The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.     

Effect of electrical stimulation of the LES on LES pressure in a canine model

Gastric electrical stimulation modulates lower esophageal sphincter pressure (LESP). High-frequency neural stimulation (NES) can induce gut smooth muscle contractions. To determine whether lower esophageal sphincter (LES) electrical stimulation (ES) can affect LESP, bipolar electrodes were implanted in the LES of four dogs. Esophageal manometry during sham or ES was performed randomly on separate days. Four stimuli were used: 1) low-frequency: 350-ms pulses at 6 cycles/min; 2) high-frequency-1: 1-ms pulses at 50 Hz; 3) high-frequency-2: 1-ms pulses at 20 Hz; and 4) NES: 20-ms bipolar pulses at 50 Hz. Recordings were obtained postprandially. Tests consisted of three 20-min periods: baseline, stimulation/sham, and poststimulation. The effect of NES was tested under anesthesia and following IV administration of l-NAME and atropine. Area under the curve (AUC) and LESP were compared among the three periods, by ANOVA and t-test, P < 0.05. Data are shown as means +/- SD. We found that low-frequency stimulation caused a sustained increase in LESP: 32.1 +/- 12.9 (prestimulation) vs. 43.2 +/- 18.0 (stimulation) vs. 50.1 +/- 23.8 (poststimulation), P < 0.05. AUC significantly increased during and after stimulation. There were no significant changes with other types of ES. With NES, LESP initially rose and then decreased below baseline (LES relaxation). During NES, N(G)-nitro-l-arginine methyl ester increased both resting LESP and the initial rise in LESP and markedly diminished the relaxation. Atropine lowered resting LESP and abolished the initial rise in LESP. In conclusion, low frequency ES of the LES increases LESP in conscious dogs. NES has dual effect on LESP: an initial stimulation, cholinergically mediated, followed by relaxation mediated by nitric oxide.     

Effects of herbivory, nutrient levels, and introduced algae on the distribution and abundance of the invasive macroalga Dictyosphaeria cavernosa in Kaneohe Bay, Hawaii

Since the 1960s, and possibly earlier, the macroalga Dictyosphaeria cavernosa has overgrown and displaced corals on reef slopes and outer reef flats in Kaneohe Bay, Oahu. This shift in reef community composition is generally attributed to nutrient enrichment resulting from sewage discharge. Following the diversion of most of the sewage effluent in 1977-1978, it was expected that D. cavernosa growth would become nutrient-limited and its abundance would consequently decline, but the alga remains abundant in much of the bay. One explanation for its persistence is that nutrients are once again high enough to support the alga's growth. An alternative explanation is that there has been a reduction in grazing intensity in the bay. In this study we resurveyed the distribution and abundance of D. cavernosa at 120 reef slope sites originally surveyed in 1969. We conducted additional surveys to estimate the biomass of herbivores and the areal coverage of D. cavernosa and other macroalgae on reef slopes and flats. Field experiments were used to determine spatial and temporal patterns of grazing intensity on and growth rates of D. cavernosa and the introduced macroalga Acanthophora spicifera. Laboratory experiments were used to examine preferences among herbivores for some of the most abundant macroalgae on Kaneohe Bay reefs. Twenty years after sewage diversion, D. cavernosa cover on reef slopes has decreased substantially in southern Kaneohe Bay, the site of most of the historical sewage discharge. D. cavernosa cover has changed less in other regions, remaining high in the central bay and low in the north bay. D. cavernosa thalli protected by grazer exclusion cages sustained positive growth rates on reef slopes and flats throughout the bay. Reduced nutrient concentrations may have caused a reduction in D. cavernosa growth rates, and a consequent reduction in D. cavernosa abundance in the south bay shortly after sewage diversion. Measurements of grazing intensity and surveys of herbivorous fish abundance suggest that the continued abundance of D. cavernosa is the result of a reduction in grazing intensity. Reduced grazing intensity on D. cavernosa may in turn be the result of a historical reduction in herbivore biomass or the establishment of several introduced macroalgae on reef flats. The introduced species are preferred by herbivorous fishes over D. cavernosa, as indicated by preference tests. The hypothesis that reduced grazing pressure on D. cavernosa is related to the establishment of introduced species is supported by the observation that D. cavernosa cover is highest on reef slopes where the cover of preferred introduced macroalgae on the adjacent outer reef flat is also high. Conversely, D. cavernosa cover is low or zero on reef slopes where the cover of introduced macroalgae on the adjacent reef flat is low or zero     

Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.     

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster

In invertebrates and vertebrates, innate immunity is considered the first line of defense mechanism against non-self material. In vertebrates, cytokines play a critical role in innate immune signalling. To date, however, the existence of genes encoding for invertebrate helical cytokines has been anticipated, but never demonstrated. Here, we report the first structural and functional evidence of a gene encoding for a putative helical cytokine in Drosophila melanogaster. Functional experiments demonstrate that its expression, as well as that of the antimicrobial factors defensin and cecropin A1, is significantly increased after immune stimulation. These observations suggest the involvement of helical cytokines in the innate immune response of invertebrates.