Binding of fatty acid ethyl esters to albumin for transport to cells in culture

Fatty acid ethyl esters (FAEE) are non-oxidative products of ethanol metabolism that have been proposed to mediate pathological changes in various organs and tissues resulting from excessive ethanol consumption. Evidence supporting this proposal is scant, however, mainly because of the lack of adequate methods with which to solubilize the highly hydrophobic FAEE in aqueous medium for testing under physiological conditions. In this report we describe a simple and practical method for solubilizing FAEE in aqueous medium by binding them to albumin. We also report that the albumin-bound FAEE are readily taken up by rat alveolar macrophages in culture. The availability of FAEE bound to albumin, their main physiological carrier in vivo, will facilitate the investigation of the role that these metabolites may have in mediating pathological changes associated with excess ethanol consumption.     

Interaction between CD44 and hyaluronic acid regulates human prostate cancer development

PURPOSE: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. MATERIALS AND METHODS: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. RESULTS: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. CONCLUSIONS: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.     

Novel screening technique for dissemination potential of ovarian cancer cells to peritoneum

Invasiveness to the peritoneum reconstituted with a mesothelial cell line and Engelbreth-Holm-Swam extract by metastatic cancer cell lines of the uterine cervix, endometrium, and ovary was always higher than that by primary cell lines. The invasiveness by metastatic ovarian cancer cell lines was significantly stronger than that by the other gynecological primary or metastatic cell lines. In the clinical ovarian cancers studied, cancer cells from the metastatic lesion were more invasive than those from the primary lesion. This suggests that metastatic ovarian cancer cells might inherently possess strong invasiveness to the peritoneum. The assay system used in the present study is useful in investigating the clinical behavior and basic biology of peritoneal dissemination.     

Binding of apoB-containing lipoproteins from unfractionated human blood sera to immobilized LDL receptor

Binding of apoB-containing lipoproteins from unfractionated human blood sera to the immobilized bovine receptor of low density lipoproteins (LDL receptor) was studied. Peroxidase-labeled anti-human apoB antibodies were used to evaluate the lipoprotein binding. The equilibrium dissociation constant (Kd) of the interaction between apoB-containing lipoproteins from unfractionated human sera from healthy donors and the immobilized LDL receptor varied from 1 to 20 microg apoB/ml. To describe the binding of lipoproteins to the LDL receptor, a parameter of relative binding affinity (RBA) was used. RBA is inversely related to value of Kd and equal to unity for the standard serum. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera to the immobilized LDL receptor were found to correlate with the RBA values for the binding of isolated VLDL (r = 0.76, p < 0.001) and fail to correlate with the RBA values for the binding of isolated LDL. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera correlated with the RBA values for the binding of apoE-containing lipoproteins from unfractionated sera (r = 0.92, p < 0.001) and with values of triglyceride concentration in the sera (r = 0.93, p < 0.001). The RBA values for the binding of apoB-containing lipoproteins from sera of patients with FDB whose LDL were unable to bind to the LDL receptor did not significantly differ from the RBA values for the normal sera. However, the removal of VLDL from the normal sera significantly decreased the RBA values for the binding of apoB-containing lipoproteins from unfractionated sera. The results indicate that the different binding of apoB-containing lipoproteins to the immobilized LDL receptor mainly depended on the different binding of VLDL and not of LDL.     

Serum hyaluronic acid in Down''s syndrome

Changes in the emission intensities of ultraweak biophoton emission during the cell proliferation of human carcinoma cell culture (TE9 cell line) were detected using a highly sensitive and low noise measurement apparatus coupled with a flow culture system. In the sampling period of 93 h, the biophoton emission intensity from the culture followed a similar course as that of the growth curve. Spectral analysis of the biophoton emission from the cell culture demonstrated a significant peak at around 530 nm. Our results suggest that the emission intensity mainly depends on the cell population and that this noninvasive technique has a potential role in cancer diagnosis.     

Depolymerization reactions of hyaluronic acid in solution

Samples of microbial sodium hyaluronate were degraded by heating, ultrasonication ultraviolet (UV) and gamma-ray irradiation and enzymatic treatment. The weight-average molecular weight, Mw, of hyaluronate in 0.15 M NaCl and 0.06 M Na2HPO4 was determined by gel filtration with UV detection. The Mw of the degraded samples varied from 8 x 10(4) to 1.38 x 10(6). Depolymerization processes can be described by linear relationship (1/Mw)2 = f(t) in the case of ultrasonic treatment and by non-linear relationships in the cases of heating and UV irradiation at 257 nm. Gamma-ray irradiation and enzymatic treatment caused chemical degradation and depolymerization to oligosaccharides, respectively.     

