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Biomedical subjects

L Vaughan

Publications and source records attributed to L Vaughan.

At least 19 recordsLinked to original sources
PubMed1992

Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains.

Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains.

Amino Acid Sequence↗
PubMed1991

J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth.

The influence of J1/tenascin adsorbed to polyornithine-conditioned plastic (substrate-bound J1/tenascin) and J1/tenascin present in the culture medium (soluble J1/tenascin) on neurite outgrowth was studied with cultured single cells from hippocampus and mesencephalon of embryonic rats. Neurons at low density grew well on J1/tenascin substrates and extended neurites that were approximately 40% longer than on the polyornithine control substrate after 24 h in vitro. The neurite outgrowth promoting effect of substrate bound J1/tenascin was largely abolished in the presence of mAb J1/tn2, but not by mAb J1/tn1. In contrast to the neurite growth-promoting effects of substrate bound J1/tenascin, neurite outgrowth on polyornithine, laminin, fibronectin, or J1/tenascin as substrates was inhibited by addition of soluble J1/tenascin to the cultures. Neither of the two mAbs neutralized the neurite outgrowth-inhibitory properties of soluble J1/tenascin. In contrast to their opposite effects on neurite outgrowth, both substrate-bound and soluble J1/tenascin reduced spreading of the neuronal cell bodies, suggesting that the neurite outgrowth-promoting and antispreading effects are mediated by two different sites on the molecule. This was further supported by the inability of the mAb J1/tn2 to neutralize the antispreading effect. The J1/tn2 epitope localizes to a fibronectin type III homology domain that is presumably distinct from the putative Tn68 cell-binding domain of chicken tenascin for fibroblasts, as shown by electronmicroscopic localization of antibody binding sites. We infer from these experiments that J1/tenascin contains a neurite outgrowth promoting domain that is distinguishable from the cell-binding site and presumably not involved in the inhibition of neurite outgrowth or cell spreading. Our observations support the notion that J1/tenascin is a multifunctional extracellular matrix molecule.

Animals↗
PubMed1991

Aging and energy expenditure.

Whether sedentary energy expenditure is normal or lower in elderly people has not yet been clearly established. Twenty-four-hour energy expenditure (24EE) and its different components were measured by use of a respiratory chamber in elderly (17 male, 21 female; 71 +/- 6 y, mean +/- SD; 71.2 +/- 13.5 kg; 32 +/- 8% fat) and young (33 male, 31 female; 24 +/- 4 y; 84.5 +/- 23.1 kg; 25 +/- 13% fat) subjects. The elderly subjects had lower mean height (P less than 0.001), weight (P less than 0.01), and fat-free mass (P less than 0.001) but higher percent body fat (P less than 0.01) than did the young adults. Absolute 24EE, basal metabolic rate (BMR), and sleeping metabolic rate were significantly lower (P less than 0.01) in the elderly subjects than in the young subjects. However, after differences in fat-free mass, fat mass, and sex were adjusted for, only BMR was found to be lower in the elderly subjects (P less than 0.01). Despite a reduced adjusted BMR in older subjects, sedentary 24EE was decreased only in proportion to their reduced body size, suggesting that the lower energy intake reported in elderly people might be mainly related to lower physical activity in free-living conditions.

Adipose Tissue↗
PubMed1991

Tenascin Mr 220,000 isoform expression correlates with corneal cell migration.

