Live-cell analysis of mitotic spindle formation in taxol-treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.     

Effects of parity, season, gonadotropin releasing hormone and altered suckling intensity on the interval to rebreeding in sows

Two experiments were conducted to determine the effects of a) parity and season of the year on the interval from weaning to rebreeding in sows and b) altered suckling intensity (ASI) and gonadotropin releasing hormone (GnRH) on the postpartum interval in primiparous and multiparous sows. Experiment I included 406 weaning-to-rebreeding intervals in 172 primiparous and multiparous sows. Primiparous sows returning to estrus during the spring and summer months had a longer (P<0.001) rebreeding interval (11.0 d) than the other groups (5.7 d). In Experiment II, 32 sows were assigned to a factorially designed experiment. The factors were ASI, GnRH and parity. Treatments were begun 7 d before weaning (about 4 wk of age). The ASI was accomplished by separating the sow from her litter for 12 h each day. The average interval from the beginning of treatment to the onset of estrus was 13.4 d and was not affected (P>0.33) by ASI, GnRH, parity or their interactions. None of the factors was found to affect the average weaning weight of the piglets (P>0.05); however, piglets in the ASI group were heavier (P<0.03) at 1-wk postweaning than those in the no-ASI group. The results showed that primiparous sows returning to estrus during the spring and summer months had the longest rebreeding interval. Additionally, neither GnRH nor ASI, separately or in conjunction, decreased the postpartum interval in sows.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.     

Effects of composition and density of the group on the performance,behaviour and age at puberty in swine

Two hundred and eighteen cross-bred pigs (Duroc × Hampshire × Landrace × Yorkshire) were assigned to a 3 × 2 factorial experiment; the factors being composition of the group and density. The composition of the group factor consisted of pigs not reaggregated (NR), re-aggregated below the median body weight (RBMW), and re-aggregated above the median body weight (RAMW) at the time of transfer to the finishing pens. The density factor consisted of groups of 6 or 12 pigs/pen. Two trials with 2 replications of each trial were conducted. Twenty-six gilts from Trial I were used to determine their age and ovulation rate at the pubertal oestrus. During the nursery phase, composition of the group had no effect (P>0.05) on average daily feed intake (ADFI) or feed efficiency (F/G). Pigs in the NR and RAMW groups had a similar (P>0.05) average daily gain (ADG), whereas, those pigs in the RBMW group gained (P<0.05) at a slower rate. Pigs housed in groups of 6 had a higher (P<0.05) ADFI, had similar (P>0.05) ADG and poorer (P<0.05) F/G than those housed in groups of 12. During the finishing phase, composition of the group had no effect (P>0.05) on ADFI, ADG or F/G. Pigs housed in groups of 6 had a greater (P<0.05) ADFI and ADG, but similar (P<0.05) F/G compared to those housed in groups of 12. Correlation coefficients between the various behaviours and the performance variables suggested that the more active pens of pigs gained less per day and were less efficient. Gilts housed in the NR groups of 6 were younger (P<0.05) at puberty than gilts in the other groups of 6 or 12. Also, gilts housed in the RBMW groups of 6 were younger (P<0.05) at puberty than those housed in the RAMW groups of 6, whereas those housed in the RBMW groups of 12 were older (P<0.05) at puberty than those housed in the RAMW groups of 12. Ovulation rate was not affected (P>0.05) by composition of the group, density or their interaction. In summary, composition and density of the group affected the performance of pigs and was correlated to some of the behavioural measurements. The most noticeable effect was that these same factors caused a delay in the onset of puberty in gilts.     

Time of onset of estrus in gilts

An experiment was conducted to determine when, during a 24-h period, gilts show the onset of behavioral estrus. Beginning on Day 16 of their first estrous cycle, 42 crossbred gilts were observed at 0600, 1200, 1800, and 2400 h for the onset of their second estrus. Fifty-five percent of the gilts had shown onset of estrus by 0600 h. None of the gilts showed the onset of estrus from 0600 to 1200 h, whereas 24% and 21% of the gilts had shown onset of estrus by 1800 and 2400 h, respectively. Chi-square analysis demonstrated that more (P < 0.025) gilts had shown onset of estrus by 0600 than by 1200, 1800, and 2400 h. When the data were combined for estrous checks by 0600 and 1800 h, 76% of the gilts had their onset of estrus by 0600 h as compared to 24% of the gilts by 1800 h (P < 0.005). In conclusion, more gilts had shown onset of estrus by 0600 h than at any other 6-h period.     

Classification of videocapsule endoscopy image patterns: comparative analysis between patients with celiac disease and normal individuals

Background  

Quantitative disease markers were developed to assess videocapsule images acquired from celiac disease patients with villous atrophy, and from control patients.     

Regulation of mitochondrial compartment volumes in rat adrenal cortex by ether stress

In vivo ether stress of rats causes release of pituitary adrenocorticotropin (ACTH) leading to activation of steroidogenesis in adrenal cortex mitochondria. The present studies show that this treatment also induces a decrease in the volume of the intermembrane space in isolated adrenal mitochondria. This decrease is accompanied by an increase in the volume of the matrix, thus leaving the total mitochondrial volume approximately constant. These effects are prevented by the protein synthesis inhibitor, cycloheximide, and are specific to the adrenal gland. The decrease in the intermembrane space (or increase in the matrix volume) is correlated with activation of the cholesterol side chain cleavage reaction (the regulated step in steroidogenesis). We propose as a working hypothesis that these changes reflect a hormonally regulated alteration in the relationship between the outer and inner mitochondrial membranes, which may facilitate the rate-limiting movement of cholesterol from the outer to the inner membrane where the side chain cleavage enzyme is located.