Absence Of Fetal Bovine Serum Research Articles

Expression of PRV-1 Decreases Dependency of Cells on Growth Factors for Proliferation.

Polycythemia rubra vera-1 (PRV-1) is a GPI-linked protein expressed on surface of neutrophils. Polycythemia vera and essential thrombocythemia were found to be associated with an increase in the level of PRV-1 mRNA; however, the function of PRV-1 remains unknown. We have studied the functional effect of expression of PRV-1, both in heterologous cell line and in neutrophils. cDNA encoding PRV-1 was subcloned from a leukocyte cDNA library and expressed in Chinese hamster ovary cells (CHO). After seeding equal numbers of CHO cells stably transfected with PRV-1 or empty plasmid, cell count was conducted at different time intervals and under different concentration of fetal bovine serum (FBS) in growth media. The numbers of PRV-1 transfected cells were higher than sham-transfected cells at 24, 48, 96 and 144 hrs after seeding, and this difference was enhanced with cells growing in a medium with a reduced concentration of serum with the maximal effect seen in serum-free medium. The percentage of apoptotic cells was similar between PRV-1 and sham-transfected cells. These results are suggestive of that CHO cells expressing PRV-1 proliferate faster than sham-transfected cells. The difference in proliferation rate was more significant at lower concentrations of serum and was the highest in serum-free medium. An immunoblot analysis with anti-phosphotyrosine antibody on the whole cell lysate demonstrated that the presence of PRV-1 alters cell signaling. A 60kDa protein band that disappeared after removal of serum in growth medium of sham-transfected cells continued to be present in the PRV-1 transfected cell lysate regardless of the presence or absence of FBS in the media. In order to understand the function of PRV-1 in neutrophils, we used the fact that in 85% of individuals PRV-1 is expressed only by a subgroup of neutrophils. We separated PRV-1 expressing neutrophils of an individual from those lacking this protein in the same donor, using anti-PRV-1 monoclonal antibody. We then compared the gene expression profile of the two groups of neutrophil using DNA microarray technique. Expression of PRV-1 was associated with several folds increase in expression of growth factors (fracture callus-1, X 14.9 times; Insulin-like 3, X 6.7; neural proliferation differentiation, and control-1 or NPDC1, X 3.4 times) and post-translational modifying enzyme of cell surface protein (N-acetylase N sulfotransferase-1, X 18.9 times). Under physiologic conditions, transcriptional regulation of the PRV-1 gene limits its expression to only a certain percentage of neutrophils. In order to investigate the role of epigenetic factors in regulation of transcription of the PRV-1 gene, we analyzed methylation of CpG dinucleotide in promoter of the PRV-1 gene in the genomic DNA obtained from neutrophils expressing PRV-1 to those that don't express this protein in the same individual. Expression of PRV-1 was associated with a decrease in methylation of the CpG dinucleotide located 2937 bp before initiation codon (45% methylated in PRV-1 negative neutrophils compared to 30% methylated in PRV positive neutrophils). We are currently studying the methylation pattern of CpG islands inside the PRV-1 gene. Studying the effect of DNA methylation on expression of PRV-1 in physiologic conditions, may shed light to the mechanism of overexpression of PRV-1 in MPD.

Evaluation of the Akt kinase activation in a PTEN deficient canine osteosarcoma cell line

