Inflammatory characteristics of premature coronary artery disease.

Although inflammation is a key process in atherogenesis, little is known about the inflammatory characteristics of culprit plaque in premature coronary artery disease (CAD). We investigated inflammation in coronary atheroma from subjects who died of premature CAD. From 2001-2005, we collected coronary plaque samples from consecutive cases of CAD (n=23) reported to the Department of Forensic Medicine which led to unexpected death in men aged <45 years. Coronary plaque from younger CAD decedents (<35 years, n=12) had lower levels of T cells (CD3+) (p=0.03), higher macrophage (CD 68+) (p=0.01) and T regulator cells (FOXP3+) (p=0.03) infiltration when compared to older CAD decedents (>35 years, n=11). Interestingly, there was no significant age-related difference between groups in the smooth muscle cell, apoA-I, myeloperoxidase and MMP-2 content within plaque. Hence, we demonstrate that higher expression of FOXP3 is associated with younger age at the time of fatal outcomes from CAD. These findings may have implications for plaque pathophysiology and thus warrant further investigation.

The yeast centrin, cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity.

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21- activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST-Kic1p and it bound to GST-Kic1p in gel overlay assays. Furthermore, GST-Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo.

We have isolated cold-sensitive mutations in two genes of the yeast Saccharomyces cerevisiae, BIN2 and BIN3, that cause aberrant chromosome segregation in vivo. BIN2 and BIN3 encode essential proteins that are similar to each other and to TCP-1. TCP-1 and TCP-1-like proteins are components of the eukaryotic cytoplasmic chaperonin that facilitates folding of tubulins and actin in vitro. Mutations in BIN2 and BIN3 cause defects in microtubule and actin assembly in vivo and confer supersensitivity to the microtubule-destabilizing drug benomyl. Overexpression of TCP1, BIN2, BIN3, or ANC2, a fourth member of the TCP-1 family in yeast, does not complement mutations in the other genes, indicating that the proteins have distinct functions. However, all double-mutant combinations are inviable; this synthetic lethality suggests that the proteins act in a common process. These results indicate that Bin2p and Bin3p are components of a yeast cytoplasmic chaperonin complex that is required for assembly of microtubules and actin in vivo.

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae.

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.

Use of predator odors as repellents to reduce feeding damage by herbivores : III. Montane and meadow voles (Microtus montanus andMicrotus pennsylvanicus).

This study investigated the influence of the major anal-gland compounds from the stoat (Mustela erminea) and fecal and urine compounds from the red fox (Vulpes vulpes) in generating an avoidance response by montane voles (Microtus montanus), as well as suppressing feeding by montane and meadow (M. pennsylvanicus) voles on apple trees in orchards. In trap bioassays, a 1∶1 mixture of 2-propylthietane and 3-propyl-1,2-dithiolane significantly reduced vole captures. Other mixtures of stoat compounds reduced the number of new voles captured but not total individuals. 2,5-Dihydro-2,4,5-trimethylthiazoline, a component of fox feces, significantly reduced vole captures in one of two bioassays. Deer mice (Peromyscus maniculatus) did not show a negative response to any predator odor. In overwinter field bioassays, mixtures of 2-propylthietane and 3-propyl-1,2-dithiolane clearly reduced vole feeding on apple trees in four test blocks. 2,5-Dihydro-2,4,5-trimethylthiazoline and a synthetic fox urine mixture also significantly reduced vole attack in respective orchard blocks. Similarly, the intensity of vole feeding, in terms of amount of bark and vascular tissues removed from trees, was reduced by 60% to 97% in predator odor treatments compared with the control. Our study reports the first long-term (four to five months) use of synthetic semiochemicals as area repellents for crop protection from vole feeding damage.

Use of predator odors as repellents to reduce feeding damage by herbivores : IV. Northern pocket gophers (Thomomys talpoides).

