The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site.

The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.     

Development and characerization of cell lines from subhuman primates

Summary Seven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro. Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence. All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties. These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells. The research upon which this publication is based was performed pursuant to Contract No. NIH-69-100 with the Division of Biologics Standards of the National Institutes of Health.     

Therapeutic gain factors for fractionated radiation treatment of spontaneous murine tumors using fast neutrons, photons plus O2(1) or 3 ATA, or photons plus misonidazole

Therapeutic gain factors (TGFs) have been determined for three spontaneous tumors of the C3H mouse treated by photons + normobaric oxygen (O2(1) ATA), photons + hyperbaric oxygen (O2 3 ATA), photons + misonidazole, or fast neutrons. The tumors were early generation isotransplants of spontaneous tumors: MCaIV, a mammary carcinoma; FSaII, a fibrosarcoma; and SCCVII, a squamous cell carcinoma. The tumors, transplanted to the right leg, were 6 mm at start of treatment. Normal tissue responses studied were acute reaction of normal skin (all treatment modalities) and LD50 following irradiation of the upper abdomen (in test of photons + O2 at 1 or 3 ATA). Thus both the tumor and normal tissues would be classified as "acute responding." All subject tissues were at congruent to 34.5-35 degrees C at irradiation. Treatments were based on d(25)Be or p(43)Be fast neutron beams, 60Co and 137Cs photon beams. Treatments were given in 5 or 15 equal doses in 5 days. For photon treatments, TGFs (air/O2 3 ATA) were substantially and significantly larger than 1 for all three tumor systems treated at small or large doses per fraction when related to skin or abdominal tissue responses. The TGFs (air/O2 1 ATA) were greater than 1 at small doses per fraction for MCaIV and FSaII for skin as the normal tissue; the TGFs for all three tumors and at all doses per fraction would be greater than 1 when related to upper abdominal tissues. TGFs (O2 1 ATA/O2 3 ATA) for photon irradiation greater than 1 were found only for SCCVII and that obtained for both large and small doses per fraction. Misonidazole achieved impressive TGFs (air/air + miso or air/O2 1 ATA + miso); the drug was tested only at 10-12 Gy/fraction and relative to skin. RBEs(FN) for the three tumors were lower at 1.5-2 Gy(FN)/fraction than at 5-6 Gy(FN)/fraction, i.e. the opposite to that reported for normal tissue (RBE increases with decreasing dose per fraction). A TGF (relative to skin reaction) greater than 1 for fast neutron therapy was found only for SCCVII when treated at large doses/fraction; this was true for air or O2 1 ATA conditions.     

Immunotherapy of spontaneous mammary tumors in mongrel dogs with autologous tumor cells and neuraminidase

Summary The effect produced on tumor progression by the injection of either VCN-treated tumor cells or tumor cells mixed with VCN to dogs with spontaneous mammary tumors was investigated. Dogs of different breeds and ages with at least two palpable spontaneous mammary tumors were selected. One tumor was left in the animal for further clinical examination, whereas the other tumor(s) was (were) excised for histologic diagnosis and for preparation of a single-cell suspension. Autologous M-cells were treated with VCN, subsequently extensively washed and injected SC into the neck of the dog on the day of operation and on the next day or different numbers of autologous M tumor cells (10⁵, 10⁶, 10⁷, 5×10⁷, 10⁸) were mixed with different amounts of VCN (0, 0.65, 6.5, 65 mU), and these various mixtures were injected ID at different sites to each dog on the day of operation. This procedure has been called chessboard vaccination (Seiler and Sedlacek, 1978). Altogether 79 dogs were blindly distributed into six groups in three consecutive studies. The results show that the therapeutic effect of the injection of VCN-treated autologous tumor cells depends on the number of tumor cells injected: injection of 2×10⁷ tumor cells repeatedly induced regression of the residual tumor mass (Studies I, II, and III) in most dogs and prevention of metastasis (Study I), while the application of 1×10⁸ tumor cells caused enhanced tumor proliferation in all and early metastasis in most of the dogs (Study I). The injection of 2×10⁶ tumor cells induced only a transient regression, with subsequent progression of the residual mammary tumor (Study II). Repetition of the injection of 2×10⁶ tumor cells three times every 4 weeks did not improve this effect (Study II). The chessboard vaccination proved to be at least as effective as the injection of 2×10⁷ VCN-treated tumor cells (Study III), although 1×10⁸ or more tumor cells had been injected; this number of cells caused tumor enhancement when the cells were treated with VCN only and injected SC (Study I). Moreover, the DTH reaction after ID injection of autologous tumor cells could be increased by the addition of VCN: low numbers of tumor cells and high amounts of VCN or high numbers of tumor cells and low amounts of VCN caused the most pronounced skin response. The relevance of these data to overcoming the risk of tumor enhancement after injection of an inadequate number of VCN-treated tumor cells and the possible diagnostic and therapeutic relevance of the DTH response after chessboard vaccination will be discussed.The abbreviations used in this work are: VCN, vibrio cholerae neuraminidase; M, mitomycin-treated; M-VCN, treated with mitomycin and VCN; DTH, delayed-type hypersensitivity; PBS, phosphate-buffered saline     

