Martin B. Mathews

A new in vitro system for studying cell response to mechanical stimulation. Different effects of cyclic stretching and agitation on smooth muscle cell biosynthesis.

An experimental model has been devised to permit morphologic and metabolic characterization of cells subjected to a range of cyclic mechanical stimuli similar to those which may prevail in blood vessel walls. A unique feature is the use of purified elastin membranes prepared from bovine aortas as extensible substrates for cell growth. Cells attached firmly to such membranes which could then be subjected to continuous stretching and relaxation or displacement without stretching by a motor coupled to a movable supporting frame. Various combinations of frequencies, amplitudes and rates of deformation have been used over extended periods with minimal fatigue or disruption of the elastin substrate. The effects of cyclic stretching on [14C]proline incorporation into protein and collagen and [3H]thymidine incorporation into DNA by rabbit aortic smooth muscle cells were distinct from those attributable to agitation without stretching, indicating that cells responded differently to these modes of stimulation. Increases in rate of protein or DNA synthesis induced by stretching were just as marked after 48 h of stimulation as they were at the outset of an experimental period. Since the system permits observations of cell response to independently variable components of pulsatile stress over extended periods and under a variety of culture conditions, it may be expected to provide new data concerning the interaction of mechanical with hormonal and genetic factors in the elaboration of connective tissue components.

Sodium chondroitin sulfate-protein complexes of cartilage. I. Molecular weight and shape.

Abstract 1. 1. Sodium chondroitin sulfate (SCS)-protein complexes of hyaline cartilage contain protein markedly different in amino acid composition from that of collagen. 2. 2. Light-scattering and viscosity data support the existence of a rodlike basic molecular unit. The molecular weight is 4.0 × 106, the length is 3700 A., the linear density is about 1000 avog./A. The structure proposed conceives the protein moiety as a core 3700 A. in length along which are distributed about 62 units of SCS, each of 50,000 molecular weight. Each linear SCS molecule, attached by one or more bonds to the protein core, retains its flexibility which allows an increase in average lateral chain extension with decrease in ionic strength. 3. 3. Aggregates, which have molecular weights as great as 50 × 106, are possibly formed by both lateral and end-to-end, hydrogen-bonded association of basic units. Additional protein may also be involved. 4. 4. The physiological significance of the results is discussed.

The determination of chondroitin sulfate C-type polysaccharides in mixture with other acid mucopolysaccharides

Chondroitin sulfate C-type polysacchrides may be directly determined in mixtures with chondroitin sulfate A and keratosulfate on a microscale by a procedure using hyaluronidase. This method furnishes a more reliable criterion for purity of chondroitin sulfate A or chondroitin sulfate C-type polysaccharides than does either infrared analysis of measurement of optical rotation.

Morphologic Alteration of Cultured Arterial Smooth Muscle Cells by Cyclic Stretching

Cyclic stretching of smooth muscle cells in culture resulted in a two- to fivefold increase in protein and collagen synthesis. The same in vitro system was utilized to relate changes in smooth muscle cell morphology to mechanical stress. Smooth muscle cells, grown in culture from rabbit aorta explants, were transferred to purified elastic membranes derived from bovine aorta. The membranes were either subjected to stretching and relaxation 52 times per minute or stretched and held stationary for 8, 48, or 56 hr. Profiles of rough endoplasmic reticulum (RER) were counted and myofilament content estimated from electron micrographs of 100 cells for each experiment. Cells from cyclically stretched preparations were compared with stationary cells derived from the same subculture. Myofilaments were largely replaced by RER in cyclically stretched cells and there was a reciprocal relationship between RER and myofilament content in individual cells. In cells from stationary preparations, myofilament content also diminished with time but RER profiles were few. At 56 hr, RER profiles numbered 16.7 +/- 1.7 in stretched cells compared with 3.6 +/- 1.3 in stationary cells (P less than 0.05). Cyclically stretched cells formed numerous intercellular contacts and showed little evidence of cytoplasmic degradation while stationary cells showed few contacts and contained numerous cytosomes and lamellar bodies. The results suggest that cyclic stretching resulted in the formation of RER or the preservation of myofilaments and that immobility resulted in the disappearance of myofilaments and cytoplasmic degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular weight of sodium chondroitin sulfate by light scattering

Abstract 1. 1. The light-scattering technique has been successfully applied to determination of the molecular weight of sodium chondroitin sulfate (SCS). 2. 2. SCS purified from bovine hyaline cartilage and treated with proteolytic enzymes to remove trace impurities has a molecular weight of 50,000 and appears to be monodisperse or nearly so. 3. 3. Previous estimates of the molecular weight of SCS were too high, probably due to contamination of preparations by SCS-protein complexes with molecular weights of 106 and higher. The SCS in such complexes also has a molecular weight of 50,000. 4. 4. Extraction of cartilage by strong alkali leads to SCS of low molecular weight and a high degree of polydispersity. Such preparations may be fractionated as to molecular weight by alcohol precipitation. Intrinsic viscosity and weight-average molecular weight are correlated by the expression: [η] = 3.1 × 10−4M0.74. 5. 5. The possible physiological significance of the existence of SCS in different molecular weight states is discussed.

