Effect of physical restraint on oxidative stress in mice fed a selenium and vitamin E deficient diet.

Physical restraint has been associated with increased oxidative damage to lipid, protein, and DNA. The purpose of this experiment was to determine whether physical restraint would further exacerbate oxidative stress in mice fed a selenium (Se) and vitamin E (VE) deficient diet. Three-week- old mice were fed a Torula yeast diet containing adequate or deficient Se and VE. Menhaden oil was added to the deficient diet to impose an additional oxidative stress. After 4 wk feeding, half the mice in each group were restrained for 5 d in well-ventilated conical tubes for 8 h daily. Mice fed the Se and VE deficient diets had increased liver thiobarbituric acid-reactive substance (TBARS) levels and decreased liver glutathione peroxidase (GPX1) activity and alpha-tocopherol levels. Plasma corticosterone levels were elevated in restrained mice fed the deficient diet compared to unrestrained mice fed the adequate diet. Restraint had no effect on liver TBARS or alpha-tocopherol levels. Liver GPX1 activity, however, was lower in restrained mice fed the adequate diet. In addition, liver superoxide dismutase (SOD) activity was lower in the restrained mice fed the adequate or deficient diet. Thus, under our conditions, Se and VE deficient diet, but not restraint, increased lipid peroxidation in mice. Restraint, however, decreased antioxidant protection in mice due to decreased activities of GPX1 and SOD enzymes.

Extracellular FGF-1 inhibits cytoskeletal organization and promotes fibroblast motility.

Previous efforts from this laboratory have established that acidic fibroblast growth factor (FGF-1), either added exogenously or secreted as a biologically active protein, induces a transformed phenotype in primary murine fibroblasts. Experimental studies described here demonstrate that constitutive exposure to extracellular FGF-I results in reduced cell attachment to multiple ligands, inhibition of cytoskeletal organization, and reduced collagen contraction, despite no detectable change in integrin cell surface expression. In addition, FGF-1-transduced fibroblasts demonstrated a > 10-fold increase in migration, an observation correlated with increased tyrosine phosphorylation of p125FAK and p130CAS. Collectively, these results suggest that FGF-1-induced fibroblast transformation includes the involvement of specific FGF receptor-mediated signal transduction cascades targeted to cytoskeletal and focal adhesion structures.

Tractional force generation by porcine Müller cells: paracrine stimulation by retinal pigment epithelium.

PURPOSE: To examine the ability of retinal pigment epithelial (RPE) cells to modulate Müller cell extracellular matrix contraction through secreted promoters. METHODS: Freshly isolated RPE cells were maintained in continuous culture until the morphologic and immunocytochemical changes associated with myofibroblastic dedifferentiation were complete. Secretory products collected from these cells during extended incubations in serum-free medium and at different stages of dedifferentiation were examined for the ability to promote extracellular matrix contraction by Müller cells. The contributions of specific growth factors to RPE-secreted activity were examined with growth factor-neutralizing antibodies. RESULTS: Secretory products from RPE cells throughout dedifferentiation contained biologically active quantities of Müller cell contraction promoters. Secretory activity increased during extended incubation in serum-free medium and during myofibroblastic dedifferentiation. Growth factor-specific neutralizing antibodies enabled the determination that insulin-like growth factor- and platelet-derived growth factor-related proteins were the secreted species to which Müller cells responded. Finally, gene expression of insulin-like growth factor 1 and platelet-derived growth factor A chain by porcine RPE cells was confirmed using reverse transcription-polymerase chain reaction. CONCLUSIONS. RPE cells are a viable source of biologically active quantities of two growth factors that stimulate extracellular matrix contraction by Müller cells. This secretory profile persists for extended periods in an otherwise serum-free environment and is enhanced during myofibroblastic dedifferentiation.

Aurothioglucose inhibits murine thioredoxin reductase activity in vivo.

