Apparatus for skidding sensitivity testing of energetic materials.

A remote-operated apparatus for testing the detonation sensitivity of energetic materials is detailed. Using an air ram and rotating disk, the normal force and transverse velocity of the impact plane are controlled independently, enabling the exploration of varying impact conditions over a wide parameter space. A microcontroller local to the apparatus is used to automate apparatus operation and ensure temporal alignment of the impacting ram head with the rotating disk. Calculation of the firing parameters and issuing of operational commands are handled by a remote computer and relayed to the local microcontroller for execution at the hardware level. Impact forces are taken from fast strain measurements obtained from gauges incorporated into the ram head. Infrared imaging of explosive samples provides insight into the peak thermal temperatures experienced at the sample surface during the impact event.

Proteoglycan synthesis in cultures of murine retinal neurons and photoreceptors.

1. In recent years, a number of histochemical and immunocytochemical studies have suggested that proteoglycans, particularly those in the interphotoreceptor matrix, exhibit altered distributions in several murine models for retinal degenerations. We are using a cell culture system to characterize the proteoglycans synthesized by neurons and photoreceptors derived from mouse retina, with the long-term goal of analyzing their role in retinal degenerations. 2. In this study we describe initial studies using cells derived from the retinas of normal mice. Cultures of retinal neurons and photoreceptors, which were free of glial, epithelia, or endothelial cells, were labeled with 3H-glucosamine and 35SO4. Proteoglycans isolated from the medium and cell layer were analyzed on the basis of charge, relative hydrodynamic size, and glycosaminoglycan content. 3. The studies indicate that the cultures actively synthesize proteoglycans. The medium contained predominantly chondroitin sulfate/dermatan sulfate, while the cell layer had a higher proportion of heparan sulfate, indicating a differential distribution between the two compartments.

Photoreceptor survival-promoting activity in interphotoreceptor matrix preparations: characterization and partial purification.

It is well established that many types of cells are dependent on trophic factors for their survival. We are investigating whether photoreceptor survival may also be dependent upon such a factor, as well as the possibility that the interphotoreceptor matrix (IPM), which lies between the outer retina and the RPE, may be a source of photoreceptor survival-promoting activities. Well-characterized cultures of embryonic chick retinal neurons and photoreceptors, in which the photoreceptors spontaneously degenerate between 7 and 10 days in culture, were used to test this hypothesis. Crude extracts of IPM were found to support three- to fourfold increases in photoreceptor survival in 10-day cultures. This response was dose-dependent, saturable, and specific for photoreceptors, since there was no difference in the number of non-photoreceptor neurons between treated and control cultures. This photoreceptor survival-promoting activity (PSPA) may function when present either as a substratum-bound molecule or as a medium supplement. PSPA is heat labile, sensitive to freeze-thawing, and stable only within a very narrow pH range. The activity binds to heparin affinity columns but, in contrast to the behavior of growth factors such as FGF, it can be eluted from the columns at low salt concentrations (0.5 M NaCl). PSPA is eluted from a Sephacryl S-200 gel filtration column in two regions with Mr = 400-450 kDa and 33 kDa, respectively. SDS-gel electrophoresis suggests that the high molecular weight activity is composed of aggregates of lower molecular weight molecules. While this factor has not been purified to homogeneity, the combination of heparin-agarose affinity chromatography and hydrophobic interaction chromatography on phenyl-Sepharose columns results in 720-fold purification of the activity, with one unit of PSPA activity corresponding to 40 ng protein. This partial purification is accompanied by substantial reduction in the complexity of protein patterns in silver-stained SDS-gels. These findings are potentially important since a macromolecular factor promoting photoreceptor survival could play a role in the normal development of the retina, and could also further our understanding of retinal degenerations such as retinitis pigmentosa.

Proteoglycan biosynthesis by chick embryo retina glial-like cells.

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.

Analysis of newly synthesized Bruch's membrane proteoglycans.

Bruch's membrane may provide a selective filtration barrier for nutrients coming from the choriocapillaris to the outer retina. Because proteoglycans have been shown to have structural and filtration properties in other tissues, we have been investigating the deposition of newly synthesized proteoglycans into Bruch's membrane and how these may be affected by aging and pathology. Proteoglycans deposited in human Bruch's membrane were metabolically labelled with 35SO4 and 3H-glucosamine using a whole-eye organ culture system. Labeled proteoglycans were extracted from dissected Bruch's membranes with 4 M guanidine and isolated by ion-exchange column chromatography on DEAE cellulose using a linear salt gradient. These molecules were subsequently chromatographed on Sepharose CL-4B and glycosaminoglycan content characterized by enzymatic and chemical degradation. The elution profiles for proteoglycans remains relatively unchanged with age, although in eyes from donors over age 70 there is a small change in size distribution toward higher molecular weights. However, the proportions of newly synthesized glycosaminoglycans remain unchanged with age, being approximately 75% chondroitin sulfate/dermatan sulfate and 25% heparan sulfate. Bruch's membrane proteoglycans from donors with different retinal pathologies, however, exhibited an increased proportion of heparan sulfate. Considering the structural and filtration properties of proteoglycans, such alterations could result in abnormal functioning of Bruch's membrane that could ultimately affect the maintenance of the outer retina.

