[Influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis].

A number of publications contain contradictory data about influence of mesenchymal stromal cells (MSC) on B-lymphocyte growth, differentiation and production of immunoglobulins (Ig). The aim of the study was to investigate the influence of MSC derived from adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of lymphoblastoid cell line Namalva and myeloma cell line U266. Co-cultivation of Namalva cells with MSC stimulated their proliferation, decreased the doubling time and the minimal effective seeding dose and therefore made cloning of these lymphoblastoid cells possible. The presence of MSC supported the survival and proliferation of Namalva cells cultivated in growth factor deficient medium. MSC also stimulated proliferation of U266 myeloma cells. Both MSC derived from adipose tissue from the healthy donors and from patients with breast cancer effectively stimulated B-cell lines proliferation. Presence of MSC in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells respectively. Co-cultivation of Namalva or U266 with MSC resulted in the formation of close intercellular contacts between cells of both types.

Gene expression array testing of FFPE archival breast tumor samples: an optimized protocol for WG-DASL sample preparation.

Archived formalin-fixed, paraffin embedded (FFPE) tissues constitute a vast, well-annotated, but underexploited resource for the molecular study of cancer progression, largely because degradation, chemical modification, and cross-linking, render FFPE RNA a suboptimal substrate for conventional analytical methods. We report here a modified protocol for RNA extraction from FFPE tissues which maximized the success rate (with 100% of samples) in the expression profiling of a set of 60 breast cancer samples on the WG-DASL platform; yielding data of sufficient quality such that in hierarchical clustering (a) 12/12 (100%) replicates correctly identified their respective counterparts, with a high self-correlation (r = 0.979), and (b) the overall sample set grouped with high specificity into ER+ (38/40; 95%) and ER- (18/20; 90%) subtypes. These results indicate that a large fraction of decade-old FFPE samples, of diverse institutional origins and processing histories, can yield RNA suitable for gene expression profiling experiments.

[Monoclonal antibodies against human secretory component: epitope specificity and utility for immunoanalysis].

AIM: To develop and characterize by immunochemical methods the panel of monoclonal antibodies (MAbs) recognizing different antigenic determinants ofhuman secretory component (SC) molecule. MATERIALS AND METHODS: sIgA and SC were obtained from colostrum by combination of ion-exchange chromatography and gel filtration. Recombinant SC was expressed in Escherichia coli cells transformed by construction that contained fragment of gene coding extracellular domain of receptor for polymeric Ig. MAbs were produced and studied using hybridoma technologies and different methods of immunoenzyme analysis respectively. RESULTS: Panel comprising 10 MAbs against human SC of which 4 types of antibodies recognize cryptic epitopes of free SC and other 6 types recognize epitopes exposed both on SC and sIgA. MAbs panel contains antibodies interacting with conformational and linear epitopes of antigen. Three from obtained MAbs bind to SC epitopes which structure is determined by presence of carbohydrate residues in the molecule of antigen. Immunometric systems were developed which allow to differentially detect free SC and sIgA. CONCLUSION: Developed and characterized MAbs panel recognizing different epitopes of SC molecule opens new opportunities for laboratory and basic research of human secretory immunity.

Differential patterns of allelic loss in estrogen receptor-positive infiltrating lobular and ductal breast cancer.

The two main histological types of infiltrating breast cancer, lobular (ILC) and the more common ductal (IDC) carcinoma are morphologically and clinically distinct. To assess the molecular alterations associated with these breast cancer subtypes, we conducted a whole-genome study of 166 archival estrogen receptor (ER)-positive tumors (89 IDC and 77 ILC) using the Affymetrix GeneChip(R) Mapping 10K Array to identify sites of loss of heterozygosity (LOH) that either distinguished, or were shared by, the two phenotypes. We found single nucleotide polymorphisms (SNPs) of high-frequency LOH (>50%) common to both ILC and IDC tumors predominately in 11q, 16q, and 17p. Overall, IDC had a slightly higher frequency of LOH events across the genome than ILC (fractional allelic loss = 0.186 and 0.156). By comparing the average frequency of LOH by chromosomal arm, we found IDC tumors with significantly (P < 0.05) higher frequency of LOH on 3p, 5q, 8p, 9p, 20p, and 20q than ILC tumors. We identified additional chromosomal arms differentiating the subtypes when tumors were stratified by tumor size, mitotic rate, or DNA content. Of 5,754 informative SNPs (>25% informativity), we identified 78 and 466 individual SNPs with a higher frequency of LOH (P < 0.05) in ILC and IDC tumors, respectively. Hierarchical clustering of these 544 SNPs grouped tumors into four major groups based on their patterns of LOH and retention of heterozygosity. LOH in chromosomal arms 8p and 5q was common in higher grade IDC tumors, whereas ILC and low-grade IDC grouped together by virtue of LOH in 16q.

