Functional molecular mass of the 14C-azidobenzamidotaurocholic acid binding proteins in hepatocellular bile acid transport systems.

The apparent target size of 14C-azidobenzamidotaurocholate binding proteins in basolateral rat liver plasma membranes (blPm) was determined by analysis of the radiation induced decrease of the binding of this photoreactive taurocholate analog to blPm. Radiation causes a dose-dependent mono-exponential reduction of binding of ABATC to the protein subunits with molecular masses of 48-50 and 52-54 kDa in SDS-PAGE. The minimal functional molecular mass of the 48-50 and 52-54 kDa ABATC binding proteins was determined to be 99 +/- 8.2 and 93.2 +/- 7 kDa, respectively.     

A combined radioimmunoassay for the measurement of unconjugated and sulfoconjugated pregnenolone, 17 hydroxypregnenolone, dehydroepiandrosterone, and estrone applied to fetal and maternal ovine plasma

This report describes a radioimmunoassay (RIA) method for the combined measurement of four steroid sulfoconjugates and their four unconjugated counterparts in maternal and fetal ovine plasma: pregnenolone (delta 5P), 17-hydroxypregnenolone (17 delta 5P), dehydroepiandrosterone (DHEA), and estrone (E1). In the procedure a preliminary ether extraction is utilized to isolate the unconjugated steroids followed by salting out, ethyl acetate extraction, and mineral acid solvolysis of the remaining sulfated steroids. The hydrolyzed sulfoconjugates are then separated chromatographically and measured in a manner identical to their unconjugated counterparts. The combined measurement of these eight steroids in single samples of fetal and maternal ovine plasma has not been reported previously and plasma concentrations of these steroids were heretofore unknown. Since no previous data was available for comparison, rigorous specificity evaluation of this RIA system was required prior to its use for physiologic studies and the reporting of concentrations in this species.     

Anomalous subdiffusion is a measure for cytoplasmic crowding in living cells

Macromolecular crowding dramatically affects cellular processes such as protein folding and assembly, regulation of metabolic pathways, and condensation of DNA. Despite increased attention, we still lack a definition for how crowded a heterogeneous environment is at the molecular scale and how this manifests in basic physical phenomena like diffusion. Here, we show by means of fluorescence correlation spectroscopy and computer simulations that crowding manifests itself through the emergence of anomalous subdiffusion of cytoplasmic macromolecules. In other words, the mean square displacement of a protein will grow less than linear in time and the degree of this anomality depends on the size and conformation of the traced particle and on the total protein concentration of the solution. We therefore propose that the anomality of the diffusion can be used as a quantifiable measure for the crowdedness of the cytoplasm at the molecular scale.     

Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.     

Nestmate recognition in burying beetles: the "breeder's badge" as a cue used by females to distinguish their mates from male intruders

Burying beetles use small vertebrate carcasses as food for theirlarvae and defend these carcasses against intra- and interspecificcompetitors. Breeding associations on carcasses can consistof single females, heterosexual pairs, or various combinationsof males and females. When a heterosexual pair collaborate ina breeding attempt, they do not typically exhibit aggressivebehavior toward each other, but do attack newly arrived conspecificsthat attempt to usurp the carcass. We investigated the cuesinvolved in discrimination between breeding partners and intrudersby female burying beetles. We found that resident females toleratemales that have cared for a brood, as well as males that havenot cared for a brood but have been on a carcass for a day ortwo. Males that have had no prior contact with a carcass areattacked. Females appear to use a chemical cue, the "breeder'sbadge," an apolar substance on the male's cuticle that can beremoved by washing with pentane. This cue is reliably correlatedwith recent male experience with a carcass that is suitablefor reproduction. The breeder's badge develops as a result ofprolonged contact with such a carcass, and disappears on removalfrom the carcass; its presence does not require contact witha female or with larvae. Female recognition of their male partnersin burying beetles thus does not involve individual recognition,but rather recognition of reproductive condition.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Intrinsic potential for immediate biodegradation of toluene in a pristine,energy-limited aquifer