Gold (I)-containing compounds, including aurothioglucose (ATG), are potent in vitro inhibitors of several selenocysteine-containing enzymes. Gold compounds have also been shown to potentiate the virulence of several viruses in mice, including coxsackievirus, implicated as a possible infectious agent in Keshan disease. One possible mechanism by which gold compounds may be increasing the virulence of viral infections in mice is by acting as a selenium antagonist in vivo and inducing oxidative stress. To investigate the possible role of gold compounds in inducing oxidative stress in mice, we assessed the ability of ATG administered in vivo to inhibit the activity of the selenocysteine-containing enzymes thioredoxin reductase (TR) and glutathione peroxidase (GPX1). Doses as low as 0. 025 mg ATG/g body weight caused significant and prolonged inhibition of TR activity in all tissues examined. No such inhibition of GPX1 activity was seen, indicating differential in vivo sensitivity of the enzymes to inhibition by ATG. In liver and heart, some recovery of TR activity was observed after a 7-d period, but no recovery was observed in pancreas or kidney. Because TR is involved in several important cellular redox functions, its inhibition most likely will affect multiple cellular processes. These results indicate that in vivo administration of ATG results in significant and long-lasting inhibition of TR activity. Such inhibition of TR could lead to increased levels of oxidative stress in vivo, thereby increasing the virulence of several viruses including the coxsackievirus.

Dietary soy isoflavones inhibit activation of rat platelets.

Isoflavones (isoflavonoids) have been proposed to be the active compounds that contribute to decreased mortality from chronic diseases in populations that consume large amounts of soy products. Diets containing soy protein with and without isoflavones were fed to rats to determine if these compounds could exert in vivo effects on physiologic markers of platelet activation. Three methods were employed to monitor platelet activation: measurement of electronic mean platelet volume, which is an indicator of shape change; monitoring of collagen-induced production of reactive oxygen signals (hydrogen peroxide); and determination of increases in phosphorylation of protein tyrosine residues after collagen stimulation. Apparent volumes were significantly smaller for platelets from rats fed isoflavones, suggesting that these platelets were in a more disc-like, quiescent state compared with platelets from rats fed the isoflavone-reduced diet (means +/- SEM, 5.37 +/- 0.08 vs. 5.70 +/- 0.06 fL, n = 6/group, P < 0.008). Results from the other functional tests were consistent with this finding. Platelet production of hydrogen peroxide was found be significantly lower 1, 3, and 5 minutes after addition of collagen for rats fed isoflavones versus rats fed the isoflavone-reduced diet (n = 6/group, P < 0.004). Phosphorylated tyrosine residues in platelet proteins after stimulation also were shown to be significantly lower in the platelets exposed to dietary isoflavones (n = 5/group, P < 0.047). These combined results indicate that soy isoflavones can alter early-event signaling networks that result in less activated platelets and may partially explain the beneficial effects of dietary soy against human heart disease.

Antigenic changes of rabbit retinal Müller cells in culture.

PURPOSE: To determine whether dissociated and cultured Müller cells from the avascular rabbit retina undergo the same phenotypic changes as Müller cells that are dissociated and cultured from a vascular retina. METHODS: Müller cells were dissociated from adult rabbit retinas by using an enzymatic digestion-mechanical trituration technique and a cell attachment method that provided Müller cell- enriched cell cultures. Indirect immunofluorescence localization of vimentin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), beta-amyloid precursor protein (beta-APP), and (alpha-smooth muscle actin (alpha-SMA) was carried out on Müller cells that were freshly dissociated, on those that had been in culture 2 and 6 days, and on confluent primary cultures and late-passage cultures. The specificity of the antibodies and changes in protein expression were examined by western blot analysis. RESULTS: The expression of vimentin, GFAP, GS, and beta-APP was present 2 days after dissociation and was retained through 6 days in culture, at which time alpha-SMA began to be expressed in a small number of cells. The confluent, primary cultures no longer expressed GS, but vimentin and beta-APP were still expressed, and the expression of alpha-SMA was increased. During the late-passage stage, the morphologic appearance of the Müller cell cultures was large and amorphous, with additional changes in antigenicity. Although there was loss of expression of the intermediate filament proteins GFAP and vimentin, the expression of beta-APP was maintained, whereas alpha-SMA was increased and appeared to be a major cytoskeletal protein. CONCLUSIONS: Dissociated Müller cells that were maintained in culture underwent phenotypic changes that included a large, amorphous appearance; the loss of detectable vimentin, GFAP, and GS expression; the persistent presence of beta-APP; and the de novo appearance of alpha-SMA. The phenotypic and antigenic changes that occurred in cultured Müller cells from an avascular retina were similar but not identical to the changes observed in cultured Müller cells from a vascular retina.

Tractional force generation by porcine Müller cells: stimulation by growth factors in human vitreous.

