Jan Lakota

Amino acid incorporation by isolated rat liver mitochondria into two protein components of mitochondrial ATPase complex: Lack of incorporation into dicyclohexylcarbodiimide binding proteolipid

The role of products of mitochondrial protein synthesis in the biogenesis of the mitochondrial ATPase complex of yeast and fungi is well documented. In Saccharomyces cerevisiae four [ 1 ] and in Neurospora crassa two [2] protein subunits of the complex are synthesized intramitochondrially. Special attention has been paid to the elucidation of the site of the synthesis of DCCD binding proteolipid of the mitochondrial ATPase complex. In yeast [3] this component is synthesized within mitochondria and in fungi [4,5] outside the organelles. Less information exists on the participation of mitochondrial translation products in the formation of mitochondrial ATPase complex and on the site of the synthesis of DCCD binding proteolipid of the complex in animal cells. The intramitochondrial formation of three protein components of mitochondrial ATPase complex in oocytes [6] and in regenerating rat liver [7] has been indicated by selective invivo inhibition of cytoplasmic or mitochondrial protein synthesis. Incorporation of labeled amino acids by isolated rat [8] and mouse [9] liver mitochondria into DCCD binding proteolipid has been described and it was concluded that the DCCD binding component of rat [8] and mouse [9] liver mitochondrial ATPase complex represents a product of mitochondrial protein synthesis. On the other hand, selective in vivo inhibition of mitochondrial protein synthesis in regenerating rat liver did not affect significantly amino

ADP, ATP TRANSLOCATOR PROTEIN OF RAT HEART, LIVER AND HEPATOMA MITOCHONDRIA EXHIBITS IMMUNOLOGICAL CROSS-REACTIVITY

Introduction Differences in the content and kinetic properties of the ADP, ATP translocator in mitochondria from various sources have been demonstrated (reviewed [ 1,2] ). Recently the translocator protein was isolated from several organs [3-51 and data were reported suggesting differences between immunological properties of this protein isolated from heart, liver and kidney mitochondria [5]. In this study a comparison of immunological properties of translocator protein of rat heart, liver and hepatoma by a radioimmunological technique revealed that the same antigenic determinant(s) related to ADP, ATP translocator protein are present in the mitochondria of all the above tissues. In addi- tion, the data obtained suggest that antigenic proper- ties of translocator protein isolated from different sources are unequally modified during the isolation. 2. Materials and

Mitochodrial ATPase of Zajdela hepatoma: Presence of F1-specific antigenic determinants outside mitochondria

The properties and the content of ATPase in mitochondria of Zajdela hepatoma differ from those in rat liver mitochondria. ATPase activity of coupled Zajdela hepatoma mitochondria is insensitive to uncouplers [I] and the content of the enzyme in hepatoma mitochondria represents only about half the amount of ATPase present in mitochondria of rat liver [2]. It is conceivable that the low ATPase content in Zajdela hepatoma mitochondria may be due not to a dinlinished synthesis of the enzynte in tumor cells but rather to a disbalance between the formation of the soluble (Fi) ATPase which is synthesized in the cytosol (reviewed [3,4]) and its integration into the mitochondrial membrane. In an attempt to check this possibility the presence of Fr-specific antigenic determinants in postribosomal fraction of Zajdela hepatoma and rat liver has been investigated in the present study. Using competition radioimmunoassay it was found that postribosomal fraction of Zajdela hepatoma contains significant amounts of antigenically active F, components, whereas no Fispecific antigenic determinants were detected in the corresponding fraction of rat liver.

Intracoronary mesenchymal stem cell transplantation in patients with ischemic cardiomyopathy

overcome during the implantation of this stent. Passage of the MGS through tortuous, calcified or small diameter vessels is very challenging, if not impossible. The few cases, where we failed to advance the stent through the lesion, were shepherds crook type of RCA anatomy. Another important issue is the compromising of side branches by the presence of polymer mesh sleeve on its external surface. In addition, there are no data about its behavior in high-thrombotic volume, especially when no other EPD were used. In our study, there were a few cases with thrombus load grade five, where M-Guard was used with the aspiration catheter and the effectiveness of their combination was impressive, resulting in TIMI 3 flow with satisfactory blush score. The limitations of our study concern the relatively small number of patients, taking mostly into consideration the subgroup of patients from theMGS group that TAwas not performed. Another aspect is also the lack of a randomized design. In conclusion, in the setting of pPCI, MGS proved to be effective and safe with low TVR and low thrombosis rates at 1 and 12 months, considering the high risk group of STEMI patients. In addition, final TIMI flow and MBG are higher and the incidence of no-reflow is less. That benefit from its use is independent from the performance of TA in pPCI. The authors report no relationships that could be construed as a conflict of interest.

Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia.

High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes--DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon.

Antibodies against Carbonic Anhydrase in Patients with Aplastic Anemia

Background/Aims: Antibodies against carbonic anhydrase (CA) have been detected in patients with an aplastic anemia (AA)-like syndrome after autologous stem cell transplantation. Methods: We analyzed sera of 53 bona fide AA patients before and after treatment with anti-thymocyte globulin (ATG) or bone marrow transplantation for the presence of anti-CA antibodies. Results: Anti-CA antibodies were detected in 20 patients (38%) and were associated with older age at diagnosis of AA. Antibody-positive patients showed poor response to ATG treatment (complete response 14%) and inferior long-term survival (36% at 10 years), when compared to antibody-negative patients (complete response and 10-year survival both 64%). Two thirds of patients with antibodies at diagnosis of AA became antibody negative after treatment with ATG. Clearance of the antibody did not appear to be associated with hematological improvement. Conclusion: Antibodies against CA are detected frequently at diagnosis of AA, and their presence identifies a subset of patients with poor response to immunosuppressive treatment.

