Collagens,collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis

Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.     

Active remodeling in idiopathic interstitial pneumonias: evaluation of collagen types XII and XIV.

Fibril-associated collagens with interrupted triple helices (FACITs) XII and XIV act as fibril organizers and assist in the maintenance of uniform fibril size. We investigated the spatial expression patterns of collagens XII and XIV in cryptogenic organizing pneumonia (COP)/organizing pneumonia (OP) and in idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) and compared them to normal human lung. Study subjects included 10 patients with COP/OP, 10 patients with IPF/UIP, and 8 control subjects. Immunostaining for collagens XII and XIV was carried out in paraffin-embedded human lung tissue sections. Picrosirius red histochemical staining for collagen I expression and electron microcopy to evaluate fibril diameter were also performed. In normal lung, collagens XII and XIV were expressed in perivascular and subpleural connective tissue. In COP/OP, both collagens showed intense staining in perivascular connective tissue, thickened alveolar septae, and subpleural areas. In IPF/UIP, XII and XIV were expressed in perivascular connective tissue, in areas of established fibrosis, and in areas of subpleural thickening. Only collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblastic foci in IPF/UIP. Collagen type I was overexpressed in fibrotic areas. Electron micrographs revealed obvious fibril diameter alteration and fusion in the same areas. FACITs XII and XIV are expressed in normal and fibrotic lung. Unlike collagen XIV, collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblast foci in IPF/UIP. This may suggest a possible distinct role for both collagens in the modulation of the extracellular matrix during the onset of fibrotic process.     

Serum thyroxine and triiodothyronine radioimmunoassay values in the normal ferret

The European ferret, Mustela putorius furo, has become increasingly popular as an animal model in biomedical research. However, certain important normal clinical data have not been established for the ferret. In this study, serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) values were obtained from ferrets by the use of commercial radioimmunoassays. Sera from 44 animals, 31 males (27 intact and 4 castrated) and 13 females (10 intact and 3 spayed) were assayed. Serum T4 values ranged from 1.01-8.29 micrograms/dl for males (mean = 3.24 +/- 1.65 micrograms/dl), and 0.71-3.43 micrograms/dl for females (mean = 1.87 +/- 0.79 micrograms/dl). Serum T4 values of adult female ferrets, juvenile ferrets (less than 1 year old) of either sex, and castrated males were similar to the normal T4 values of the cat, 1.20-3.80 micrograms/dl. Intact adult male ferrets had higher serum T4 values which were more comparable to those of the normal dog 1.52-3.60 micrograms/dl. Serum T3 values ranged from 0.45-0.78 ng/ml for males (mean = 0.58 +/- 0.09 ng/ml), and 0.29-0.73 ng/ml for females (mean = 0.53 +/- 0.13 ng/ml). These values are comparable to those of dogs and cats which are 0.50-1.50 ng/ml.     

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.     

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushing's syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Differential immunoregulation of granulomatous inflammation, portal hypertension, and hepatic fibrosis in murine schistosomiasis mansoni

The present study investigates the immunoregulation of hepatic fibrosis in experimental murine schistosomiasis. Disease parameters measured were portal pressure, hepatic granuloma area, hepatic interstitial collagen, and glycosaminoglycans. C57BL/6 mice were infected with 25 Schistosoma mansoni cercariae and administered splenocytes or serum derived from uninfected mice or chronically infect syngeneic mice at 6 and 7 wk of infection. Immunologically mediated modulation was noted in animals receiving splenocytes derived from chronically infected mice. Both a reduction in portal pressure and hepatic granuloma areas were noted. Hepatic collagen content but not glycosaminoglycan content was reduced by the administration of either lymphoid cells or serum from chronically infected mice. The isotypic profile of hepatic interstitial collagens was modulated by both the administration of serum or lymphoid cells. Augmented levels of type III collagen was noted on administration of serum derived from chronically infected mice, whereas type I collagen levels were relatively elevated on administration of splenocytes. The data indicate that immunomodulation of inflammation and hepatic fibrosis can occur in murine schistosomiasis but that fibrotic events and inflammatory processes are independently modulated.     