PURPOSE: To examine the levels of Müller cell contraction-stimulating activity in human vitreous, correlate these levels with clinical presentation, and identify, the causative growth factors. METHODS: Human vitreous was collected from patients undergoing pars plana vitrectomy (n = 84). Müller cells were isolated from porcine retina and maintained in tissue culture. Tractional forces generated by cells incubated on three-dimensional collagen gels were measured as changes in gel thickness. Contraction-stimulating activity in vitreous (VA) was calculated from the close-response profiles of gel contraction to vitreous protein. The contributions of individual growth factors to vitreous activity (n = 10) were assessed by inhibition with specific neutralizing antibodies. RESULTS: The mean VA of patients with retinal detachment (3.65) and proliferative vitreoretinopathy stages A, B, and C (2.06) were elevated above that of patients without retinal pathology (vitreous activity = 0.23) or retinal defects alone (0.57). Mean activities in patients with epimacular proliferation (1.22) and vitreous hemorrhage (1.40) were also significantly elevated. The percentage of this activity attributable to insulin-like growth factor 1 (IGF-1) varied from 9.2% to 84.5% with a mean of 61.3%. Similarly, the percent contribution of platelet-derived growth factor (PDGF) ranged from 6.8% to 49.0% with a mean of 26.5%. CONCLUSIONS: The vitreous of patients with retinal detachment, proliferative retinal disease, and vitreous hemorrhage contain varying amounts of growth factors that stimulate tractional force generation by Müller cells. The majority of the activity can be attributed to IGF-1 and a smaller proportion to PDGF.

Racial differences in the renal response to blood pressure lowering during chronic angiotensin-converting enzyme inhibition: a prospective double-blind randomized comparison of fosinopril and lisinopril in older hypertensive patients with chronic renal insufficiency.

This study was undertaken to compare the effects of chronic angiotensin-converting enzyme (ACE) inhibition on blood pressure (BP) and renal hemodynamics in older black and nonblack hypertensive patients with chronic renal insufficiency. A multicenter, placebo lead-in double-blind, parallel group study was performed to compare the antihypertensive efficacy and renal hemodynamic response to the once-daily ACE inhibitor fosinopril (n = 14) and lisinopril (n = 13) over a 22-week period. The study goal was to lower diastolic blood pressure (DBP) to 90 mm Hg or less. Furosemide was added after 6 weeks if blood pressure goal was not achieved. At outpatient clinics at university medical centers, 27 older hypertensive patients (> or = 45 years; 12 blacks, 15 nonblacks; 19 male, eight female) with DBP of 95 mm Hg or higher and 4-hour creatinine clearance 20 to 70 mL/min/1.73 m2 were studied. Changes (delta) from baseline in BP, glomerular filtration rate (GFR), and renal plasma flow (RPF) were measured. Mean systolic blood pressure (SBP) and DBP decreased significantly and to a similar extent in randomized groups: fosinopril (mean +/- SEM) delta DBP at 6 weeks was -13 +/- 2 (P < 0.0001; 95% CI, -16 to -9) and at 22 weeks was -12 +/- 2 (P < 0.0001; 95% CI, -16 to -9); lisinopril delta DBP at 6 weeks was -14 +/- 6 (P < 0.0001; 95% CI, -10 to -18) and at 22 weeks was -16 +/- 2 (P < 0.0001; 95% CI, -12 to -21). GFR and RPF did not change significantly in either group. BP was significantly reduced and to a similar extent in blacks and nonblacks: for blacks, delta DBP at 6 weeks was -11 +/- 3 (P < 0.05; 95% CI, -0.01 to -9) and at 22 weeks was -16 +/- 2 (P < 0.0001; 95% CI, -11 to -20); for nonblacks, delta DBP at 6 weeks was -14 +/- 1 (P < 0.0001; 95% CI, -12 to -17) and at 22 weeks was -12 +/- 2 (P < 0.0001; 95% CI, -16 to -8). Eight patients (five blacks and three nonblacks) required an addition of furosemide after 6 weeks to reach the DBP goal of < or = 90 mm Hg at 22 weeks. GFR was not significantly altered for either racial group at 6 weeks; however, at 22 weeks; however, at 22 weeks, GFR decreased significantly in blacks (delta GFR, -16 +/- 5; P < 0.006; 95% CI, -26 to -5) and tended to increase in nonblacks (delta GFR, 7 +/- 6; P > 0.25). delta GFR correlated directly with the delta RPF (delta GFR = 0.0611* delta RPF -2.35 +; r = 0.68; P < 0.003). There was no correlation between delta MAP and delta GFR or delta RPF in blacks or nonblacks. We conclude that chronic ACE inhibition with fosinopril and lisinopril alone or in combination with furosemide lowers BP in older blacks and nonblacks with hypertension and chronic renal insufficiency. Racial differences in the renal hemodynamic response to chronic ACE inhibition were noted and appear to be independent of diuretic use and the magnitude of BP lowering.

