PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Chemical Composition and Insecticidal Activity against Tribolium Castaneum of Thapsia garganica L. Seed Essential Oil

Due to the several side effects of synthetic pesticides, including environmental pollution, threats to human health, and the development of pest resistance to insecticides, the use of alternative healthy, available and efficient agents in pest management strategies is necessary. Recently, the use of essential oil obtained from aromatic plants has shown significant potential for insect pest management. For this reason, the essential oil isolated from seeds of Thapsia garganica L. was investigated for the first time for its chemical profile, and its toxicity and repellency effects against Tribolium castaneum adults. Qualitative and quantitative analyses of the chemical composition by gas chromatography coupled to mass spectrometry (GC/MS) revealed the presence of 18 organic volatiles representing 96.8 % of the total constituents. The main compounds were 1,4-dimethylazulene (51.3 %) followed by methyl palmitate (8.2 %), methyl linoleate (6.2 %) and costol (5.1 %). Concerning the repellent effect, results revealed that SEO (Seed Essential Oil) was very repellent towards T. castaneum adults, with 100 % repellency after 2 h of exposure. Furthermore, the essential oil exhibited remarkable contact toxicity against T. castaneum (93.3 % of mortality) at the concentration of 10 % (v/v). The median lethal dose (LD₅₀) of the topical application of the seed essential oil was 4.4 %. These encouraging outcomes suggested that the essential oil from T. garganica seeds could be considered a potent natural alternative to residual persistent and toxic insecticides.     

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Reactions of the haloalcohols HO(CH2)nCl (n = 2, 3) with platinum(II) - alkynyl and -alkyne complexes. X-ray crystal structure of trans-[Pt(Me) {C(OCH2CH2Cl) (CH2Ph)} (PPh3)2] [BF4]

The chloro complexes trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄, react with 1-alkynes HC---C---R in the presence of NEt₃ to afford the corresponding acetylide derivatives trans-[Pt(Me) (C---C---R) (PPh₃)₂] (R = p-tolyl (1), Ph (2), C(CH₃)₃ (3)). These complexes, with the exception of the t-butylacetylide complex, react with the chloroalcohols HO(CH₂)_nCl (n = 2, 3) in the presence of 1 equiv. of HBF₄ to afford the alkyl(chloroalkoxy)carbene complexes trans-[Pt(Me) {C[O(CH₂)_nCl](CH₂R) } (PPh₃)₂][BF₄] (R = p-tolyl, N = 2 (4), N = 3 (5); R=Ph, N = 2 (6)). A similar reaction of the bis(acetylide) complex trans-[Pt(C---C---Ph)₂(PMe₂Ph)₂] with 2 equiv. HBF₄ and 3-chloro-1-propanol affords trans-[Pt(C---CPh) {C(OCH₂CH₂CH₂Cl)(CH₂Ph) } (PMe₂Ph)₂][BF₄] (7). T alkyl(chloroalkoxy)-carbene complex trans-[Pt(Me) {C(OCH₂CH₂Cl)(CH₂Ph) } (PPh₃)₂][BF₄] (8) is formed by reaction of trans-[Pt(Me)(Cl)(PPh₃)₂], after treatment with AgBF₄ in HOCH₂CH₂Cl, with phenylacetylene in the presence of 1 equiv. of n-BuLi. The reaction of the dimer [Pt(Cl)(μ-Cl)(PMe₂Ph)]₂ with p-tolylacetylene and 3-chloro-1-propanol yields cis-[PtCl₂{C(OCH₂CH₂CH₂Cl)(CH₂C₆H₄-p-Me}(PMe₂Ph)] (9). The X-ray molecular structure of (8) has been determined. It crystallizes in the orthorhombic system, space group Pna2₁, with a = 11.785(2), B = 29.418(4), C = 15.409(3) Å, V = 4889(1) ų and Z = 4. The carbene ligand is perpendicular to the Pt(II) coordination plane; the PtC(carbene) bond distance is 2.01(1) Å and the short C(carbene)-O bond distance of 1.30(1) Å suggests extensive electronic delocalization within the Pt---C(carbene)---O moietry.     

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     

Hematology and plasma chemistry values in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

To establish baseline hematologic and plasma biochemistry values in free-ranging Humboldt penguins (Spheniscus humboldti), heparinized blood samples were collected from 51 apparently healthy, adult Humboldt penguins residing at two colonies off the Chilean coast. Thirty samples were collected in April, 1992, from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September, 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Hematologic values measured include packed cell volume, leucocyte count, leucocyte differential, and the presence of blood parasites. Plasma biochemistry values measured include glucose, blood urea nitrogen, creatinine, uric acid, calcium, inorganic phosphorous, sodium, potassium, chloride, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and creatine kinase. Only the mean values for chloride and for the number of eosinophils differed significantly between the two sample groups. No blood parasites were seen. © 1995 Wiley-Liss, Inc.     

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.