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.

Two-dimensional protein patterns of Arabidopsis wild-type and auxin insensitive mutants, axr1, axr2, reveal interactions between drought and hormonal responses.

In order to detect gene products involved in Arabidopsis drought adaptive strategy, 2D-PAGE protein patterns of two auxin-insensitive mutants, axr1, axr2, differentially affected in specific drought responses, were compared to the wild-type Columbia ecotype, in well-watered and drought-stressed conditions. Coupled to computer analysis of polypeptide amounts, 2D-electrophoresis revealed subtle changes in protein expression induced by progressive drought stress and/or mutations affecting the auxin response pathway. The differential protein patterns of axr1 and axr2 were consistent with their contrasting drought responses. The specific leaf and root protein patterns of axr1 showed that this mutation disrupts drought responses related to auxin regulation. In particular, the near absence of drought rhizogenesis in axr1 was associated with a root protein pattern closer to the well-watered than to the water-stressed axr2 and Columbia wild-type root protein patterns. Also, the largely different effects of axr1 and axr2 mutations suggest that they affect different pathways in auxin response. Several sets of polypeptides, whose regulation was affected by drought and/or mutation, were thus detected. These polypeptides could play a role both in the auxin and the drought response pathways. Their identification, through microsequencing, should be most informative.

Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana.

Four clones corresponding to Arabidopsis thaliana transcripts regulated by progressive drought stress were isolated. Abundance of the AtDi8, AtDi19 and AtDi21 mRNAs increased in both roots and leaves during progressive drought. The AtDr4 mRNA was expressed in a root-specific manner in regularly watered plants, and became undetectable under drought conditions. In all cases, the drought-induced modifications of mRNA abundance could be reversed by subsequent rehydration. The predicted AtDr4 protein displays extensive similarity to various members of the Künitz protein family, suggesting that AtDr4 might be a root-specific protease inhibitor. Of these four genes, only AtDi8 and AtDi21 responded to an exogenous supply of abscisic acid (ABA). Analysis of the ABA-deficient aba mutant demonstrated that endogenous ABA indeed participates in the drought regulation of these two transcripts. This ABA-dependent response was differentially affected in the various classes of ABA-insensitive Arabidopsis mutants. The AtDi19 and AtDr4 mRNAs both responded to drought in an ABA-independent manner, but at distinct thresholds of the progressive drought stress. Regulation of these four target genes by progressive drought stress thus appears to be mediated by at least three distinct signals, only one of which is ABA.

Current advances in abscisic acid action and signalling.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Drought Rhizogenesis in Arabidopsis thaliana (Differential Responses of Hormonal Mutants).

Drought rhizogenesis is an adaptive strategy that occurs during progressive drought stress and is characterized in the Brassicaceae and related families by the formation of short, tuberized, hairless roots. These roots are capable of withstanding a prolonged drought period and give rise to a new functional root system upon rehydration. The kinetics of drought rhizogenesis during progressive water shortage was analyzed in the Arabidopsis thaliana wild-type ecotypes Landsberg erecta and Columbia. In both genotypes, this response started from a similar threshold of soil humidity (about 2%). The intensity of drought rhizogenesis was compared in various A. thaliana hormonal mutants. The wild-type lines and most of the mutants achieved a similiar drought rhizogenetic index (DRI), defined as the maximum number of short roots produced per mg of root biomass, after progressive drought stress. However, this DRI was dramatically reduced in the abscisic acid (ABA)-deficient aba, ABA-insensitive abi1-1, and auxin-resistant axr1-3 mutants. These data indicate that endogenous ABA and auxin play a promotive role in drought rhizogenesis. The DRI was highly increased in the gibberellin (GA) biosynthetic mutant ga5, suggesting that some GAs might also participate in this process. The possible role and identity of the GA species involved is discussed in view of the unaltered DRI values of the ga2, ga3, and ga4 mutants. The present analysis also allowed further discrimination among the various ABA-insensitive (abi1 versus abi2 and abi3) and auxin-resistant (axr1 versus aux1) mutants tested. In particular, drought rhizogenesis is the first physiological response shown to be differentially affected by the abi1-1 and abi2-1 mutations.

