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Biomedical subjects

D Fan

Publications and source records attributed to D Fan.

At least 109 records · Page 6Linked to original sources
PubMed1993

Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activation.

Calmodulin (CaM) and the cardiac isoform of troponin C (cTnC) are close structural homologs, but cTnC cannot activate most CaM target enzymes. To investigate structure-function relationships, we constructed a series of CaM.cTnC chimeras and determined their ability to bind Ca2+ and activate CaM target enzymes. Previously, we exchanged domain 1 and found that the chimeras exhibited profoundly impaired activation of smooth muscle myosin light chain kinase (smMLCK) and had differential effects on other CaM target enzymes (George, S. E., VanBerkum, M. F. A., Ono, T., Cook, R., Hanley, R. M., Putkey, J. A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). One of the domain 1 chimeras was a potent competitive inhibitor of smMLCK. We now extend our study of CaM.cTnC chimeras by exchanging all of the remaining functional domains of CaM and cTnC. We determined the ability of the chimeras to bind Ca2+ and activate phosphodiesterase (PDE) and smMLCK. Chimeras containing both domains 3 and 4 of cTnC exhibited high affinity Ca2+ binding that was indistinguishable from cTnC, whereas chimeras containing either domain 3 or 4 of cTnC demonstrated Ca2+ affinity that was intermediate between CaM and cTnC. All of the CaM.cTnC chimeras showed near-maximal PDE activation but required 5-775-fold higher concentrations than CaM to produce half-maximal PDE activation. In contrast, all of the chimeras showed impaired ability to activate smMLCK, and some were potent competitive inhibitors of smMLCK activation by CaM.

Amino Acid Sequence↗
PubMed1993

Cytotoxic T cells isolated from ovarian malignant ascites recognize a peptide derived from the HER-2/neu proto-oncogene.

The HER-2/neu proto-oncogene encodes a transmembrane receptor protein whose expression is enhanced in a number of breast and ovarian tumors and correlates with tumor aggressiveness, suggesting that it may play an important role in tumor growth. Recent evidence suggests that HER-2/neu may be a potential candidate for targeted immune intervention. In this report we show that cytotoxic T lymphocytes (CTL) expanded from tumor-associated lymphocytes with HLA-A2+ and HER-2/neu+ tumors can specifically recognize synthetic peptides corresponding to amino acids 971-980 of HER-2/neu protein. This sequence includes a potential amphiphilic area containing both Rothbard's epitode motifs and HLA-A2 anchor residues. Our study provides the first direct evidence of HER-2/neu-reactive CTL in humans. The fact that these HER-2/neu peptide-reactive CTL show significantly lower reactivity with corresponding EGF-R peptides offers new perspectives for understanding the recognition of self-antigens by tumor-reactive T cells.

Amino Acid Sequence↗
PubMed1993

A simultaneous study of Doppler-echo and catheterization in noninvasive assessment of the left ventricular dp/dt.

To confirm the feasibility and accuracy of the method for the noninvasive measurement of the left ventricular dp/dt, 53 patients with mitral regurgitation underwent simultaneous determination of left ventricular dp/dt by continuous-wave Doppler echocardiography and cardiac catheterization. Doppler-determined left ventricular dp/dt is derived from the Doppler mitral regurgitant spectrum by dividing the magnitude of the left ventricular-atrial pressure gradient rise between 1 and 3 m/s of the mitral regurgitant velocity signal by the time taken for this change. Left ventricular dp/dt by Doppler ranged from 629 to 3494 mmHg/s (mean +/- SD, 1971 +/- 785 mmHg/s), and that by catheterization varied between 716 and 3650 mmHg/s (mean +/- SD, 1974 +/- 727 mmHg/s). There was a high correlation (r = 0.93, y = 0.862x + 274.77, SEE = 271 mmHg/s, p < 0.001) of left ventricular dp/dt between the two techniques. It is concluded that left ventricular dp/dt is one of the most commonly used parameters for the evaluation of left ventricular systolic function and that Doppler echocardiography provides a new, accurate and noninvasive method of evaluation.

Adolescent↗
PubMed1993

Noninvasive evaluation of right ventricular and pulmonary artery systolic pressures in patients with ventricular septal defects: simultaneous study of Doppler and catheterization data.