Tractional force generation by porcine Müller cells. Development and differential stimulation by growth factors.

PURPOSE: To assess the ability of retinal Müller cells to generate tractional forces during dedifferentiation in culture and to assess their responsiveness to contraction-stimulating growth factors. METHODS: Müller cells were isolated from papain-DNase-digested porcine retina. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and alpha smooth muscle actin (alpha SMA). Tractional force generation was assessed as a function of Müller cell contraction of collagenous extracellular matrices in vitro. The effects of potential promoters were assessed by addition directly to culture medium. The contributions of specific promoting to the contraction-promoting activity in serum were assessed by adding neutralizing antibodies and measuring loss of stimulatory activity. RESULTS: Freshly isolated Müller cells did not generate substantial matrix contraction. However, this activity increased 150-fold within 12 days in culture and continued to increase during the next 21 days. Development of the capacity for extracellular matrix contraction coincided with the acquisition of immunodetectable alpha SMA and loss of GFAP. Matrix contraction by Müller cells was stimulated in a dose-dependent fashion by human serum, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Müller cells were not stimulated by transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), or endothelin-1 (E1). Neutralizing antibodies against PDGF and IGF-I reduced the activity in human serum by 37% and 58%, respectively, and 87% when added together. CONCLUSIONS: Porcine Müller cells in culture acquire the ability to contract extracellular matrices and thus generate tractional forces. Acquisition of this activity coincides with alpha SMA expression and loss of GFAP. Further, this activity is dependent on the presence of exogenous promoters, including PDGF or IGF-I.

Quantitative vitreous collection using a modified infusion fluid during vitrectomy.

PURPOSE: To develop a vitreous sampling method that increases yield while reducing risk of harm to the patient. METHODS: Potential negative effects of fluoresceinated BSS infusion solution on biologic activity were assessed by monitoring changes in fibroblast contraction of collagen gels in response to serum or vitreous. Paired vitreous samples were collected from ten patients before and during infusion of fluoresceinated BSS solution. The extent to which the vitreous was diluted was calculated by comparing the levels of fluorescence in the samples with that in fluoresceinated BSS. Protein concentrations and levels of contraction-stimulating activity were measured for each sample. RESULTS: Fluoresceinated BSS did not alter fibroblast morphology, rate, or extent of gel contraction. Differences in corrected protein concentrations and biologic activities of the undiluted and diluted homogenous vitreous samples were not statistically significant. CONCLUSIONS: The use of fluoresceinated BSS infusion yields larger vitreous samples from which the native biochemical characteristics can be determined. Patient safety during collection is enhanced because ocular hypotony and collapse can be avoided.

[Contraction of extracellular matrix by transdifferentiated retinal pigment epithelial cells, inducers and inhibitors]. / Kontraktion extrazellularer Matrix durch transdifferenzierte retinale Pigmentepithelzellen: Induktoren und Inhibitoren.

Transdifferentiated retinal pigment epithelial cells (RPE) display enhanced contractile potentials and have been implicated in the development of tractional retinal detachment. This study determines the activity of contraction-promoting factors, examines some involved mechanisms and evaluates inhibitors. Using an in vitro contraction assay, we demonstrated that collagen matrix contraction by transdifferentiated RPE cells is effectively stimulated by serum, platelet-derived growth factor and insulin-like growth factor-1. Endothelin-1 and transforming growth factor-beta 1 and -beta 2 have a more discrete or marginal effect. Tractional forces promoted by these peptides are completely protein synthesis dependent. Contraction stimulated by serum is only partly dependent on de novo protein synthesis, suggesting different active factors and/or pathways. Staurosporine, a broad-spectrum kinase inhibitor, effectively inhibited collagen matrix contraction by transdifferentiated RPE cells regardless of the promoter.

Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.