A new dermatan polysulfate, chondroitin sulfate H, from hagfish notochord

Abstract A novel oversulfated mucopolysaccharide was isolated from the notochord of hagfish, which belongs to the more primitive branch of cyclostomes. It is concluded that the mucopolysaccharide is a new type of dermatan polysulfate consisting mainly of (1 → 4)-α-L-idopyranuronosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-galactopyranosyl 4,6-disulfate unit. The name of chondroitin sulfate H is proposed for this dermatan polysulfate.

Macromolecular properties of isomeric chondroitin sulfates

Abstract 1. 1. Three isomeric chondroitin sulfates of connective tissue, CS-A, CS-B, and CS-C, are found to be linear polyelectrolytes with similar chain configuration-charge relationships. 2. 2. Clear distinction of these substances as molecular individuals with isomeric repeating units is established by comparison of molecular constants and physical properties and confirmed by infra-red analyses. 3. 3. Molecular weight, intrinsic viscosity, solubility, electrophoretic mobility, and potentiometric titration data are reported.

The molecular weight and viscosity of chondroitin-sulfuric acid ☆

Abstract 1. 1. The molecular weights of different preparations of chondroitin-sulfuric acid from bovine cartilage have been determined by osmotic pressure measurements. Preparations of high purity were obtained by use of Co(NH3)6Cl3 as a precipitant. 2. 2. Extraction with strong alkali yielded material of low MN and [η]. Use of CaCl2 gave material of higher MN and [η], while extraction of stored cartilage with KCl resulted in material with constants of intermediate range. 3. 3. The preparations were polydisperse in molecular size and data did not fit the Mark relation, [η] = KMα. 4. 4. The value of MN of CaCl2-extracted material was 43,300 as compared to a value of 160,000 estimated by interpolation from viscosity and molecular length (flow birefringence) data of Blix and Snellman for similar preparations. This discrepancy may be due to the presence of an asymmetric impurity in the latter preparations since neither the Co(NH3)6Cl3-purified preparations nor a preparation containing a chondroitin-protein complex showed any flow birefringence in buffer solution with gradients up to 26,000 sec.−1. The same preparations, however, showed birefringence when dissolved in pure water. This is evidence that the shape of the chondroitin molecule depends upon the ionic environment.

Characterization of notochord collagen as a cartilage-type collagen☆

Abstract Sturgeon notochord and cartilage collagens have been characterized with respect to chromatographic properties, amino acid composition, carbohydrate content, and cyanogen bromide cleavage products of the component α chains. The data show that the collagen of both tissues is comprised of a single type of α chain and that the notochord and cartilage chains are identical. Further, the sturgeon chains bear a striking resemblance to previously characterized α1(II) chains from avian and mammalian hyaline cartilages. These observations strongly suggest that the data may be extrapolated to higher organisms and indicate that during development, a cartilage-type collagen is synthesized by notochord cells prior to the appearance of tissues classically identified as cartilage on the basis of morphology.

Chondroitinsulfuric acid—a linear polyelectrolyte☆

Abstract 1. 1. Chondroitinsulfuric acid exhibits the typical viscosity behavior of linear polyelectrolytes in pure water. Preparations of different molecular weight were found to conform to the Fuoss relation: η sp C = A (1 + BC 1 2 ) + D . The data are consistent with the assumption of a highly extended configuration of the polymer at high degrees of ionization. 2. 2. Increasing electrolyte concentration reduces the viscosity of solutions of chondroitinsulfuric acid. The effect is a function of the charge and concentration of the cations present. 3. 3. Data on potentiometric titration of chondroitinsulfuric acid are in agreement with the theory of Katchalsky and Gillis which considers the effect of ionization upon the degree of coiling. The p K 0 of the carboxyl groups in the polymer is close to the p K 0 of free glucuronic acid. 4. 4. Some biological implications of configurational change and ionbinding by molecules of chondroitinsulfuric acid are discussed.