Altered proteoglycans in cultured human retinitis pigmentosa retinal pigment epithelium.

Proteoglycans are involved in a variety of cell-cell and cell-matrix interactions. These include cell adhesion, growth regulation and a number of developmental processes. Their involvement in such interactions may be of particular importance in retinitis pigmentosa (RP) because of the detachment and migration of retinal pigment epithelial (RPE) cells often associated with this condition. Because of these important functions in cell behavior, we have been studying the proteoglycans produced by human RPE and how these may be altered in RP. Confluent cultures of RPE from normal donors and from two donors with dominantly inherited RP were labeled with 3H-glucosamine and 35SO4 and the proteoglycans isolated from the medium, substratum and two cell membrane-associated compartments, designated "EDTA-released" and "cell-associated." The proteoglycans were analyzed for size distribution by Sepharose CL-4B chromatography and for glycosaminoglycan (GAG) composition based on enzymatic and chemical susceptibilities. Differences in size distribution and GAG composition were found between the two cell-associated compartments on normal cells. Retinitis pigmentosa proteoglycans differed from their normal counterparts in corresponding compartments both in size distribution and GAG composition. Most affected were those proteoglycans released from the cell surface by EDTA. These findings may be of importance in retinitis pigmentosa since alterations in these molecules could influence the way RPE cells interact with their microenvironment.

Proteoglycan synthesis in flat cell-free cultures of chick embryo retinal neurons and photoreceptors.

Extracellular matrix and cell surface proteoglycans are thought to play important roles in neural development and regeneration. Central nervous system proteoglycans have been isolated and characterized from rat and sheep brain and from chick neural retina. An experimental advantage offered by the latter tissue is that it is avascular and can be isolated free of connective tissue and pigment epithelium. Therefore, proteoglycans synthesized by this tissue are derived exclusively from neural cells. However, it has not yet been determined whether neurons and photoreceptors contribute to proteoglycan synthesis or whether these molecules are largely glial in origin. In the present study we have addressed this question using cultures of chick neural retinal cells free of flat, glial-like cells. Proteoglycans synthesized by cultures of retinal neurons, photoreceptors, and undifferentiated, process-free round cells from 8-day embryonic chick neural retina were metabolically labeled in vitro using [35S]sulfate and [3H]glucosamine as precursors. Radiolabeled proteoglycans accumulated in the medium, and could also be extracted from the cell layer by sequential treatments with Triton X-100 and with guanidine HCl. The proteoglycans were isolated by ion-exchange chromatography, and characterized by gel filtration chromatography and by susceptibility to degradation by enzymatic and chemical treatments. Overall, heparan sulfate proteoglycans were the predominant type of proteoglycan synthesized in vitro by the cultured neural retinal cells at this developmental stage. The medium and the Triton extract contained different proportions of both chondroitin sulfate and heparan sulfate proteoglycans, while heparan sulfate was the only proteoglycan recovered from the guanidine extract. These studies demonstrate that heparan sulfate and chondroitin sulfate proteoglycans are actively synthesized by cultures of neural retinal cells free of flat, glial-like cells.

Detection of extracellular matrix molecules synthesized in vitro by monkey and human retinal pigment epithelium: influence of donor age and multiple passages.

The retinal pigment epithelium of various species, including man, can synthesize extracellular matrix components in vitro. We studied extracellular matrix molecule production by human, rhesus and cynomolgus monkey pigment epithelium in vitro, using a panel of specific antibodies to collagen types I-V, fibronectin, laminin, the basement membrane heparan sulfate proteoglycan, human alpha elastin, glial fibrillary acidic protein (GFAP), and Factor VIII in a modified indirect immunofluorescence reaction. Results were similar between human and both monkey species in early passage cultures with respect to the positive detection of collagen types I, III, IV, V laminin, basement membrane proteoglycan, and fibronectin. The fluorescence was strongest in the center of confluent monolayers and was absent in border or isolated cells. Collagen type II, elastin, GFAP, or Factor VIII were not detected. Based on these findings, human and monkey pigment epithelium in vitro should be useful for the study of the synthesis, deposition and supramolecular interactions of a variety of extracellular matrix molecules.

Detection of specific extracellular matrix molecules in drusen, Bruch's membrane, and ciliary body.

We used specific antibodies to localize a variety of extracellular matrix components by indirect immunofluorescence reactions on cryostat sections of drusen-bearing human donor eye tissue. These included collagen types I through V, fibronectin, laminin, heparan sulfate-containing basement membrane proteoglycan as well as antibodies to human IgG and IgM. The composition of drusen with respect to these specific molecules varied greatly, even within the same region of an eye. Hard or nodular drusen were more likely to show a discrete organized layer of specific basement membrane molecules over their apical surfaces than were diffuse type drusen. Diffuse but not nodular drusen generally contained fibronectin. Immunohistochemical reactivity of Bruch's membrane showed age-related accumulation of type I collagen and localized changes associated with some drusen.

Altered glycoconjugates in cultures of retinitis pigmentosa retinal pigment epithelium.

