Schwannoma-Derived Growth Factor Interacts with the Epidermal Growth Factor Receptor

Abstract: Schwannoma-derived growth factor (SDGF) is a potent mitogen and neuronal differentiation factor. Because of its relationship to epidermal growth factor (EGF) and the heregulins, it was asked if SDGF interacts with the EGF receptor or HER2/neu. SDGF binds to and causes the phosphorylation on tyrosine of the EGF receptor but not HER2/neu.     

Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product

We have identified a nodD gene from the wide host-range Rhizobium strain MPIK3030 (termed nodD1) which is essential for nodulation on Macroptilium atropurpureum (siratro). Experiments with nodA–lacZ gene fusions demonstrate that the MPIK3030 nodD1 regulates expression of the nodABC genes. Additionally, we used nodC–lacZ fusions of Rhizobium meliloti to show that the MPIK3030 nodD1 gene induces expression of these fusions by interacting with plant factors from siratro and from the non-host Medicago sativa (alfalfa). The R. meliloti nodD genes, however, only interact with alfalfa exudate. In line with these results, no complementation of MPIK3030 nodD1 mutants could be obtained on siratro with the R. meliloti nodD genes, while the MPIK3030 nodD1 can complement nodD mutants of R. meliloti on alfalfa. Furthermore, R. meliloti transconjugants harbouring the MPIK3030 nodD1 efficiently nodulate the illegitimate host siratro. When compared with other nodD sequences, the amino acid sequence of the MPIK3030 nodD1 shows a conserved aminoterminus, whereas the carboxy-terminus of the putative gene product diverges considerably. Studies on a chimeric MPIK3030/R. meliloti nodD gene indicates that the carboxy-terminal region is responsible for the interaction with plant factor(s) and may have evolved in different rhizobia specifically to interact with plant–host factors.     

Evolution,pollination, and systematics of the tribeNeottieae (Orchidaceae)

The various classifications of the orchid tribeNeottieae are reviewed and a new classification is proposed that divides the tribe into three subtribes,Neottiinae, Limodorinae, andCephalantherinae, based primarily on characters of the column (gynostemium). A cladistic analysis illustrates that these three subtribes are more closely related to one another than either is to any other group in subfam.Neottioideae, although there are very few apomorphic characters for the tribe. Pollination biology is also discussed showing links between breeding systems and distribution. There is also a possible role between column and labellum morphology and the emergence of a deceptive pollination syndrome from one of reward.