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PubMed1991

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

Animals↗
PubMed2004

Direct contact between enamel matrix derivative (EMD) and osteoblasts is not required for EMD-induced cell proliferation.

OBJECTIVE: The purpose of this study was to determine whether the effect of enamel matrix derivative (EMD) on osteoblast proliferation is dependent on direct contact between EMD and the cells. STUDY DESIGN: MC3T3-E1 cells were seeded onto 6-well culture plates at an initial density of 5000/cm(2) in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). Serum was removed from the culture medium after 24 hours with or without the addition of EMD. Four groups were evaluated: group 1, DMEM only; group 2, DMEM with 100 microg/mL EMD directly added to the culture medium; group 3, DMEM with a culture plate insert (30-mm diameter; 0.4-microm pore size) only; group 4, DMEM with 100 microg/mL EMD added onto a culture plate insert. The porous membrane of the insert prevented direct contact between EMD and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted and analyzed using 1-way ANOVA. RESULTS: EMD formed precipitated aggregates on the membrane of the culture insert with the same appearance as when it was added directly to the medium. The culture plate insert alone did not cause any changes in cell morphology or proliferation. The addition of EMD significantly increased cell number regardless the presence of the culture plate insert. CONCLUSION: This study suggests that direct contact between EMD and osteoblasts is not required to induce cell proliferation. Soluble peptides released from EMD may contribute to the stimulating effects of EMD on cell proliferation.

3T3 Cells↗
PubMed2002

Analysis of genetic diversity and population structure within Florida coconut (Cocos nucifera L.) germplasm using microsatellite DNA, with special emphasis on the Fiji Dwarf cultivar.

Using 15 simple sequence repeat (SSR) microsatellite DNA loci, we analyzed genetic variation within Cocos nucifera germplasm collections at two locations in south Florida, representing eight cultivars. The loci were also used in a parentage analysis of progeny of the 'Fiji Dwarf' variety at both locations. A total of 67 alleles were detected, with eight the highest number at any one locus. These loci identified 83 of the 110 individual palms. Gene diversity of the 15 loci ranged from 0.778 to 0.223, with a mean of 0.574. 'Fiji Dwarf', 'Malayan Dwarf', 'Green Niño' and 'Red Spicata' cultivars resolve as distinct clusters in a neighbor joining tree using modified Rogers distance, while the tall varieties form two aggregates. The highest gene diversity was found in the tall cultivars (H = 0.583 cumulatively), and the lowest in the 'Malayan Dwarf' (H = 0.202). After the tall coconuts, the 'Fiji Dwarf' was most genetically diverse (H = 0.436), and had the largest number of unique alleles. Genetic identity is highest among the 'Malayan Dwarf' phenotypes, and between the tall varieties. The 'Red Malayan Dwarf' is genetically distinct from the 'Green' and 'Yellow Malayan Dwarf' phenotypes, which cannot be distinguished with the SSR loci used. Off-type 'Malayan Dwarf' phenotypes (putative hybrids with talls) can be identified genotypically. Parentage analyses of 30 'Fiji Dwarf' progeny propagated from five adults surrounded by other cultivars estimate that only 20% of the progeny were out-crossed to the other varieties, while 40-46% were possible selfs. This suggests that a seed-production orchard of the variety maintained at reasonable distance from other varieties, will likely yield only 'Fiji Dwarf' genotypes. Our data are discussed in the context of hypotheses of coconut dissemination around the world.

Alleles↗
PubMed1990

Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina.

When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. This supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5 micron) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters.

Animals↗
PubMed2000

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells. Absence of peroxisomal reticulum but evidence of close spatial association with the endoplasmic reticulum.

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3'-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.

Humans↗
PubMed2003

[Collagen membrane as scaffold for the three-dimensional cultivation of cardiac cells in vitro].

