Immortalization of human urothelial cells by human papillomavirus type 16 E6 and E7 genes in a defined serum-free system.

UNLABELLED: Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract. OBJECTIVE: Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum-free culture system. METHOD AND RESULTS: Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7. CONCLUSION: The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.

Combinación de clorimipramina, imipramina y sertralina para el tratamiento de la eyaculación precoz / Combination of clorimipramine, imipramine and sertraline in the treatment of premature ejaculation

El presente trabajo se encuentra compuesto por 30 (treinta pacientes), que consultaron en la Cátedra de Urología, Sección Sexología Clínica del Hospital de Clínicas José de San Martín. Los hombres poseían edades entre 21 y 63 años y consultaron en nuestro Servicio entre abril y septiembre de 2001 por EP. La totalidad de los mismos tenían pareja estable, con una duración de la relación igual o mayor a seis meses. La edad promedio de la muestra es de 38,8 años, siendo la del grupo que recibió placebo, de 32,6 años y la de los pacientes que recibieron la combinación de antidepresivos (AD) de 35,6 años. La administración de los medicamentos antidepresivos utilizados para el tratamiento de la eyaculación precoz se efectuó por receta magistral con la finalidad de que no contuviera el prospecto acompañante habitual de las monodrogas, porque su presencia, en pacientes altamente sugestionables, podía interferir en el resultado. Los tres componentes se administraron en un solo comprimido. Tanto en el grupo placebo como en el grupo tratado con combinación de antidepresivos, 4 pacientes eran eyaculadores precoces verdaderos (26,6 por ciento) y los 11 restantes eran eyaculadores rápidos (73,3 por ciento). Once de los 15 pacientes que recibieron medicación antidepresiva, manifestaron mejoría moderada o significativa (73,33 por ciento) y 2 de los 15 (13 por ciento), a los cuales se les había administrado placebo, presentaron mejoría significativa en la eyaculación. Luego del mes de tratamiento con placebo, 10 (67 por ciento) de los 15 pacientes refirieron no haber notado cambio alguno. De los 5 (33,3 por ciento) restantes 3 (20 por ciento) refirieron mejoría leve y 2 (13 por ciento) mejoría moderada. En cuanto a los efectos adversos, sólo 2 (13,2 por ciento) pacientes del grupo placebo, refirieron tenerlos, uno de ellos cefalea y el otro, malestar gastrointestinal inespecífico. Del grupo tratado con AD, 5 (33,3 por ciento) de los pacientes presentaron efectos adversos. Tres de ellos, (20por ciento) tuvieron somnolencia. Uno solo de ellos (6,6 por ciento) náuseas y el restante (66,6 por ciento), una leve pérdida de la función eréctil, que no le impidió la penetración. Se acompaña, en el final del trabajo, una bibliografía pertinente de uso de antidepresivos en síndromes eyaculatorios precoces.

Combinación de clorimipramina, imipramina y sertralina para el tratamiento de la eyaculación precoz / Combination of clorimipramine, imipramine and sertraline in the treatment of premature ejaculation