The three isoforms of chicken tenascin, an extracellular matrix glycoprotein, are generated by alternatively spliced fibronectin type III domains. The resulting proteins migrate as bands of Mr 220,000 (ten220), Mr 200,000 (ten200) and Mr 190,000 (ten190) on SDS-PAGE. We describe here two monoclonal antibodies, one specific for ten220 (mAb T17) and another that recognizes all isoforms (mAb T16). These were used to examine the differential expression of isoforms during development. Most impressive is the close correlation between ten220 expression and cell migration in the embryonic cornea. Initially (stage 18), ten190/200 can be detected within the corneal epithelium and along the basement membranes of the lens and sclera. Ten220 appears within the primary stroma immediately prior to the invasion by neural-crest-derived cells. This expression is maintained during the subsequent migration of fibroblasts from the conjunctiva into the primary stroma. With the completion of migration and the marked increase in matrix synthesis by corneal fibroblasts, ten220 disappears. Ten190/200 remains in the region adjoining the endothelium, the Bowman's membrane and the adjacent stroma. The cell-migration-associated isoform is isolated from extracts of embryonic tissues as a homohexamer. Low molecular weight forms appeared absent but a new tenascin band of Mr 210,000 could be detected in brain extracts which may be a new isoform. We conclude that the synthesis of tenascin isoforms is under tight developmental control and speculate that a function of the additional domains is to facilitate cell migration.

Animals↗
PubMed1989

Cartilage contains mixed fibrils of collagen types II, IX, and XI.

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.

Acetates↗
PubMed1988

Isolation and sequence analysis of the glycosaminoglycan attachment site of type IX collagen.

Type IX collagen from chick embryonic cartilage is unique among the collagens in that it contains chondroitin sulfate covalently linked to the alpha 2(IX) polypeptide chain. We have isolated and sequenced the glycosaminoglycan-containing peptide released by collagenase digestion from type IX collagen, labeled biosynthetically with [35SO4] and 3H-aminoacids. This peptide was purified by gel filtration and, following chondroitinase ABC digestion, by reverse-phase high performance liquid chromatography. The amino acid sequence obtained for this peptide has 23 residues, beginning and ending with a collagenous sequence, indicating that it spans an internal noncollagenous domain. Comparison of this sequence with the one predicted from cDNA clone pYN 1738 for the alpha 1(IX)chain and pYN 1731 and pDM 222 for the alpha 2(IX)chain revealed the peptide to be the noncollagenous NC3 domain of alpha 2(IX). The glycosylated sequence Val-Glu-Gly-Ser*-Ala-Asp- of type IX collagen does not have the Ser-Gly normally functioning as the attachment sequence but does have an acidic residue preceding the serine which should improve the acceptability of this sequence for the xylosyltransferase. That it is an adequate acceptor can be inferred from the observation that type IX collagen carries a glycosaminoglycan chain on over 70% of the molecules isolated.

Amino Acid Sequence↗
PubMed1988

Comparative studies of collagens in normal and keratoconus corneas.

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.

Adult↗
PubMed1988

D-periodic distribution of collagen type IX along cartilage fibrils.

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.

Animals↗
PubMed1987

A major, six-armed glycoprotein from embryonic cartilage.

Here we describe the isolation and identification of a major cartilage glycoprotein which is co-extracted along with typical hyaline cartilage components such as collagen types II and IX from chicken embryo sternum. In polyacrylamide gel electrophoresis it migrates as a high molecular mass protein (greater than 10(6) daltons) which on reduction gives rise to a prominent doublet at 205/195 kd and minor bands at 220 and 170 kd. The intact molecule sediments as a 13S component in rate zonal centrifugation, indicative of a highly extended conformation in solution. In these properties it closely resembles myotendinous antigen, a glycoprotein recently detected in a number of embryonic tissues, including cartilage. This identity was confirmed by immunoblotting using a monoclonal antibody (M1) specific for myotendinous antigen. Electron micrographs of the rotary shadowed molecule revealed an unusual six-armed structure, indistinguishable in form and dimensions from hexabrachion, a recently discovered contaminant of cellular fibronectin preparations. These structures could be decorated with the M1-antibody, demonstrating that hexabrachion is myotendinous antigen. This extended, potentially multivalent molecule could provide an ideal substrate to connect widely spaced components in a highly hydrated tissue such as cartilage.

Animals↗
PubMed1986

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s).

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.

Amino Acids↗
PubMed1985

Proteoglycan Lt from chicken embryo sternum identified as type IX collagen.

