Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen.

S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the influence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme beta-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-lac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain 536WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain 536WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate had a strong influence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.     

Species effects on nitrogen cycling: a test with perennial grasses

Summary To test for differing effects of plant species on nitrogen dynamics, we planted monocultures of five perennial grasses (Agropyron repens, Agrostis scabra, Poa pratensis, Schizachyrium scoparium, and Andropogon gerardi) on a series of soils ranging from sand to black soil. In situ net N mineralization was measured in the monocultures for three years. By the third year, initially identical soils under different species had diverged up to 10-fold in annual net mineralization. This divergence corresponded to differences in the tissue N concentrations, belowground lignin concentrations, and belowground biomasses of the species. These results demonstrate the potential for strong feedbacks between the species composition of vegetation and N cycling. If individual plant species can affect N mineralization and N availability, then competition for N may lead to positive or negative feedbacks between the processes controlling species composition and ecosystem processes such as N and C cycling. These feedbacks create the potential for alternative stable states for the vegetation-soil system given the same initial abiotic conditions.     

Carbon and nitrogen cycling during old-field succession: Constraints on plant and microbial biomass

Soil C and N dynamics were studied in a sequence of old fields of increasing age to determine how these biogeochemical cycles change during secondary succession. In addition, three different late-successional forests were studied to represent possible "steady state" conditions. Surface soil samples collected from the fields and forests were analyzed for total C, H₂O-soluble C, total N, potential net N mineralization, potential net nitrification, and microbial biomass. Above-and belowground plant biomass was estimated within each of the old field sites.Temporal changes in soil organic C, total N and total plant biomass were best described by a gamma function [y =at ^b e ^ctd +f] whereas a simple exponential model [y =a(l – e^–bt ) + c] provided the best fit to changes in H₂O-soluble C, C:N ratio, microbial C, and microbial N. Potential N mineralization and nitrification linearly increased with field age; however, rates were variable among the fields. Microbial biomass was highly correlated to soil C and N pools and well correlated to the standing crop of plant biomass. In turn, plant biomass was highly correlated to pools and rates of N cycling.Patterns of C and N cycling within the old field sites were different from those in a northern hardwood forest and a xeric oak forest; however, nutrient dynamics within an oak savanna were similar to those found in a 60-yr old field. Results suggest that patterns in C and N cycling within the old-field chronosequence were predictable and highly correlated to the accrual of plant and microbial biomass.     

Osmotic regulation of MG-132-induced MAP-kinase phosphatase MKP-1 expression in H4IIE rat hepatoma cells.

BACKGROUND/AIMS: Proteasome inhibitors such as MG-132 are considered as potential therapeutical tools in different clinical settings. The dual specificity MAP-kinase phosphatase MKP-1 plays a role in balancing signals mediating cell death or survival. Here the effect of cell hydration on MG-132-induced MKP-1 expression was investigated in H4IIE rat hepatoma cells. RESULTS: Hyperosmolarity (405mosmol/l) increased MKP-1 expression by MG-132, which was accompanied by an induction of c-Fos, c-Jun, cJun Ser73 phosphorylation, and AP-1 DNA binding. MKP-1 induction by MG-132 plus hyperosmolarity was sensitive to inhibition of p38(MAPK) and c-Jun-N-terminal kinases (JNKs) but not extracellular signal-regulated kinases Erk-1/Erk-2, and was accompanied by a decline of MAP-kinase activities. Although hyperosmolarity increased overall protein ubiquitination in presence of MG-132, ubiquitination of MKP-1 was found under normo-, but not hyperosmotic conditions. Hyperosmolarity also enabled MG-132 to induce poly-ADP-ribose polymerase (PARP) cleavage which was sensitive to inhibition of p38(MAPK) and JNKs but not Erk-1/Erk-2. PARP cleavage and caspase-3 activation in H4IIE cells treated with hyperosmolarity plus MG-132 was further increased by vanadate, consistent with a contribution of MKP-1 to counterbalance proapoptotic MAP-kinase signals. CONCLUSION: The findings suggest that among other factors cell hydration critically determines the cellular response to proteasome inhibitors.     

MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma

Objective

Metastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases.

Methods

We analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR.

Results

All three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01).

Conclusion

In the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases.     

Artificial insemination in cattle with DNA‐treated sperm

Abstract

We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/10⁶ sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×10⁶ sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.