Introduction: Osteosarcoma is a devastating disease in both human and veterinary medicine. The dog has been recognized as an important model system for the study of this disease, with a high incidence of tumor development noted annually. The cure rates in veterinary oncology are between 20 and 30% of patients seen. Akt is a serine/threonine kinase that primarily protects the cell from apoptosis and may prompt proliferation and growth. Akt activation involves sequential phosphorylation of threonine at position 308 (T308) and serine at position 473 (S473). While for the T308 phosphorylation responsible is PDK1 kinase, for S473 phosphorylation no definitive mechanism has yet been identified. The tumor suppressor PTEN, which has been reported to be absent in canine osteosarcoma, negatively regulates Akt. In our study we evaluated the Akt activation sequence in response to serum deprivation.Methods: “Buck,” an immortalized PTEN deficient canine OSA cell line established and characterized in our laboratory was used. Buck cells were grown in the absence of fetal bovine serum for 8, 24, and 48 hours. Activity of Akt was assayed directly by a method described by Kang et al. The phosphorylation status of Akt at T308 and S473, together with serine at position 241 (S241) of PDK1 was assessed by immunoblotting.Results: We noted marked decrease of the Akt kinase activity with the nadir at the 24‐hour time point and subsequent increase of Akt activity at the 48‐hour time point. Decoupling of the two Akt phosphorylation sites at the 24, and 48‐hour time points was observed (p < 0.01). The phosphorylation status of T308 declined steadily throughout the experiment. The phosphorylation status of S473 declined until the 24‐hour time point, and then increased in parallel with the results noted in the kinase activity assay. Phosphorylation of PDK1 S241 was examined and found to decrease at 24 hours, with subsequent increase after 48 hours of serum starvation, in parallel with the phosphorylation pattern of Akt S473 (p < 0.01).Conclusions: We report parallel patterns of PDK1 S241 and Akt S473 phosphorylation in the absence of growth factors. PDK1 activity is not assessed directly, but its phosphorylation status indicates that more PDK1 is in an activated from at the 48‐hour time point than at 24 hours. The increase of Akt activity and S473 phosphorylation at the 48‐hour time point is presumably a response to the apoptogenic conditions of the serum free medium. The same response of PDK1 may indicate the presence of a regulatory autophosphorylation mechanism that members of the AGC kinase family share. Surprisingly, the increased amount of activated PDK1 does not correlate with the phosphorylation pattern of Akt T308, and could be attributed to cytoplasmic sequestration of PDK1.

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures.

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures.

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.

A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins.

We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialoglycoproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production.

Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.

A jet impingement investigation of osteoblastic cell adhesion.

When designing dental and orthopedic implants, it is important to consider phenomena occurring at the microscopic level, particularly at the bone-implant interface. The presence of hard tissue at this interface is essential to implant viability. The integrity of this tissue-biomaterial interface is dependent on appropriate osteoblast functions (adhesion, matrix deposition, etc.) in the immediate area. Researchers have modified various materials with cell-adhesive peptides with the ultimate goal of controlling osteoblast functions. This study used microjet impingement to compare the strength of adhesion of osteoblastic cells (at varying populations) and fibroblasts to peptide-modified substrates in the presence and absence of fetal bovine serum. In the presence of the serum, there was no significant difference in cellular adhesion strength between substrates. In the absence of serum, all cells tested adhered more strongly to underlying substrates, and the strength of cellular adhesion was greater on modified surfaces than on plain glass surfaces. In the absence of serum, second-passage osteoblastic cells generally adhered to substrates more strongly than first-passage osteoblastic cells; fibroblasts adhered similarly to second-passage osteoblastic cells. Fundamental studies such as the present increase the understanding of cell adhesion to various substrates--knowledge that may be ultimately useful in creating an optimal bone-implant interface.

Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aorta.

1 We investigated the effects of prolonged exposure to copper (Cu(2+)) on vascular functioning of isolated rat aorta. 2 Aortic rings were exposed to CuSO(4) (3-24 h) in Dulbecco's modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence of Cu(2+). 3 Exposure to 2 micro M Cu(2+) in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 micro M PE by 30% (P<0.05) and impaired the relaxant response to 3 micro M ACh or 1 micro M A23187 (ACh, from 65.7+/-7.1 to 6.2+/-1.1%, n=8; A23187, from 74.6+/-8.2 to 12.0+/-0.8%, n=6; P<0.01 for both). Cu(2+) exposure did not affect the relaxant response to NO-donors. 4 Impairment of vasorelaxation appeared 3 h after incubation with 2 micro M Cu(2+) and required 12 h to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mM tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2+/-6.4%, n=10, P<0.01 vs Cu(2+) alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective. 5 Twenty-four hour-exposure to 2 micro M Cu(2+) did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8+/-1.1 to 18.9+/-2.9 ng mg(-1) wet weight, n=8; P<0.01. 6 Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations of Cu(2+) impaired endothelium-dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions. British Journal of Pharmacology (2002) 136, 1185-1193

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells.

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.

