Standardization of the CFU-GM assay using hematopoietic growth factors

BACKGROUND: The recently discovered LRP protein has been shown to be involved in drug resistance and possibly in detoxification processes. MATERIALS AND METHODS: To study the relation between LRP expression and exposure to cigarette smoke, LRP immunoreactivity was evaluated in 39 paraffin embedded normal lung tissues derived from patients operated on for pneumothorax, and related to amount of pack years smoked. We also studied the LRP protein expression in 36 non-small-cell lung cancer (NSCLC) samples and related the expression to patient characteristics and survival. Furthermore 17 lung tumor samples (10 NSCLC and 7 SCLC) derived from patients treated with chemotherapy were analysed in order to investigate the relation between LRP or MRP expression and the patient's response to chemotherapy. RESULTS: In the normal lung tissues, LRP intensity levels were not correlated to the amount of pack years smoked, although a trend was seen for higher LRP intensity levels in patients who smoked more than 10 pack years. LRP expression was significantly higher in NSCLC samples than in SCLC samples, and all SCLC samples displayed very low LRP expression. Within NSCLC, squamous cell and adenocarcinomas had higher LRP expression than large cell undifferentiated and mixed tumors. In NSCLC patients LRP expression was not a prognostic factor for survival. At initial analysis LRP expression levels did not predict for the response to chemotherapy. Only 3 out of 17 patients expressed MRP, and all SCLC samples were MRP negative. CONCLUSIONS: Striking different expression levels were seen between NSCLC and SCLC for both LRP and MRP. In a preliminary analysis LRP expression was not predictive for response to chemotherapy in lung cancer patients. In pneumothorax patients LRP levels were not correlated with the amount of pack years smoked.     

T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signals that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in signal transduction pathway of the CD43 molecule are only beginning to be understood. We have shown recently that cross-linking CD43 on the cell surface of human T lymphocytes with the anti-CD43 monoclonal antibody L10 leads to CD43-Fyn kinase interactions and to Fyn phosphorylation on tyrosine residues. This interaction seems to be mediated by the SH3 domain of Fyn and a proline-rich sequence located in the cytoplasmic domain of CD43. Here we show that CD43-specific activation of human T lymphocytes induced tyrosine phosphorylation of the adaptor protein Shc and of the guanine exchange factor Vav, as well as the formation of a macromolecular complex that comprises Shc, GRB2, and Vav. CD43 ligation resulted in enhanced formation of Vav.SLP-76 complexes and in the activation and nuclear translocation of ERK2. Cross-linking of the CD43 molecule in 3T3-CD43(+) cells induced luciferase activity from a construct under the control of the Fos serum responsive element. Altogether, these data suggest that the mitogen-activated protein kinase pathway is involved in CD43-dependent interleukin-2 gene expression.     

Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes

BACKGROUND & AIMS: Bile salt-induced apoptosis is mediated by a trypsin-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced hepatocyte apoptosis. METHODS: Rats, isolated rat hepatocytes, and a rat hepatoma cell line stably transfected with a bile salt transporter (McNtcp.24) were used for this study. RESULTS: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in trypsin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as cathepsin B. Specific, structurally dissimilar cathepsin B inhibitors blocked glycochenodeoxycholate (GCDC)-induced apoptosis in cultured rat hepatocytes. Furthermore, stable transfection of McNtcp.24 cells with the complementary DNA for cathepsin B in the antisense orientation reduced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, cathepsin B cellular localization during apoptosis was determined by immunoblot analysis of nuclear cell fractions, immunocytochemistry, and by determining the compartmentation of expressed cathepsin B fused to green fluorescent protein. All three approaches showed translocation of cathepsin B from the cytoplasm to the nucleus during GCDC-induced apoptosis. CONCLUSIONS: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bile salt-induced apoptosis of hepatocytes.     

Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.     

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.     

Severe malformation of one foot from amniocentesis needle injury

An 11-year-old boy is described who was born with a poorly developed right foot. At 16 weeks gestation his mother had had amniocentesis without direct ultrasound guidance. After the insertion of the amniocentesis needle, she felt strong abdominal resistance, which disappeared with slight withdrawal of the needle. Then, real-time ultrasound was used to determine the position of the needle, which was reported to be up against the feet. Nine days later deep purple amniotic fluid was removed by amniocentesis. At birth there was a scab over an oozing hole in the lateral aspect of his right 'foot', a poorly formed structure with five digit-like structures at the tip. It seems most likely that the deformity was caused by tissue injury from needle puncture at 16 weeks gestation.     

Leiomyosarcoma of the kidney in an HIV-infected child

Spindle cell tumors (leiomyoma and leiomyosarcoma) have been described in immunocompromised children after transplantation and in association with HIV infection. Previous reports have described tumors of respiratory (lung, bronchi), gastrointestinal (stomach, bowel, liver), and subcutaneous origins. We report a case of leiomyosarcoma of the kidney in an HIV-infected child.     

Effect of sodium bicarbonate ingestion on exhaustive resistance exercise performance

Six weight trained males were studied prior to, during, and in recovery from exhaustive resistance exercise, 105 min after ingesting 300 mg.kg-1 of either a placebo or NaHCO3. The exercise test consisted of four sets of 12 repetitions with a fifth set to volitional fatigue on a Universal leg press machine at a resistance equaling approximately 70% of the subjects 1-repetition maximum. Arterialized venous blood was analyzed for lactate concentration, blood gas, and acid-base parameters. The ingestion of NaHCO3 produced a significant increase in resting pH (7.39 to 7.46), HCO3- (22.9 to 28.3 mEq.l-1), and oxygenated base excess (-1.3 to 4.4 mEq.l-1). With the completion of each exercise set, a progressive decline in the acid-base status of both groups was observed (pH set 1-5: NaHCO3, 7.40 to 7.31; placebo, 7.34 to 7.25; HCO3- set 1-5: NaHCO3, 25.3 to 17.9; placebo, 21.7 to 15.3 mEq.l-1; base excess set 1-5: NaHCO3, 3.7 to -7.1; placebo, -1.4 to -10.7 mEq.l-1); however, the NaHCO3 condition was significantly more alkaline than the placebo condition. Blood lactate concentration [La] progressively increased with the completion of each exercise set ([La] set 1-5: NaHCO3, 1.37 to 11.15; placebo, 1.31 to 9.81 mM); but were not significantly different between treatments. Repetitions performed in the final exercise set were not significantly different between groups (NaHCO3: 19.6 +/- 1.6, placebo: 18.2 +/- 1.1 repetitions).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic and immunomorphologic study of the placenta in genital mycoplasmosis]

The authors have made an analysis of 1500 operations using a unique technology of closure without perfusion of the interventricular septum defects of the heart under conditions of hypothermia. Lethality was 1.7%, frequency of recanalization of the defects was 0.9%.