Testing cyclin specificity in the exit from mitosis

Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis. Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated. Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division. CLB2-Deltadb overexpression that was quantitatively comparable to CLB5-Deltadb overexpression with respect to Clb protein production and Clb-associated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA. Simultaneous overexpression of CLB2-Deltadb and CLB5-Deltadb overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-Deltadb overexpression alone. These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion. We speculate that CLB5-Deltadb overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle.     

In vivo ozone exposure induces antioxidant/stress-related responses in murine lung and skin

Lung and skin are the organs directly exposed to environmental pollution. Ozone (O(3)) is a toxic, oxidant air pollutant, and exposure has been shown to induce antioxidant depletion as well as oxidation of lipids and proteins within the outermost skin layer (stratum corneum) and the lung respiratory tract lining fluids (RTLFs). To further define skin and lung responses to O(3) exposure, SKH-1 hairless mice were exposed to either 0.8 ppm of O(3) (a level occasionally reached in very polluted areas) or ambient air 6 h/day for 6 consecutive days. O(3) exposure resulted in the depletion of alpha-tocopherol in lung and plasma and induction in both skin and lung of heme oxygenase 1, cyclooxygenase 2, and proliferating cell nuclear antigen. O(3)-exposed animals showed a similar extent of upregulation of COX-2 and PCNA in lung and skin, whereas HO-1 was more responsive in skin than in lung (7-fold induction vs. 2-fold induction). In addition to these measures of response to oxidative stress, O(3) exposure led to the activation of nuclear factor kappaB measured as IkappaBalpha phosphorylation in both tissues. We conclude that in this model, O(3) at high pollutant levels is able to affect both lung and skin biology, inducing depletion of alpha-tocopherol and inducing stress-related responses in both skin epidermis and respiratory tract epithelium.     

Using stable isotope analysis to assess the effects of hypolimnetic oxygenation on diet in a mixed cold- and warmwater fish community

Line-diffuser hypolimnetic oxygenation was initiated in North Twin Lake, Washington, in 2009 to mitigate reductions in Rainbow Trout (Oncorhynchus mykiss) and Brook Trout (Salvelinus fontinalis) habitat due to temperature-dissolved oxygen “habitat squeeze”. Previous studies demonstrated that trout populations rapidly expanded into increased hypolimnetic habitat within the first few years of oxygenation and previous short-term diet analyses indicated an effect on fish diet; however, the long-term effects on fish ecology have yet to be established. In this study, stable isotope analysis of fish liver tissue suggests relatively few differences in feeding ecology of principal coldwater fish species in North Twin Lake compared to unoxygenated South Twin Lake. When compared between lakes, Rainbow Trout and Brook Trout diets contained similar proportions of Daphnia, Chironomidae, and Chaoboridae. Littoral and epilimnetic-focused Golden Shiner (Notemigonus crysoleucas) diets were also similar between lakes. Observed similarities between Golden Shiner and trout diets suggest the effects of interspecific competition between salmonids and non-salmonids may be limiting trout growth and survival. Fisheries managers should therefore consider both habitat limitations and interspecific competition when managing for coldwater fish species in mesotrophic, dimictic lakes.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Mitotic Exit in the Absence of Separase Activity

In budding yeast, three interdigitated pathways regulate mitotic exit (ME): mitotic cyclin–cyclin-dependent kinase (Cdk) inactivation; the Cdc14 early anaphase release (FEAR) network, including a nonproteolytic function of separase (Esp1); and the mitotic exit network (MEN) driven by interaction between the spindle pole body and the bud cortex. Here, we evaluate the contributions of these pathways to ME kinetics. Reducing Cdk activity is critical for ME, and the MEN contributes strongly to ME efficiency. Esp1 contributes to ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity, we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. We defined the first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor. This measure indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. We also found a novel role for the MEN: activating Cdc14 nuclear export, even in the absence of Net1.     

Telemetric monitoring of body temperature in the horse mare

The objective of this study was to develop a radiotelemetric system capable of frequent monitoring of mare body temperature. A radio transmitter was implanted in the flank of each of four mares. Telemetered data were received by a pair of antennae placed at right angles in a 3.3 x 6.6-m stall and stored on a computer hard disk. The data were recorded every 5 minutes except when mares were out of the stall for a 1- to 2- hour exercise period. No effect of environmental temperature, ranging from 5 degrees C to 30 degrees C, on mare body temperature was apparent. The radiotelemetric system used in this study was effective for frequent measurement of mare body temperature.     

The use of two populations of hepatocytes with different triacylglycerol contents as a model to study the accumulation of liver lipid in the laying hen.

(1) A method has been developed to separate hepatocytes, isolated from laying hens, according to their densities, using discontinuous density-gradient centrifugation on Nycodenz. (2) The hepatocytes recovered from the interface of the 5% and 10% Nycodenz layers were rich in triacylglycerol and were termed 'fatty' hepatocytes: 'non-fatty' hepatocytes were obtained from the interface of the 15% and 30% Nycodenz layers and contained less than one-quarter as much triacylglycerol. (3) 'Fatty' hepatocytes incorporated radiolabelled glucose and glycerol into total lipid at more than twice the rate of 'non-fatty' cells: the corresponding increases in the incorporation of radiolabelled choline and valine into phospholipid and protein respectively were smaller and not statistically significant. (4) A higher proportion of glycerol and glucose incorporated into total lipid was found to be phospholipid in the 'non-fatty' hepatocytes. (5) A higher proportion of radiolabelled lipid or protein formed from glycerol or valine respectively was secreted into the medium by the 'non-fatty' hepatocytes. (6) The use of these hepatocytes as a model to study fatty liver syndromes is discussed.     

Fitness reduction and potential extinction of wild populations of Atlantic salmon, Salmo salar, as a result of interactions with escaped farm salmon

The high level of escapes from Atlantic salmon farms, up to two million fishes per year in the North Atlantic, has raised concern about the potential impact on wild populations. We report on a two-generation experiment examining the estimated lifetime successes, relative to wild natives, of farm, F(1) and F(2) hybrids and BC(1) backcrosses to wild and farm salmon. Offspring of farm and "hybrids" (i.e. all F(1), F(2) and BC(1) groups) showed reduced survival compared with wild salmon but grew faster as juveniles and displaced wild parr, which as a group were significantly smaller. Where suitable habitat for these emigrant parr is absent, this competition would result in reduced wild smolt production. In the experimental conditions, where emigrants survived downstream, the relative estimated lifetime success ranged from 2% (farm) to 89% (BC(1) wild) of that of wild salmon, indicating additive genetic variation for survival. Wild salmon primarily returned to fresh water after one sea winter (1SW) but farm and 'hybrids' produced proportionately more 2SW salmon. However, lower overall survival means that this would result in reduced recruitment despite increased 2SW fecundity. We thus demonstrate that interaction of farm with wild salmon results in lowered fitness, with repeated escapes causing cumulative fitness depression and potentially an extinction vortex in vulnerable populations.