Tumor-associated hyaluronic acid: a new sensitive and specific urine marker for bladder cancer

Hyaluronic acid (HA), a glycosaminoglycan, is known to promote tumor cell adhesion and migration, and its small fragments stimulate angiogenesis. We compared levels of HA in the urine of normal individuals and patients with bladder cancer or other genitourinary conditions, using a sensitive ELISA-like assay. Among the 144 specimens analyzed, the urinary HA levels of bladder cancer patients with G1 (255 +/- 41.7 ng/mg), G2 (291.8 +/- 68.3 ng/mg) and G3 (428.4 +/- 67 ng/mg) tumors are 4-9-fold elevated as compared to those of normal individuals (44.7 +/- 6.2 ng/mg) and patients with other genitourinary conditions (69.5 +/- 6.8 ng/mg; P < 0.001). Urinary HA measurement by the ELISA-like assay shows a sensitivity of 91.9% and specificity of 92.8% to detect bladder cancer. Thus, urinary HA measurement is a simple, noninvasive yet highly sensitive and specific method for bladder cancer detection. The increase in urinary HA concentration is a direct correlate of the elevated tumor-associated HA levels, because the HA levels are also elevated (3-5-fold) in bladder tumor tissues (P < 0.001). The profiles of urinary HA species of normal individuals and bladder cancer patients are different. Although only the intermediate-size HA species are found in the urine of normal and low-grade bladder tumor patients, the urine of high-grade bladder cancer patients contains both the high molecular mass and the small angiogenic HA fragments. These urinary HA fragments stimulate a mitogenic response (2.4-fold) in primary human microvessel endothelial cells, suggesting that the small HA fragments may regulate tumor angiogenesis by modulating endothelial cell functions.     

Increased urinary hyaluronic acid and interstitial cystitis

PURPOSE: We compared urinary levels of hyaluronic acid in patients who met the National Institute for Diabetes, and Digestive and Kidney Diseases criteria for interstitial cystitis and in age matched healthy female controls. MATERIALS AND METHODS: Urinary hyaluronic acid was measured by solid phase radiometric assay using hyaluronic acid binding protein. Hyaluronic acid and symptom scores were compared in interstitial cystitis patients who gave multiple urine samples during treatment. Since hyaluronic acid changed with treatment in some patients, 17 samples from untreated interstitial cystitis patients were selected and compared with 17 control samples. RESULTS: Mean plus or minus standard deviation urinary hyaluronic acid concentrations were similar in the 2 groups (interstitial cystitis group 574 +/- 496, controls 512 +/- 324 ng./ml., p = 0.77). When normalized to creatinine urinary hyaluronic acid was significantly higher in interstitial cystitis patients (interstitial cystitis group 674 +/- 220, controls 446 +/- 220 ng./mg. creatinine, p = 0.0019). Urinary creatinine concentrations did not differ significantly (interstitial cystitis group 842 +/- 715, controls 1,162 +/- 516 mg./l., p = 0.12). CONCLUSIONS: Urinary hyaluronic acid was higher in interstitial cystitis patients than healthy controls. Since bladder hyaluronic acid is below the epithelium, this finding may indicate leakage across the epithelium into the urine in interstitial cystitis patients.     

Mobilization of peripheral blood stem cells in advanced ovarian cancer

High-dose chemotherapy with hematopoietic support has been expected to improve the survival of advanced ovarian cancer patients in recent years. An essential component of such treatment has been the ability to collect and reinfuse a large number of peripheral blood stem cells (PBSCs) following high dose therapy. This study was designed to determine which clinical and hematological factors would be better indicators to collect the proper volume of PBSCs. Thirteen patients received a total of 24 courses of induction chemotherapy and 69 of apheresis. We usually mobilized stem cells using CEP chemotherapy (cisplatin 50-70 mg/m2, epirubicin 50 mg/m2 and cyclophosphamide 1.5 g/m2) with G-CSF and CEE regimen (cyclophosphamide 2.0 g/m2, epirubicin 50 mg/m2, and etoposide 50 mg/m2) as a salvage for mobilization. We obtained an average 5 x 10(6)/kg of CD34+ cells for 3 days as one course. The number of CD34+ cells collected significantly depended on the platelets and reticulocytes on the first day of apheresis, but not a nadir of WBCs. It is concluded that apheresis should be started on recovery of WBCs to 5,000-10,000/microliters, of immature granulocytes to > or = 10% and of reticulocytes to > or = 20%. This study confirmed the feasibility of collecting enough PBSCs to use standard chemotherapy of ovarian cancer patients.     