The method for evaluating right ventricular and pulmonary artery systolic pressures by subtracting the systolic pressure gradient across the ventricular septal defect from the cuff systolic blood pressure is documented. To confirm the reliability and accuracy of this method, simultaneous continuous wave Doppler echocardiography and left and right cardiac catheterization were performed in 66 patients with congenital ventricular septal defects. The comparisons between the Doppler-derived right ventricular or pulmonary artery systolic pressures and those measured by catheterization yielded an excellent correlation (r = 0.969, y = 1.035x - 3.627, SEE = 8.2 mm Hg, p < 0.001 and r = 0.972, y = 1.012x - 2.904, SEE = 7.6 mm Hg, p < 0.001), respectively. There was a close agreement between the Doppler-derived right ventricular or pulmonary artery systolic pressures and those measured by catheters, and the differences between the two measurements did not differ significantly from zero. It is concluded that Doppler echocardiography offers a reliable and accurate method for noninvasive assessment of right ventricular and pulmonary artery systolic pressures in patients with ventricular septal defects.

Adolescent↗
PubMed1992

Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil.

We determined the effects of organ environment on the response of murine CT-26 colon carcinoma cells to 2 structurally and pharmacologically distinct chemotherapeutic agents. CT-26 cells were injected i.v. (to produce lung lesions), s.c., into the cecal wall, and into the spleen (to produce spleen and liver lesions). Doxorubicin (DXR) at 10 mg/kg, 5-fluorouracil (5-FU) at 20 mg/kg, or saline (control) was injected intravenously on different schedules after tumor-cell implantation. The in vivo responses of the tumors growing in the cecum, spleen, liver, lung and subcutis were compared. Colon carcinomas growing in the subcutis were most sensitive to DXR. Tumors growing in the spleen and cecum were most sensitive to 5-FU and less so to DXR. Tumors in the liver were highly resistant to both drugs, whereas experimental lung metastases were sensitive to 5-FU but resistant to DXR. The differential responses of the tumors to the drugs were not due to drug distribution. The level of protein-kinase-C activity was elevated in the spleen, liver and cecum tumors as compared with s.c. tumors and correlated with the in vivo DXR resistance of the tumor cells. This correlation suggested that organ environment may modulate the chemosensitivity of tumor cells, at least in part, by perturbing signal transduction pathways. Collectively, the data indicate that the organ environment has profound effects on the response of tumor cells to chemotherapy. A molecular understanding of this phenomenon should facilitate the design of more effective systemic chemotherapy for cancer metastases.

Animals↗
PubMed1992

Pulmonary artery diastolic pressure: a simultaneous Doppler echocardiography and catheterization study.

Pulmonary hypertension is an important determinant of the clinical presentation of and surgical approach to patients with heart disease. To confirm the utility of continuous wave Doppler echocardiography in assessing the pulmonary artery diastolic pressure in patients with pulmonary regurgitation, 51 patients representing the wide hemodynamic spectrum of pulmonary artery pressure underwent simultaneous determination of pulmonary artery diastolic pressure by continuous wave Doppler echocardiography and cardiac catheterization. Pulmonary artery diastolic pressure was estimated from the Doppler recordings by the end-diastolic pressure gradient obtained by the modified Bernoulli equation plus the estimated right atrial pressure. A correlation was observed (r = 0.935, SEE = 7.4 mmHg) between Doppler and catheterization pulmonary artery diastolic pressure. In addition, comparison between the mean diastolic pressure gradient across the pulmonary valve by Doppler and pulmonary artery diastolic pressure at catheterization yielded a high correlation (r = 0.947, SEE = 5.1 mmHg). These data demonstrate that continuous wave Doppler echocardiography is a useful noninvasive technique for evaluating the pulmonary artery diastolic pressure in patients with pulmonary regurgitation.

Adolescent↗
PubMed1992

Takayasu arteritis in China: a report of 530 cases.

A total of 530 patients with Takayasu arteritis were studied. Among 346 patients who were diagnosed by aortography, the female to male ratio was 2.9 to 1, and the age of onset ranged from 5 to 45 years. Three hundred and eighteen (60%) patients with secondary hypertension, including renovascular hypertension in 281, and 197 (37.2%) with pulseless extremities were found in the series. All the patients were treated with medical or surgical procedures. Surgical treatment is preferred if clinical status of the patient permits. The patients were followed for 1-29 years (average 7.8 years). There were 55 deaths (10.4%) in this series. Cerebral hemorrhage was found as a common cause of death. Five-year and ten-year survival rates were 93.1% and 91.1%, respectively.