[Distribution of alleles of DRB, DQB and DQA loci of major histocompatibility complex in healthy donors of Saint Peterburg]. / Osobennosti raspredeleniia allelei lokusov DRB, DQB i DQA glavnogo kompleksa gistosovmestimosti u zdorovykh donorov Sankt-Peterburga.

The distribution of alleles of DRB, DQB and DQA loci was investigated in 118 healthy donors from St. Petersburg. Said alleles were identified by the restriction fragment length polymorphism method using DRB, DQB and DQA probes after TaqI digestion and Southern blotting. The frequencies of said alleles were compared with those of a cohort of healthy donors living in Germany. A significantly higher frequency of DRB-17-2 was identified in the Russian donors, as compared with the German counterparts. Also, the Russian donors revealed a decrease in the frequency of DQB-1 (P = 0.01) and an increase in that of DQB-X allele (P = 0.05). An analysis of DRB, DQB and DQA locus a alleles adhesion in a sample of donors from St. Petersburg showed it to agree, in a large proportion of cases, with the literature data. A parallel study of immunological specificity of DR antigen was undertaken to compare the results of genetic and serologic typing; errors in DR specificity identification were found to have been made in 17.0%.

Characterization of a Novel Protein Induced by Progressive or Rapid Drought and Salinity in Brassica napus Leaves.

Under progressive drought stress, Brassica napus displays differential leaf modifications. The oldest leaves, developed before the onset of water deficit, wilt gradually, whereas the youngest leaves harden. Hardening was distinguished by leaf turgor and bluish wax bloom when the shoot water potential was below -3 MPa and the leaf water saturation deficit was about 60%. This adaptive change was accompanied by modifications in two-dimensional protein profiles. Ten percent of the polypeptides had altered abundance or were unique to drought-stressed plants. Two-dimensional analysis of in vitro translation products did not reveal a general decrease in mRNA population. A 22-kD double polypeptide was increased by progressive or rapid water stress and salinity and disappeared upon rehydration. These polypeptides have a common N-terminal sequence, which does not reveal homology with any known water-stress protein but which contains the signature motif of soybean Künitz trypsin inhibitors. Immunoprecipitation allowed these polypeptides to be identified on two-dimensional gels of in vitro translation products. They appeared to be synthesized as a 24-kD precursor, and their transcript was present in the control well-watered leaves, where the polypeptides were never detected, indicating a possible translational regulation. A putative function of this protein, named BnD22, in the retardation of drought-induced leaf senescence is discussed.

A Brassica napus transcript encoding a protein related to the Künitz protease inhibitor family accumulates upon water stress in leaves, not in seeds.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.

[Effect of antilymphocyte globulin on growth and erythroid differentiation of K-562 cells]. / Vliianie antilimfotsitarnogo globulina na rost i éritroidnuiu differentsirovku kletok linii K-562.

The influence of antilymphocytic globulin (ALG) on the proliferative activity and erythroid differentiation of erythromyeloid K-562 cells was studied. It has been established that ALG produces a dose-dependent inhibiting effect (20-150 micrograms of ALG/ml) on the proliferative activity of K-562 cells and induces erythroid differentiation of these cells that does not depend on the concentration of ALG used in the test. Pretreatment of K-562 cells with ALG increases their sensitivity to the following growth-inhibiting action of cytosine-arabinoside.

[Changes in the functional properties of NK-cells and the cytostatic activity of granulocytes in acute leukemia patients]. / Izmenenie funktsional'nykh svoistv NK-kletok i tsitostaticheskoi aktivnosti granulotsitov u bol'nykh ostrym leikozom.