PURPOSE: To characterize phenotypic and antigenic changes in isolated Müller cells during proliferation in extended culture. METHODS: Müller cells were isolated from porcine retina by sequential papain and DNase digestion, trituration, and density gradient centrifugation. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and delta smooth muscle actin (alpha SMA). Continuously proliferating cells established in culture were examined for changes in the expression of these antigens. RESULTS: Primary cultures of purified Müller cells, incubated under routine culture conditions, were proliferative and lost immunodetectable CRALBP within 2 weeks. The expression of CA-II also diminished with time, but at an apparently lower rate than that of CRALBP. Loss of GFAP expression was even more gradual and was complete by passage 5. De novo expression of alpha SMA was detectable in some cells within 12 days in culture and by all cells by passage 5. During this period, vimentin expression remained qualitatively unchanged. CONCLUSIONS: Isolated porcine Muller cells in culture undergo a phenotypic dedifferentiation to a fibroblast-like cell, which includes loss of detectable CRALBP, CA-II, and GFAP, and they acquire expression of the myoid marker alpha SMA.

Pathologic human vitreous promotes contraction by fibroblasts. Implications for proliferative vitreoretinopathy.

OBJECTIVE: To establish and quantify the presence of contraction-stimulating activity in pathologic vitreous and correlate this activity with clinical presentation and outcome, especially with proliferative vitreoretinopathy. METHODS: Contraction-stimulating activity of vitreous collected during surgery was quantified with a tissue culture assay using fibroblasts as target cells. The activity of each sample was correlated with patient history, clinical presentation, risk factors, proliferative disease, and postoperative proliferation. RESULTS: Pathologic vitreous contained measurable quantities of contraction-stimulating activity and stimulated contraction in vitro, with elevated activities in samples from patients with proliferative vitreoretinopathy, epimacular proliferation, retinal detachment, retinal defects, pigmented cells in the vitreous, hemorrhage, or uveitis. Patients with postoperative proliferation had significantly elevated mean activities. CONCLUSIONS: Levels of contraction-stimulating activity in pathologic vitreous correlate with some risk factors for the development of proliferative vitreoretinopathy and may ultimately be useful in the assessment of disease severity and the prediction of postoperative proliferation.

Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype.

PURPOSE: To describe and evaluate retinal pigment epithelial (RPE) cell transdifferentiation in vitro and to determine its importance to the development of proliferative vitreoretinal disorders. METHODS: Porcine RPE cells from single animals were examined at different passages in culture. The authors examined cellular morphology, contraction of a collagenous matrix, and adhesion to fibronectin and type I collagen-coated substrata. These activities were correlated with loss of epithelial characteristics, redistribution of the actin cytoskeleton, and expression of alpha-smooth muscle actin (alpha-SMA), a marker of myoid differentiation. RESULTS: During routine culture on tissue culture plastic, porcine RPE cells lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The ability of cultured porcine RPE cells to adhere to and exert tractional forces on an extracellular matrix increases with continued passage in vitro and transdifferentiation. This correlates with the loss of the differentiated epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton, and de novo expression of alpha-SMA. CONCLUSION: Results indicate that RPE transdifferentiate in culture and that this transition is accompanied by a shift in biologic activities. Therefore, morphologic and behavioral transdifferentiation of these cells in culture are influencing factors in experimental pathology. The potential relevance of these extensive changes to the biology of proliferative vitreoretinal disorders is discussed.

Isolation and HPLC of N-epsilon-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives.

N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum. NELL was isolated on a reversed phase Sep-Pak C18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors. The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate. Using this procedure we have demonstrated the presence of TBL in native histones.

Extracellular matrix contraction by choroidal fibroblasts: inhibition by staurosporine.

PURPOSE: To examine the potential of a broad-spectrum kinase inhibitor as a means of neutralizing the effects of contraction promoters on ocular cells. METHODS: The inhibitory effects of a broad-spectrum kinase inhibitor were examined and characterized using an in vitro assay of extracellular matrix contraction by choroidal fibroblasts. RESULTS: Staurosporine effectively inhibited collagen matrix contraction by choroidal fibroblasts. The inhibitory effects of staurosporine were rapid in onset and reversible upon removal of the inhibitor. Inhibition was observed when fibroblasts were stimulated with serum, transforming growth factor beta 1, transforming growth factor beta 2, platelet-derived growth factor, and endothelin-1. We also observed that platelet-derived growth factor and endothelin-1 stimulated only modest amounts of matrix contraction compared to transforming growth factor beta. CONCLUSIONS: Matrix contraction by cells, as observed in the development of tractional forces, can be modulated by a broad-spectrum kinase inhibitor. The marginal contractile responses of choroidal fibroblasts to platelet-derived growth factor and endothelin-1, both potent promoters of dermal fibroblast contraction, suggest that there are substantive difference in the responses of these two cell types to growth factors.

