Müllerian duct regression in the embryo correlated with cytotoxic activity against human ovarian cancer.

A significant cytotoxicity index was obtained when human ovarian cancer cells in a microcytotoxicity assay were exposed during the S (DNA-synthesizing) phase of the cell cycle to purified fractions of testis exhibiting high Müllerian inhibiting substance bioactivity. The same effect was not observed when these fractions were tested against human glioblastoma or fibroblast lines. Most human ovarian cancers are said to resemble Müllerian tissues histologically. Müllerian inhibiting substance may thus deserve further study as a potential chemotherapeutic agent.

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.

The altered fibrous form of vitreous collagen following solubilization with pepsin.

Collagen occurs in the extracellular matrix of the bovine vitreous as fibers which have a fairly uniform diameter of approximately 195 A and exhibit an indistinct axial periodicity. Following treatment with pepsin approximately three quarters of the collagen was rendered soluble and by gel electrophoresis and comparison with calf skin collagen was shown to be composed of alpha1 chains, a high molecular weight alpha chain component, beta, as well as other high order components. No alpha2 chains were detected. The amino acid composition of the pepsin soluble collagen was different from that of other collagens composed only of alpha1 chains which suggests that it is composed of either a distinct type or mixture of alpha chains. When fibers were reconstituted from the pepsin solubilized collagen they differed markedly from the native fibers and exhibited a pronounced axial periodicity (approximately 640 A) and had diameters up to 1500 A. The difference between the reconstituted and native fibers suggests that the presence of the peptides cleaved by pepsin may be one of the factors which determines the particular fibrous form of collagen in the bovine vitreous.

Kinetics of the chondrocyte biosynthetic response to compressive load and release.

To gain insight regarding the rate at which cartilage tissue can sense and respond to a dynamic mechanical stimulus, we have examined the time-course of changes in biosynthetic activity following both the application and release of a static compressive stress. Cartilage harvested from the reserve zone of calf epiphyseal plate was subjected to unconfined static compressive stresses of 0, 0.25 and 0.5 MPa. Incorporation of [35S]sulfate and [3H]proline was measured during loading periods of less than 1 to 26 h and after preloading periods of 0.5, 2 or 12 h. During loading, total incorporation decreased to steady levels with time constants estimated to be 0.25-4 h (proline) and 1-5 h (sulfate). Proline incorporation exceeded control levels for 3 h after release of a 2 or 12 h preload. Sulfate incorporation remained depressed for at least 4 h after release of a 12 h preload and remained at control levels following release of 0.5 and 2 h preloads. We conclude that the modulation of proline incorporation by both loading and load release is faster than the modulation of sulfate incorporation. Furthermore, the response to unloading is not just the inverse of the response to loading; this nonlinearity suggests that the response to dynamic loading would not be determined simply by the time average component of the dynamic load.

A comparison of glycosaminoglycan synthesis by human fibroblasts from normal skin, normal scar, and hypertrophic scar.

Fibroblasts isolated from normal skin, normal scar, and hypertrophic scar tissues were compared with respect to their growth curves, protein contents, and abilities to synthesize glycosaminoglycans (GAGs). While no significant differences were found with respect to protein content or population doubling times, we did find significant differences in the proportions of radiolabel incorporated into the various GAGs among the 3 groups of cell lines. Using a dual-label technique to label both hyaluronic acid and the sulfated GAGs, we isolated labeled constituents from the extracellular, the pericellular, and the cellular fractions by pronase digestion and gel filtration and identified the various GAGs by electrophoresis and selective digestion with enzymes. Of the GAGs isolated from the extracellular fraction, hypertrophic scar fibroblasts incorporated proportionately more 35S into chondroitin sulfate and less into heparan sulfate and more [3H]glucosamine into hyaluronic acid than did normal skin fibroblasts. Of the GAGs isolated from the cellular fraction, hypertrophic scar fibroblasts incorporated proportionately more 35S into heparin and less into dermatan sulfate and more [3H]glucosamine into hyaluronic acid than did normal skin fibroblasts. These differences in biosynthesis may help to explain the differences in GAG content in skin and scars found in vivo and to give insight into the development of hypertrophic scars.