The objective of this study was to develop research of cardiac cells to reestablish 3D tissue architecture in vitro, we performed studies using collagen membrane as three-dimensional scaffold for cardiac cells culture with the principles and methods of tissue engineering. The polymer scaffold provides a 3-D substrate for cell attachment and tissue formation. Cardiac cells isolated by enzymatic digestion from 1d old neonatal rats were seeded to three-dimensional collagen scaffolds and tissue culture plates. The morphology, beating rate and the metabolic indexes, including specific consumption rate of glucose (q(glu)) , specific production rate of lactate (q(lac)), lactate transform rate ( Y(lac/glu)), specific creatine kinase (CK) and lactate dehydrogenase (LDH) activities of cardiac cells cultured on three-dimensional collagen membrane and tissue culture plates were compared. It was found that cells shape and cells' CK and LDH activity was no differences between 3D and 2D cultures and cell beat rate on cell culture cluster was slower than those cells cultured on collagen membrane, However the cell glucose consumption and lactate yield rate of cells cultured on cluster was higher than those cells cultured on collagen membrane. After 5 days of cultivation, cardiac cells cultured on collagen membrane scaffolds organized into three-dimensional (3D) aggregates as opposed to the two-dimensional (2D) aggregates mosaic pattern seen in tissue culture plates, and spontaneous and rhythmical contractile 3D cultures in unison were visible to the naked eye and the area of synchronous contract three-dimensional (3D) aggregates reaches 80cm2. The mean value of q(glu), q(lac) and Y(lac/glu) of cultured on three-dimensional collagen scaffold was 7.37 micromol/10(6) cells/d, 2.92 micromol/10(6) cells/ d and 0.38 micromol/micromol, versus 7.59 micromol/10(6)cells/d, 3.83 micromol/10(6) cells/d and 0.51 micromol/micromol in tissue culture plates. These results demonstrate that cardiac cells immobilized on collagen membrane in 3D cultures maintain similar metabolic activity and contractile function when compared with native cardiac cells. The above results support the idea that engineered cardiac tissue can be used as a model of native tissue for studies of tissue development and function in vitro and eventually for tissue repair in vivo.

Animals↗
PubMedDate not supplied

Comparison of different bioreactor systems for the production of high titer retroviral vectors.

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).

Bioreactors↗
PubMed2006

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model.

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.

Animals↗
PubMed2006

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model.

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.

Animals↗
PubMed1988

Effect of dietary alpha-linolenate/linoleate balance on mean survival time, incidence of stroke and blood pressure of spontaneously hypertensive rats.

Following the suckling period, stroke-prone spontaneously hypertensive rats (SHR-SP) were fed semi-purified diets supplemented either with safflower seed oil (rich in linoleic acid) or with perilla seed oil (rich in alpha-linolenic acid). The mean survival time of male SHR-SP fed the perilla diet was longer than that fed the safflower diet by 17% (p less than 0.001) while the difference was 15% in female SHR-SP (p less than 0.05). The mean survival times of female SHR-SP were more than 40% longer than those of male SHR-SP in both dietary groups. Post-mortem examinations of brains revealed apoplexy-related symptoms as the major cause of the death in both dietary groups. The systolic blood pressure was lower by ca. 10% (21 mmHg) in the perilla group than in both the safflower group and conventional diet group. The eicosapentaenoate (20:5 n-3)/arachidonate (20:4 n-6) ratio of platelet phospholipids in spontaneously hypertensive rat (SHR), a measure of platelet aggregability, was much higher in the perilla group than in the safflower group. Thus, increasing the dietary alpha-linolenate/linoleate ratio resulted in an increased mean survival time of SHR-SP rats, possibly by lowering blood pressure and platelet aggregability.

Animals↗
PubMed1975

Lectin-receptor interactions in liposomes.

