While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Influence of Seeding Ratio,Planting Date,and Termination Date on Rye-Hairy Vetch Cover Crop Mixture Performance under Organic Management

Cover crop benefits include nitrogen accumulation and retention, weed suppression, organic matter maintenance, and reduced erosion. Organic farmers need region-specific information on winter cover crop performance to effectively integrate cover crops into their crop rotations. Our research objective was to compare cover crop seeding mixtures, planting dates, and termination dates on performance of rye (Secale cereale L.) and hairy vetch (Vicia villosa Roth) monocultures and mixtures in the maritime Pacific Northwest USA. The study included four seed mixtures (100% hairy vetch, 25% rye-75% hairy vetch, 50% rye-50% hairy vetch, and 100% rye by seed weight), two planting dates, and two termination dates, using a split-split plot design with four replications over six years. Measurements included winter ground cover; stand composition; cover crop biomass, N concentration, and N uptake; and June soil NO₃ ^--N. Rye planted in mid-September and terminated in late April averaged 5.1 Mg ha^-1 biomass, whereas mixtures averaged 4.1 Mg ha^-1 and hairy vetch 2.3 Mg ha^-1. Delaying planting by 2.5 weeks reduced average winter ground cover by 65%, biomass by 50%, and cover crop N accumulation by 40%. Similar reductions in biomass and N accumulation occurred for late March termination, compared with late April termination. Mixtures had less annual biomass variability than rye. Mixtures accumulated 103 kg ha^-1 N and had mean C:N ratio <17:1 when planted in mid-September and terminated in late April. June soil NO₃ ^--N (0 to 30 cm depth) averaged 62 kg ha^-1 for rye, 97 kg ha^-1 for the mixtures, and 119 kg ha^-1 for hairy vetch. Weeds comprised less of the mixtures biomass (20% weeds by weight at termination) compared with the monocultures (29%). Cover crop mixtures provided a balance between biomass accumulation and N concentration, more consistent biomass over the six-year study, and were more effective at reducing winter weeds compared with monocultures.     

Anti-malarial market and policy surveys in sub-Saharan Africa

Background

Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia.

Methods

A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections.

Results

The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%).

Conclusions

The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.     

Synthesis and biological evaluation of 4-amino derivatives of benzimidazoquinoxaline, benzimidazoquinoline, and benzopyrazoloquinazoline as potent IKK inhibitors

We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC50 = 0.30 microM), which however was considerably less potent against IKK-1 (IC50 = 4.0 microM). In order to further explore the SAR around the imidazoquinoxaline tricyclic structure of 1, we prepared a series of tetracyclic analogues (7, 13, and 18). The synthesis and biological activities of these potent IKK inhibitors are described.     

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.     

Identification of DNA repair gene Ercc1 as a novel target in melanoma

Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments.     

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.     

The behavioural responses of badgers (Meles meles) to exclusion from farm buildings using an electric fence

Behavioural investigations into the transmission of bovine tuberculosis (Mycobacterium bovis) between badgers and cattle suggest that badger activity in farm buildings may incur a significant risk of cross-infection. However, measures to exclude badgers from buildings have not been systematically field-tested. In the present study, remote surveillance and radio-tracking were used to monitor the effect of electric fencing manipulations on the frequency of badger incursions into feed stores and cattle housing, and on badger ranging behaviour. Electric fencing was effective in preventing access to the farm buildings where it was installed and also significantly reduced incursions into unfenced buildings. Badger home range and core activity areas tended to increase in size when the fencing was installed, although they did not extend beyond the boundaries of the relevant social group territories. We discuss the logistical constraints of using electric fencing in this context and conclude that it is a potentially useful method of reducing contact between badgers and cattle, within farm buildings and yards.     

Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.