Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this limit, which may explain why it has the smallest known mitotic spindle that still manifests the classic congression architecture.     

Tracking Public Beliefs About Anthropogenic Climate Change

A simple question about climate change, with one choice designed to match consensus statements by scientists, was asked on 35 US nationwide, single-state or regional surveys from 2010 to 2015. Analysis of these data (over 28,000 interviews) yields robust and exceptionally well replicated findings on public beliefs about anthropogenic climate change, including regional variations, change over time, demographic bases, and the interacting effects of respondent education and political views. We find that more than half of the US public accepts the scientific consensus that climate change is happening now, caused mainly by human activities. A sizable, politically opposite minority (about 30 to 40%) concede the fact of climate change, but believe it has mainly natural causes. Few (about 10 to 15%) say they believe climate is not changing, or express no opinion. The overall proportions appear relatively stable nationwide, but exhibit place-to-place variations. Detailed analysis of 21 consecutive surveys within one fairly representative state (New Hampshire) finds a mild but statistically significant rise in agreement with the scientific consensus over 2010–2015. Effects from daily temperature are detectable but minor. Hurricane Sandy, which brushed New Hampshire but caused no disaster there, shows no lasting impact on that state’s time series—suggesting that non-immediate weather disasters have limited effects. In all datasets political orientation dominates among individual-level predictors of climate beliefs, moderating the otherwise positive effects from education. Acceptance of anthropogenic climate change rises with education among Democrats and Independents, but not so among Republicans. The continuing series of surveys provides a baseline for tracking how future scientific, political, socioeconomic or climate developments impact public acceptance of the scientific consensus.     

A purine permease in Candida glabrata

Abstract Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative mutant. The C. glabrata 4 cytosine permease negative mutant was isolated using 5-fluorocytosine selection. This mutant no longer transported cytosine, but transported adenine and guanine. A transport system for hypoxanthine was not detected. Hence, in addition to the cytosine permease, a purine permease exists in C. glabrata . This differs from the purine cytosine permeases in Saccharomyces cereuisiae and Candida albicans which transport adenine, cytosine, guanine and hypoxanthine.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.     

Persistence of Epstein-Barr Virus in Self-Reactive Memory B Cells

Epstein-Barr virus infection has been epidemiologically associated with the development of multiple autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis. Currently, there is no known mechanism that can account for these associations. The germinal-center (GC) model of EBV infection and persistence proposes that EBV gains access to the memory B cell compartment via GC reactions by driving infected cells to differentiate using the virus-encoded LMP1 and LMP2a proteins, which act as functional homologues of CD40 and the B cell receptor, respectively. The ability of LMP2a, when expressed in mice, to allow escape of autoreactive B cells suggests that it could perform a similar role in infected GC B cells, permitting the survival of potentially pathogenic autoreactive B cells. To test this hypothesis, we cloned and expressed antibodies from EBV⁺ and EBV⁻ memory B cells present during acute infection and profiled their self- and polyreactivity. We find that EBV does persist within self- and polyreactive B cells but find no evidence that it favors the survival of pathogenic autoreactive B cells. On the contrary, EBV⁺ memory B cells express lower levels of self-reactive and especially polyreactive antibodies than their uninfected counterparts do. Our work suggests that EBV has only a modest effect on the GC process, which allows it to access and persist within a subtly unique niche of the memory compartment characterized by relatively low levels of self- and polyreactivity. We suggest that this might reflect an active process where EBV and its human host have coevolved so as to minimize the virus''s potential to contribute to autoimmune disease.     

Effect of domestic cattle on the condition of female white-tailed deer in southern pine-bluestem forests,USA

Effect of domestic cattle stocking on the nutritional condition of white-tailed deerOdocoileus virginianus (Zimmermann, 1780) was assessed using physiological indices of collected specimens. Three study areas were delineated in McCurtain County, Oklahoma (heavy cattle stocking), and Howard (moderate to light cattle stocking) and Pike (no cattle stocking) counties, Arkansas that were similar with respect to soils and vegetation but differed with respect to cattle stocking rate. Female white-tailed deer were collected from study areas in February and August 1987–1988 to assess nutritional condition. Deer collected from study areas exposed to cattle grazing in February had lower carcass weights, fat attributes (femur marrow and kidney fat), and reproductive rates (fetuses/doe) than deer that were not exposed to cattle grazing. In August, deer collected from the moderate cattle area had heavier eviscerated carcass weights, serum glucose, albumin, and albumin/globulin ratios than deer collected from the heavy cattle area. Results suggest that if cattle are removed from managed forests in winter, nutritional condition of deer would be improved because of reduced competition for food.