The major sialoglycoprotein of mammalian erythrocytes has been incorporated into phosphatidylcholine membranes to generate a model system, glycoprotein-liposomes. Electron microscopic examination revealed these structures to be vesicles, approximately 300 A in diameter. An aqueous compartment inside the glycoprotein-liposomes has been identified by trapped volume studies with [14C]sucrose. These glycoprotein-liposomes were found to interact with the lectins, wheat germ agglutinin, and phytohemagglutinin, to form aggregates of mainly unfused vesicles. The aggregation process has been studied by electron microscopy, 90 degrees light scattering, and differential ultracentrifugation analysis. Hapten inhibitors of the lectins were found to inhibit the lectin-induced aggregation of the glycoprotein-liposomes. Binding of 125I-labeled wheat germ agglutinin to glycoprotein-liposomes was studied by differential ultracentrifugation. Hapten inhibitors of wheat germ agglutinin were also found to inhbit the binding of 125I-labled wheat germ agglutinin to the glycoprotein-liposomes. The characteristics of the lectin interactions with glycoprotein-liposomes appeared to be phenomenologically similar to lectin-cell interactions.

Acetylglucosamine↗
PubMed1979

Mannose-contaminating agglutinin for Actinomyces viscosus and Actinomyces naeslundii.

Rapid agglutination of Actinomyces viscosus and Actinomyces naeslundii cells by D-mannose solutions was observed during studies of their attachment to mammalian cells in vitro. The specificity of the agglutination reaction was studied by slide agglutination tests and by measuring the rate of decrease in optical density of bacterial phosphate buffer suspensions caused by the setting of bacterial aggregates. Actinomyces cells were agglutinated by protein-containing mannose solutions of several chemical suppliers. Solutions of sugars other than D-mannose and solutions of mannitol and mannan all failed to agglutinate A. viscsus and A. naeslundii. "Mannose-enhanced" agglutination was impaired by boiling or autoclaving the mannose but was not affected by heating the bacteria, the presence of chloramphenicol, running the assay in the cold, or incorporating any of several commercially purchased sugars in the reaction mixture. During these hapten inhibition experiments, only 6-deoxy-L-talcose-containing extracts of an A. viscosus strain retarded the rate of mannose-enhanced agglutination. Protein-containing fractions of D-mannose mother liquors also agglutinated cells of A. viscosus and A. naeslundii. Other species of oral gram-positive rods were not agglutinated by mannose solutions. Together the data indicate that plant seed-derived D-mannose contains a protein-associated agglutinin for A. viscosus and A. naeslundii which may function via a "lectin-like" selective affinity for the unique cell wall sugar 6-deoxy-L-talose.

Actinomyces↗
PubMed1999

Evaluation of a novel bioartificial liver in rats with complete liver ischemia: treatment efficacy and species-specific alpha-GST detection to monitor hepatocyte viability.

BACKGROUND/AIMS: There is an urgent need for an effective bioartificial liver system to bridge patients with fulminant hepatic failure to liver transplantation or to regeneration of their own liver. Recently, we proposed a bioreactor with a novel design for use as a bioartificial liver (BAL). The reactor comprises a spirally wound nonwoven polyester fabric in which hepatocytes are cultured (40 x 10(6) cells/ml) as small aggregates and homogeneously distributed oxygenation tubing for decentralized oxygen supply and CO2 removal. The aims of this study were to evaluate the treatment efficacy of our original porcine hepatocyte-based BAL in rats with fulminant hepatic failure due to liver ischemia (LIS) and to monitor the viability of the porcine hepatocytes in the bioreactor during treatment. The latter aim is novel and was accomplished by applying a new species-specific enzyme immunoassay (EIA) for the determination of porcine alpha-glutathione S-transferase (alpha-GST), a marker for hepatocellular damage. METHODS: Three experimental groups were studied: the first control group (LIS Control, n = 13) received a glucose infusion only; a second control group (LIS No-Cell-BAL, n = 8) received BAL treatment without cells; and the treated group (LIS Cell-BAL, n = 8) was connected to our BAL which had been seeded with 4.4 x 10(8) viable primary porcine hepatocytes. RESULTS/CONCLUSIONS: In contrast to previous comparable studies, BAL treatment significantly improved survival time in recipients with LIS. In addition, the onset of hepatic encephalopathy was significantly delayed and the mean arterial blood pressure significantly improved. Significantly lower levels of ammonia and lactate in the LIS Cell-BAL group indicated that the porcine hepatocytes in the bioreactor were metabolically activity. Low pig alpha-GST levels suggested that our bioreactor was capable of maintaining hepatocyte viability during treatment. These results provide a rationale for a comparable study in LIS-pigs as a next step towards potential clinical application.