The effects of substance P and vasoactive intestinal peptide on interleukin-6 synthesis in cultured human keratinocytes

Interleukin-6 (IL-6) is a cytokine implicated as a key mediator of immune and inflammatory responses in psoriasis. Recent studies have shown that neuropeptides, substance P (SP) and vasoactive intestinal peptide (VIP), can modulate a production of IL-6 from cells, such as monocytes and astrocytes, participating in an immune reaction. The aim of this study was to assess the role of the neuropeptides on cytokine production of keratinocytes in physiologic or pathologic conditions. Cultured human keratinocytes derived from normal foreskin and psoriatic lesions were treated with various concentrations of SP or VIP, in the presence or absence of fetal bovine serum. The secretion of IL-6 by the treated keratinocytes was analyzed by enzyme-linked immunosorbent assay. Although neither SP nor VIP, by itself, was able to induce IL-6 synthesis in cultured human keratinocytes, we have found that SP, not VIP, significantly reduced 5% fetal bovine serum-induced IL-6 production in time- and dose-dependent fashion. This down-regulatory effect of SP was reversed by spantide, a SP antagonist. Lesional psoriatic keratinocytes showed a similar, but weaker, response when compared with normal keratinocytes. These data suggested that SP might modulate IL-6 synthesis of keratinocytes in either physiologic or pathologic conditions such as psoriasis.

Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture.

We investigated whether vascular endothelial growth factor (VEGF) production by human pulp cells (HPC) is regulated by lipopolysaccharide (LPS) in relation to the pathogenesis of pulpitis. Although HPC incubated with medium alone only marginally expressed VEGF mRNA and produced a low level of VEGF as detected by enzyme-linked immunosorbent assay, the VEGF mRNA expression and VEGF production were markedly enhanced upon stimulation with LPS from Escherichia coli. Prevotella intermedia LPS, phorbol 12-myristate 13-acetate, and interleukin-6 also induced VEGF mRNA expression in HPC. A simian virus 40-infected HPC line also exhibited increased VEGF mRNA expression in response to E. coli LPS, but lung and skin fibroblasts did not. Fetal bovine serum (FBS) increased the sensitivity of HPC to LPS in a dose-dependent manner. HPC did not express membrane CD14 on their surfaces. However, the anti-CD14 monoclonal antibody MY4 inhibited VEGF induction upon stimulation with LPS in HPC cultures in the presence of 10% FBS but not in the absence of FBS. LPS augmented the VEGF production in HPC cultures in the presence of recombinant human soluble CD14 (sCD14). To clarify the mechanisms of VEGF induction by LPS, we examined the possible activation of the transcription factor AP-1 in HPC stimulated with LPS, by a gel mobility shift assay. AP-1 activation in HPC was clearly observed, whereas that in skin fibroblasts was not. The AP-1 inhibitor curcumin strongly inhibited LPS-induced VEGF production in HPC cultures. In addition, a protein synthesis inhibitor, cycloheximide, inhibited VEGF mRNA accumulation in response to LPS. These results suggest that the enhanced production of VEGF in HPC induced by LPS takes place via an sCD14-dependent pathway which requires new protein synthesis and is mediated in part through AP-1 activation.

The Pleckstrin Homology Domain of Protein Kinase D Interacts Preferentially with the η Isoform of Protein Kinase C

The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta. The presence of PKCeta in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCeta was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCepsilon, and did not bind PKCzeta. When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCeta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon and eta, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium

In the present study reassortant influenza A viruses of both the H1N1 and H3N2 type were generated in Madin Darby Canine Kidney cells grown in the absence of fetal bovine serum (MDCK-SF1 cells). To this end, MDCK-SF1 cells were simultaneously infected with one of the high-growth laboratory strains A/Puerto Rico/8/34 (H1N1) or A/Hong Kong/2/68 (H3N2) and recent H3N2 and H1N1 vaccine strains, respectively. Reassortant viruses obtained from these mixed infections were genetically characterized by RT-PCR and restriction enzyme analysis and their growth properties were compared to those of the corresponding field strains. Reassortant H3N2 viruses inherited the matrix and polymerase pa gene whilst H1N1 reassortant viruses inherited the matrix and polymerase pb1 gene of the high-growth parent. Reassortant viruses generally gave higher viral yields, as measured by a haemagglutination assay, than their wild type counterparts. The procedure followed results in the generation of high-growth reassortant viruses in weeks. The use of MDCK-SF1 cells together with these reassortants for generating influenza virus antigens can significantly speed up the vaccine production procedure.

In vitro Cultivation and Sporulation of Neozygites parvispora (Zygomycetes: Entomophthorales)

Summary Neozygites parvispora was isolated from Tbrips tabaci infesting leek plants. In vitro cultivation of two isolates was achieved using supplemented Grace's insect tissue culture medium complemented with fetal bovine serum (FBS) and pre-treated lepidopteran hemolymph. This medium composition enabled exponential cell growth as rod-shaped hyphal bodies with a length over diameter ratio of 4.5-5.0. Both FBS and hemolymph were mandatory for sustained growth. The growth requirements indicate the presence of one or several growth factors in both the hemolymph and FBS. Absence of FBS resulted in growth arrest with subsequent cell lysis. Omission of hemolymph led to rounding and size decrease of the hyphal bodies, some of which formed septated germ tubes in a later stage. Eventually, cell death occurred under such conditions. Hyphal bodies from in vitro culture were capable of capilliconidia formation after entrapment in alginate pellets. This suggests the requirement of a liquid/air interface, which triggers primary sporulation and subsequent capilliconidium formation.