Pristine and energy-limited aquifers are considered to have a low resistance and resilience towards organic pollution. An experiment in an indoor aquifer system revealed an unexpected high intrinsic potential for the attenuation of a short-term toluene contamination. A 30 h pulse of 486 mg of toluene, used as a model contaminant, and deuterated water (D₂O) through an initially pristine, oxic, and organic carbon poor sandy aquifer revealed an immediate aerobic toluene degradation potential. Based on contaminant and tracer break-through curves, as well as mass balance analyses and reactive transport modelling, a contaminant removal of 40 % over a transport distance of only 4.2 m in less than one week of travel time was obtained. The mean first-order degradation rate constant was λ = 0.178 day^?1, corresponding to a half-life time constant T_1/2 of 3.87 days. Toluene-specific stable carbon isotope analysis independently proved that the contaminant mass removal can be attributed to microbial biodegradation. Since average doubling times of indigenous bacterial communities were in the range of months to years, the aerobic biodegradation potential observed is assumed to be present and active in the pristine, energy-limited groundwater ecosystems at any time. Follow-up experiments and field studies will help to quantify the immediate natural attenuation potential of aquifers for selected priority contaminants and will try to identify the key-degraders within the autochthonous microbial communities.     

Genetic analysis of the LEW.1AR1-iddm rat: an animal model for spontaneous diabetes mellitus

The LEW.1AR1-iddm/Ztm rat is a new animal model of type 1 diabetes mellitus, which shows an autosomal recessive mode of inheritance for the diabetes-inducing gene. The aim of this study was to define predisposing loci of the diabetic syndrome by linkage analysis using microsatellite markers. A backcross population of 218 rats (BN × LEW.1AR1-iddm) × LEW.1AR1-iddm was analyzed using 157 polymorphic microsatellite markers covering the entire genome. Three genomic regions showed a significant linkage to the diabetic syndrome. The first susceptibility locus on rat Chromosome (RNO) 1 (LOD score 4.13) mapped to the region 1q51–55, which codes for potential candidate genes like Ins1 and Nkx2-3. The second susceptibility locus was also localized on RNO1 in the centromeric region 1p11 (LOD score 2.7) encompassing the Sod2 gene. The third quantitative trait loci (LOD score 2.97) was located on RNO20 within the major histocompatibility complex region. Comparative mapping revealed that the homologous regions in the human genome contain the IDDM loci 1, 5, 8, and 17. The identification of diabetes susceptibility regions of the genetically uniform LEW.1AR1-iddm rat strain will pave the way toward a detailed characterization of the loci conferring diabetes development as well as their functional relevance for the pathogenesis of type 1 diabetes mellitus.     

A Statistical Model for Regional Tornado Climate Studies

Tornado reports are locally rare, often clustered, and of variable quality making it difficult to use them directly to describe regional tornado climatology. Here a statistical model is demonstrated that overcomes some of these difficulties and produces a smoothed regional-scale climatology of tornado occurrences. The model is applied to data aggregated at the level of counties. These data include annual population, annual tornado counts and an index of terrain roughness. The model has a term to capture the smoothed frequency relative to the state average. The model is used to examine whether terrain roughness is related to tornado frequency and whether there are differences in tornado activity by County Warning Area (CWA). A key finding is that tornado reports increase by 13% for a two-fold increase in population across Kansas after accounting for improvements in rating procedures. Independent of this relationship, tornadoes have been increasing at an annual rate of 1.9%. Another finding is the pattern of correlated residuals showing more Kansas tornadoes in a corridor of counties running roughly north to south across the west central part of the state consistent with the dryline climatology. The model is significantly improved by adding terrain roughness. The effect amounts to an 18% reduction in the number of tornadoes for every ten meter increase in elevation standard deviation. The model indicates that tornadoes are 51% more likely to occur in counties served by the CWAs of DDC and GID than elsewhere in the state. Flexibility of the model is illustrated by fitting it to data from Illinois, Mississippi, South Dakota, and Ohio.     

INTERNODE ELONGATION IN XANTHIUM PLANTS TREATED WITH GIBBERELLIC ACID PRESENTED IN TERMS OF RELATIVE ELEMENTAL RATES

Xanthium plants were grown vegetatively and their developmental stages were designated by a previously described plastochron index (PI). Internodes of plants, both treated with gibberellic acid (GA₃) and untreated, were marked with India ink and photographed during 3 successive days. The relative elemental rates of elongation d(dX/dt)/dX were estimated between 15.7 and 19.0 plastochrons. The rate of growth of the GA₃-treated internodes was at least twice that of the control. The emerging pattern of acropetal internode elongation was similar in both GA₃-treated and control plants. Only rates of growth were significantly higher in the GA₃-treated plants. The acropetal pattern of internode elongation was the opposite of the basipetal pattern observed in Xanthium leaves but followed the acropetal pattern observed in Helianthus and Phaseolus internode growth.