Autogenous arteriovenous elbow fistula for haemodialysis and upper extremity ischemia

BACKGROUND: The autogenous brachiocephalic or brachiobasilic arteriovenous elbow fistula is not considered to be only the secondary haemodialysis access. In patients with an unsuitable forearm vessel bundle, it is indicated as primary access and it is the method preferred to the fistula creation using a vascular prosthesis. Its rather rare complication is the development of upper extremity ischemia. AIM: To summarise current knowledge of this fistula type and its associated complications METHODS: Review of the literature. RESULTS: The creation and maturation of the fistula and occurrence of the steal syndrome is influenced by a number of factors. The analysis and awareness of such factors will provide for creation of a suitable fistula as well as for timely complication diagnostics and treatment. CONCLUSIONS: The autogenous elbow fistula utilising the brachial artery and the cephalic or basilic vein in the upper extremity represents a high-quality haemodialysis access. Its potential complication is the occurrence of the steal syndrome. Its occurrence and manifestations do not constitute indications for ligation of the access. The gathered information shows that a suitable surgical procedure can help meet the basic rule for haemodialysis access--resolving the ischemia and maintaining the access.     

Peripheral T-cell lymphoma, unspecified--the analysis of the data from the Czech Lymphoma Study Group (CLSG) registry

BACKGROUND: Peripheral T-cell lymphoma, unspecified (PTCL-US) is one of the entities from the infrequent family of nodal mature T-cell lymphomas. The clinical course is aggressive, and despite multiagent chemotherapy, the median survival is about 2 years. Published data are limited to retrospective, mostly single-center studies or reviews and usually include more lymphoma subtypes. AIM: To evaluate the current treatment modalities, clinical outcome and prognostic factors in unselected, new diagnosed patients with PTCL-US in the population of the central european region (Czech Republic). METHOD: Czech Lymphoma Study Group is a national scientific organization which provides an on-line database registry which collects a data about almost all new diagnosed lymphoma patients since year 2000. All diagnostic biopsies were reviewed by a reference pathologist. RESULTS: We analyzed 63 patients with new diagnosis of PTCL-US. The median age was 59 years (25-81), chemotherapy (CHT) was administered in 56 of the 63 patients: anthracyclin-based CHT in 51%, intensive CHT in 21% and non-anthracyclin regimen was applied in 13% of the patients. The overall response rate was 74.4%, (CR in 57.4%). After a median follow-up of 19.6 months, 41% of the patients were in CR, 3.4% in PR or stable disease and 55% of the patients died. The estimated survival probability in 3 years was 36%. Clinical stage (IV) and CR achievement were found to be independent survival predictors in a multivariate analysis. CONCLUSIONS: Although the current treatment modalities are mostly ineffective in PTCL-US, appropriate intensive treatment may lead to prolonged remission but not survival.     

Cytotoxicity of colchicine derivatives in primary cultures of human hepatocytes

BACKGROUND: Colchicine has been used to treat gout for centuries. However, owing to its toxicity it displays a variety of side effects. The replacement of colchicine by less toxic but still active derivatives would solve this drawback. AIM: The aim of this study was to examine the cytotoxicity of 17 colchicine derivatives. METHODS: Primary cultures of human hepatocytes were the model of choice. Prior to testing, we measured the biochemical parameters of liver donors and the toxicological response of the hepatocytes cultures. For toxicity testing, cells were treated for 24 h with tested compounds in concentrations 1-100 microM. We monitored lactate dehydrogenase (LDH) leakage into the medium, mitochondrial activity (MTT test) and secretion of albumin. RESULTS: Our data show that LDH and MTT were less sensitive parameters compared to albumin secretion for monitoring the toxicity of colchicine derivatives. Compounds with lower antimitotic activity displayed lowered toxicity. CONCLUSION: Since human hepatocytes in culture are quiescent cells, they are not as susceptible to tropolone alkaloids as proliferating cells. Screening for colchicine derivatives with lowered cytotoxicity revealed that 10-O-demethylated compounds might be the substances of choice.