The rhodium(III) trisacetonitrile complexes: a re-investigation of the synthesis of fac- and mer- [RhCl3(CH3CN)3], their characterization by 2D NMR spectroscopy and the X-ray crystal structure of mer-[RhCl3(CH3CN)3] · CH3CN

It is shown that the reaction of RhCl₃·3H₂O with acetonitrile normally produces mixtures of mer- and fac-[RhCl₃(CH₃CN)₃] (1a and 1b, respectively). The IR and ¹H NMR spectra of these isomers were re-investigated. Their two-dimensional (¹⁰³Rh,¹H) NMR spectra were also recorded. Equilibrium and exchange studies of 1a and 1b in CD₃C were performed. It was found that in 1a the exchange rate of the nitrile molecule trans to Cl is much faster than those of mutually trans nitriles. Also the nitrile molecules in 1b underwent fast exchange in CD₃CN; however, their rate was slightly faster than that of the more labile CH₃CN in 1a. The X-ray crystal structure of mer-[RhCl₃(CH₃CN)₃]·CH₃CN (1c) was determined. Crystal data: triclinic space group

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Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

In vitro mimicry of CaMBr1 tumor-associated antigen by synthetic oligosaccharides

The murine monoclonal antibody (Mab) MBr1, raised against thebreast cancer cell line MCF7, recognizes a saccharidic epitopeoverexpressed on a high percentage of human breast, ovary, andlung carcinomas. This antigen was originally identified on theimmunogen as a globo-series glycosphingolipid with an H-likedeterminant at its terminus (globo-H). We report here the biologicalcharacterization of the entire globo-H hexasaccharide and fivesynthetic oligosaccharides representing fragments of the entirestructure andlor different anomeric configurations. Using competitivebinding assays on live cells, we identified the residues andthe linkages essential for mimicry of the cellular antigensrecognized by Mab MBr1 on the breast carcinoma cell line MCF7and small cell lung cancer cell line POVD. The terminal tetrasaccharidicfragment of globo-H is the oligosaccharide that most resemblesthe MBr1-defined epitope both on glycolipids and on glycoproteins.This information will help in the rational design of a highlyspecific reagent for active specific immunotherapy of carcinomasoverexpressing the MBr1-defined antigen. CaMBr1 immunotherapy monoclonal antibody oligosaccharides tumor-associated antigen     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996     

Genetic differentiation and detection of cryptic species in the genus Lepismachilis (Insecta: Microcoryphia) from the Western Mediterranean region

Different species of the bristletail genus Lepismachilis were collected in 14 localities in Italy and Spain and an allozyme electrophoretic survey was carried out to estimate the degree of genetic variability and differentiation at intra- and interspecific levels. Four morphological species were initially identified (L osellai, L. y-signata, L. affinis, L. targionii), but the electrophoretic analysis demonstrated the presence of two additional species among the individuals of L. targionii (Lepismachilis spl and sp2). The validity of these species and their differentiation from L targionii were demonstrated by the fixation of alternative allelic patterns at several loci (7 in Lepismachilis spl and 8 in Lepismachilis sp2), coupled with fixed, previously undetected, morphological differences. In addition, Lepismachilis sp2 was sympatric with L. targionii in three collecting sites, where the fixation of alternative allelic patterns unequivocally demonstrated reproductive isolation. Genetic variability did not seem to be correlated with local ecological factors, and differences between species should rather be explained by different historical factors. Low levels of gene flow, estimated with two different indirect methods, were observed in L. targionii and L. y-signata, and were due to high levels of structuring among populations. Genetic differentiation among conspecific populations was not correlated to their geographical arrangement and the presence of loci fixed for different alleles among them suggested that stochastic factors (such as genetic drift) may have played a role in determining genetic differentiation of geographically isolated populations. Genetic divergence values indicated that the six species are well differentiated and allozyme profiles were diagnostic for all of them. On the other hand, allozyme data did not provide adequate information to resolve evolutionary relationships among the species, nor did they confirm the validity of the two subgenera (Lepismachilis and Berlesilis) in which the genus Lepismachilis is traditionally divided.     

Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.