Mechanical analysis of hypertrophic scar tissue: structural basis for apparent increased rigidity.

The mechanical behavior of normal human skin and hypertrophic scar tissue (HST) is compared using constant-strain-rate and successive stress-relaxation uniaxial loading programs in vitro. HST is less extensible, requires more energy to be stretched in the physiologic range, and stores strain energy less efficiently than normal skin. The explanations for the differences observed between the mechanical behavior of normal skin and HST are based on the differences in their composition and structure. We suggest that the collagen fiber network is partially "prealigned" in a crimped tendon-like organization in HST, which reduces its extensibility and raises the strain energy required to stretch it. It is further hypothesized that an incomplete elastic fiber network, an abnormal glycosaminoglycan content, and/or abnormal collagen fiber slippage are responsible for the reduced capacity to return strain energy in the hypertrophic scar tissue. The results of these studies indicate that although HST has been described as stiffer than normal skin, the maximum stiffness of skin and HST are similar. The "apparent" increased rigidity of HST is a result of reduced extensibility rather than a change in its stiffness. This inexensibility may manifest itself by limiting joint mobility in the patient with HST.

Studies on human scar tissue proteoglycans.

The proteoglycans (PGs) in pooled normal scars and pooled hypertrophic scars were extracted with 4 M guanidinium chloride and isolated by DEAE-cellulose chromatography. The PG samples were then fractionated further by dissociative CsCl density gradient sedimentation. Following cleavage of the density gradient PG fractions with alkaline NaB3H4, the glycosaminoglycan (GAG) constituents were isolated and analyzed by quantitative cellulose acetate electrophoresis. In addition, single samples of normal skin and a keloid scar were also analyzed. The results showed that the hypertrophic scars had a higher average content of extractable and also residual PGs than did the normal scars but a wide range of values was obtained for each type of scar. Some differences were noted in the amounts and distribution of the GAGs in CsCl gradient fractions, in the different types of scar tissue. The PGs in tissues were distributed in low-, medium-, and high density fractions and are, therefore, heterogeneous. Dermatan sulfate (DS) was the major GAG in each tissue and smaller quantities of chondroitin sulfate (CS), heparan sulfate (HS), and heparin (HP) were also present. In addition, 2 other GAG constituents were also detected. Based on the susceptibility of these GAGs to enzymes and nitrous acid treatments, one was a HS or HP while the second was a DS. The major differences in the PG composition of the scar tissues were the higher proportions of low-density CS-PGs in the keloid scar and of low density DS-PGs in hypertrophic and keloid scars.

The protein composition of the ocular zonules.

Bovine and human zonules were found to be composed of noncollagenous acidic glycoprotein with a high cysteine content, double that previously reported. In reduced zonular fractions the most prominent peptide had a molecular weight (MW) of approximately 70,000. Lesser quantities of 170,000, 50,000, and 35,000 dalton peptides were also present and a variable number of lower MW bands, depending upon the degree of reduction and denaturation. A fraction of bovine zonules soluble in low ionic strength buffers contained primarily a peptide of approximately 50,000 daltons, often present as a doublet. Amino acid and hexosamine content of these two fractions was consistent with the presence of at least two different glycoconjugates, one a proteoglycan. Carbohydrate analysis of whole zonules suggested that these glycoconjugates include a sialofucose-containing glycoprotein and a lesser quantity of xylose-containing proteoglycan. The amino acid profile and peptide content of the zonules resembled that of elastic tissue microfibrils, increasing further the possibility of a close relationship between these two fibrils.

Vitreous substitution with gases.

The effects of vitreous substitution with air, a sulfur hexafluoride-air mixture, an octafluorocyclobutane-air mixture, and physiological saline were compared in owl monkeys. Each gas caused an increase in ocular vascular permeability greater than that caused by saline, as measured by vitreous inflow of serum protein labeled with iodine I 131 and discgel electrophoresis. The duration of increased vascular permeability closely paralleled the time each gas remained in the vitreous cavity.

