Regulation of sulphur amino acids metabolic enzymes in Cephalosporium acremonium strains differing in antibiotic production

Three strains of Cephalosporium acremonium with different potential of cephalosporin C production show differences in the levels of sulphur amino acid metabolic enzymes. The regulation of these enzymes is also different in the strains. In the superior producing strain two enzymes directly involved in cysteine synthesis are the least susceptible to repression by methionine. In this strain cystathionine metabolizing enzymes seem to favour cysteine synthesis. Antibiotic producing strains, in contrast to the non-producing strain, are highly sensitive to chromate.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.     

LCA as an element in environmental management systems—comparison of conditions in selected organisations in Poland, Sweden and Germany

Purpose

In this two-part paper (Background and Initial Assumptions (part 1) and Results of Survey Research (part 2)), we present surveys whose main objective is to determine whether, and to what extent, the life cycle assessment (LCA) technique is used for the identification and assessment of environmental aspects in environmental management systems (EMS) and whether there are any differences in this respect between the companies and countries analysed.

Methods

The survey research was carried out using the computer assisted self-administered interviewing method among selected Polish, German and Swedish organisations which implement EMS in accordance with the requirements of ISO 14001 and/or the EMAS regulation.

Results

The organisations investigated, regardless of their country, are dominated by qualitative and semi-quantitative techniques of assessment and identification of environmental aspects. LCA was used sporadically, although some differences can be observed between the countries analysed.

Conclusions

The environmental managers accustomed to traditional qualitative and semi-quantitative solutions have not been given preparation to enable them to understand and adopt different approaches such as LCA. On the other hand, representatives of the organisations investigated declared that they were ready to accept an even longer timescale for the identification and assessment processes relating to environmental aspects, which represents a potential opportunity for LCA. The more precise understanding and definition of environmental problems that are precisely defined in LCA would represent a novelty for environmental managers. In practice, environmental problems are defined in a general sense and rather ambiguously, as this level of detail is sufficient in the context of qualitative and semi-quantitative techniques commonly used for the identification and assessment of environmental aspects.