VCAM-1 expression on bone marrow stromal cells from patients with myelodysplastic syndromes.

We studied the expression of VCAM-1 adhesion molecules on stromal cells from the bone marrow of patients with myelodysplastic syndromes, healthy donors, and patients with chronic myeloproliferative diseases and acute leukemias. Expression of adhesion molecule on mesenchymal stromal cells from the bone marrow of patients and healthy donors was evaluated after 2-4 passages by the methods of immunoprecipitation and electrophoresis. VCAM-1 expression in the majority of patients with myelodysplastic syndromes was lower than in healthy donors. At the same time, VCAM-1 expression was not identified on mesenchymal cells from acute leukemia patients. VCAM-1 expression on cells from patients with chronic myeloproliferative diseases did not differ from that in healthy donors. We conclude that VCAM-1 synthesis in bone marrow stromal cells is impaired in patients with myelodysplastic syndromes and acute leukemias. These changes can be followed by the loss of relationships between hemopoietic cells and stromal microenvironment in bone marrow niches. Hemopoietic cells gain the ability for uncontrolled growth, which results in progression of the disease.

Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts.

METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.

[Limited proteolysis of integrin alpha v beta3 from human placenta]. / Ogranichennyi proteoliz integrina alpha v beta3 iz platsenty cheloveka.

Purification of alpha v beta 3 integrin from human placenta with successive usage of two affinity sorbents--immobilized monoclonal antibodies to alpha v beta 3 integrin and immobilized RGD-containing decapeptide allowed to purify this integrin's partially degraded fraction, that was nevertheless able to interact with its ligand. During the incubation of partially degraded alpha v beta 3 integrin at 37 degrees C its further degradation went on. Addition of serine proteinase inhibitors: (phenylmethilsulfonyl fluoride, leupeptin and aprotinin) completely suppressed integrin further degradation of alpha v beta 3. In preparations of intact and partially degraded alpha v beta 3 integrin specific activity of two serine proteinases--urokinase and dipeptidilpeptidase IV--was discovered. alpha v beta 3 integrin, undergoing limited proteolysis, had lesser affinity towards RGD peptide, that intact integrin. The results show, that alpha v beta 3 integrin from human placenta co-purifies with serine proteinases. It is suggested that a definite part of functionally active alpha v beta 3 integrin, extracted from human placenta by triton X-100, forms a stable complex with serine proteinases.

[Aminopeptidases in human leukemia cells]. / Aminopeptidazy v leikoznykh kletkakh cheloveka.

Aminopeptidase activity in lymphoid cells of patients with various lymphoproliferative diseases was studied. The enzyme activity was detected in lysates of all leukemic B- and T-cells. The lymphoid cells contained aminopeptidases of at least two classes: metallo- and SH-dependent enzymes. The SH-dependent aminopeptidase with pH optimum 8.5-9.0 was revealed in lymphoid cells for the first time, and it seems to belong to a poorly studied aminopeptidase family.

Proteolytic enzymes in human leukemic lymphoid cells. III. Aminopeptidases, angiotensin-converting enzyme, and its inhibitor in cells of different immunological phenotype.

Activities of plasma membrane proteinases such as angiotensin-converting enzyme (ACE), aminopeptidases, and dipeptidyl peptidase IV (DPP-IV) were determined in lymphoid cells of various immunological phenotype which were obtained from 30 patients with lymphoproliferative diseases. The enzyme activities significantly varied depending on the immunological phenotype and stage of cell differentiation, but no correlation was found between activities of ACE, DPP-IV, and aminopeptidases in the cells of different type. The cell lysates studied contained at least two classes of aminopeptidases: metal- and sulfhydryl-dependent enzymes. A sulfhydryl-dependent aminopeptidase with activity optimum at pH 8. 5-9.0 was found for the first time and is suggested to be from a poorly studied aminopeptidase family. In addition to ACE, lysates of leukemic T- and B-cells were found to contain an inhibitor of ACE which was not previously described for these cells.

[A comparative study of aspartyl and cysteine proteinases and their inhibitors in human B- and T-cell leukemias]. / Sravnitel'noe izuchenie aspartil'nykh, tsisteinovykh porteinaz i ikh ingibitorov v leikoznykh B- i T-limfotsitakh cheloveka.

A procedure was developed for simultaneous isolation of aspartyl and cysteine proteinases as well as of the cysteine-proteinase inhibitors. Affinity chromatography using pepstatin-Sepharose enabled one to isolate aspartyl proteinases, while inhibitors of cysteine-proteinases were isolated by affinity chromatography on CM-papain-Sepharose; further purification of the enzymes was carried out using ion exchange chromatography and gel filtration. Partially purified preparations of cathepsin D as well as of cysteine-proteinases and their inhibitors were obtained. Some physicochemical and enzymatic properties of the enzymes and inhibitors obtained were studied.

Dipeptidylpeptidase IV in human leukemic B- and T-cells.