Animals↗
PubMed1990

Maintenance on extracellular matrix and expression of heparanase activity by human ovarian carcinoma cells from biopsy specimens.

A routine procedure has been developed for the isolation and maintenance in culture of human ovarian carcinoma cells derived from biopsy specimens. Cell attachment, plating efficiency and initial outgrowth were greatly improved by seeding the cells on a basement-membrane-like extracellular matrix (ECM) deposited by cultured corneal endothelial cells. These effects were most significant in serum-free conditions which markedly reduced the rate of cell attachment and growth on regular tissue culture plastic. In 60-80% of the cases and regardless of the patient's age, cells cultured on ECM in the absence of serum divided actively and formed a tightly packed epithelial cell monolayer. Fibroblast overgrowth and cell detachment often occurred on ECM in the presence of serum. Incubation of the human ovarian carcinoma cells with sulfate-labelled ECM, resulted in the release of heparan sulfate degradation fragments, 4- to 7-fold smaller than intact heparan sulfate side chains. This degradation was brought about by endoglycosidase (heparanase) activity expressed to a higher extent by cells that were first maintained in primary cultures as compared with cell aggregates taken directly from the biopsy specimen. In most cases, cells derived from metastatic tumors expressed a higher heparanase activity than cells from the primary ovarian tumor. This result corroborates previous studies, performed with cell lines, on the possible involvement of heparanase in tumor cell invasion and metastasis.

Autoradiography↗
PubMed2001

Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit.

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells during seed development. Although gliadins are mainly monomeric, glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. These polymers are among the largest protein molecules known in nature and are the most important determinants of the viscoelastic properties of gluten. As a first step toward the elucidation of the folding and assembly pathways that lead to glutenin polymer formation, we have exploited an in vitro system composed of wheat germ extract and bean microsomes to examine the role of disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. When conditions allowing the formation of disulfide bonds were established, the in vitro synthesized low molecular weight glutenin subunit was recovered in monomeric form containing intrachain disulfide bonds. Conversely, synthesis under conditions that did not favor the formation of disulfide bonds led to the production of large aggregates from which the polypeptides could not be rescued by the post-translational generation of a more oxidizing environment. These results indicate that disulfide bond formation is essential for the conformational maturation of the low molecular weight glutenin subunit and suggest that early folding steps may play an important role in this process, allowing the timely pairing of critical cysteine residues. To determine which cysteines were important to maintain the protein in monomeric form, we prepared a set of mutants containing selected cysteine to serine substitutions. Our results show that two conserved cysteine residues form a critical disulfide bond that is essential in preventing the exposure of adhesive domains and the consequent formation of aberrant aggregates.

Base Sequence↗
PubMed1990

Antiproliferative action of retinoic acid in cultured human brain tumour cells Gl-As-14(S).

We have employed human Gl-As-14(S) brain tumour cell line to study antiproliferative action of retinoic acid (RA). RA (20 microM) caused a time-dependent, dose-dependent, cell seeding density-dependent reduction in cell proliferation in liquid medium and inhibited growth in agar. Growth inhibitory effects of RA were also affected by the concentration of fetal bovine serum (PBS) in the medium. All these effects could be reversed within 48 h after removal of RA from the growth medium. RA-treated cells also displayed reduced concanavalin A (Con A) binding ability by microhemadsorption technique. The results demonstrated that RA can suppress in this tumour cell line the expression of some properties frequently associated with transformed cells.

Antineoplastic Agents↗
PubMed2006

7S globulins from Phaseolus vulgaris L.: Impact of structural aspects on the nutritional quality.

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS-PAGE showed major bands corresponding to the phaseolin subunits (43-53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 degrees C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of beta-sheet and beta-turn structures, together with a contribution at 1687 cm(-1) that was assigned to intramolecular beta-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.

Amides↗