Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Kinetics and stereochemistry of the microsomal epoxide hydrolase-catalyzed hydrolysis of cis-stilbene oxides

The microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of cis-4,4′–dimethylstilbene oxide ( 1a ), cis-4,4′-diethylstilbene oxide ( 1b ), cis-4,4′-diisopropylstilbene oxide ( 1c ), and cis-4,4′-dichlorostilbene oxide ( 1d ) have been investigated using rabbit liver microsomal preparations. The kinetic parameters, K_m and V_max, and the absolute stereochemistry of the reactions have been determined and compared with those of cis-stilbene oxide ( 1e ). All epoxides 1a – d are hydrolyzed by mEH with high product enantioselectivity to give (R,R)-(+)-diols with ee ≥ 90%. The presence of the substituents on the phenyl rings markedly reduces the rates of mEH catalyzed hydrolysis with respect to cis-stilbene oxide, by increasing K_m and reducing V_max in the cases of 1a , 1b , and 1d , or reducing only the V_max in the case of 1c . The very low V_max, together with a persistent ability to fit into the mEH active site, make all these epoxides, and particularly 1c , inhibitors of cis-stilbene oxide hydrolysis. The kinetic and stereochemical results are interpreted on the basis of the proposed topology of the mEH active site. © 1994 Wiley-Liss, Inc.     

Induction of the murine “W phenotype” in long-term cultures of human cord blood cells by c-kit antisense oligomers

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley-Liss, Inc.     

Non-linear loss of suitable wine regions over Europe in response to increasing global warming

Evaluating the potential climatic suitability for premium wine production is crucial for adaptation planning in Europe. While new wine regions may emerge out of the traditional boundaries, most of the present-day renowned winemaking regions may be threatened by climate change. Here, we analyse the future evolution of the geography of wine production over Europe, through the definition of a novel climatic suitability indicator, which is calculated over the projected grapevine phenological phases to account for their possible contractions under global warming. Our approach consists in coupling six different de-biased downscaled climate projections under two different scenarios of global warming with four phenological models for different grapevine varieties. The resulting suitability indicator is based on fuzzy logic and is calculated over three main components measuring (i) the timing of the fruit physiological maturity, (ii) the risk of water stress and (iii) the risk of pests and diseases. The results demonstrate that the level of global warming largely determines the distribution of future wine regions. For a global temperature increase limited to 2°C above the pre-industrial level, the suitable areas over the traditional regions are reduced by about 4%/°C rise, while for higher levels of global warming, the rate of this loss increases up to 17%/°C. This is compensated by a gradual emergence of new wine regions out of the traditional boundaries. Moreover, we show that reallocating better-suited grapevine varieties to warmer conditions may be a viable adaptation measure to cope with the projected suitability loss over the traditional regions. However, the effectiveness of this strategy appears to decrease as the level of global warming increases. Overall, these findings suggest the existence of a safe limit below 2°C of global warming for the European winemaking sector, while adaptation might become far more challenging beyond this threshold.     

Aqueous plant extracts as stimulators of laccase production in liquid cultures of Lentinus edodes

Summary Total and specific activities of extra-cellular laccases from Lentinus edodes were enhanced by adding corn straw and chestnut juice to the liquid growth medium. The aqueous extracts were chemically characterized and revealed the presence of several phenolic and non-phenolic compounds. Extensive extraction of these components from the tested extracts completely annulled their stimulating properties on laccase production, suggesting that these compounds can act at micromole levels.     

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997