This study investigated the influence of the major anal-gland compounds from the stoat (Mustela erminea) and ferret (M. putorius) in generating an avoidance response by northern pocket gophers (Thomomys talpoides) in tree fruit orchards in the Okanagan Valley of British Columbia, Canada. A secondary objective assessed the impact of additional predator odors on gopher avoidance behavior in laboratory bioassays. In field bioassays, a 1: 1 mixture of 2-propylthietane and 3-propyl-1,2-dithiolane, as well as 3,3-dimethyl-1,2-dithiolane, placed in gopher burrows did not reduce the number of gophers colonizing treatment versus control grids in orchard blocks. However, these predator gophers did dramatically alter the distribution of gophers. Significantly more gophers were captured at perimeter than nonperimeter trap stations on treatment versus control grids in two of three orchards. In all orchards, significantly more gophers were captured at perimeter stations after the predator odors had been placed in burrows than prior to the start of the experiment. Gophers clearly avoided 2,5-dihydro-2,4,5-trimethylthiazoline, a component of fox (Vulpes vulpes) feces, but did not avoid 2,2-dimethylthietane from the mink (M. vison) or 3-methyl-3-butenyl methyl sulfide from fox urine in laboratory bioassays. Poor avoidance was also recorded for 3,3-dimethyl-1,2-dithiolane, although this may be due to the state of polymerization of this compound. An improved formulation is required to dispense these semiochemicals in controlled-release devices within orchards and other forest-agricultural areas.

Use of predator odors as repellents to reduce feeding damage by herbivores : I. Snowshoe hares (Lepus americanus).

The effectiveness of predator odors (fecal, urine, and anal scent gland) in suppressing feeding damage by snowshoe hares was investigated in pen bioassays at the University of British Columbia Research Forest, Maple Ridge, British Columbia, Canada. A total of 28 bioassay trials tested the effects of these odors on hare consumption of willow browse and coniferous seedlings. Lynx and bobcat feces, weasel anal gland secretion, and lynx, bobcat, wolf, coyote, fox, and wolverine urines resulted in the most effective suppression of hare feeding damage. Novel odors of domestic dog urine and 2-methylbutyric acid did not reduce feeding. A field bioassay with lodgepole pine seedlings and weasel scent provided significant results comparable to the pen bioassays. The short-term (up to seven days) effectiveness of these treatments was more likely due to evaporative loss of the active repellent components of a given odor than habituation of hares to the stimulus. Predator odors as repellents have a biological basis compared with the anthropomorphic origins of commercial repellents. When encapsulated in weather-proof controlled-release devices, these odors could provide long-term protection for forestry plantations and agricultural crops which experience hare/rabbit feeding damage.

Use of predator odors as repellents to reduce feeding damage by herbivores : II. Black-tailed deer (Odocoileus hemionus columbianus).

The effectiveness of predator odors (fecal and urine) in suppressing feeding damage by black-tailed deer was investigated in pen bioassays at the University of British Columbia Research Forest, Maple Ridge, British Columbia, Canada. A total of eight bioassay trials tested the effects of these odors on deer consumption of salal leaves and coniferous seedlings. Cougar, coyote,and wolf feces as well as coyote, wolf, fox, wolverine, lynx, and bobcat urines provided the most effective suppression of deer feeding damage. Novel odors of ammonia and human urine did not reduce feeding. Predator fecal odor formulations in direct foliar application, adhesive application, and in plastic vials were all effective in suppressing deer feeding. Of all urines tested, coyote provided the most consistent suppression of deer browsing on salal. Deer consumed significantly more untreated Douglas fir and western red cedar seedlings than those protected by coyote urine odor. The active repellent components of predator odors which suppress deer feeding may be suitable for encapsulation in controlled-release devices which could provide long-term protection for forest and agricultural crops.

Experimental studies of rainbow trout populations exposed to field applications of Roundup herbicide.

This paper reports the effects of Roundup herbicide (MON 02139) on rainbow trout viability and behavior in several field experiments at the University of British Columbia Research Forest. Laboratory and field 96-hr LC50 values were similar: 54.8 and 52.0 mg/L. Avoidance-preference data indicated that fish would avoid lethal levels of Roundup. Operational application of Roundup at the recommended field dose of (2.2 kg a.e./ha), as well as 10x and 100x field dose resulted in no mortality to rainbow trout in field streams. Results indicate that operational spraying with this herbicide for weed control should not be detrimental to rainbow trout populations. Improper use or accidental spills of Roundup could be avoided by rainbow trout and should not be lethal if diluted in a moderately-flowing stream.