RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.     

Proteomic analysis of resistance mediated by Rcm 2.0 and Rcm 5.1, two loci controlling resistance to bacterial canker of tomato

Two quantitative trait loci from Lycopersicon hirsutum, Rcm 2.0 and Rcm 5.1, control resistance to Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato. Lines containing Rcm 2.0 and Rcm 5.1 and a susceptible control line were compared at 72 and 144 h postinoculation, using 2-dimensional gel electrophoresis to identify proteins regulated in response to C. michiganensis subsp. michiganensis infection. A total of 47 proteins were subjected to tandem mass spectrometry. Database queries with resulting spectra identified tomato genes for 26 proteins. The remaining 21 proteins were either identified in other species or possessed no homology to known proteins. Spectra were interpreted to deduce peptide amino acid sequences that were then used to query publicly available data. This approach identified tomato genes or expressed sequence tags for 44 of the proteins analyzed. Three superoxide dismutase (SOD) enzymes were differentially regulated among genotypes, and patterns of hydrogen peroxide accumulation were genotype- and tissue-specific, indicating a role for oxidative stress in response to C. michiganensis subsp. michiganensis. Steady-state mRNA and protein levels for SOD, thioredoxin M-type, S-adenosylhomocysteine hydrolase, and pathogenesis-related proteins demonstrated similar patterns of differential regulation. Lines containing Rcm 2.0 and Rcm 5.1 accumulate different proteins and steady-state mRNAs in response to inoculation, suggesting that the two loci may confer resistance through distinct mechanisms.     

Pollen assemblages as paleoenvironmental proxies in the Florida Everglades

Analysis of 170 pollen assemblages from surface samples in eight vegetation types in the Florida Everglades indicates that these wetland sub-environments are distinguishable from the pollen record and that they are useful proxies for hydrologic and edaphic parameters. Vegetation types sampled include sawgrass marshes, cattail marshes, sloughs with floating aquatics, wet prairies, brackish marshes, tree islands, cypress swamps, and mangrove forests. The distribution of these vegetation types is controlled by specific environmental parameters, such as hydrologic regime, nutrient availability, disturbance level, substrate type, and salinity; ecotones between vegetation types may be sharp. Using R-mode cluster analysis of pollen data, we identified diagnostic species groupings; Q-mode cluster analysis was used to differentiate pollen signatures of each vegetation type. Cluster analysis and the modern analog technique were applied to interpret vegetational and environmental trends over the last two millennia at a site in Water Conservation Area 3A. The results show that close modern analogs exist for assemblages in the core and indicate past hydrologic changes at the site, correlated with both climatic and land-use changes. The ability to differentiate marshes with different hydrologic and edaphic requirements using the pollen record facilitates assessment of relative impacts of climatic and anthropogenic changes on this wetland ecosystem on smaller spatial and temporal scales than previously were possible.     

The GTP-binding protein G(ialpha) translocates to kinetochores and regulates the M-G(1) cell cycle transition of Swiss 3T3 cells

The receptor-generated signals that are responsible for driving the cell cycle are incompletely characterised in mammalian cells. It is clear, however, that the cellular messenger systems that stimulate DNA synthesis and mitosis are separable. These are interwoven with biochemical checkpoints that ensure that processes, such as chromosomal replication and microtubule attachment to duplicated chromosomes, are complete before the following phase of the cell cycle is initiated. In some cells, activation of DNA synthesis by factors such as LPA and serum has been shown to require the GTP-binding protein G(i). We have found that G(i) plays an additional role in mitosis activated by both 7-transmembrane receptors and tyrosine kinase receptors, and that this involves the translocation of the alpha-subunit of G(i) (G(ialpha)) to the nucleus. Here we show by confocal microscopy that G(ialpha)migrates to the nucleus near the onset of mitosis in serum-activated Swiss 3T3 cells and binds to the kinetochore region of replicated chromosomes. Inhibition of G(i) function with pertussis toxin had no effect on the induction of DNA synthesis by serum, but cell proliferation was inhibited. Flow cytometric analysis showed that this resulted from retardation of the transition through mitosis and into G(1). Additionally, pertussis toxin impaired the activity of p34(cdc2), a cyclin-dependent kinase involved in the transition from M-phase to G(1), but not the S-phase cyclin, cyclin E. These data show that the G-protein G(i) has a key role in the regulation of mitosis in fibroblasts.     