Adolescent↗
PubMed1992

Noninvasive evaluation of interventricular pressure gradient across ventricular septal defect: a simultaneous study of Doppler echocardiography and cardiac catheterization.

Simultaneous continuous-wave Doppler echocardiography and left- and right-sided cardiac pressure measurements were performed during cardiac catheterization in 64 patients with a congenital ventricular septal defect (VSD). The peak-to-peak pressure gradient across the VSD on cardiac catheterization ranged from 0 to 109 mm Hg (61 +/- 31.7 mm Hg). The peak shunt velocity and peak pressure gradient across the VSD on Doppler ultrasound imaging ranged from 0.96 to 5.21 m/sec (3.75 +/- 1.16 m/sec) and from 4 to 105 mm Hg (62 +/- 29.8 mm Hg), respectively. Doppler measurements of the peak interventricular pressure gradient correlated well with measurements obtained by cardiac catheterization (r = 0.98, standard error of estimate = 6.3 mm Hg, p less than 0.001). Doppler ultrasound imaging yielded information comparable to that obtained by catheterization and provided an accurate method of measuring the pressure gradient across the VSD, which is a useful parameter for the assessment of pulmonary artery systolic pressure in patients with a VSD, without a left or right ventricular outflow tract obstruction.

Adolescent↗
PubMed1992

Simultaneous measurement of left atrial pressure by Doppler echocardiography and catheterization.

Simultaneous, continuous wave Doppler echocardiography, left ventricular systolic and mean pulmonary capillary wedge pressure measurements were performed during cardiac catheterization in 54 patients with mitral regurgitation. Doppler-derived left atrial pressure, which was calculated by subtracting mitral regurgitant gradient from brachial artery systolic pressure, correlated well with mean pulmonary capillary wedge pressure by catheter (r = 0.933, SEE = 2.9 mmHg, P < 0.001); a comparison between non-invasive and invasive systolic gradients across the mitral valve yielded a high correlation (r = 0.91, SEE = 6.0 mmHg, P < 0.001); and there was also a high correlation between brachial artery and left ventricular systolic pressures (r = 0.93, SEE = 4.9 mmHg, P < 0.01). It is concluded that Doppler echocardiography provides a reliable and accurate method for complete non-invasive assessment of left atrial pressure in patients with mitral regurgitation.

Adolescent↗
PubMed1991

Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor.

Recombinant desulfatohirudin (r-hirudin), a highly specific inhibitor of thrombin, was examined to determine whether it would inhibit production of experimental lung metastasis by B16-F10 melanoma cells. In in vitro assays using mouse plasma, the high level of procoagulant activity in B16-F10 cells was significantly inhibited by r-hirudin in a dose-dependent manner. From 15 to 120 min after s.c. administration into C57BL/6 mice, r-hirudin (10 mg/kg) markedly prolonged clotting time in a time course pattern that directly correlated with that of blood distribution of 125I-labeled r-hirudin. The production of experimental lung metastasis by B16-F10 cells was significantly inhibited by r-hirudin administered s.c. at time points ranging from 120 min before to 60 min after tumor cell inoculation with the most significant effects found in mice given r-hirudin 15 or 2 min before the i.v. injection of tumor cells. The organ distribution of [125I]IdUrd-labeled tumor cells demonstrated a clear difference in the lungs of mice treated with r-hirudin and the lungs of control mice, and these differences directly correlated with the number of lung tumor colonies found 3 weeks later. The inhibition of lung metastasis was not due to direct antitumor effects of r-hirudin. These results suggest that inhibition of coagulation events by r-hirudin significantly inhibit experimental lung metastasis during a critical time of 60 min after the entry of tumor cells into the circulation.

Animals↗
PubMed1991

Transient enhancement of multidrug resistance by the bile acid deoxycholate in murine fibrosarcoma cells in vitro.