El presente trabajo se encuentra compuesto por 30 (treinta pacientes), que consultaron en la Cátedra de Urología, Sección Sexología Clínica del Hospital de Clínicas José de San Martín. Los hombres poseían edades entre 21 y 63 años y consultaron en nuestro Servicio entre abril y septiembre de 2001 por EP. La totalidad de los mismos tenían pareja estable, con una duración de la relación igual o mayor a seis meses. La edad promedio de la muestra es de 38,8 años, siendo la del grupo que recibió placebo, de 32,6 años y la de los pacientes que recibieron la combinación de antidepresivos (AD) de 35,6 años. La administración de los medicamentos antidepresivos utilizados para el tratamiento de la eyaculación precoz se efectuó por receta magistral con la finalidad de que no contuviera el prospecto acompañante habitual de las monodrogas, porque su presencia, en pacientes altamente sugestionables, podía interferir en el resultado. Los tres componentes se administraron en un solo comprimido. Tanto en el grupo placebo como en el grupo tratado con combinación de antidepresivos, 4 pacientes eran eyaculadores precoces verdaderos (26,6 por ciento) y los 11 restantes eran eyaculadores rápidos (73,3 por ciento). Once de los 15 pacientes que recibieron medicación antidepresiva, manifestaron mejoría moderada o significativa (73,33 por ciento) y 2 de los 15 (13 por ciento), a los cuales se les había administrado placebo, presentaron mejoría significativa en la eyaculación. Luego del mes de tratamiento con placebo, 10 (67 por ciento) de los 15 pacientes refirieron no haber notado cambio alguno. De los 5 (33,3 por ciento) restantes 3 (20 por ciento) refirieron mejoría leve y 2 (13 por ciento) mejoría moderada. En cuanto a los efectos adversos, sólo 2 (13,2 por ciento) pacientes del grupo placebo, refirieron tenerlos, uno de ellos cefalea y el otro, malestar gastrointestinal inespecífico. Del grupo tratado con AD, 5 (33,3 por ciento) de los pacientes presentaron efectos adversos. Tres de ellos, (20por ciento) tuvieron somnolencia. Uno solo de ellos (6,6 por ciento) náuseas y el restante (66,6 por ciento), una leve pérdida de la función eréctil, que no le impidió la penetración. Se acompaña, en el final del trabajo, una bibliografía pertinente de uso de antidepresivos en síndromes eyaculatorios precoces. (AU)

Novel CPR with periodic Gz acceleration.

The effects of periodic Gz acceleration (pGz) on cardiovascular function and hemodynamics were determined in a pig model of acute cardiopulmonary resuscitation (CPR). The application of pGz (horizontal head-to-foot oscillations) at 2 Hz increased cardiac output in fibrillated animals proportional to the amplitude of the applied acceleration force that plateaued at 0.7 G. Cardiac output in fibrillating animals was restored to 20% of the values obtained before fibrillation with pGz-CPR and arterial blood gas values were normal during this period. The central vascular pressure gradient driving blood flow was only about 6 mmHg, suggesting low vascular resistance during pGz-CPR. In another study, capillary blood flow was determined before and after pGz-CPR using colored microspheres. Capillary perfusion was detected in all tissue beds studied during pGz-CPR. Significant capillary blood flow was detected in the endocardium and brain stem during pGz-CPR that represented 39 and 197% of control values before fibrillation, respectively. Thus, the cardiac output during pGz-CPR was preferentially distributed to the myocardial and brain tissues. In a final group, animals were successfully resuscitated with return of spontaneous circulation (ROSC) after pGz-CPR for 15 min following cardiac fibrillation with a 3-min non-intervention period. Following ROSC, blood pressure was maintained at pre-arrest values for 2 h without any pharmacological or mechanical support. Arterial blood gases during the pGz-CPR and the ROSC periods were normal and not different from values obtained before fibrillation. None of the control animals (18 min of fibrillation without pGz-CPR) survived the experimental protocol and only two of these six animals briefly returned to spontaneous circulation (<20 min). In conclusion, experimental pGz-CPR produces cardiac output, capillary blood flow, and ventilation sufficient to maintain fibrillating animals for 18 min with ROSC for 2 h without support.

Regional blood flow during periodic acceleration.

OBJECTIVE: To determine whether a motion platform that imparts noninvasive periodic acceleration (pGz) forces to the body causes systemic vasodilation and changes local organ blood flow. DESIGN: Prospective paired blocked design. SETTING: Medical center research laboratory. SUBJECTS: Juvenile Yorkshire pigs. INTERVENTIONS: Juvenile pigs (12 kg) were anesthetized, paralyzed, and placed on a motion platform that oscillated at a frequency of 4 Hz and a force of approximately 0.4 G. MEASUREMENTS AND MAIN RESULTS: Regional blood flows, as assessed by colored microspheres, increased during pGz relative to values obtained before pGz. Blood flow (mL.min-1.100 g-1) significantly increased to the epicardium (71%), endocardium (93%), cerebrum (183%), brain stem (177%), renal cortex (53%), ileal mucosa (69%), gastric antral mucosa (72%), and liver (86%). Spleen and skeletal muscle blood flow increased without statistical significance, 38% and 158% with pGz, relative to paired control values. Regional blood flows returned to baseline 10 mins after discontinuation of pGz, except in the myocardial layers, where blood flow remained significantly elevated. There was no difference compared with baseline in heart rate, arterial blood gases, and blood pressure, but serum nitrite concentration was significantly higher (58%) during pGz. In another series of animals, pGz increased pulmonary artery blood flow directly proportional to the magnitude of the applied acceleration force with frequency held constant. CONCLUSIONS: Periodic sinusoidal inertial forces in the spinal axis increase blood flow to tissues. The increased blood flow is reversible and may be caused by vasodilation secondary to local mediator release. These effects may be desirable in clinical conditions of low tissue oxygen delivery and perfusion.

