Experimental reduction of eggshell conductance during incubation. I. effect on the susceptibility to ascites syndrome.

Two hundred-four fertile broiler chicken eggs were obtained from a commercial source and divided into three equal groups. On day 1 of incubation, 68 eggs were selected randomly and four strips of vinyl tape were applied to the shell below the air cell (tape-day 1) to reduce eggshell conductance. This procedure was repeated with an additional group of 68 eggs on day 14 (tape-day 14). Sixty-eight eggs were incubated without treatment, as controls. One week after hatch, 20 chickens from each treatment group and control group were placed into a hypobaric chamber (simulated altitude of 2500 m) for 5 weeks. The remaining chickens in each group were maintained under normobaric conditions. The hematocrit and the mean frontal resultant electrical vector (MRV) of the heart were measured following 1 week, 3 weeks, and 5 weeks of hypobaric or normobaric exposure. Surviving chickens were euthanized at the end of 5 weeks. The weight ratio of right ventricle to left ventricle plus septum (the hypertrophy index [HI]) and the cardiac index, the HI divided by body weight, were determined. All mortality during the study was subjectively scored for the presence of ascites syndrome lesions. The percentage of chickens dying during, or exhibiting ascites syndrome at the completion of, the 5-week hypobaric exposure was 16.7%, 66.7%, and 58% for control, tape-day 1, and tape-day 14 treatments, respectively. MRV values of birds following hypobaric exposure were significantly different between treatment and control groups of the hypobaric exposure and between the two tape treatments. These results suggest that reducing conductance of the eggshell during incubation significantly potentiates the development of ascites syndrome in the broiler chicken.

The large non-collagenous domain (NC-1) of type VII collagen is amino-terminal and chimeric. Homology to cartilage matrix protein, the type III domains of fibronectin and the A domains of von Willebrand factor.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical segment flanked by non-collagenous domains, NC-1 and NC-2. In this study, we examined the domain organization of human type VII collagen through analysis of deduced amino acid sequences derived from cloned complementary and genomic DNAs, as compared to peptide segments derived from amniotic membrane type VII collagen. The results revealed that the peptide segments derived from the NC-1 domain of type VII collagen could be assigned to the 5' portion of the composite cDNA, indicating that NC-1 resides at the amino terminal end of the molecule. Several sub-domains with homology to adhesive molecules were also identified within NC-1. These protein domains may confer adhesive properties to NC-1, thereby facilitating the binding of type VII collagen to the lamina densa in the cutaneous basement membrane and the anchoring plaques within the dermis.

Evaluation of vectorelectrocardiographic analysis of young broiler chickens as a predictive index for susceptibility to ascites syndrome.

Vectorelectrocardiographic (VCG) analysis was performed on 50 male broiler chicks (1 week of age) before placement in a hypobaric chamber. During 5 weeks of exposure to hypobaric hypoxia (simulated altitude of 2900 m), all recorded mortality (38%) was due to the development of ascites syndrome. There was a positive correlation (r = 0.74, P less than 0.01) between the increment in the frontal plane mean resultant vector magnitude divided by body weight (designated as the cardiac index [CI]), with the severity of right ventricular enlargement. Chicks developing ascites syndrome had a greater CI (P less than 0.05) at 1 week of age when compared with chicks that did not develop the syndrome. Therefore, the CI calculated by VCG analysis recognizes right ventricular enlargement, suggesting that a pre-hatch or early post-hatch functional cardiac stress has occurred, predisposing the 1-week-old broiler chick to ascites syndrome. Application of the CI has a physiological index may prove useful in future studies targeted for selection of ascites syndrome resistance in broiler chickens.

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils.

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain.

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.

Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture.

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Massive perioperative polyuria.

A 25-year-old man with a spinal cord transection secondary to a gunshot wound underwent surgical repair of a perforated prepyloric ulcer on the eighth hospital day. He received 68 liters and excreted 43 liters of intravenous fluid during the perioperative period. Causes of this massive polyuria during and after the second operation are unknown, although iatrogenically induced glycosuria and natriuresis may have contributed to its severity. The problems and their management are discussed.