Dipeptidylpeptidase IV (DPP-IV) activity in lymphoid cells of patients with various lymphoproliferative diseases has been assayed. The enzyme activity was detected in lysates of all leukemic T- and B-cells studied. High DPP-IV activity levels (5-7 fold higher than those in mature Th and Ts) were found in T-cells with phenotypes corresponding either to the early thymic stages of maturation, or to activated T-lymphocytes, or to some atypic T-cells. In most leukemic B-cells with phenotypes of mature and late B-lymphocytes, the DPP-IV activity was lower than in mature T-cells; however, in some B-cells carrying the activation markers CD 25 and CD 30 the activity was rather high. High DPP-IV activity was seen in the majority of leukemic B-cells expressing CD 25 (87%) and CD 11c (82%) antigens. Experiments with intact cells have shown that DPP-IV is present in the plasma membrane of leukemic B- and T-cells studied. Thus DPP-IV cannot be considered as a marker of activated T-cells alone because a significant DPP-IV activity was also detected in a number of activated leukemic B-cells and in early T-cells.

[Properties and specificity of cathepsin H from the bovine spleen]. / Izuchenie svoistv i spetsifichnosti katepsina H iz selezenki byka.

Action of cathepsin H from bovine spleen on tuftsin, enkephalin, the oxidized B chain of insulin and a variety of synthetic substrates was studied. Cathepsin H splits off only one N-terminal amino acid from each tuftsin and enkephalin, which, according to the literature, led to inactivation of peptides. The enzyme acts on the oxidized B chain of insulin as an aminoendopeptidase: it splits off the N-terminal phenylalanine and the centrally located bond(s). Km and Vmax for the cathepsin H catalyzed hydrolysis of LeuNA, ArgNA LysNA and BANA were determined. Substrates with the free NH2 group were hydrolyzed at a higher rate. Based on the data obtained and the previously reported results on conversion of kallidin into bradykinin, the specificity of cathepsin H and its possible biological functions were discussed. Cathepsin H appears to participate in formation and inactivation of physiologically active peptides. Using the antiserum to spleen cathepsin H it was found that liver, kidney and lung tissues contained the enzymes identical and/or partially identical to cathepsin H from spleen. The data on the properties of cathepsin H from various sources are summarized.

[Immunochemical study of bovine spleen thiol proteinases: cathepsinS B, H and L]. / Immunokhimicheskie issledovaniia tiolovykh proteinaz selezenki byka: katepsinov B, H i L.

Rabbit antisera were prepared against three highly purified enzymes from bovine spleen: proteinase I (cathepsin L), proteinase II (cathepsin H), and cathepsin B. The Ouchterlony double diffusion test shows that each antiserum specifically reacts with the corresponding antigen and does not cross react with other proteinases. These data provide evidence that the three proteinases are distinct with respect to their antigenic properties. Using specific antisera, the identity of two preparations of proteinase I isolated by different methods was demonstrated. Analysis of the fractions obtained in the course of isolation procedure revealed a component reacting with antisera against proteinase I. It had a greater molecular mass than proteinase I (30 000-40 000), was richer in antigenic respect and had a lower proteolytic activity as compared with proteinase I. The effect of various inhibitors and denaturation conditions on antigenic properties of proteinases was also studied.

[Characteristics of two thiol proteinases from spleen active in neutral media]. / Kharakteristika dvukh aktivnykh v neitral'noi srede tiolovykh proteinaz selezenki.

Two SH-dependent proteinases (I and II) active in neutral media were isolated from bovine spleen and purified to apparent homogeneity. The histone-hydrolyzing activity of proteinase I was increased 3500-fold as compared to that of the original extract. Proteinase I hydrolyzed a variety of proteins (histones, azocasein, hemoglobin, collagen) but did not hydrolyze low molecular weight synthetic substrates, such as BAPA, BANA, BAEE, ATEE, Leu-beta-NA, Arg-beta-Na and Ala-beta-NA. The molecular weight of the enzyme as determined by SDS electrophoresis was found to be about 23,000. Isoelectrofocusing of the enzyme resulted in one major component with pI of 6.05 and in two minor components with pI of 6.2 and 6.4. Proteinase II hydrolyzed Leu-beta-NA, Arg-beta-NA and Ala-beta-NA but did not hydrolyze beta-naphthylamides of dicarboxylic acids and Gly-Phe-beta-Na. This proteinase split BANA and histone and very slowly split azocasein and collagen. Proteinase II was found to have a molecular weight of 30 000 and a pI of 6.8-6.9. Proteinase I inactivated fructose-1.6-diphosphate aldolase, partly inactivated glucose-6-phosphatase dehydrogenase and caused activation of phosphodiesterase of cyclic nucleotides. Proteinase II had no effect on the activity of the above enzymes. A comparison of proteinase I and II with enzymes described in literature demonstrated that the former was cathepsin L, while the latter was cathepsin H from spleen.