Modeling the clonal heterogeneity of stem cells

Recent experimental studies suggest that tissue stem cell pools are composed of functionally diverse clones. Metapopulation models in ecology concentrate on collections of populations and their role in stabilizing coexistence and maintaining selected genetic or epigenetic variation. Such models are characterized by expansion and extinction of spatially distributed populations. We develop a mathematical framework derived from the multispecies metapopulation model of Tilman et al (1994) to study the dynamics of heterogeneous stem cell metapopulations. In addition to normal stem cells, the model can be applied to cancer cell populations and their response to treatment. In our model disturbances may lead to expansion or contraction of cells with distinct properties, reflecting proliferation, apoptosis, and clonal competition. We first present closed-form expressions for the basic model which defines clonal dynamics in the presence of exogenous global disturbances. We then extend the model to include disturbances which are periodic and which may affect clones differently. Within the model framework, we propose a method to devise an optimal strategy of treatments to regulate expansion, contraction, or mutual maintenance of cells with specific properties.     

Exploration of the P1 SAR of aldehyde cathepsin K inhibitors

The synthesis and biological activity of a series of aldehyde inhibitors of cathepsin K are reported. Exploration of the properties of the S(1) subsite with a series of alpha-amino aldehyde derivatives substituted at the P(1) position afforded compounds with cathepsin K IC(50)s between 52 microM and 15 nM.     

Artificial insemination following observational versus electronic methods of estrus detection in red deer hinds (Cervus elephus)

The objectives were to determine the efficacy of the HeatWatch (HW) electronic estrus detection system for monitoring behavioral estrus (including duration and intensity) in red deer hinds and to evaluate pregnancy rate to AI after detected estrus. Red deer hinds (Cervus elephus; n = 50) were allocated into two treatment groups: AI following synchronization (CIDR/PMSG) and observed estrus (induced estrus group: IE; n = 25) or AI following the detection of natural estrus (NE; n = 25) without hormonal treatment. Hinds were fitted with two HeatWatch (HW) electronic estrus detection transmitters, one above the tail (bottom) and one between the tuber coxae of the pelvic girdle (top), and visual observations for mounting activity began with the aid of young sterile red deer stags (18 months old) fitted with marking harnesses. Hinds in both groups were inseminated (10-12h after observed estrus) with frozen-thawed red deer semen using a transvaginal/cervical AI approach. Following a 26-day period of AI, hinds were placed with a mature fertile stag for an additional 30-day natural breeding period. Pregnancy diagnosis was performed 57 and 86 days after the start of AI. While the hinds were housed with the young stags, 82% were detected in estrus by visual appraisal of stag crayon marks, but only 32% of these were detected by HW. In contrast, in the hinds housed with the mature stag, 93% detected in estrus by crayon marks were also detected by HW. The top HW transmitter consistently recorded more mounts (P < 0.05) than the bottom transmitter. The pregnancy rate was numerically better in IE versus NE hinds (42% versus 29%, P > 0.10). In summary, there were no differences (P > 0.10) in the intensity (number) or duration of mounts (detected by HW) during estrus in IE versus NE hinds, and HW was most effective in detecting estrus in the presence of a heavier, mature stag versus a younger stag. When used in combination with transvaginal AI, an overall first-service pregnancy rate of 36.6% was achieved with AI of frozen-thawed semen in red deer hinds following detected estrus.     

A Review of: “Progress in Separation and Purification Vol. 3, E. S. Perry and C. J,van Oss,eds., Willy/Intersciencc,New York, 1970; xi + 316 pares; hard cover; 515.95”

The optimal culture conditions of exopolysaccharides (EPS) production in submerged culture medium by Pleurotus geesteranus 5 ^# were determined using an orthogonal matrix method. The optimal defined medium (per liter) was 60.0 g maltose, 5.0 g tryptone, 1 mM NaCl, 5 mM KH₂PO₄, and initial pH 6.0 at 28 °C. In the optimal culture medium, the maximum EPS production was 16.97 g/L in a shake flask. Two groups of EPSs (designated as Fr-I and Fr-II) were obtained from the culture filtrates by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The approximate weight-average molar masses of the Fr-I and Fr-II of EPS were determined to be 3.263 × 10⁴ and 5.738 × 10³ g/mol, respectively. The low values of polydispersity ratio (1.176 and 1.124 for Fr-I and Fr-II, respectively) of EPSs mean that these EPS molecules exist much less dispersed in aqueous solution without forming large aggregates. Furthermore, the experiments in vitro indicated that P. geesteranus 5^# EPS exhibit high antitumor and antioxidative effects.     

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.