Effector activity of natural killers (NK) was studied, and functional state of NK and granulocytes was evaluated in 23 normal subjects and 28 acute leukemia patients (12 of them being in clinico-hematological remission up to 2 years). In acute leukemia significant disorders are observed in the system of nonspecific antitumor defence that is evidenced by a drastic decrease of NK activity due to changes in the functional properties of NK--their capacity for producing conjugates and blast cell lysis, low sensitivity to exogenous interferon, and lowered spontaneous cytostatic of granulocytes.

[Functional changes in the natural killer cells of patients with acute leukemia]. / Funktsional'nye izmeneniia estestvennykh killernykh kletok u bol'nykh ostrym leikozom.

The results of study of NKC activity (51Cr release assay, conjugate-forming and lytic activity) in healthy persons and patients with acute leukemia (AL) in active phase and in remission are presented. It has been shown that the depression of the NKC activity in acute phase of AL (6 times as low as in norm) and in remission (3 times as low as in norm) depended upon disturbances of the processes of conjugate formation, lytic and recycling activity. Correlation has been found between percentage of lytic conjugates and that of cytotoxicity. The increase of NKC activity in remission seemed to be due to tendency to normalization of functional state of NK-cells (conjugate-forming and recycling activity).

Differential Two-Dimensional Protein Patterns as Related to Tissue Specificity and Water Conditions in Brassica napus var oleifera Root System.

Differential two-dimensional protein patterns as related to tissue specificity and water conditions were investigated within Brassica napus var oleifera root system. The different parts of the root system (tap root, lateral roots, and drought-induced short roots) were analyzed under various moisture regimes (regular watering at field capacity, progressive drought stress, and rewatering). Tissue specificity was evident from 25 differences in protein patterns (qualitative and quantitative) between well-watered lateral and tap roots. Twice as many polypeptides (52) were drought-affected and the response to the water stress was shown to be similar in both root types. In addition, more than half of the polypeptides detected as organ-specific were affected by drought. Based upon the trend of variation observed under drought and rehydration, three categories of polypeptides could be defined that might be differently involved in drought susceptibility or tolerance. A highly differentiated protein pattern characterized the drought-induced short roots. This pattern appeared as far from the watered as from the water-stressed normal roots. In particular, 13 unique polypeptides were detected which could be relevant to their adaptive morphogenesis and/or their specific drought tolerance induction. Upon rehydration, their polypeptide pattern and their specific morphology returned to a normal well-watered lateral root type.

[Cytostatic activity of granulocytes in patients with acute leukemia]. / Izuchenie tsitostaticheskoi aktivnosti granulotsitov u bol'nykh ostrym leikozom.

The in vitro spontaneous cytostatic activity of peripheral blood granulocytes against human tumour cell of erythromyeloid line K-562 has been studied in healthy persons and in patients with acute leukemia. It has been found that with the target cell K-562: granulocyte ratio of 1:20, the normal granulocytes inhibit the K-562 cell growth by 44.3 +/- 3.1% at 37 degrees C within 42 h; in patients with acute leukemia in the acute phase of the disease the cytostatic activity is greatly lowered, while in remission up to two years it increases, however, without reaching the normal values. In remissions of more than three years, this physiological function of granulocytes is normalized.

[Activity of natural killers in acute leukemia in children]. / Aktivnost' estestvennykh killerov pri ostrom leikoze u detei.

Natural killer (NK) activity was studied in healthy children, (16) and those with acute leukemia at different phases of the disease (63). Patients' age ranged 3-11 years. NK activity was measured by release of 51Cr from the labelled K 562 target cells. A sharp decrease in NK activity was found in the acute phase, irrespective of the morphologic pattern of acute leukemia, the degree of blastosis and therapy. 75% of patients achieving remission revealed a decreased NK activity within the first 24 months, while in 25% it was normal. At 3 years and later, the majority of responders showed normal NK activity.

Drought-Induced Changes in Protein Patterns of Brassica napus var. oleifera Roots.

Drought-induced changes in two-dimensional silver stained protein patterns of Brassica napus L. var. oleifera M. root system were detected both at quantitative and qualitative levels. Particularly, 13 new polypeptides of low molecular weight were evidenced in the drought-stressed tap root, 12 of which were also present in the short tuberized roots, a specific drought-induced root type. The reversibility of these modifications, observed after 3 days rehydration, suggests that they might be involved in drought tolerance.