Proteoglycan Lt (PG-Lt), isolated from 17-day-old chicken embryo sterna, appeared to differ from its counterpart from tibia and femur (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The intact disulfide-bonded molecule of approximately 300 kDa was separable into three chains of 115, 84, and 68 kDa on reduction, the molecular masses being relative to those of collagen standards on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is in contrast to tibial cartilage PG-Lt, from which there was no observed release of a 68-kDa chain (100 kDa relative to globular protein standards) after reduction. The 115-kDa chain of sternum PG-Lt consists of a core 68-kDa polypeptide to which the chondroitin sulfate chains are attached. The ratio of 4-sulfated to 6-sulfated disaccharides released after either chondroitinase ABC or AC digestion is 3:1. Identity of PG-Lt with type IX collagen was indicated by their similar elution profiles on DEAE-Trisacryl and by the presence in both proteins of co-migrating 84- and 68-kDa bands on SDS-PAGE. This identity was confirmed by immunoblotting PG-Lt after SDS-PAGE, with affinity-purified polyclonal antibodies specific for a triple helical domain (HMW) of type IX collagen. The nonreduced high molecular mass material and all three bands of the reduced PG-Lt were immunoreactive, giving immunostaining patterns similar to autoradiographs from the [14C]glycine-labeled protein.

Animals↗
PubMed1985

Type IX collagen from sternal cartilage of chicken embryo contains covalently bound glycosaminoglycans.

Type IX collagen was isolated as a native protein from chicken embryo sternal cartilages and purified to homogeneity. Chondroitin and/or dermatan sulfate were bound covalently to one of the three polypeptide chains present in this protein containing collagenous and noncollagenous domains. Type IX collagen could be metabolically labeled with both radioactive sulfate and glycine. The protein containing either of these labels was sensitive to digestion by bacterial collagenase as well as chondroitinase ABC. Besides the glycosaminoglycans, type IX collagen contains asparagine-linked carbohydrate chains because the protein could be labeled with radioactive mannose and no glycosaminoglycans other than those mentioned above were present. The melting curve indicated that, in contrast to interstitial collagens, this molecule contains at least two disulfide-bonded collagenous domains with distinct thermal stabilities.

Animals↗
PubMed1985

Food-induced "dose-dumping" from a once-a-day theophylline product as a cause of theophylline toxicity.

Three slow-release preparations of theophylline have received approval from the U.S. Food and Drug Administration (FDA) for "once-daily" dosing indications, amid controversy regarding the appropriateness of this decision. Because of specific concerns regarding data submitted to the FDA in support of the first of these products to be approved, Theo-24, we examined the absorption characteristics of this newly marketed formulation. Eight healthy volunteers received, in a crossover manner, single doses of a theophylline reference solution and Theo-24, taken both fasting and after a breakfast of bacon and eggs. The concentrations of theophylline were measured up to 60 hours after the dose. Absorption of Theo-24 after an overnight fast was very slow, with only 71 +/- 6 percent (mean +/- SE) of the dose ultimately absorbed. In contrast, food caused precipitous "dose-dumping," resulting in dose-normalized peak levels in the serum that averaged 2.3 times higher than after a fasting dose. About half of the dose was absorbed in a four-hour period, generally beginning six to eight hours after the postprandial dose, and complete absorption was then attained within 24 hours (p less than 0.001). Toxic effects of theophylline occurred in four subjects when they took the dose with food whereas no toxic effects occurred during the fasting regimen. Consequently, doses of Theo-24 that would have attained a predicted peak concentration of 15 micrograms/ml after multiple dosing taken without food would, if taken with food, have resulted in larger fluctuations and in peak concentrations in the potentially toxic range for six of the eight subjects.

Adult↗
PubMed1975

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ +K+, and 5'-adenylylimidodiphosphate.

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate, (AMP-P(NH)P). This compound, in which the oxygen connecting the beta and gamma phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg2+-ATPase activities. The K1 of AMP-P(NH)P for Mg2+ ATPase activity was 2.0 - 10-4 M and, while the Km of ATP for this activity was also 2.0 - 10-4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na+, K+ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 - 10-3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-free porcine erythrocyte ghosts were studied in order to characterize the system more adequately.

Acetates↗