An analysis of two fibril systems in the mammalian vitreous.

The vitreous of most mammals contains low concentrations of three polymeric macromolecules, collagen, hyaluronan and beaded fibrils. To determine whether the beaded fibrils may have a localized function, or whether all three of these polymers could contribute to the structure and/or strength of the vitreous gel, the distribution of collagen and beaded fibrils in the central and cortical regions of the bovine vitreous was studied. The polymers were separated by isopycnic centrifugation and analyzed to determine the protein ratios. Both collagen and beaded fibrils are present in each region, but the concentration of the beaded fibril is greatest in the posterior cortical volume.

Development and characterization of monoclonal antibodies against a mucin-type glycoprotein.

The preparation of greater than 30 different hybridomas, all secreting IgM class antibodies against epiglycanin, a glycoprotein at the surface of the mouse mammary carcinoma cell line TA3-Ha, is described. The specificities of 10 of the antibodies, with affinity constants in the range of 10(8)-10(10) l/mol were compared in an enzyme competitive binding assay. The affinity of epiglycanin was strongly reduced for all antibodies tested by incubation with periodate (10 mM, 4 degrees C) and was reduced for most of the antibodies by endo-alpha-N-acetyl- D-galactosaminidase. This suggested that carbohydrate, and specifically the Gal beta (1----3)GalNAc disaccharide, formed an integral part of the epitopes of most of the antibodies. The isolated disaccharide, however, exhibited 250,000 times less inhibitory activity in the competitive binding assay than epiglycanin. The binding capacity of epiglycanin was also reduced by incubation with trypsin or pronase, suggesting a high molecular weight dependency for binding. Incubation with sialidase increased its affinity for the antibodies. The binding of the antibodies to epiglycanin was strongly inhibited by peanut agglutinin, and to a lesser extent by lectins from Triticum vulgaris, Ricinus communis, Pisum sativum and Phaseolus vulgaris. None of the antibodies bound to any of eight different gangliosides immobilized on HPTLC plates. Mono- (Fab) and divalent [F(ab')2] fragments of the antibodies possessed very low affinity for epiglycanin. The results demonstrated that the specificities of the antibodies are related, but distinguishable, and they suggest that this epiglycanin-IgM model may be useful for studies on the general principles of the interaction between IgM antibodies and mucin-type glycoproteins.

Collagens of the bovine vitreous.

The predominant structural components of the vitreous are collagenous fibrils. Prior biochemical analyses have been limited by incomplete solubilization of the constituent collagen types. The techniques described enable an effective separation and nearly complete solubilization of calf vitreous fibrils. Semiquantitative analysis has led to the detection of types II, V, and IX collagen in a ratio of 69:24:7, respectively. Qualitatively similar ratios were found in vitreous from other mammals, including rabbits, monkeys, and humans. Electron microscopic evidence suggests that type VI collagen may be distributed selectively near the vitreous base. These observations are pertinent to vitreous gel stabilization, aging, and disease.

Diamines and aminoalcohols: neutral solvents for native collagen.

A substantial fraction of interstitial collagens (types I, III, V and VI) can be dissolved from finely divided, connective tissues by homogenization in 0.5-1 M ethylene diamine and certain other organic amines neutralized by hydrochloric or other acids to pH 7-8.5. The amount of collagen solubilized from the tendons and other tissues of young animals is similar to that extracted by dilute acetic acid. In ethylene diamine hydrochloride solutions, native type I collagen denatures at 38 degrees and it has a sedimentation coefficient similar to that measured in acidic citrate solutions. From these amine hydrochloride solutions native collagen fibrils can be regenerated simply by tenfold dilution with water or by dialysis. The form of these precipitates is determined by temperature, pH, enzyme pretreatment of the collagen, and the dialysate salt concentrations and composition. Weak gels can be formed with less than 0.1 mg collagen per ml. Regenerated matrices containing types V and VI collagens, and with different fibril sizes can be used to support cell growth or to form sheets or hydrated gels possibly suitable for prosthetic uses.

The contribution of labile crosslinks to the tensile behavior of tendons.