Caspases (Interleukin-1β-Converting Enzyme Family Proteases) Are Involved in the Regulation of the Survival of Osteoclasts

Osteoclast-like multinucleated cells (OCLs) were prepared on collagen gels in a coculture system of mouse bone marrow cells and osteoblasts, and purified by collagenase and a subsequent pronase treatment. More than 80% of the purified OCLs were found to undergo apoptotic cell death by 48 h during the culture in a culture medium containing 10% fetal bovine serum (FBS). Withdrawal of FBS from the culture medium accelerated the cell death, which induced more than 80% of OCLs to undergo apoptotic cell death by as early as 18 h. Two peptide inhibitors of caspases (interleukin-1β-converting enzyme family proteases), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), extended the survival time of OCLs in the presence and absence of 10% FBS, but the effect was rather limited in the absence of FBS. Because interleukin-1α (IL-1α) and the macrophage colony stimulating factor (M-CSF) are known to promote the survival of osteoclasts, we examined the effect of the peptide inhibitors and these cytokines. Combinations of the peptide inhibitors and IL-1α, or the peptide inhibitors and M-CSF, were more effective than the inhibitors alone. When endogenous caspase activities of OCLs were analyzed using fluorescence peptide substrates, the activities, in particular, caspase-3 (CPP32)-like activity, were markedly increased in OCLs by the withdrawal of FBS from the culture medium. IL-1α and M-CSF suppressed the activation of the caspases. In addition, western blot analysis revealed that the expression of Bcl-2, which inhibits the activation of caspases, was very weak or even negligible in OCLs. Taken together, these results suggest that the caspases are involved in the regulation of survival and apoptotic cell death of osteoclasts.

Effects of troglitazone and pioglitazone on cytokine-mediated endothelial cell proliferation in vitro.

We examined whether troglitazone and pioglitazone, antidiabetic thiazolidinediones, would directly induce endothelial cell proliferation or influence cytokine-driven proliferation in vitro. Monolayers of Balb/c mouse aortic endothelial cells were treated with troglitazone or pioglitazone in the absence of fetal bovine serum. Endothelial cells also were exposed to varying concentrations of basic fibroblast growth factor (bFGF) or insulin with or without either thiazolidinedione. After 48 h, 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays were performed to quantitate endothelial cell proliferation by using the various treatment regimens. The data demonstrate that the antidiabetic thiazolidinediones troglitazone and pioglitazone negligibly affect direct endothelial cell proliferation in vitro. Furthermore, troglitazone and pioglitazone significantly inhibit bFGF-induced endothelial cell mitogenesis, whereas only high concentrations of troglitazone affect insulin-mediated proliferation.

Effects of epidermal growth factor on the [3H]-thymidine uptake in the SK-N-SH and SH-SY5Y human neuroblastoma cell lines.

The studies on the factors that regulate the biology of the neuroblastoma cell lines may offer important information on the development of tissues and organs that derive from the neural crest. In the present paper we study the action of epidermal growth factor (EGF) on two human neuroblastoma cell lines: SK-N-SH which is composed at least of two cellular phenotypes (neuroblastic and melanocytic/glial cells), and its pure neuroblastic subclone SH-SY5Y. The results show that EGF (10 ng/ml) significantly stimulates the incorporation of [3H]-thymidine in the SK-N-SH cells only in the presence of fetal bovine serum (FBS) (control = 58,285 +/- 9327 cpm; EGF = 75,523 +/- 4457, p < 0.05). Such effect is not observed in the presence of a chemical defined medium, that is, in the absence of FBS (control = 100,997 +/- 4375; EGF = 95,268 +/- 4683; NS) In the SH-SY5Y cells the EGF does not modify the incorporation of [3H]-thymidine either in the presence of 10% of BFS (control = 113,838 +/- 6978; EGF = 119,434 +/- 9441; NS) or in its absence (control = 46,197 +/- 3335; EGF = 44,472 +/- 3493; NS). The results here reported suggest that: a) EGF may affect the proliferation of cells derived from a primary human neuroblastoma; b) this is evident by the EGF-induced increase of [3H]-thymidine incorporation in SK-N-SH cells; c) it is required the presence of other growth factors, present in the FBS, for the mitogenic action to be accomplished; d) since the pure neuroblastic SH-SY5Y cell line are refractory to the EGF, the effects observed in SK-N-SH cells probably occur on the melanocytic/glial cell subpopulation.