Sera of patients with spontaneous tumour regression and elevated anti-CA I autoantibodies change the gene expression of ECM proteins.

Spontaneous tumour regression after high‐dose therapy and autologous stem cell transplantation is associated with the aplastic anaemia‐like syndrome and the presence of polyclonal autoantibodies against carbonic anhydrase I (CA I). When tumour cells were grown in vitro in the presence of patients’ sera positive for anti‐CA I autoantibodies, their morphological pattern was altered. These changes were accompanied by modifications in the gene expression profile. We observed downregulation of genes of the basal lamina assembly (collagen type IV alpha 4, the laminin subunit gamma 2), the extracellular matrix (collagen type I alpha 1), the cytoskeleton (keratin 14 type I), the collagen triple helix repeat containing 1 and the proto‐oncogene WNT7B. On the other hand, the expression of the CA 1 gene was increased in the tumour cells. It was also noticed that the presence of anti‐CA I autoantibodies did not impair tumour cell proliferation and cell viability in vitro. These findings were observed only in the presence of patients’ sera positive for anti‐CA I autoantibodies.

Silencing of CA1 mRNA in tumour cells does not change the gene expression of the extracellular matrix proteins

We report the silencing of CA1 mRNA in PC3 and MDA cells. The levels of mRNA coding CA1 protein in the knock‐down mRNA (CA1 siRNA) cells have been measured by RT‐PCR and were approximately 5% (PC3) and 20% (MDA‐MB‐231), respectively, of the level of control (Mock siRNA) used during silencing. In PC3 and MDA‐MB‐231 cells, the mRNAs for COL1A1 and COL4A4 were up‐regulated. The mRNAs for CTHRC1, LAMC2, and WNT7B were not changed when compared to the control. The morphology of the cells during the treatments remained the same. On the Western blots, the lysate from the silenced cells showed lower levels of CA I as well.

Fate of human mesenchymal stem cells (MSCs) in humans and rodents-Is the current paradigm obtained on rodents applicable to humans?

Dear Editor, Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types (osteoblasts, chondrocytes, myocytes and adipocytes). MSCs are found in bone marrow, adipose fat tissue, umbilical cord, dental pulp and other tissues [for review, see]. They are able to regenerate different damaged tissues. There is an intensive research of the use of MSCs in the tissue regeneration and the disease treatment, which is performed on animals, mainly rodents. Moreover, it has been shown that in rodents (mice, rats), the MSCs migrate directly to the damaged, inflamed areas, including tumours. This phenomenon has been used to prepare “therapeutic” MSCs. The “therapeutic” MSCs are genetically modified MSCs that contain stable gene coding for protein or enzyme product able to kill the tumour cells. An example of such construct is the yeast enzyme cytosine deaminase, which converts the rather non-toxic 5-fluorocytosine to the cytostatic agent 5fluorouracil. In the presence of 5-fluorocytosine, the MSCs, which (in rodents) invade in the tumour, were able to kill the tumour cells and to “cure” the animals. This therapeutic concept became known as the “stem cell-based cancer gene therapy”. The main “ideological” obstacle of the MSC use (normal or genetically modified) is the observation that the intravenous infusion of MSCs generally leads to their entrapment in the lung, liver and spleen. However, this experimental fact is based on the data obtained on rodents (mice, rats). We used the “therapeutic” MSCs to treat a patient with squamous carcinoma of the tongue who developed lung metastases. There was no sign of any therapeutic effect after intravenous (not local, ie intratumour) administration of the “therapeutic” MSCs. (The CT scan performed on day +6 (the “therapeutic” MSCs were applied on the day 0) showed no difference in the size or density of the patients’ pulmonary metastases compared to the CT scan on day 1. On the day +40, there were signs of a progression of the metastases on the CT scan.) After the intravenous administration, the “therapeutic” MSCs were “homing” in the bone marrow. Even a rather low cell count was able to cause grade 2 thrombocytopenia and grade 3 neutropenia, respectively. We conclude that there has not been any entrapment of the “therapeutic” MSCs in the lungs. Nor the cells were homing in tumour metastases. However, even a small number (60 9 10 cells) of the “therapeutic” MSCs were able to cause a deep temporal bone marrow suppression. We have performed a treatment of another patient with metastatic (liver, retroperitoneum, abdominal wall) ovarian carcinoma. The “therapeutic” MSCs were applied locally into two metastases in the abdominal wall. We have observed only partial necrosis of these two metastases. The other metastases progressed without any sign of temporal regression, and the patient finally died of liver failure. There was no sign of any therapeutic effect of the “therapeutic” MSCs on other patients’ metastases. We concluded that the “therapeutic” MSCs did not migrate to any “neighbour” metastases (liver, retroperitoneum, abdominal wall) (Lakota J, unpublished observation). The indirect confirmation that the MSCs (or “therapeutic” MSC) are in humans not entrapped in the lung, liver and spleen comes from thousands haematopoietic stem cell transplantations performed annually in patients with malignant disease. A considerable part of these transplants is performed with the stem cells obtained from bone marrow. The bone marrow is rich in haematopoietic as well as mesenchymal stem cells. So far, nobody observed any entrapment of these cells in the patient’s lungs. In our opinion, the requisitioned data of the MSCs, mechanically taken from mice and rats, could negatively influence the trends of the research in the novel treatment(s) of human diseases.