Influence of bilateral cryptorchidism on rat pituitary luteinizing hormone charge microheterogeneity

Chromatofocusing was used to characterize the isohormones of rat luteinizing hormone (rLH) in extracts of pituitaries from intact and bilaterally cryptorchid animals. Pituitary extracts contained at least seven isoelectric variants (isohormones) or rLH: one eluted in the column void volume (Isohormone, I, pI greater than 9.8), five exhibited apparent pI's in the range of 9.25 to 8.97 (designated as Isohormones II-VI) and one bound to the column but eluted with 1.0 M NaCl (Isohormone VII, pI less than 7.0). In both intact and cryptorchid rats, a large percentage of the rLH was present as Isohormone I. Furthermore, Isohormone I was present in greater absolute and relative amounts in cryptorchid rats. All seven rLH isohormones exhibited activity in an in vitro bioassay and the biological to immunological assay (B:I) ratios decreased with their apparent pI's but were not significantly affected by cryptorchidism. When the data were expressed as the product of the amount of rLH and the B:I ratio (termed Bio-Index), Isohormone I was the predominant form of rLH in both cryptorchid and intact rats, with the Bio-Index of Isohormone I being significantly greater for cryptorchid rats. These results suggest that cryptorchidism alters the pattern of rLH isohormones in the pituitary, yielding a greater percentage of the most basic rLH isohormone, which also has the highest B:I ratio. Thus, cryptorchidism significantly alters the qualitative pattern as well as the quantity of rLH in the pituitary.     

In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells

While caspases play an established role as intracellular executors of apoptosis, little is known about extracellular activities of this ubiquitously expressed family of proteases. We demonstrate here that recombinant caspase-3 retained enzymatic activity in various extracellular fluids. Experiments with cell lines, primary cells, and mice with fulminant CD95-triggered hepatitis showed that significant amounts of DEVD-aminofluoromethylcoumarine-cleaving activity, indicative of active effector caspases, were released into the medium/plasma during apoptosis. Furthermore, caspase activities were detected in liquor samples from human head trauma patients. These findings warrant closer investigation of DEVDase activity as a diagnostic marker, and of potential extracellular substrates for caspases.     

Informing climate models with rapid chamber measurements of forest carbon uptake

Models predicting ecosystem carbon dioxide (CO₂) exchange under future climate change rely on relatively few real‐world tests of their assumptions and outputs. Here, we demonstrate a rapid and cost‐effective method to estimate CO₂ exchange from intact vegetation patches under varying atmospheric CO₂ concentrations_. We find that net ecosystem CO₂ uptake (NEE) in a boreal forest rose linearly by 4.7 ± 0.2% of the current ambient rate for every 10 ppm CO₂ increase, with no detectable influence of foliar biomass, season, or nitrogen (N) fertilization. The lack of any clear short‐term NEE response to fertilization in such an N‐limited system is inconsistent with the instantaneous downregulation of photosynthesis formalized in many global models. Incorporating an alternative mechanism with considerable empirical support – diversion of excess carbon to storage compounds – into an existing earth system model brings the model output into closer agreement with our field measurements. A global simulation incorporating this modified model reduces a long‐standing mismatch between the modeled and observed seasonal amplitude of atmospheric CO₂. Wider application of this chamber approach would provide critical data needed to further improve modeled projections of biosphere–atmosphere CO₂ exchange in a changing climate.     

A survey of single nucleotide polymorphisms identified from whole‐genome sequencing and their functional effect in the porcine genome,

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole‐genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire–Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss‐of‐function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss‐of‐function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses.     

Viscometric analysis of the interaction of bisphenanthridinium compounds with closed circular supercoiled and linear DNA.

The interaction with closed circular supercoiled and linear DNA of bisphenanthridinium compounds substituted through both the meta and para positions of the 6-phenyl group, along with appropriate monomer intercalators as controls, has been investigated by viscometric titration. When CPK models for the phenanthridinium rings of the three bis-compounds are oriented in a parallel manner as a model for intercalation, their ring plane to ring plane distances are approximately 7 to 8 A (SR 2430), 11 A (SR 2193), and 15 A (SR 2166). In SR 2430 the two phenanthridines are linked through the para positions of the 6-phenyl group; this chain allows intercalation of the two rings at adjacent binding sites in DNA, but is not long enough to accommodate an excluded site. The viscometric titrations with both superhelical and linear DNA clearly indicate that SR 2430 gives results close to those of the monomer control compounds while SR 2193 and SR 2166 have approximately twice the unwinding angle and DNA length increase on binding to DNA as the monomer compounds. These phenanthridinium compounds, therefore, are capable of bisintercalation only if their linking groups are of sufficient length to allow an excluded binding site between base pairs. This conclusion is supported by DNA thermal denaturation experiments in the presence of these compounds.     

Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells.

We investigated the ability of EGF to stimulate the phosphorylation (i.e. activation) of extracellular signal-regulated kinases (ERKs) in freshly isolated porcine granulosa cells (pGC) held in suspension. pGCs were isolated from the ovaries of prepubertal pigs at slaughter, and equilibrated for 24 h at 37 degrees C in 12 x 75 mm culture tubes. The cells were then treated with 0-10 ng/ml EGF for 1-240 min. Treatments were terminated, and the total cell lysates were subjected to SDS-PAGE and Western analysis. The Westerns were blotted with anti-panERK and with anti-phosphoERK, antibodies that recognize all forms of ERKs and the phosphorylated (i.e. activated) forms of ERKs, respectively. Western blot analysis with the panERK antibody revealed a gel shift of ERKs, suggesting hyperphosphorylation after treatment with as little as 0.1 ng/ml of EGF. Phosphorylation of the ERKs was confirmed by using the phosphoERK antibody, which indicated increased phosphorylation of ERKs above control with 0.1 ng/ml EGF and maximal phosphorylation of ERK with 5-10 ng/ml EGF. Activation of ERK by EGF, as measured by both gel shift analysis and active ERK blotting, in the freshly isolated pGC was rapid, increasing above controls after 1 min of treatment, maintaining high levels through 40 min, and declining from 60 to 240 min. These data indicate that EGF stimulates active ERK in a time- and concentration-dependent manner in freshly isolated pGCs and that this experimental approach represents an effective manner with which to evaluate the role of EGF and the ERK signal transduction pathway in freshly harvested pGC.     

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.