Recent studies have implicated protein kinase C (PKC) activation in drug resistance in vitro. PKC can be activated directly by phorbol-ester tumor promoters as well as by the bile acid deoxycholate. In this report, we demonstrate that deoxycholate, at concentrations that are chronically present in the lumen of the colon in vivo, mimicked phorbol-ester tumor promoters by protecting Adriamycin (ADR)-sensitive and multidrug-resistant (MDR) murine fibrosarcoma UV-2237M cells from ADR cytotoxicity. Deoxycholate also enhanced the resistance of the MDR cell line UV-2237M-ADRR to the cytotoxic effects of vincristine and vinblastine. In contrast to cytotoxic drug-selected MDR phenotypes, deoxycholate-induced drug resistance was transient and required continuous exposure to the bile acid. The protein kinase inhibitor H7 completely reversed the protection against ADR cytotoxicity conferred on UV-2237M-ADRR cells by deoxycholate, providing evidence that deoxycholate exerts its protective effects by a mechanism that involves stimulation of protein phosphorylation and not merely by detergent effects on membrane permeability. PKC consists of a family of at least seven isozymes with distinct modes of activation and substrate specificities. We previously reported that MDR UV-2237M cell lines contain higher levels of PKC activity than the parental ADR-sensitive UV-2237M cell line (O'Brian et al., FEBS Lett 246: 78-82, 1989). The present report shows that PKC-III is a major PKC isozyme in ADR-sensitive and MDR UV-2237M cell lines. Thus, the resistance to ADR induced by the phorbol esters in UV-2237M cell lines provides strong evidence that PKC-III activation confers protection against ADR on ADR-sensitive and MDR UV-2237M cell lines. Furthermore, since deoxycholate is an endogenous molecule in the colonic epithelium, our finding that physiological concentrations of deoxycholate can render cells more resistant to chemotherapeutic drugs in vitro may have implications for the biology and therapy of intestinal cancers.

Animals↗
PubMed1991

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene.

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.

Cell Division↗
PubMed1991

A novel N-myristylated synthetic octapeptide inhibits protein kinase C activity and partially reverses murine fibrosarcoma cell resistance to adriamycin.

This report shows that N-acylation of the protein kinase C (PKC) substrate Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) provides it with a potent inhibitory activity against PKC. N-myristoyl-RKRTLRRL inhibited Ca2(+)- and phosphatidylserine (PS)-dependent histone phosphorylation catalyzed by PKC with a 50% inhibitory concentration (IC50) of 5 microM, whereas neither RKRTLRRL nor myristic acid inhibited PKC-catalyzed histone phosphorylation at concentrations as high as 50 microM. A fully active, Ca2(+)- and PS-independent catalytic fragment of PKC can be generated by limited proteolysis. N-myristoyl-RKRTLRRL inhibited histone phosphorylation catalyzed by the catalytic fragment of PKC (IC50 = 80 microM), but neither myristic acid nor the nonmyristylated peptide inhibited the activity of the catalytic fragment at concentrations up to and including 200 microM. The Km app and Vmax app for N-myristoyl-RKRTLRRL were similar to those of RKRTLRRL. Thus, N-myristylation provided the octapeptide with an inhibitory activity against PKC but had only minor effects on its Km app and Vmax app. Kinetic analysis provided evidence that the peptide inhibited PKC noncompetitively with respect to ATP. Previously, we reported that the protein kinase inhibitor H7 partially reverses Adriamycin resistance in the multidrug resistant (MDR) murine fibrosarcoma line UV-2237M-ADRR. In this report, we show that N-myristoyl-RKRTLRRL also partially reverses Adriamycin resistance in UV-2237M-ADRR cells. These results suggest that potent and selective cell permeable PKC inhibitors may be designed by N-acylating small PKC peptide substrates.

Amino Acid Sequence↗
PubMed1991

Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages.