Degradation process of hyaluronic acid by Streptomyces hyaluronidase

Saturated and unsaturated hyaluronate oligosaccharides were prepared from human umbilical cord hyaluronic acid by partial digestion with bovine testicular and Streptomyces hyaluronidase, respectively. After treatment with Streptomyces hyaluronidase, the distribution of degradation products from these oligosaccharides was determined. Octasaccharides, either saturated or unsaturated, were substrates of the minimum size. Tetra- and hexasaccharides were not degraded further and remained as final products. This indicates that at least four succeeding N-acetylhyalobiuronosyl residues were required for this enzymatic degradation. Since unsaturated oligosaccharides were more susceptible to this enzyme than saturated ones of the same polymerization degree, and inner glucosaminidic linkages were cleaved in preference to those at the outermost sites, some groups adjacent to this segment seemed to influence the susceptibility of the oligosaccharide to this enzyme.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers. pH 3.5-7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

BAD partly reverses paclitaxel resistance in human ovarian cancer cells

Although paclitaxel is an important chemotherapy agent for the treatment of patients with epithelial ovarian cancer, its utility is significantly limited by the frequent development of drug resistance. Recent evidence suggests that resistance to chemotherapy may be partly related to defects in the apoptotic pathway. In this study we have investigated whether enhancement of apoptotic pathway function through stable expression of the BAD protein is capable of sensitizing human epithelial ovarian cancer cells to the effects of chemotherapy. Expression of HA-BAD in six separate clonal transfectants from two different ovarian cancer cell lines was found to significantly enhance the cytotoxic effects of paclitaxel, vincristine, and, to a lesser extent, etoposide. Enhancement of paclitaxel-induced apoptosis in HA-BAD-expressing clones was demonstrated by trypan blue exclusion, clonogenic cell assay, and flow cytometric evaluation. Importantly, this effect was associated with binding of HA-BAD to BCL-xL and concomitant disruption of BAX:BCL-xL interaction. Taken together, these data suggest that the development of small molecules which mimic the effects of BAD may represent a new class of drugs capable of preventing or reversing resistance to chemotherapy agents such as paclitaxel.     

A 340 kDa hyaluronic acid secreted by human vascular smooth muscle cells regulates their proliferation and migration

The formation of atherosclerotic lesions is characterized by invasion of vascular smooth muscle cells (VSMC) into the tunica intima of the arterial wall and subsequently by increased proliferation of VSMC, a process apparently restricted to the intimal layer of blood vessels. Both events are preceded by the pathological overexpression of several growth factors, such as platelet-derived growth factor (PDGF) which is a potent mitogen for VSMC and can induce their chemotaxis. PDGF is generally not expressed in the normal artery but it is upregulated in atherosclerotic lesions. We have previously shown that PDGF-BB specifically stimulates proliferating VSMC to secrete a 340 kDa hyaluronic acid (HA-340). Here, we present evidence regarding the biological functions of this glycan. We observed that HA-340 inhibited the PDGF-induced proliferation of human VSMC in a dose-dependent manner and enhanced the PDGF-dependent invasion of VSMC through a basement membrane barrier. These effects were abolished following treatment of HA-340 with hyaluronidase. The effect of HA-340 on the PDGF-dependent invasion of VSMC coincided with increased secretion of the 72-kDa type IV collagenase by VSMC and was completely blocked by GM6001, a hydroxamic acid inhibitor of matrix metalloproteinases. HA-340 did not exert any chemotactic potency, nor did it affect chemotaxis of VSMC along a PDGF gradient. In human atheromatic aortas, we found that HA-340 is expressed with a negative concentration gradient from the tunica media to the tunica intima and the atheromatic plaque. Our findings suggest that HA-340 may be linked to the pathogenesis of atherosclerosis, by modulating VSMC proliferation and invasion.     

Binding of the Helicobacter pylori vacuolating cytotoxin to target cells

The vacuolating cytotoxin of Helicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.     

Binding of diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions in vitro and in mouse foetal cells in culture

Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.