Insulin-like growth factor-1, a component of serum that enables porcine cumulus cells to expand in response to follicle-stimulating hormone in vitro.

The present study was aimed at determining whether insulin-like growth factor-1 (IGF-1) is a component of fetal bovine serum (FBS) that enables porcine cumulus cells to expand in response to FSH in vitro. Cumulus-oocyte complexes (COC) obtained from 4- to 6-mm follicles of prepubertal gilts were cultured at 39 degrees C for 24 h in media that contained human recombinant IGF-1 (50 ng/ml), FBS (15% v:v), or their combination, with or without FSH (1.5 microg/ml), and cumulus expansion was scored microscopically. Expansion was FSH dependent and was observed only when IGF-1, FBS, or both were present. The proportion of FSH-stimulated COC exhibiting full expansion in response to IGF-1 alone did not differ significantly (p > 0.05) from the proportion in those cultured with FBS or IGF-1+FBS (79 +/- 2.8% vs. 84 +/- 2.2% or 76 +/- 6.2%, respectively). In a concentration-response study, FSH-stimulated expansion was observed in a significant proportion of COC (32 +/- 2.8% vs. 0% control) at 1 ng/ml IGF-1, with the proportions increasing dose-dependently to maximal values between 10 and 75 ng/ml IGF-1, and decreasing at higher IGF-1 concentrations. Exposure of COC to an IGF-1 receptor (IGF-1R)-neutralizing antibody (Ab) for 90 min before addition of FSH and FBS dose-dependently inhibited cumulus expansion, with maximal inhibition at 10 microg/ml (1 +/- 1.0% vs. 68 +/- 1.1% control). In the absence of FBS or IGF-1, some COC had a tendency toward slight expansion when cultured with FSH, and the Ab completely inhibited that effect, suggesting that this may be due to endogenous IGF-1 production by the COC. The Ab effect was reversible and was eliminated by washing twice with fresh medium followed by culture for an additional 24 h in the presence of FSH and FBS. Expression of IGF-1R mRNA in the isolated oocyte and cumulus cells was determined by reverse tramscriptase polymerase chain reaction using sequence-specific primers. The IGF-1R message was detected in both the oocyte and cumulus cells. Collectively, these observations suggest that IGF-1 is a component of serum that enables cumulus cells to expand in response to FSH in vitro, and that the effect is receptor mediated. Since IGF-1 is present in the follicle in vivo, it may have a physiological role during gonadotropin-induced cumulus expansion.

Regulation of osteoclast function.

Regulation of osteoclast function.

Hyporesponsiveness of inflamed human gingival fibroblasts from patients with chronic periodontal diseases against cell surface components of Porphyromonas gingivalis

Inflamed human gingival fibroblasts (HGF) of patients with chronic periodontal diseases have less active interleukin-8 (IL-8) production compared with normal HGF of volunteers with healthy gingival tissues, after stimulation with Porphyromonas gingivalis surface components such as fimbriae, lipopolysaccharide (LPS) and its lipid A, but not LPS or lipid A from other bacterial species. A decrease in number of specific binding sites for P. gingivalis fimbrial molecules in inflamed HGF is also observed by Scatchard plot analysis. A short exposure (6 h) to P. gingivalis LPS resulted in significant potentiation of the LPS-dependent IL-8 production in normal HGF, whereas a long exposure (48 h) to the LPS significantly reduced IL-8 production. Tyrosine phosphorylation of proteins of 127 kDa and 186 kDa in inflamed HGF stimulated with P. gingivalis fimbriae or its LPS was observed by immunoblotting, and these two phosphoproteins were termed tolerance-induced protein, TIP. Protein bands of 45 kDa which bound to radioiodinated P. gingivalis fimbriae in the presence and absence of fetal bovine serum (FBS), and major 73-kDa and minor 30-kDa and 45-kDa bands which bound to radioiodinated P. gingivalis LPS in the presence of FBS in normal and inflamed HGF were observed by using photocrosslinking. These findings suggest that the hyporesponsiveness of HGF induced by a prolonged exposure to P. gingivalis may emerge because of HGF damage or result from host defense in chronic periodontal lesions.