We examined the activation to the tumoricidal state of normal mouse peritoneal exudate macrophages, bone marrow macrophages, and human blood monocytes by liposomes containing either lipophilic muramyl tripeptide (CGP 19,835) or a new synthetic analogue of lipoprotein from gram-negative bacteria outer wall, CGP 31,362, or combinations of the two. The superiority of liposomes containing the synthetic lipopeptide over liposomes containing lipophilic muramyl tripeptide for in vitro activation of monocytes and macrophages was demonstrated in several experiments. First, liposome-CGP-19,835 activated monocytes only in the presence of interferon-gamma, whereas activation with liposome-CGP 31,362 was interferon-independent. Second, activation of both mouse macrophages and human blood monocytes by liposome-CGP 31,362 occurred at a lower liposomal concentration than that by liposome-CGP 19,835. Third, monocytes incubated with liposome-CGP 31,362 released both tumor necrosis factor (TNF) and interleukin-1 activities, whereas monocytes treated with liposome-CGP 19,835 (in the absence of interferon-gamma) released only TNF activity. These data suggest that liposomes containing the synthetic lipopeptide CGP 31,362 are superior to liposomes containing CGP 19,835 for systemic activation of macrophages.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Overcoming murine tumor cell resistance to vinblastine by presentation of the drug in multilamellar liposomes consisting of phosphatidylcholine and phosphatidylserine.

We determined whether vinblastine (VLB) encapsulated within multilamellar vesicle-liposomes (MLV) would reverse target cell resistance to the drug exhibited by the UV-2237M murine fibrosarcoma and its Adriamycin (ADR)-selected multidrug resistant (MDR) variants. Resistant fibrosarcoma cells were grown in medium containing 1 and 10 micrograms/ml ADR to yield the MDR lines UV-2237M/ADRR (ADR-1) and UV-2237M/ADRRR (ADR-10), respectively. VLB encapsulated in MLV composed of phosphatidylcholine (PC) and phosphatidylserine (PS) (7:3 molar ratio) was hydrophobic, occupied an internal space equivalent of 6.13 microliters/mumol, and was stable in medium at 37 degrees C for up to 6 days. The 50% inhibitory concentrations (IC50) of VLB were 2, 25, and 70 ng/ml for the parent, ADR-1, and ADR-10 cell lines, respectively. VLB in MLV significantly enhanced sensitivity of tumor cells to VLB. The respective IC50 of liposomal VLB were 0.5, 5.7, and 12 ng/ml for the parent, ADR-1, and ADR-10 lines. MLV containing saline were not toxic to the cells. These data indicate that presentation of VLB entrapped in PC:PS MLV provides a method to overcome tumor cell resistance to this drug.

Animals↗
PubMed1991

Cytotoxic potential of liposomes containing tumor necrosis factor-alpha against sensitive and resistant target cells.

The purpose of these studies was to determine whether recombinant tumor necrosis factor (TNF) incorporated into liposomes produced enhanced antitumor effects against TNF-sensitive and TNF-resistant target cells. The lipid composition of liposomes influenced their binding to and endocytosis by target cells. Liposomes consisting of phosphatidylcholine and phosphatidylserine (7:3 molar ratio) bound to L929 cells and A375 human melanoma cells, albeit to different degrees. Liposomes retained encapsulated TNF for up to 48 h of incubation. TNF in liposomes lysed the TNF-sensitive A375 melanoma and L929 cells at levels similar to that mediated by free, unencapsulated TNF. Cells selected for resistance against free TNF were not sensitive to TNF in liposomes. Since liposomes concentrate in organs with high levels of reticuloendothelial activity, and TNF in liposomes retains antitumor activity, this delivery system may prove to be useful for treatment of lymph node and hepatic metastases.

Animals↗
PubMed1991

Optimization of the liposomes encapsulating a new lipopeptide CGP 31362 for efficient activation of tumoricidal properties in monocytes and macrophages.

The purpose of this study was to optimize a suitable liposomal carrier for CGP 31362, a new synthetic lipopeptide analogue of gram-negative bacterial cell walls. CGP 31362 was inserted into the membranes of different liposomes with different phospholipid composition. We determined the ability of these liposomes to activate tumoricidal properties in mouse peritoneal and bone marrow macrophages, and in human monocytes. The ideal liposome carrier for CGP 31362 consisted of phosphatidylcholine and phosphatidylserine in a 7:3 molar ratio. Subsequent to efficient binding and endocytosis, CGP 31362 in liposomes of this composition rendered mouse macrophages and human monocytes highly tumoricidal. Moreover, even in the absence of interferon-gamma, human monocytes released significant levels of tumor necrosis factor and interleukin-1. These data show that in a suitable liposomal carrier, the new synthetic lipopeptide in liposomes is a potent activator of tumoricidal properties in macrophages.

Animals↗