Hyaluronic acid vitreous substitute. A six-year clinical evaluation.

Hyaluronic acid vitreous substitute (HYVISC) was used during 347 operations performed on 294 eyes of 286 patients. It was injected intraocularly in 266 operations (73 scleral bucklings, 175 open-sky vitrectomies, among others) on eyes with complex retinal detachments that were considered inoperable by ordinary surgical techniques. During 81 closed vitrectomies, it was applied extraocularly between the surgical contact lens and the cornea. Hyaluronic acid was found to be well tolerated by human eyes. In eyes that had been given a poor prognosis, scleral buckling with hyaluronic acid tamponade produced reattachment in 16% of the eyes; open-sky vitrectomy, scleral buckling, and hyaluronic acid salvaged 18%. It also appeared helpful in the preservation of corneal epithelial integrity and clarity during closed vitrectomy.

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I, a glycoprotein present in articular lubricating fraction of bovine synovial fluid.

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly alpha-(2 leads to 3) to D-galactose residues. The resistance of 2-acetamido-2-deoxy-D-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-1 with alkaline sulfite, approximately 80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal leads to GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-alpha-D-galactosidase (which suggests an alpha-D-GalNAc leads to L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan-Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae beta-D-galactosidase, which hydrolyzes only galactosides linked beta-D-(1 leads to 4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes beta-D-galactosidase released greater than 90% of D-galactose. These results provide evidence for beta-D-Galp-(1 leads to 3)-alpha-D-GalNAcp-(1 leads to 3) alpha-D-GalNAcp-(1 leads to 3)-L-Ser or -L-Thr and alpha-NeuAc-(2 leads to 3)-beta-D-Galp-(1 leads to 3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.

Isolation and some structure analyses of a copolymeric chondroitin sulfate-dermatan sulfate proteoglycan from post-burn, human hypertrophic scar.

A D-glucuronic acid rich, copolymeric chondroitin sulfate (CS)-dermatan sulfate (DS) proteoglycan (PG) from post-burn hypertrophic scar tissue (HSc) was obtained by DEAE-cellulose chromatography and differential ethanol fractionation, and further purified on a Sepharose CL-6B column. CS-DS-PG protein content was 14% (w/w). The amino-terminal amino acid sequence of the first ten residues was as follows: NH2-Asp-Glu-Ala-B-Gly-Ile-Gly-Pro-Glu-Val. This sequence is identical to that of human embryonic fibroblast cell (IMR-90) CS-DS-PG, as well as to human HSc-DS-PG. After chondroitinase ABC treatment, two peptides (Mr 22,000 and 16,000 daltons) were detected by sodium dodecyl sulfate-(polyacryl)amide gel electrophoresis (SDS-PAGE). ELISA analysis using rabbit antiserum raised against a synthetic peptide that contained 15 amino acids in the same sequence as the amino terminus of human fetal membrane PG showed significant reactivity with HSc CS-DS-PG. HSc CS-DS-PG had an apparent Mr of approximately 78,000 daltons, as determined by Sepharose CL-6B chromatography and SDS-PAGE. Alkaline borohydride treatment of CS-DS-PG liberated CS-DS glycosaminoglycan (GAG) chains having an Mr of 29,000 daltons. The conversion of xylose to xylitol indicated that the GAG chains are attached to the PG protein core at O-3 through a xylosyl-seryl linkage. CS-DS-PG also contained both N and O-linked oligosaccharides and did not aggregate with hyaluronic acid. These results, together with those reported previously, showed that HSc CS-DS-PG and DS-PG have the same A1-A15 amino acid sequence at the amino terminus but different protein cores. HSc CS-DS-PG was completely digested with chondroitinase AC and is, therefore, distinctly different from HSc DS-PG.

Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid.

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1.2x10(6) and 6.5x10(4) respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.