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.     

Locomotor Activity in the Freshwater Crab Potamon fluviatile: The Analysis of Temporal Patterns by Radio-telemetry

Locomotor activity in a field population of the freshwater crab, Potamon fluviatile, was studied during the breeding season by means of radio-telemetry and by direct counts of active animals along a transect of the stream. The basic pattern of crabs' locomotor activity can be described as a sequence of short distance movements around the shelters (foraging movements), followed by rarer long distance displacements (wandering movements). Whilst direct counting showed that the crabs exhibit a broadly nocturnal rhythm in foraging movements, no daily periodicity in wandering activity was revealed by telemetry. There is sexual difference in these latter excursions: females move farther along the stream and into the surrounding terrestrial habitat than do males. This behaviour is in contrast to observations made during the non-breeding season and is probably related to the stage of female reproduction.     

An elongation factor Tu (EF-Tu) resistant to the EF-Tu inhibitor GE2270 in the producing organism Planobispora rosea

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [α-³²P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high-affinity ATP-binding domain.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

Leaf characteristics and optical properties of different woody species

 Light partition has been examined and evaluated on five woody species (Olea europaea, Ficus carica, Pittosporum tobira, Hedera helix maculata, Persica vulgaris) in relation to their leaf morpho-histological characteristics, water and chlorophyll contents. Leaf parameters and optical properties (reflectance, transmittance, absorbance) in PAR, FR and NIR wavebands (400–1100 nm) were preliminarily submitted to a canonical correlation analysis where lamina thickness and water content showed a leading role in determining all the optical properties, while chlorophyll, influential in the PAR region, was remarkably effective only in an extreme pigment situation when green and albino patches of ivy leaves were compared. Transmittance appeared inversely related to lamina thickness in accordance with the Lambert Beer law. Significant correlations were found also between mesophyll water content and both transmittance (positive) and reflectance (negative). Olive leaves showed peculiar optical patterns because of the dense and continuous trichome layer on their abaxial surface. Received: 3 January 1997 / Accepted: 5 May 1997     

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.     

Distribution and abundance of house-dust mites (Acarina: Pyroglycphidae) in Rome,Italy

For the first time in Rome, house-dust mite infestation was studied in 90 randomly selected houses. In each house, mite infestation was assessed in three sites: mattress, bedroom and living room. In total, 87.8% of the sampled houses were positive for dust mites. In the houses infested, 11.4% showed densities of >100 mites/g of dust, 15.2% registered densities between 50 and 99, and in the remaining houses (73.4%), the densities were between 1 and 49 mites/g dust. The percentages of infested houses were positively correlated with the relative humidity (RH) values (r=0.89,P=0.02). At the lowest range of RH (between 46 and 50), the infestation was 50% and at the highest range of RH (between 73 and 78) it was 100%. The mattress was significantly the most infested (71.1%) of the tested sites. Only wool and spring mattresses were infested, and they did not show any significant differences in mite concentrations.Dermatophagoides farinae was the most abundant species (53.1%), followed byGlycyphagus domesticus (34.5%),D. pteronyssinus (5.2%), andEuroglyphus maynei (0.2%);D. farinae was also the most frequent species (56.9%). The remaining specimens (7.0%) were predator species commonly found in houses. The prevalence ofD. farinae in Rome is discussed.     

Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.     

Effects of protein perturbants on phospholipid bilayers

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.     

Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.