The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation.     

Computerized Stabilometry on a Four-Legged Platform for Comparative Analysis of Foot Positioning

Biomedical Engineering - Computerized stabilometry using a four-legged stabilometric platform allows “free” positioning of the feet to be used, which is of fundamental importance for...     

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors.     

On the possibility of the unification of drug targeting systems. Studies with liposome transport to the mixtures of target antigens

In order to make the drug targeting system more effective, simple and technological, we suggest creation of drug-bearing conjugates capable of simultaneous binding with different antigenic components of the target via specific antibodies. It is supposed that the targeted therapy should include sequential administration of the mixture of modified antibodies (or other specific vectors) against different components of affected tissue and, upon antibody accumulation in the desired region, administration of modified drugs or drug carrying systems which can recognize and bind with the target via accumulated antibodies due to the interaction between vector modifier and carrier modifier. Using as a model system monolayers consisting of the mixture of extracellular antigens and appropriated antibodies, it was shown that the treatment of the target with the mixture of biotinylated antibodies against all target components and subsequent binding with the target of biotinylated liposomes via avidin permits high liposome accumulation on the monolayer. The binding achieved is always higher than in the case of the utilization of single antibody-bearing liposomes. Besides, the system suggested is very simple and its components can be easily obtained on technological scale in standardized conditions.     

Two novel polyadenylation mutations leading to β+-thalassaemia

In an ongoing effort to identify point mutations causing beta-thalassaemia, we have found two previously unreported mutations which are located in the Poly A site of the beta-globin gene. The screening programme used amplified DNA and dot-blot hybridization with several 32P-labelled oligonucleotide probes. DNA samples which remained unidentified by this methodology were subjected to sequencing with 32P-labelled primers and modified T7 DNA polymerase. The newly discovered mutations were confirmed by the dot-blot hybridization technique. One type concerned an AATAAA----AATGAA mutation in the polyadenylation site and was found in one family from Yugoslavia (including one patient with the C----T mutation at codon 29 in trans), one from Bulgaria (the patient had the G----A mutation at IVS-I-110 in trans), and one from Greece (this patient had the C----G mutation at IVS-II-745 in trans). Haematological data for three simple heterozygotes suggested a rather mild beta(+)-thalassemia. The second type involved an AATAAA----AATAGA mutation and was found in one family from Malaysia. The propositus had the beta E mutation on the other chromosome, was originally diagnosed as mild Hb E-beta(+)-thalassaemia, and had Hb A and Hb E percentages which were nearly the same.     

Parameters of thermoelectric cooling devices in conditions of variable temperatures of the heat-exchange media

New method for optimized computing thermoelectric coolers is proposed for the case of variable temperatures within heat-transfer media. The operation of the device is analyzed when the temperature of the cooled medium is greater than the temperature of the heated one, i. e. under conditions of the negative temperature difference. The comparative analysis of the computed and experimental data in values of the cooling and electric power demonstrates fully satisfactory results.     

A new mutation in the beta-globin gene (IVS II-850 G-C) found in a Yugoslavian beta-thalassemia heterozygote.

BACKGROUND. The recent development of laboratory techniques that can rapidly characterize the molecular defects of beta-thalassemia has resulted in the discovery of more than 100 different point mutations in the beta-globin gene. These mutations are population specific. About 20 of them account for over 90% of beta-thal genes in the world. The other mutations are usually found in single families. In this paper we describe a case with a novel mutation at position IVS II-850 (G-C) as a cause of beta-thalassemia. METHODS. Direct sequencing of PCR amplified DNA was used for the detection of the mutation. ASO probes were synthesized for dot-blot hybridization. Expression of the mutated allele was evaluated through Northern blot and RNA-PCR analyses. RESULTS. This mutation was found in four members of a family, who exhibited severe microcytosis and hypochromic anemia, with an average alpha/beta ratio of 2.0. The sequencing of PCR amplified DNA showed a G-C mutation at position IVS II-850 of the beta-globin gene. Dot blot analyses confirmed the presence of this substitution in all four carriers. Northern blot and RNA-PCR analyses did not reveal any abnormally spliced mRNA species. DISCUSSION. The G-C substitution at position IVS II-850 is the third mutation in the invariant AG dinucleotide of the acceptor splice site of the second intron of the beta-globin gene. It abolishes normal splicing, which leads to abnormally processed mRNA. It is a relatively rare mutation since it was not detected among the uncharacterized beta-thal chromosomes from Yugoslavia.     

Hb Icaria–Hb H disease: identification of the Hb Icaria mutation through analysis of amplified DNA

Hb Icaria-Hb H disease was observed in a Yugoslavian teenager who exhibited moderate anaemia with severe microcytosis and hypochromia and 16% Hb H. Four of his relatives were Hb Icaria heterozygotes; their haematological data were comparable to those with a deletional type of alpha-thalassaemia-2. The patient also had an additional alpha-thalassaemia-1 deletion, an approximately 20.5 kb deletion, common among Mediterranean populations. The Hb Icaria mutation, i.e. the TAA----AAA mutation at codon 142, was identified by hybridization of amplified DNA with specific probes. The mutation is located on the alpha 2-globin gene; the one remaining alpha 1-globin gene is apparently able to compensate sufficiently for the loss of the three alpha-globin genes to maintain a haemoglobin level of 8-9 g/dl.