Identification of Tuber magnatum Pico DNA markers by RAPD analysis

Summary Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species.     

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.     

Photomorphogenic effects on in vitro rooting of Prunus roostock GF 655-2

The morphogenic effect of different light wavelengths on in vitro rooting of Prunus insititia GF655-2 in relation to the presence of napthaleneacetic acid (NAA) in the culture medium was investigated. Results of experiments in which plantlets were rooted in NAA enriched medium showed that the presence of auxin induced rooting even in the dark after an initial lag period. Illumination of the cultures with Red light was as effective in promoting rooting as treatment with 0.5 M NAA; Red was more active in stimulating rooting in the short term than was NAA. The pattern of root formation resulting from the addition of NAA appeared to dominate development under White, Blue and Far Red treatments. Although it was possible to correlate the rooting response to the phytochrome photoequilibrium induced by the light treatments used, there arises a possible interference of specific Blue absorbing photoreceptors.Abbreviations B Blue - FR Far Red - HIR High Irradiance Response - P_fr active (far-red absorbing) form of phytochrome - P_tot total phytochrome - R Red - W White - NAA -naphtaleneacetic acid - BA benzyladenine - IAA indole 3-acetic acid     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

A karyotype analysis of the genus Dicentrarchus by different staining techniques

This study presents a cytogenetic analysis of the genus Dicentrarchus , represented by two species, D. labrax and D. punctatus . The karyotypes are very similar, even after staining with different techniques. Both species show 48 subtelocentric and acrocentric chromosomes, gradually decreasing in size. One pair of small size chromosomes has heteropycnotic and heteromorphtc short arms of longer size. These short arms are C- and Ag-positive, i.e. nucleolar organizer regions, NORs, are located there. Constitutive heterochromatin is also evident as a subcentromeric band on the long arms of a large chromosome pair. CMA₃-staining confirms the location and the heteromorphism of NORs. DAPI and quinacrine produce homogeneous staining of chromosomes. A review of cytogenetic studies on 'serranid' species is also presented.     

Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion.     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.