The FMR1 CGG repeat and linked microsatellite markers in two Basque valleys

Fragile X syndrome is associated with an unstable CGG repeat sequence in the 5' untranslated region of the first exon of the FMR1 gene. The present study involved the evaluation of factors implicated in CGG repeat stability in a normal sample from two Basque valleys (Markina and Arratia), to discover whether the Basque population shows allelic diversity and to identify factors involved, by using the data in conjunction with previous findings. The study was based on a sample of 204 and 58 X chromosomes from the Markina and Arratia valleys, respectively. The CGG repeat, the AGG interspersion and two flanking microsatellite markers, FRAXAC1 and DXS548, were examined. In the Markina valley, gray zone alleles (> or =35 CGG repeats) were associated with anchoring AGGs, with the longest 3' pure CGG repeats of the valley (=15), with the 5' instability structure 9+n and with one principal fragile X FRAXAC1-DXS548 haplotype 42-50. In the Arratia valley, gray zone alleles (> or =35 CGG repeats) showed the highest frequency among the Basque samples analyzed, and were associated with anchoring AGGs, with the longest 3' pure repeats (> or =20), with the 5' instability structure 9+n and with one "normal" FRAXAC1-DXS548 haplotype 38-40 (these data from Arratia suggest the existence of a "protective" haplotype). The results showed, on the one hand, differences between Markina and Arratia in factors implicated in CGG repeat instability and, on the other hand, a great similarity between the general Basque sample from Biscay and the Markina valley.     

A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes

Membranes of cellular organelles and plasma membranes of some type of cells are not accessible to the high-resolution recordings that the conventional patch-clamp technique allows. However, when these purified membranes are dehydrated together with small lipid vesicles and hydrated again, cell-size vesicles (5-100 micron diameter) are obtained, on which single-channel recordings are possible. This approach, which has been proven successful with about ten different membrane preparations of varied origin, is further illustrated with two examples. First, a known conductivity of the sarcoplasmic reticulum membrane is compared with data obtained by using other techniques. Second, a new sodium current, present at purified postsynaptic membranes from the Torpedo electric organ, is described.     

Thiourea derivatives and their nickel(II) and platinum(II) complexes: antifungal activity

We have synthesized two thiourea derivatives of methyl anthranilate (1, 2) and their complexes with nickel (3) and platinum(II) (4). We have also prepared the complexes of nickel(II) with two benzoylthiourea derivatives (5, 6). The obtained compounds were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-vis, NMR), mass spectrometry and thermal analysis. Compound 1, C(20)H(23)N(3)O(2)S, crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=8.8042(4) A, b=7.6608(3) A, c=28.834(2) A, alpha=gamma=90 degrees, beta=90.94(1) degrees. Compound 2, C(20)H(21)N(3)O(3)S, crystallizes in monoclinic space group P21/c, with Z=4, and unit cell parameters, a=7.7345(4) A, b=8.6715(4) A, c=29.113(2) A, alpha=gamma=90 degrees, beta=90.67(1) degrees. Compound 5, C(24)H(30)N(4)NiO(2)S(2), crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=10.4317(8) A, b=18.517(2) A, c=13.299(1) A, alpha=gamma=90 degrees, beta=104.53(1) degrees. Compound 6, C(25)H(28)Cl(2)N(4)NiO(4)S(2), crystallizes with a molecule of CH(2)Cl(2) in triclinic space group P-1, with Z=2, and unit cell parameters, a=10.362(1) A, b=11.849(2) A, c=12.536(2) A, alpha=90.04(2) degrees, beta=84.73(1) degrees, gamma=113.43(2) degrees. Compounds 1 and 2 show antifungal activity against the major pathogens responsible for important plant diseases (Botrytis cinerea, Colletotrichum fragariae, Fusarium oxysporum and Phoma betae). The antifungal activity is practically the same for morpholine and ethyl derivatives.     

Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ε-subunit DNA

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of -,β-, and δ-subunits [1]. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ε-subunit, which is believed to replace the γ-subunit in forming the adult AChR subtype [4], BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR ε-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ε-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant ε-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ε-subunit may assemble with mouse muscle -, β- and δ-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ε-subunit mRNA. Thus, the notion that replacement of the γ-subunit by the ε-subunit during development is the result of the postnatal rise in the level of ε-subunit specific mRNA is further supported.     

Purification and Chemical Characterization of a W2 Protein from Brain Myelin

Abstract: Starting from a pellet of beef brain myelin insoluble in chloroform/ methanol (2:1, vol/vol) (Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N-terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross-reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin-like protein nature that is associated with the myelin membrane and could play a role in the myelination process.     

Synthesis and characterization of sodium cis-dichlorochenodeoxycholylglycinato(O,N) platinum(II)--cytostatic activity.

With a view to using bile acids as shuttles for delivering platinum-related cytostatic drugs to liver tumors, a chenodeoxycholylglycinato(CDCG)-derivative of platinum(II) has been synthesized. The complex--named Bamet-M2--was chemically characterized by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopy. The results indicate the following composition: C26H42N2O5Cl2NaPt(II), the metal Pt(II) being bound to two Cl- and one bidentate CDCG moiety, i.e., Na[Pt CDCG(N,O) Cl2]. The compound is highly soluble (up to 20 mM) in water and (up to 35 mM) in ethanol, methanol and DMSO. Hydrolysis was investigated because this is assumed to be an important step in intracellular activation and interaction with DNA of this type of compounds. The reaction kinetics of this complex in aqueous solution show unusual behaviour; the substitution process with the displacement of two Cl- was almost instantaneous, and the resulting species were found to be very stable. Kinetic studies carried out in the presence of different NaCl concentrations (up to 500 mM) revealed similar fast and nonreversible aquations of Bamet-M2. This contrasts with the slow aquation of cisplatin in extracellular-line solutions (i.e., at high NaCl concentrations) as compared with fast hydrolysis in cells. This difference may partly account for the low cytostatic activity observed here for Bamet-M2 against several tumor cell-lines.     

Differential mobility analysis-mass spectrometry coupled to XCMS algorithm as a novel analytical platform for metabolic profiling

The development of additional analytical instruments is of great interest to expand metabolome coverage. Differential mobility analyzers (DMAs) are a type of ion mobility spectrometers that can be straightforwardly interfaced with commercial mass spectrometers. In this pilot study, we explored the capabilities of an ion mobility-mass spectrometry platform, based on interfacing a Differential Mobility Analyzer with a commercial quadrupole time of-flight mass spectrometer (DMA-QTOF), to phenotype the metabolic urinary fingerprint of a cohort of prostate cancer patients (n = 8) and a group of healthy counterparts (n = 20). The resolving power of the DMA and the QTOF was ∼55 and ∼6,500, respectively. The transmission efficiency of the DMA was 50%. We illustrate the benefits of incorporating the DMA through the separation of isobaric species according to their electrical mobility, which were not fully resolved by the high resolution QTOF. In addition, we show that the bidimensional electrical mobility-mass spectra obtained can be successfully processed with the XCMS routine, extending its potential to ion mobility-mass spectrometry-based platforms. Data mining with XCMS revealed seven features significantly down-regulated in cancer patients (P < 0.05). These peaks were the input of principal component analysis, showing a clear separation tendency from prostate cancer patients and healthy controls. NIST MS search algorithm was used to classify the samples according to their class, with a resulting 75% sensitivity and 80% specificity. We pursued further fragmentation experiments for structural elucidation of the most discriminant metabolites, thereby illustrating the full potential of this analytical platform for the task. In summary, DMA-MS/MS provides an additional level of separation as compared to traditional mass spectrometry-based methods, thereby increasing the array of multi-analytical platforms available to global metabolite profiling and metabolite identification.