleation at −5 °C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell-cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell “viability” does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.     

An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS

The application of LC-MS for untargeted urinary metabolite profiling in metabonomic research has gained much interest in recent years. However, the effects of varying sample pre-treatments and LC conditions on generic metabolite profiling have not been studied. We aimed to evaluate the effects of varying experimental conditions on data acquisition in untargeted urinary metabolite profiling using UPLC/QToF MS. In-house QC sample clustering was used to monitor the performance of the analytical platform. In terms of sample pre-treatment, results showed that untreated filtered urine yielded the highest number of features but dilution with methanol provided a more homogenous urinary metabolic profile with less variation in number of features and feature intensities. An increased cycle time with a lower flow rate (400mul/min vs 600mul/min) also resulted in a higher number of features with less variability. The step elution gradient yielded the highest number of features and the best chromatographic resolution among three different elution gradients tested. The maximum retention time and mass shift were only 0.03min and 0.0015Da respectively over 600 injections. The analytical platform also showed excellent robustness as evident by tight QC sample clustering. To conclude, we have investigated LC conditions by studying variability and repeatability of LC-MS data for untargeted urinary metabolite profiling.     

Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii

Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model. By high-throughput screening, we have isolated 165 temperature sensitive parasite growth mutants. Phenotypic analysis of these mutants suggests regulated progression through the parasite cell cycle with defined phases and checkpoints. These analyses also highlight the critical importance of the peculiar intranuclear spindle as the physical hub of cell cycle regulation. To link these phenotypes to parasite genes, we have developed a robust complementation system based on a genomic cosmid library. Using this approach, we have so far complemented 22 temperature sensitive mutants and identified 18 candidate loci, eight of which were independently confirmed using a set of sequenced and arrayed cosmids. For three of these loci we have identified the mutant allele. The genes identified include regulators of spindle formation, nuclear trafficking, and protein degradation. The genetic approach described here should be widely applicable to numerous essential aspects of parasite biology.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.     

SCF TrCP1 activates and ubiquitylates TAp63gamma

p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and also has an important role in cancer progression. Currently, the molecular mechanisms governing regulation of p63 function remain largely unclear. This study identifies a unique E3 ubiquitin ligase for p63, SCF(betaTrCP1). SCF(betaTrCP1) is able to bind p63gamma isoforms, with a higher affinity for the TAp63gamma isoform. Strikingly, co-expression of TAp63gamma and betaTrCP1 leads to the stabilization of TAp63gamma. This stabilization of TAp63gamma leads to up-regulation of p21 at the mRNA and protein level by increased binding of TAp63gamma at the p21 promoter. The up-regulation of p21 causes a subsequent increase in G(1) phase cell cycle arrest. Last, SCF(betaTrCP1) is able to ubiquitylate TAp63gamma, and this ubiquitylation, as well as the increased activity of TAp63gamma, is ablated with the expression of a ubiquitin-deficient mutant of betaTrCP1 (DeltaFbetaTrCP1). Therefore, our study reveals that SCF(betaTrCP1) is an E3 ligase that activates p63 through ubiquitylation.     

Cortisol and GH secretory dynamics, and their interrelationships, in healthy aged women and men

We studied 130 healthy aged women (n = 57) and men (n = 73), age 65-88 yr, with age-related reductions in insulin-like growth factor I and gonadal steroid levels to assess the interrelationships between cortisol and growth hormone (GH) secretion and whether these relationships differ by sex. Blood was sampled every 20 min from 8:00 PM to 8:00 AM; cortisol was measured by RIA and GH by immunoradiometric assay, followed by deconvolution analyses of hormone secretory parameters and assessment of approximate entropy (ApEn) and cross-ApEn. Cortisol mass/burst, cortisol production rate, and mean and integrated serum cortisol concentrations (P < 0.0005), and overnight basal GH secretion (P < 0.05), were elevated in women vs. men. Integrated cortisol concentrations were directly related to most measures of GH secretion in women (P < 0.01) and with mean and integrated GH concentrations in men (P < 0.05). Integrated GH concentrations were directly related to mean and integrated cortisol levels in women (P < 0.005) and men (P < 0.05), with no sex differences. There were no sex differences in cortisol or GH ApEn values; however, the cross-ApEn score was greater in women (P < 0.05), indicating reduced GH-cortisol pattern synchrony in aged women vs. men. There were no significant relationships of integrated cortisol secretion with GH ApEn, or vice versa, in either sex. Thus postmenopausal women appear to maintain elevated cortisol production in patterns that are relatively uncoupled from those of GH, whereas mean hormone outputs remain correlated.     

Moisture retention properties of a mycorrhizal soil

The water relations of arbuscular mycorrhizal plants have been compared often, but virtually nothing is known about the comparative water relations of mycorrhizal and nonmycorrhizal soils. Mycorrhizal symbiosis typically affects soil structure, and soil structure affects water retention properties; therefore, it seems likely that mycorrhizal symbiosis may affect soil water relations. We examined the water retention properties of a Sequatchie fine sandy loam subjected to three treatments: seven months of root growth by (1) nonmycorrhizal Vigna unguiculata given low phosphorus fertilization, (2) nonmycorrhizal Vigna unguiculata given high phosphorus fertilization, (3) Vigna unguiculata colonized by Glomus intraradices and given low phosphorus fertilization. Mycorrhization of soil had a slight but significant effect on the soil moisture characteristic curve. Once soil matric potential (_m) began to decline, changes in _m per unit change in soil water content were smaller in mycorrhizal than in the two nonmycorrhizal soils. Within the range of about –1 to –5 MPa, the mycorrhizal soil had to dry more than the nonmycorrhizal soils to reach the same _m. Soil characteristic curves of nonmycorrhizal soils were similar, whether they contained roots of plants fed high or low phosphorus. The mycorrhizal soil had significantly more water stable aggregates and substantially higher extraradical hyphal densities than the nonmycorrhizal soils. Importantly, we were able to factor out the possibly confounding influence of differential root growth among mycorrhizal and nonmycorrhizal soils. Mycorrhizal symbiosis affected the soil moisture characteristic and soil structure, even though root mass, root length, root surface area and root volume densities were similar in mycorrhizal and nonmycorrhizal soils.