Noninvasive motion ventilation (NIMV): a novel approach to ventilatory support.

A motion platform was developed that oscillates an animal in a foot-to-head direction (z-plane). The platform varies the frequency and intensity of acceleration, imparting periodic sinusoidal inertial forces (pG(z)) to the body. The aim of the study was to characterize ventilation produced by the noninvasive motion ventilator (NIMV) in animals with healthy and diseased lungs. Incremental increases in pG(z) (acceleration) with the frequency held constant (f = 4 Hz) produced almost linear increases in minute ventilation (VE). Frequencies of 2-4 Hz produced the greatest VE and tidal volume (VT) for any given acceleration between +/-0.2 and +/-0.8 G. Increasing the force due to acceleration produced proportional increases in both transpulmonary and transdiaphragmatic pressures. Increasing transpulmonary pressure by increasing pG(z) produced linear increases in VT, similar to spontaneous breathing. NIMV reversed deliberately induced hypoventilation and normalized the changes in arterial blood gases induced by meconium aspiration. In conclusion, a novel motion platform is described that imparts periodic sinusoidal acceleration forces at moderate frequencies (4 Hz) to the whole body in the z-plane. These forces, when properly adjusted, are capable of highly effective ventilation of normal and diseased lungs. Such noninvasive ventilation is accomplished at airway pressures equivalent to atmospheric or continuous positive airway pressure, with acceleration forces less than +/-1 G(z).

Hemodynamic effects of periodic G(z) acceleration in meconium aspiration in pigs.

The hemodynamic effects of periodic acceleration (pG(z)), induced in the spinal axis with noninvasive motion ventilation (NIMV), were studied in a piglet model of pulmonary hypertension associated with meconium aspiration. Animals (n = 12) were anesthetized, paralyzed, intubated, and supported by conventional mechanical ventilation (CMV). Thirty minutes after tracheal instillation of meconium solution (6 ml/kg), either CMV (n = 6) was continued or NIMV (n = 6) was initiated. Changes in systemic and pulmonary hemodynamics and arterial blood gases were tracked for 2 h after aspiration. Thermodilution, cardiac output, and heart rate were not significantly different after meconium aspiration in the pG(z) group relative to the CMV controls. Aortic pressure and systemic vascular resistance were significantly lower (approximately 30%) after meconium aspiration in NIMV animals relative to CMV animals. Pulmonary arterial pressure and pulmonary vascular resistance were also significantly lower, by 100%, after aspiration of meconium in the NIMV animals compared with the CMV controls. Meconium aspiration significantly decreased total respiratory compliance by approximately 50% and increased total respiratory resistance by approximately 100% in both CMV and NIMV animals, but such alterations did not differ between the two groups. Both CMV and NIMV satisfactorily supported ventilation in these paralyzed animals. In conclusion, NIMV through pG(z) in the spinal axis decreased systemic and pulmonary vascular resistance in piglets after meconium aspiration.

Expression and purification of recombinant human SPARC produced by baculovirus.

SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40), a matrix-associated protein, disrupts cell adhesion and inhibits the proliferation of many cultured cells. We report the expression of recombinant human protein (rhSPARC) in a baculovirus expression system. This procedure routinely yields approximately 1 mg of purified protein per 500 ml of culture supernate. rhSPARC produced by insect cells migrates at the appropriate molecular weight under reducing and nonreducing conditions. The rhSPARC purified from insect cell media appeared structurally similar to SPARC purified from mammalian tissue culture by the criterion of circular dichroism. In addition, a series of anti-SPARC and anti-SPARC peptide antibodies recognized insect cell rhSPARC. We also show that rhSPARC produced in this system is glycosylated and is biologically active, as assessed by inhibition of endothelial cell proliferation and induction of collagen I mRNA in mesangial cells. Significant amounts of rhSPARC can now be generated in the absence of contaminating mammalian proteins for structure/function assays of SPARC activities.