MMP mediated degradation of type VI collagen is highly associated with liver fibrosis--identification and validation of a novel biochemical marker assay

Background and Aims

During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.

Methods

Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.

Results

Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl₄-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R² = 0.58, p<0.0001) in CCl₄- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).

Conclusions

This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.     

Identification of 14-hydroxy-retro-retinol and 4-hydroxy-retinol as endogenous retinoids in rats throughout neonatal development

14-Hydroxy-retro-retinol was previously described as an in vivo and in vitro metabolite of retinol. Furthermore, the retinoid 4-hydroxy-retinol was identified as an endogenous occurring retinoid in the amphibian organism and an in vitro metabolite of retinol. We describe in the present study that 14-hydroxy-retro-retinol and 4-hydroxy-retinol are present in normal neonatal rat serum as endogenous occurring retinoids in normal non-vitamin A supplemented mammals (rats). Both retinoids were detected in serum and liver of neonatal rats at days 3 and 11 after birth. The respective concentrations at day 11 after birth were 41.8 +/- 2.8 ng/ml (serum)/ 104 +/- 6 ng/g (liver) for 4-hydroxy-retinol and 23 +/- 4.6 ng/ml (serum)/ 285 +/- 5 ng/g (liver) for 14-hydroxy-retro-retinol. Both retinoids could not be detected in adult rat serum and liver. From our experiments important physiological functions of these retinoids during postnatal development could be postulated.     

Successful targeting to rat hepatic stellate cells using albumin modified with cyclic peptides that recognize the collagen type VI receptor

The key pathogenic event in liver fibrosis is the activation of hepatic stellate cells (HSC). Consequently, new antifibrotic therapies are directed toward an inhibition of HSC activities. The aim of the present study was to develop a drug carrier to HSC, which would allow cell-specific delivery of antifibrotic drugs thus enhancing their effectiveness in vivo. We modified human serum albumin (HSA) with 10 cyclic peptide moieties recognizing collagen type VI receptors (C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) yielding pCVI-HSA. In vivo experiments showed preferential distribution of pCVI-HSA to both fibrotic and normal rat livers (respectively, 62 +/- 6 and 75 +/- 16% of the dose at 10 min after intravenous injection). Immunohistochemical analysis demonstrated that pCVI-HSA predominantly bound to HSC in fibrotic livers (73 +/- 14%). In contrast, endothelial cells contributed mostly to the total liver accumulation in normal rats. In vitro studies showed that pCVI-HSA specifically bound to rat HSC, in particular to the activated cells, and showed internalization of pCVI-HSA by these cells. In conclusion, pCVI-HSA may be applied as a carrier to deliver antifibrotic agents to HSC, which may strongly enhance the effectiveness and tissue selectivity of these drugs. This approach has the additional benefit that such carriers may block receptors that play a putative role in the pathogenesis of liver fibrosis.     

Differential interactions of decorin and decorin mutants with type I and type VI collagens.

The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.     

Bovine oviductal fluid components and their potential role in sperm cholesterol efflux

