Surface distribution and internalization of erbB-2 proteins.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.     

Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation

T-DNA integration patterns in 49 transgenic grapevines produced via Agrobacterium-mediated transformation were analyzed. Inverse PCR (iPCR) was performed to identify T-DNA/plant junctions. Sequence comparison revealed several deletions in the T-DNA right border (RB) and left border (LB), and filler DNA and duplications or deletions of grapevine DNA at the T-DNA insertion loci. In 20 T-DNA/grapevine genome junctions microsimilarities were found associated with the joining points and in all grapevine lines microsimilarities were present near the breaking points along the 30 bases of T-DNA adjacent to the two borders. Analysis of target site preferences of T-DNA insertions indicated a non-random distribution of the T-DNA, with a bias toward the intron regions of the grapevine genes. Compositional analysis of grapevine DNA around the T-DNA insertion sites revealed an inverse relationship between the CG and AT-skews and AT rich sequences present at 300–500 bp upstream the insertion points, near the RB of the T-DNA. PCR assays showed that vector backbone sequences were integrated in 28.6% of the transgenic plants analyzed and multiple T-DNAs frequently integrated at the same position in the plant genome, resulting in the formation of tandem and inverted repeats.     

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.     

Screening of turfgrass species and cultivars for NaCl tolerance

Summary Information regarding the relative levels of salt tolerance between cultivars of Kentucky bluegrass (Poa pratensis L.) is lacking. The objectives of this study were to 1) develop a simple, quick and sensitive method of screening turfgrass species for NaCl tolerance and 2) to compare the relative salt tolerance of five cultivars of Kentucky bluegrass (Ram I, Adelphi, Baron, Bensun, and Nassau) to other known salt tolerant turfgrass species such as alkalaigrass (Puccinellia distans (L.) Parl. cv. Fults) and two cultivars of red fescue (Festuca rubra L. Dawson, and Checker).Alkalaigrass and both cultivars of red fescue retained a high level of salt tolerance compared to the Kentucky bluegrass cultivars. Significant variability in salt tolerance was apparent among the Kentucky bluegrass cultivars with Adelphi and Ram I exhibiting the best overall tolerance.     

Comparative electron microscopic study on the location of ribosomal proteins S3 and S7 on the surface of the E. coli 30S subunit using monoclonal and conventional antibody

Summary Mice were immunised with 30S subunits from E. coli and their spleen cells were fused with myeloma cells. From this fusion two monoclonal antibodies were obtained, one of which was shown to be specific for ribosomal protein S3, the other for ribosomal protein S7. The two monoclonal antibodies formed stable complexes with intact 30S subunits and were therefore used for the three-dimensional localisation of ribosomal proteins S3 and S7 on the surface of the E. coli small subunit by immuno electron microscopy. The antibody binding sites determined with the two monoclonal antibodies were found to lie in the same area as those obtained with conventional antibodies. Both proteins S3 and S7 are located on the head of the 30S subunit, close to the one-third/two-thirds partition. Protein S3 is located just above the small lobe, whereas protein S7 is located on the side of the large lobe.     

Nitrogen removal with the anaerobic ammonium oxidation process

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium to N₂ with nitrite as the terminal electron acceptor in the absence of O₂. Nitritation–anammox bioreactors provide a cost-effective and environment-friendly alternative to conventional nitrification/denitrification nitrogen removal systems. Currently, this process is only applied for ammonium removal from wastewater with high ammonium load and temperature. Nevertheless, recent results obtained with laboratory-scale bioreactors suggest new possible routes of application of the Nitritation–anammox technology including (1) municipal wastewater treatment, removal of (2) methane in combination with nitrite-reducing methane-oxidizing bacteria, (3) nitrate coupled to organic acid oxidation and (4) nitrogen oxides. The current review summarizes the state-of-the-art of the application of Nitritation–anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas β-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-κB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or β-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or β-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF- or β-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.     

SEL1L and HRD1 are involved in the degradation of unassembled secretory Ig-mu chains

When expressed in the absence of light chains, secretory Ig-micro chains (micro(s)) undergo endoplasmic reticulum associated degradation (ERAD). This process involves the recognition of terminally misfolded or unassembled molecules, their retro-translocation across the ER membrane and ubiquitination for degradation by cytosolic proteasomes. The molecular components of the ERAD pathway and their coordination remain largely unknown. Here we employed co-immunoprecipitation, silencing or over-expression assays to show that SEL1L and HRD1 are involved in the degradation of unassembled Ig-micro(s), but have minor effects on another substrate, TCR-alpha. SEL1L and HRD1 localize in the early secretory apparatus and are induced by ER stress and during B cell differentiation, concomitantly with the onset of massive IgM secretion. These findings reveal a role for SEL1L and HRD1 in IgM quality control.