Review article: The molecular era of bladder research. Transgenic mice as experimental tools in the study of outlet obstruction.

PURPOSE: To review the crucial role of transgenic mice as experimental tools in the study of outlet obstruction. MATERIALS AND METHODS: We reviewed the literature for studies that have used mice as models for outlet obstruction. RESULTS: The combination of genetic manipulations and cellular physiology defines state-of-the-art experiments that explore the reciprocal mesenchymal-epithelial interactions that regulate bladder cell mechanisms. CONCLUSIONS: The use of transgenic mice in bladder research has provided important data with respect to the molecular signals that drive bladder development, homeostasis, and the response to injury.

Differential expression of osteonectin/SPARC during human prostate cancer progression.

The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP.

Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis.

UNLABELLED: Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis. BACKGROUND: SPARC has been implicated as a counteradhesive and antiproliferative protein associated with deposits of extracellular matrix in renal disease. METHOD: We have examined the effect of recombinant SPARC containing a C-terminal His tag (rSPARC) in an acute model of mesangial cell injury that is induced in the rat by an antibody against the Thy1 antigen on the mesangial cell membrane. The recombinant protein was administered 24 hours after the induction of nephritis and was infused through day 4. RESULTS: rSPARC was localized to the renal glomeruli of rats treated with anti-Thy1 antibody. Type I collagen and fibronectin, as well as transforming growth factor-beta1 (TGF-beta1), were increased at day 5 in rats treated with rSPARC (N = 4, P < 0.05 vs. delivery buffer), but only minimal effects were seen on mesangial cell and endothelial cell proliferation. In primary cultures of rat mesangial cells, infusion of rSPARC was associated with increases in TGF-beta1 mRNA and in total, secreted TGF-beta1 protein. CONCLUSIONS: rSPARC stimulates expression of TGF-beta1 both in vitro and in vivo. Given the closely regulated expression of SPARC, TGF-beta1, and type I collagen in several animal models of glomerulonephritis, we propose that SPARC could be one of the major mediators of the induction of TGF-beta1 in renal disease.

SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in mesangial cells.

The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153-1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARC gene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-beta1 (TGF-beta1) mRNA and secreted TGF-beta1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-beta1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-beta1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus.

Type IV collagen and laminin regulate glomerular mesangial cell susceptibility to apoptosis via beta(1) integrin-mediated survival signals.

Postinflammatory scarring is characterized by changes in extracellular matrix (ECM) composition and progressive loss of normal resident cells. In glomerular inflammation there is now evidence that unscheduled apoptosis (programmed cell death) of mesangial and other resident cells may mediate progression to irreversible glomerulosclerosis. In the current study we examined the hypothesis that ECM components may differ in their capacity to support mesangial cell survival by suppression of apoptosis. Using a well-established in vitro model of mesangial cell apoptosis, we found that collagen IV and laminin, components of normal mesangial ECM, protected rat mesangial cells from apoptosis induced by serum starvation and DNA damage, by a beta(1) integrin-mediated, but arg-gly-asp (RGD)-independent mechanism. In contrast, collagen I, fibronectin, and osteonectin/SPARC, which are overexpressed in diseased glomeruli, failed to promote rat mesangial cell survival. However, the survival-promoting effect of collagen IV and laminin was not associated with changes in cellular levels of apoptosis regulatory proteins of the Bcl-2 family. These experiments demonstrate that glomerular mesangial cell survival is dependent on interactions with ECM and provide insights into potential mechanisms by which resident cell loss may occur during acute inflammation and postinflammatory scarring of the kidney and other organs.