Bovine oviductal fluid (OF) was collected and analyzed throughout the estrous cycle, and the capacity of the protein and lipoprotein components to support cholesterol efflux from bovine sperm was evaluated. Blood was collected and assayed for progesterone (P4) to monitor the estrous cycle. Protein and lipoprotein separation was achieved by density gradient centrifugation. Two major bands were identified. The first (1.056 less than delta 20 less than 1.140 g/ml) corresponded to bovine and rabbit plasma high-density lipoprotein (HDL) based on distribution in the density gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second band (1.235 less than delta 20 less than 1.243 g/ml) consisted predominantly of oviductal fluid albumin (OFA). Oviductal fluid protein concentration increased as serum P4 decreased around the time of estrus. Mean OF protein concentration was 21.3 mg/ml when serum P4 was lower than 0.5 ng/ml and 6.9 mg/ml when serum P4 was greater than 0.5 ng/ml. An inverse log relationship was found between HDL protein concentration and serum P4. Unesterified cholesterol (UC), cholesteryl ester, and phospholipid (PL) content of HDL for HDL protein concentrations of 3-56.1 micrograms/ml were 1.35-46.2 micrograms/ml, 1.91-44.48 micrograms/ml, and 1.69-59.8 micrograms/ml, respectively. Phosphatidylcholine and -ethanolamine were the major PLs present in the HDL fraction and their molar ratio (4:1 mol/mol) was relatively constant through the estrous cycle. The OFA fraction of the same samples accounted for more than 90% of total protein and for most of the variation in OF protein. To determine the ability of OF components to serve as sperm cholesterol acceptors, OF samples were incubated 1:1 (v/v) with and without 4 X 10(8) bovine sperm in 1.0 ml of modified Tyrode's solution and OF for 2 hr at 39 degrees C. After incubation, HDL and OFA fractions were isolated and analyzed for changes in protein and lipid content. After OF, samples were incubated with sperm, an increase in UC was found in the HDL fractions. UC in HDL increased by 12.1 +/- 1.0 micrograms/ml (means +/- SE) when serum P4 was less than or equal to 0.5 ng/ml. For samples corresponding to higher serum P4, the increase in UC was 3.60 +/- 0.89 micrograms/ml. Values for UC in HDL were corrected for the contribution of UC from OFA of OF samples. Cholesterol efflux from sperm has been implicated in the process of sperm capacitation. These results indicate that HDL from OF is elevated during the follicular phase of the estrous cycle and can serve as an acceptor for bovine sperm cholesterol.     

A quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and 125I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and 125I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous 125I immunobinding assays which were not quantitative but were used as antigen "spot tests" or which required iodination of the antibody.     

Changes in circulating levels of immunoreactive follicle stimulating hormone, luteinizing hormone, and testosterone during sexual development in the rhesus monkey, Macaca mulatta

Basal serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) and the responsiveness of these hormones to a challenge dose of luteinizing hormone releasing hormone (LHRH), were determined in juvenile, pubertal, and adult rhesus monkeys. The monkey gonadotrophins were analyzed using RIA reagents supplied by the World Health Organization (WHO) Special Programme of Human Reproduction. The FSH levels which were near the assay sensitivity in immature monkeys (2.4 +/- 0.8 ng/ml) showed a discernible increase in pubertal animals (6.4 +/- 1.8 ng/ml). Compared to other two age groups, the serum FSH concentration was markedly higher (16.1 +/- 1.8 ng/ml) in adults. Serum LH levels were below the detectable limits of the assay in juvenile monkeys but rose to 16.2 +/- 3.1 ng/ml in pubertal animals. When compared to pubertal animals, a two-fold increase in LH levels paralleled changes in serum LH during the three developmental stages. Response of serum gonadotrophins and T levels to a challenge dose of LHRH (2.5 micrograms; i.v.) was variable in the different age groups. The present data suggest: an asynchronous rise of FSH and LH during the pubertal period and a temporal correlation between the testicular size and FSH concentrations; the challenge dose of LHRH, which induces a significant rise in serum LH and T levels, fails to elicit an FSH response in all the three age groups; and the pubertal as compared to adult monkeys release significantly larger quantities of LH in response to exogenous LHRH.     

Serum aldosterone and protein-binding variables in Yanomama Indians: a no-salt culture as compared to partially acculturated Guaymi Indians