The tensile strength of rat tail tendons, measured by the load that ruptures the tendon, varies with the weight of the animal, the vertebra to which the tendon was attached, the pH of the tendon, and the chemical treatment. Reduction with sodium borohydride multiplies the tensile strength by a factor up to 4.5 (the factor diminishing with age) and reduces creep in the strained tendon. It is concluded that strain catalyzes the hydrolysis of aldimine intermolecular crosslinks that contribute significantly to the tensile strength under physiological conditions, and that the crosslinks are stabilized by reduction or with aging. It is postulated that creep is the result of slippage between polymeric assemblies of stably crosslinked molecules. This postulate leads to a plausible description of elongation of connective tissue fibrils in growing animals.

A versatile procedure for the radioiodination of proteins and labeling reagents.

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.

Facile preparation and some applications of an affinity matrix with a cleavable connector arm containing a disulfide bond.

A versatile affinity matrix in which the ligand of interest is linked to the matrix through a connector arm containing a disulfide bond is described. It can be synthesized from any amino-substituted matrix by successive reaction with 2-imino-thiolane, 5,5'-dithiobis(2-nitrobenzoic acid), and a thiol derivative of the ligand of choice. The repertoire of ligands can be significantly increased by the appropriate use of avidin-biotin bridges. After adsorption of the material to be fractionated, elution can be effected by reducing the disulfide bond in the connector arm with dithiothreitol. Examples of the preparation and use of various affinity matrices based on amino-substituted Sepharose 6MB are given. One involves the immobilization of the Fab' fragment of a monoclonal antibody against Aspergillus oryzae beta-galactosidase and the specific binding of that enzyme to the resulting immunoaffinity matrix. Another involves the immobilization of N-biotinyl-2-thioethylamine followed by complex formation with avidin. The resulting avidin-substituted matrix was used for the selective adsorption and subsequent recovery of mouse hybridoma cells producing anti-avidin antibodies. By further complexing the avidin-substituted matrix with appropriate biotinylated antigens, it should be possible to fractionate cells producing antibodies against a variety of antigens.

Connective tissue remodeling in corneal and scleral wounds.

The fluorescent dye dichlorotriazinyl aminofluorescein will bind to amino groups of proteins covalently under physiological conditions. It has been used to dye the connective tissue around an ulcer or nonpenetrating, linear incision in the rabbit cornea and sclera, and the healing of the tissue has been examined up to 1 yr later. Sagittal sections were stained for light microscopy, and adjacent unstained sections were examined in the fluorescence microscope. The stained sections showed the reestablishment of the lamellar organization of the stromal collagen across the site of the incision; the fluorescence showed where the connective tissue that was present when the wound was made persisted, and thereby defined the limits of remodeling in the healing process. In Bowman's layer and the adjacent stroma, there was often an abrupt transition from fluorescent to new, undyed connective tissue. Deeper in the scar, and particularly around the ulcer, dark streaks were observed between the fluorescent lamellae, showing apparently new (non-fluorescent) tissue interdigitating with the old. These observations are discussed in relation to the mechanism of healing and the residual mechanical weakness that persists across scars in the cornea and sclera.

Fluorescent dyes demonstrate the uniform expansion of the growing rabbit cornea.

Thiocyanate and triazinyl chloride derivatives of fluorescent dyes have been employed for the covalent labeling of components of the connective tissue of the rabbit cornea. Collagen is the major macromolecular component that becomes dyed. Some of the stromal components that are labeled by these reagents in the first two weeks of life persist over many months, but are progressively dispersed over a greater area consistent with a uniform expansion of the tissue with growth without complete turnover. These dyes may find useful application in the morphologic study of other persistent connective tissue components during development and wound healing.

Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments.

Preparation of bispecific antibodies by the chemical reassociation of monovalent fragments derived from monoclonal mouse immunoglobulin G1 is inefficient because of side reactions during reoxidation of the multiple disulfide bonds linking the heavy chains. These side reactions can be avoided by using specific dithiol complexing agents such as arsenite and effecting disulfide formation with a thiol activating agent such as 5,5'-dithiobis(2-nitrobenzoic acid). In this way bispecific antibodies were obtained in high yield and free of monospecific contaminants from monoclonal mouse immunoglobulin G1 fragments. The bispecific antibodies were used as agents for the selective immobilization of enzymes.