Cell cycle-dependent nuclear location of the matricellular protein SPARC: association with the nuclear matrix.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M-phase: cells in metaphase and anaphase exhibited strong cytoplasmic immunoreactivity, whereas cells in telophase were devoid of labeling. Immunocytochemical analysis of embryonic chicken cells in vitro likewise showed the presence of SPARC in the nucleus. Furthermore, elution of soluble proteins and DNA from these cells indicated that SPARC might be a component of the nuclear matrix. We subsequently examined cultured bovine aortic endothelial cells, which initially appeared to express SPARC only in the cytoplasm. However, after elution of soluble proteins and chromatin, we also detected SPARC in the nuclear matrix of these cells. Embryonic chicken cells incubated with recombinant SPARC were seen to take up the protein and to translocate it to the nucleus progressively over a period of 17 h. These observations provide new information about SPARC, generally recognized as a secreted glycoprotein that mediates interactions between cells and components of the extracellular matrix. The evidence presented in this study indicates that SPARC might subserve analogous functions in the nuclear matrix.

Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein that regulates cellular adhesion and proliferation. In this report, we show that SPARC protein is restricted to epithelial cells of the murine lens and ends abruptly at the equatorial bow region where lens fiber differentiation begins. SPARC protein was not detected in the lens capsule or in differentiated lens fibers. SPARC-null mice developed cataracts at approximately 3-4 months after birth, at which time posterior subcapsular opacities were observed by slit lamp ophthalmoscopy. Histological analyses of ocular sections from 3-month old animals revealed several microscopic abnormalities present in the SPARC-null mice but absent from the wild-type animals. Fiber cell elongation was incomplete posteriorly and resulted in displacement of the lenticular nucleus to the posterior of the lens. Nuclear debris was present in the posterior subcapsular region of the lens, an indication of the abnormal migration and elongation of either fetal or anterior epithelial cells, and the bow region was disrupted and vacuolated. In the anterior lens, the capsule appeared to be thickened and was lined by atypical, plump cuboidal epithelium. Moreover, anterior cortical fibers were swollen. Polyacrylamide gel electrophoresis of the epithelial, cortical and nuclear fractions of wild-type and SPARC-null lenses indicated no significant differences among the alpha-, beta-, and gamma-crystallins. Expression of alphaB-crystallin appeared similar in fiber cells of wild-type and SPARC-null lenses, although the distribution of alphaB-crystallin was asymmetric in SPARC-null lenses as a result of abnormal lens fiber differentiation. No evidence of atypical extracellular matrix deposition in areas other than the capsule was detected in wild-type or SPARC-null lens at 3 months of age. We conclude that the disruption of the Sparc locus in mice results in the alteration of two fundamental processes of lens development: differentiation of epithelial cells and maturation of fiber cells.

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines.

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.

Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli.

Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

Differential modulation of cell adhesion by interaction between adhesive and counter-adhesive proteins: characterization of the binding of vitronectin to osteonectin (BM40, SPARC).

Heparin-binding forms of vitronectin, a multifunctional adhesive glycoprotein, are associated with the extracellular matrix (ECM) at different locations in the body and serve to promote cell adhesion and the regulation of pericellular proteolysis at sites of angiogenesis. In the present study we characterized the interactions of vitronectin with the counter-adhesive protein osteonectin (also termed SPARC or BM40). Osteonectin and vitronectin were both found associated with the ECM of cultured endothelial cells and were localized in vessel wall sections of kidney tissue. In vitro, the heparin-binding multimeric isoform of vitronectin bound to immobilized osteonectin in a saturable manner with half-maximal binding at 30-40 nM. Preincubation of plasma vitronectin with plasminogen activator inhibitor 1 (PAI-1), which provoked multimer formation, induced the binding of vitronectin to osteonectin. Binding was optimal at physiological ionic strength, and binary complexes were stabilized by tissue transglutaminase-mediated cross-linking. In a concentration-dependent fashion, PAI-1, CaCl2, heparin and heparan sulphate, but not other glycosaminoglycans, interfered with the binding of vitronectin to osteonectin. Using vitronectin-derived synthetic peptides as well as mutant forms of recombinant osteonectin, we found that the heparin-binding region of vitronectin interacted with the C-terminal region of osteonectin that contains a high-affinity Ca2+-binding site with counter-adhesive properties. Adhesion of cultured endothelial cells was partly abrogated by osteonectin and was correspondingly reversed by vitronectin in a concentration-dependent manner. These results indicate that specific interactions between vitronectin and osteonectin modulate cell adhesion and might thereby regulate endothelial cell function during angiogenesis.