Yanomama Indians from the jungles of southern Venezuela and northern Brazil excreted 1 +/- 1.5 mEq of Na and 203 +/- 109 mEq of K and had low blood pressure (BP), 102/62 mm Hg). In comparison, Guaymi Indians of Panama excreted 103 +/- 50 mEq of Na and 118 +/- 52 mEq of K and had significantly higher BP (114/75 mm Hg, p less than 0.001). Elucidating the renin-aldosterone axis, total upright serum aldosterone in 34 Yanomama was high (85.6 +/- 78 ng/100 ml). The binding capacities of thermolabile (ABG) and thermostable (ABG-Ts) serum globulins for aldosterone were elevated at 23.8 +/- 6 and 14.9 +/- 2.6%, respectively; consequently, total ABG- plus ABG-Ts- bound aldosterone was as high as 38.6 +/- 6.3%. Plasma renin activity (PRA 10.3 +/- 2.4 ng/ml/h) and urinary aldosterone 18-glucuronide (70.3 +/- 30 micrograms/24 h) in 17 Yanomama were also very high. In contrast, total serum corticosteroids and corticosteroid-binding globulin (CBG) binding capacity were normal, suggesting normal ACTH activity. PRA correlated positively with total (r = 0.47, p less than 0.05) and free (r = 0.47, p less than 0.05) serum aldosterone, which in turn showed a negative trend with Na (r = 0.33, NS) excretion. The effect of high dietary K appeared less important to aldosterone stimulation and PRA suppression. ABG-bound aldosterone (r = 0.43, p less than 0.01) as well as ABG-Ts (r = 0.56, p less than 0.05) were negatively correlated with diastolic but not systolic BP. The total ABG- and ABG-Ts-bound fraction correlated with diastolic BP (r = 0.43, p less than 0.05) in contrast to the free fraction (r = 0.08, NS) or total aldosterone (r = -0.09). Apparently, only bound serum aldosterone is important for the maintenance of diastolic BP. High serum aldosterone, with elevated excretion, indicates an increased secretion rate; increased serum protein binding suggests an increased tissular activity and alterations in aldosterone metabolism. In Guaymi Indians both total plasma aldosterone (14.5 +/- 65 ng/100 ml) and urinary aldosterone (8.1 +/- 4.8 micrograms/creatinine excretion) were normal. ABG-binding capacity for aldosterone was moderately elevated (17.8 +/- 4.8) and of ABG-Ts normal (10.2 +/- 1.2) suggesting a nearly normal aldosterone metabolism and regulation. The BP of Guaymi was significantly higher than that of the Yanomama.     

Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein.

The collagen framework of the intervertebral disc contains two major fibril-forming collagens, types I and II. Smaller amounts of other types of collagen are also present. On examination of the nature and distribution of these minor collagens within bovine disc tissue, type VI collagen was found to be unusually abundant. It accounted for about 20% of the total collagen in calf nucleus pulposus, and about 5% in the annulus fibrosus. It was discovered by serially digesting disc tissue with chondroitin ABC lyase and Streptomyces hyaluronidase that native covalent polymers of type VI collagen could be extracted. Electron micrographs of this material prepared by rotary shadowing revealed the characteristic dimensions of tetramers and double tetramers of type VI molecules, with their central rods and terminal globular domains. Molecular-sieve column chromatography on agarose under non-reducing non-denaturing conditions gave a series of protein peaks with molecular sizes equivalent to the tetramer, double tetramer and higher multimers. On SDS/polyacrylamide-gel electrophoresis after disulphide cleavage, these fractions of type VI collagen all showed a main band at Mr 140,000 and four lesser bands between Mr 180,000 and 240,000. On electrophoresis without disulphide cleavage in agarose/2.4% polyacrylamide only dimeric (six chains) and tetrameric (12 chains) forms of type VI molecules were present. The ability to extract all the type VI collagen of the tissue in 4 M-guanidinium chloride, and absence of aldehyde-mediated cross-linking residues on direct analysis, showed that, in contrast with most matrix collagens, type VI collagen does not function as a covalently cross-linked structural polymer.     

Effects of a simulated microgravity model on cell structure and function in rat testis and epididymis.

A tail-suspension (TS) rat model used to simulate microgravity was tested for its effects on the anatomy, cell structure, and function of the testis and epididymis in sexually mature male rats. Rats suspended for 7 days without inguinal canal ligation exhibited a significant (P less than or equal to 0.05) reduction in testis weight compared with controls (1.55 +/- 0.04 to 1.1 +/- 0.02 g). Except for the liver, epididymis, and adrenals of TS rats and TS rats allowed to recover for 7 days, no significant (P less than or equal to 0.05) change was observed in the weight of other body and accessory sex organs. A histological examination of the testes and epididymides of model animals revealed disorganized seminiferous tubules and accumulation of large multinucleated cells and spermatids in the lumen of the epididymis. A significant (P less than or equal to 0.05) increase in serum luteinizing hormone (53.1 +/- 6.7 to 66.2 +/- 10.1 ng/ml) and follicle-stimulating hormone (257 +/- 25 to 305 +/- 38 ng/ml) was observed in TS nonligated rats, whereas serum prolactin and testosterone levels were observed to decline from 8.3 +/- 1.3 to 5.1 +/- 0.29 and 7.1 +/- 1.3 to 3.8 +/- 0.25 ng/ml, respectively. Decreases in testis protein content and testosterone levels of the testis, interstitial fluid, and epididymis were also observed in model animals. These data demonstrate that the suspension procedure used in the National Aeronautics and Space Administration TS model results in the testis and epididymis translocating into the abdominal cavity, causing cellular degeneration and organ dysfunction.     

Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 +/- 10.3%, and type III, 271.7 +/- 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.     

Growth hormone response of bull calves to growth hormone-releasing factor

Three experiments were conducted to determine serum growth hormone (GH) response of bull calves (N = 4; 83 kg body wt) to iv injections and infusions of human pancreatic GH-releasing factor 1-40-OH (hpGRF). Peak GH responses to 0, 2.5, 10, and 40 micrograms hpGRF/100 kg body wt were 7 +/- 3, 8 +/- 3, 18 +/- 7, and 107 +/- 55 (mean peak height +/- SEM) ng/ml serum, respectively. Only the response to the 40-microgram dose was greater (P less than 0.05) than the 0-microgram dose. Concentrations of prolactin in serum were not affected by hpGRF treatment. In calves injected with hpGRF (20 micrograms/100 kg body wt) at 6-hr intervals for 48 hr, GH increased from a mean preinjection value of 3.1 ng/ml serum to a mean peak response value of 70 ng/ml serum. Differences in peak GH response between times of injection existed within individual calves (e.g., 10.5 ng/ml vs 184.5 ng/ml serum). Concentrations of GH in calves infused continuously with either 0 or 200 micrograms hpGRF/hr for 6 hr averaged 7.4 +/- 3 and 36.5 +/- 11 ng/ml serum, respectively (P less than 0.05). Concentrations of GH oscillated markedly in hpGRF-infused calves, but oscillations were asynchronous among calves. We conclude that GH response of bull calves to hpGRF is dose dependent and that repeated injections or continuous infusions of hpGRF elicit GH release, although magnitude of response varies considerably. We hypothesize that differences in GH response to hpGRF within and among calves, and pulsatile secretion in the face of hpGRF infusion may be related to the degree of synchrony among exogenous hpGRF and endogenous GRF and somatostatin.     

Decreased content in left atrium and increased plasma concentration of atrial natriuretic polypeptide in spontaneously hypertensive rats (SHR) and SHR stroke-prone

The atrial contents and concentrations, and the plasma concentrations of atrial natriuretic polypeptide (ANP) in spontaneously hypertensive rats (SHR) and SHR stroke-prone (SHRSP) were measured and compared with those of age-matched Wistar Kyoto rats (WKY) using a specific radioimmunoassay (RIA) for alpha-rat ANP (alpha-rANP). The contents of alpha-rANP-LI in the atria of SHR (19.0 +/- 0.9 micrograms, mean +/- SEM) and SHRSP (19.3 +/- 0.6 micrograms) were significantly lower than that of WKY (22.8 +/- 1.4 micrograms) (p less than 0.05). The atrial concentration of alpha-rANP-LI was also significantly lower in SHR (248.2 +/- 11.3 ng/mg, p less than 0.05) and tended to be lower in SHRSP (272.2 +/- 12.4 ng/mg) than that of WKY (300.0 +/- 14.2 ng/mg). Furthermore, the concentrations in the left auricles of SHR and SHRSP were significantly lower than that of WKY (p less than 0.01 and p less than 0.05, respectively). In contrast, no significant difference was observed in the alpha-rANP-LI concentrations in the right auricles of WKY, SHR and SHRSP. Gel filtration studies coupled with RIA showed that gel filtration profiles of the extracts from the right and left auricles of WKY, SHR and SHRSP were essentially identical. The plasma alpha-rANP-LI levels in SHR (260 +/- 34 pg/ml) and SHRSP (319 +/- 19 pg/ml) were significantly higher than that in WKY (170 +/- 17 pg/ml) (p less than 0.05 and p less than 0.01, respectively). These results suggest that the secretion of ANP from the heart is increased in SHR and SHRSP compared with WKY.