Identification of type II procollagen in rabbit vitreous.

A procollagen in the soluble fraction of rabbit vitreous was isolated by dialysis against dilute acetic acid and partially purified by Bio-Gel A 5M gel filtration. The molecule was identified to be type II procollagen by comparing its segment-long-spacing (SLS) banding pattern with that of standard type II collagen isolated from rabbit articular cartilage. Electron microscopy of the SLS of this type II procollagen revealed a fuzzy propeptide extension at the N-terminal end of the molecule. Pepsin digestion of the procollagen removed this extension, thus converting the molecule into a collagen which had mobility similar to that of pepsin-soluble cartilage type II collagen in SDS-polyacrylamide gel electrophoresis. No inter-chain disulfide bond was found in the propeptide extension when the procollagen samples were electrophoresized with or without mercaptoethanol. Comparison of the cyanogen bromide peptide map of the type II procollagen with that of the pepsin-soluble type II collagen indicated that two extra peptides were present in the digest of procollagen. All of this evidence suggested that the procollagen in the soluble vitreous body of the rabbit eye was a novel type II procollagen with a propeptide extension only at the N-terminus.

The collagens of the developing bovine cornea.

The morphology of the developing bovine eye has been examined and the collagens in fetal bovine eyes from three months' gestation to maturity have been solubilized by pepsin treatment and analyzed to determine the ratios of the predominant types of collagen. The type I collagen decreased, while the type V collagen increased with age. Type III collagen comprised less than 1% of all the corneas, except for the three-month fetal calf. The anterior to posterior thickness of the paraffin-embedded fetal calf cornea increased from the third to the seventh month, decreased from the seventh month to birth, and then increased after birth. Descemet's membrane increased in thickness with age. Analysis of dissected regions of the calf cornea showed a uniform distribution of the collagen populations from the center to the limbus (89% type I, 10% type V and less than 1% type III collagen) and uniformity through the depth of the stroma, except that type III was concentrated around Bowman's layer, and type IV in Descement's membrane. The localization of the different collagens was consistent with the immunofluorescent staining studies with anticollagen antibodies, but the ratios of the intensities of the fluorescence did not correspond to the quantitative analyses. These results are concordant with other studies that have shown that antibody binding may be masked or diminished in certain tissues and therefore immunofluorescence cannot be used reliably for quantitative measurements.

A simple vertebrate collagenase assay using soluble radioactive collagen substrate.

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.

The organization of cross-linking in collagen fibrils.

The cross-linked peptides in cyanogen bromide digests from rabbit and calf fibrils of type I collagen have been compared by gel filtration and electrophoresis. Fibrils were prepared in vitro from acid-soluble collagen, or tendons were used; both were reduced with borohydride to stabilize cross-linking adducts. The cross-linked peptides were isolated and hydrolyzed, and the reducible cross-links were analyzed. We propose that a prominent pattern of cross-linking involves in-register palisades of molecules overlapping by 27 nm and joined through the condensation of amino-terminal aldol adducts on the carboxy-terminal helical regions of overlapping molecules. Lateral association probably occurs through the carboxy-terminal aldehydes from two molecules forming tri- or quadrivalent adducts with residues in the overlapped molecule. This model favors the recently proposed quasi-hexagonal organization of molecules in fibrils in which rows of molecules occur in lateral register.

The healing of linear nonperforating wounds in rabbit corneas of different ages.

Linear nonperforating incisions were made in the corneas of 2-week-old and 2-year-old rabbits. The resulting wounds were examined by light microscopy and transmission and scanning electron microscopy. A corneal incision of a 2-week-old rabbit produced a wide gaping wound caused by retraction of the cut stromal lamellae away from the incision. The wound became wider with time as the developing eye enlarged and the cut lamellae retracted further. Polymorphonuclear leukocytes, presumably from the tear film, penetrating into the wound area before it was covered over by the sliding epithelium. Most of the leukocytes disappeared by 3 days after wounding. Three to six layers of fibroblasts appeared beneath the epithelial plug. The tissue eventually rebuilt approximately one third of the corneal depth lost to the wound. The stroma of the wounded region did not return to its normal width, but the epithelium was thicker than that of the unwounded cornea. An incision in a 2-year-old rabbit cornea produced a narrow V-shaped wound that did not change shape with time. This wound was repaired by fibroblasts resulting in collagenous repair tissue being the same depth as the normal stroma. There appears to be no evidence for wide gaping wounds in humans in the literature, as was found in this study in rabbits.

