Extraribosomal functions associated with the C terminus of the 37/67?kDa laminin receptor are required for maintaining cell viability

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G₁ phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.     

Effect of fertilizer levels on grain yield,incidence and severity of downy mildew in pearl millet

The effect of various levels of nitrogen (0.0, 30.0, 60.0, 120.0) and phosphorus (0.0, 6.5, 13.0, 36.0) on the incidence and severity of downy mildew of pearl millet and yield of two pearl millet varieties (Zango and GB8375) were studied under field conditions in 2000 and 2001 respectively. Both nitrogen and phosphorus significantly increased incidence and severity of the disease in the two varieties. Grain yield and 1000 grain weight of the varieties also increased with nitrogen and phosphorus levels.     

Interaction of Human Laminin Receptor with Sup35, the [PSI +] Prion-Forming Protein from S. cerevisiae: A Yeast Model for Studies of LamR Interactions with Amyloidogenic Proteins

The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP^C and PrP^Sc. Indeed, LamR is a receptor for PrP^C. Whether LamR interacts with PrP^Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP^Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI ⁺] and [psi ⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI ⁺] strains. The presence of [PSI ⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI ⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI ⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.     

Effects of climate change on productivity of cereals and legumes; model evaluation of observed year-to-year variability of the CO2 response

The effect of elevated [CO₂] on the productivity of spring wheat, winter wheat and faba bean was studied in experiments in climatized crop enclosures in the Wageningen Rhizolab in 1991–93. Simulation models for crop growth were used to explore possible causes for the observed differences in the CO₂ response. Measurements of the canopy gas exchange (CO₂ and water vapour) were made continuously from emergence until harvest. At an external [CO₂] of 700 μmol mol^?1 Maximum Canopy CO₂ Exchange Rate (CCERmax) at canopy closure was stimulated by 51% for spring wheat and by 71% for faba bean. At the end of the growing season, above ground biomass increase at 700 μmol mol^?1 was 58% (faba bean), 35% (spring wheat) and 19% (winter wheat) and the harvest index did not change. For model exploration, weather data sets for the period 1975-88 and 1991–93 were used, assuming adequate water supply and [CO₂] at 350 and 700 μmol mol^?1. For spring wheat the simulated responses (35–50%) were at the upper end of the experimental results. In agreement with experiments, simulations showed smaller responses for winter wheat and larger responses for faba bean. Further model explorations showed that this differential effect in the CO₂ response may not be primarily due to fundamental physiological differences between the crops, but may be at least partly due to differences in the daily air temperatures during comparable stages of growth of these crops. Simulations also showed that variations between years in CO₂ response can be largely explained by differences in weather conditions (especially temperature) between growing seasons.     

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.     

Specific cell targeting for delivery of toxins into small-cell lung cancer using a streptavidin fusion protein complex

New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity.     

Seasonal changes in the response of winter wheat to elevated atmospheric CO2 concentration grown in Open-Top Chambers and field tracking enclosures

Winter wheat was grown at ambient and elevated (ambient plus 350 μL L^–1) CO₂ concentrations in open top chambers and in field-tracking sun-lit climatized enclosures (elevated is 718 μL L^–1). There was no significant effect of CO₂ concentration on sheath, leaf and root biomass and leaf area in the early spring (January to April). 24-h canopy CO₂ exchange rate (CCER) was not significantly affected either. However, elevated CO₂ concentration increased CCER at midday, decreased evapotranspiration rate and increased instantaneous water-use-efficiency during early spring. Leaf, sheath and root nitrogen concentration per unit dry weight decreased and nonstructural carbohydrate concentration increased under elevated CO₂, and N-uptake per unit ground area decreased significantly (– 22%) towards the end of this period. These results contrast with results from the final harvest, when grain yield and biomass were increased by 19% under elevated CO₂. N concentration per dry weight was reduced by 5%, but N-uptake per unit ground area was significantly higher (+ 11%) for the elevated CO₂ treatment. 24-h and midday-CCER increased significantly more in late spring (period of 21 April to 30 May) (respectively by + 40% and 53%) than in the early spring (respectively 5% and 19%) in response to elevated CO₂. Midday evapotranspiration rate was reduced less by elevated CO₂ in the late spring (– 13%) than in early spring (– 21%). The CO₂ response of midday and 24-h CCER decreased again (+ 27% and + 23% resp.) towards the end of the growing season. We conclude that the low response to CO₂ concentration during the early spring was associated with a growth-restriction, caused by low temperature and irradiance levels. The reduction of nitrogen concentration, the increase of nonstructural carbohydrate, and the lower evapotranspiration indicated that CO₂ did have an effect towards the end of early spring, but not on biomass accumulation. Regression analysis showed that both irradiance and temperature affected the response to CO₂.     

Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus.

The murine cationic amino acid transporter is also the receptor for murine ecotropic leukemia retrovirus (MuLV-E). Recently, we have cloned a human gene (H13) homologous to the murine ecotropic retroviral receptor (ERR). Although the human homolog is very similar to murine ERR in sequence (87.6% amino acid identity) and structure (14 transmembrane-spanning domains), the human protein fails to function as a receptor for MuLV-E. To identify amino acid residues critical for MuLV-E infection, we took advantage of this species difference and substituted human H13 and murine ERR amino acid residues. Mouse-human chimeric receptor molecules were generated by taking advantage of using common restriction sites. These studies demonstrated that extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual ERR mutants containing one or two amino acids substitutions or insertions within these two extracellular domains. Substitution of as few as one amino acid residue (Tyr) at position 235 in ERR with the corresponding H13 amino acid residue Pro abrogates the ability to function as a receptor for MuLV-E infection. Conversely, substitution of just two amino acid residues at positions 240 and 242 or 242 and 244 in H13 with the corresponding amino acid residues in ERR endows H13 with the ability to function as the receptor. This observation can be utilized to significantly improve the safety of retrovirus-mediated gene therapy in humans.