Fecal bile acids and neutral sterols in the cotton-top tamarin (Saguinus oedipus).

1. The cotton-top tamarin (Saguinus oedipus), a small New World primate susceptible to spontaneous development of colon cancer, was studied for its fecal neutral sterol and bile salt composition. 2. Standardization procedures to establish the effect of exposure of the stool to room temperature air for various time-periods showed no significant effects on the neutral sterol and bile salt composition of the samples. 3. Microbial degradation of cholesterol and bile acids to secondary metabolites showed a progressive rise during the first year of life after which some degree of homeostasis was observed. 4. The proportion of cholesterol that remained unmetabolized by colonic microflora was in excess of 50%, an amount that was significantly higher than in man and other higher primates. 5. Ursodeoxycholic acid was identified as a significant (12%) component of fecal bile acids in this species. 6. Secondary bile acids formed by the action of enteric microflora were also significantly lower than levels found in man and other animals.

Fibroblast contraction of collagen gels requires activation of protein kinase C.

Fibroblasts stimulated to contract collagen gels with serum were completely inhibited by staurosporine, a broad spectrum kinase inhibitor. Further analysis demonstrated that staurosporine is potent (IC50 17 nM), rapid in onset, and completely reversible. Complete inhibition of gel contraction was also observed with calphostin C (IC50 48 nM), an inhibitor specific for protein kinase C (PKC). Similar effects were not observed with KT5926 or KT5720, inhibitors for myosin light chain kinase and cAMP-dependent kinases, respectively. These data suggested that serum-stimulated fibroblast contraction is dependent upon activation of PKC. This was also observed with fibroblast contraction stimulated with endothelin-1, platelet-derived growth factor, and transforming growth factor beta. PKC activated directly with low concentrations of phorbol ester was observed to stimulate fibroblast contraction of collagen gels, in some cases within 30 minutes of exposure.

Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus).

A study was conducted in captive baboons to determine (i) the impact of cereal dietary fibre on faecal progestogen excretion, and (ii) whether means of controlling dietary effects could be identified. Blood was collected on 3 days per week and faeces on 5 days per week from four unanesthetized cyclic female baboons, consecutively fed three diets of 5, 10 and 20% fibre for 90 days per diet. A 2 day lag time was detected before progesterone in the blood appeared in the faeces, regardless of diet (mean correlation was 0.62, P = 0.002). Increased dietary fibre had a negative effect on progestogen excretion (P < 0.004). Correspondence between blood and faecal progestogens was consistently greatest and the effect of dietary fibre least when faecal progestogens were expressed g-1 dry faeces. Several means of indexing faecal steroid excretion rates were examined including dehydroepiandrosterone (DHEA) and a number of byproducts of cholesterol metabolism. The cholesterol metabolite, cholestanone, was positively correlated with dietary fibre (r = 0.27; P < 0.04). Multiplying faecal progestogen concentration by the cholestanone g-1 dry faeces concentration increased the correlation between serum and cholestanone-indexed faecal progestogens (r = 0.78, P = 0.0001) compared with nonindexed progestogens (r = 0.71, P = 0.0001). We conclude that expressing faecal progestogens g-1 dry faeces may be sufficient and the most cost-effective method for controlling for most dietary effects when the objective is monitoring longitudinal endocrine status in baboons. However, it may be appropriate to express faecal progestogens by cholestanone concentrations when increased precision is needed to overcome the effects of profound variations in dietary fibre.

Collagen gel contraction by cells associated with proliferative vitreoretinopathy.

The capacities of porcine choroidal fibroblasts, retinal glial cells, and retinal pigment epithelial cells to contract collagen gels in vitro were compared. Experiments with varied cell numbers indicated that glial cells are the most effective, followed by choroidal fibroblasts and retinal pigment epithelial cells. Analysis of the secretory products from cultures of these cell types revealed that retinal pigment epithelial cells synthesize and secrete peptides that promote fibroblast contraction of collagen gels in vitro. The mechanism of action of the retinal pigment epithelial cell-secreted contraction promoter was compared with that found in serum (type A) and secreted by cultured endothelial cells (type B). Like the serum factor, the retinal pigment epithelial cell-secreted factor was not dependent on active protein synthesis by the target cell and must be present continuously to promote contraction.