Collagenase digestion demonstrates carboxy-terminal crosslinking in acid-soluble collagen.

Among the products of the collagenase cleavage of Type I acid-soluble collagen from calf and rabbit tendons, there can be found fragments with the lengths of half alpha-chains. Because purified collagenase cleaves the alpha-chains three-quarters of the length from the amino-terminus, the presence of half-length chains is evidence for the occurrence of crosslinks between two carboxy-terminal, quarter-length fragments, The collagen preparations were reduced with [3H]borohydride, the collagenase-cleaved fragments were separated by gel electrophoresis, and their 3H-labeled crosslink derivatives were analyzed. The major labeled components in the half-length chains were the reduced aldol condensation product and hydroxylysinonorleucine. These experiments demonstrate that the carboxy-terminal telopeptides in monomer-enriched collagen samples form aldol crosslinks which are probably intramolecular, but some intermolecular aldol and aldimine crosslinks may also be formed.

Protein catabolism in cultures of hepatocytes derived from mice of various ages.

The degradation of pulse-labeled protein was measured in cultures of hepatocytes derived from mice of 3--4, 15--16, and 28 months of age. The rates of protein degradation were determined in culture media with varying amino acid, insulin, and glucagon concentrations. No differences with age were seen. Also no difference with age was detected in the lysosomal degradation of 125I-labeled asialofetuin.

Biochemical and immunochemical evidence for the formation of a dimer of glial fibrillary acidic protein.

The glial fibrillary acidic protein isolated from calf brain white matter revealed two major protein bands with apparent molecular weights of 135 000 and 50 000, respectively. The heavy component could be enhanced by oxidation and completely converted to the light component by reduction. It was stoichiometrically established that one sulfhydryl group was present in each 50 000-dalton monomer and the disulfide linkage was involved in dimer formation. The results of the peptide mapping and the immuno-cross-reactivities of the two components indicated that they are identical proteins. We conclude that glial fibrillary acidic protein can occur as a dimeric structure which is formed by an intermolecular disulfide bond from two identical polypeptide chains.

Immunological, biochemical, ultrastructural, and electrophysiological characteristics of a human glioblastoma-derived cell culture line.

This report presents the results of a study using multiple techniques of the established human cell line, LM, which has been developed in culture medium from a patient with a right temporoparietal glioblastoma. This cell line has human subtetraploid karyotype and has several features of a transformed line in culture. These include continuous propagation for 10 years, ability to form tumor nodules when transplanted into immunologically suppressed hamsters, and pleomorphic appearance. Ultrastructurally, it is characterized by multiple nuclei, few actin cables, and numerous surface-membrane microvilli, as well as abundant 9- to 10-nm cytoplasmic filaments. By its immunological reactivity, the line can be shown to contain glial fibrillary acidic protein at low levels, consistent with its glial origin and continued nature. Dibutyryl cyclic adenosine monophosphate (db-cAMP) induces formation of long astrocytic-like processes as well. Its membrane electrical characteristics include a low resting membrane potential and short time constant. Used in a microtiter antiglioma antibody cytotoxicity assay, LM yields a positive reaction to antibodies in the sera of 80% of patients with astrocytomas and only 9% of normal blood-bank donors, suggesting that it shares common antigens with other astrocytic tumor lines. The varied characteristics of this glioblastoma-derived line emphasize the "multiforme" nature on the neoplasm and suggest that for characterization of any such line, multiple parameters are necessary to allow comparison with other long-term glioblastoma lines in the literature. The usefulness of the LM line in in vitro cell biological, immunological, chemotherapeutic, and radiobiological studies of gliomas makes such efforts very worthwhile.