These studies have shown that these are some alterations in the complex carbohydrates associated with the cell surface of RPE cells from donors with dominant retinitis pigmentosa. Differences are found both in proteoglycans and in glycoproteins. Proteoglycans of different sizes and glycosaminoglycan composition are associated with distinct cellular compartments of normal RPE cells. In the case of dominant retinitis pigmentosa studied here, there are some alterations in RPE proteoglycans. Within a given cellular compartment, there may be differences in the size distribution and/or the ratios of the proteoglycan size classes. In addition, there may be altered glycosaminoglycan content found between peaks of comparable size. There also appears to be more microheterogenity in oligosaccharides derived from surface glycoproteins of retinitis pigmentosa RPE cells. This may be due to differences in the total number of carbohydrate units or in the organization of the branching structure as suggested by altered size distribution and lectin-reactivity, respectively. While the reason for these differences is not clear, they could be due to abnormal processing and/or increased degradation. Considering their importance, alterations in cell surface glycoconjugates could affect the ability of RPE cells to interact with their environment and to maintain a healthy status.

Macular corneal dystrophy. Lack of keratan sulfate in serum and cornea.

An ELISA assay using a monoclonal antibody (ET-4-A-4) that recognizes a sulfated carbohydrate epitope in both keratan sulfate type I (corneal) and type II (skeletal) was employed to quantify keratan sulfate in serum and corneal tissue from patients with macular corneal dystrophy (MCD). This assay disclosed significant quantities of keratan sulfate in the serum in 45 healthy individuals (251 +/- 78 ng/ml), and in 66 patients with various corneal diseases (273 +/- 101 ng/ml). In contrast keratan sulfate was not detected (less than 2 ng/ml) in the serum of 16 patients with histopathologically confirmed MCD. Keratan sulfate was also detected in extracts of normal corneas and corneal tissue with a variety of pathologic conditions, but was virtually absent in corneal tissue from five patients with MCD. In corneas with MCD the chondroitin sulfate/keratan sulfate ratio was considerably higher than that of all normal and pathologic corneas studied. Since keratan sulfate in the serum appears to be derived predominantly from the normal turnover of cartilage these studies strongly suggest that the defect in keratan sulfate synthesis in MCD is not restricted to corneal cells and that MCD is one manifestation of a systemic disorder of keratan sulfate. The cartilage changes, however, do not have clinical significance. Moreover, since keratan sulfate can be detected in the blood of newborns it should be possible to diagnose MCD prior to corneal opacification.

Macular degeneration and elevated serum ceruloplasmin.

Macular degeneration associated with age and drusen, an important cause of visual loss, is associated clinically with alterations in the retinal pigmented epithelium. Because the pigmented epithelium is a copper-rich tissue with antioxidant properties, the copper economy in patients and controls were studied by measuring ceruloplasmin. Ceruloplasmin, a multifunctional, copper-binding alpha-globulin, was significantly elevated in non-related patients as compared with controls (691 +/- 153 mg/L vs 312 +/- 64; P less than .001), both by the p-phenylenediamine oxidation technique and radial immunodiffusion assay. When 53 members of a large family were divided clinically into persons with and without macular degeneration, the ceruloplasmin concentrations were not significantly different from each other, but were elevated as compared with non-related controls (P less than .001). These differences were not due to an intragroup age mismatch. A group of patients with retinitis pigmentosa had normal serum ceruloplasmin concentrations. This study suggests a relationship between serum ceruloplasmin, trace metals, and the tissue alterations associated with macular degeneration that deserves further investigation.

Absence of normal keratan sulfate in the blood of patients with macular corneal dystrophy.

We measured levels of sulfated keratan sulfate in serum using a monoclonal antibody in an enzyme-linked immunosorbent assay. Sulfated keratan sulfate was not detected in the serum of 16 patients with macular corneal dystrophy, but was present at normal levels in 66 patients with other corneal diseases. There were no differences with respect to age, sex, and other ocular findings. This monoclonal antibody recognizes a sulfated carbohydrate epitope present in both corneal and skeletal keratan sulfate. Since most serum keratan sulfate is derived from the cartilages, the defect in keratan sulfate synthesis in macular corneal dystrophy may not be restricted to corneal cells. This assay should prove useful in the diagnosis of macular corneal dystrophy, particularly in children at risk before the appearance of opacification.

Chondronectin interactions with proteoglycan.

We have investigated whether proteoglycans are involved in the attachment of embryonic chick chondrocytes to type II collagen. Chondroitin sulfate proteoglycan, when added exogenously, promotes the binding of chondronectin, the chondrocyte attachment factor, to type II collagen substrates and thereby stimulates chondrocyte adhesion. Blockage of endogenous proteoglycan synthesis with beta-xylosides prevents chondronectin-mediated chondrocyte attachment, confirming that proteoglycan is required. The intact proteoglycan must be present since chondroitin sulfate glycosaminoglycans did not promote chondronectin-mediated cell attachment but, rather, inhibited it in a dose-dependent manner. This inhibition, however, could be overcome with excess exogenous proteoglycan. Consequently, chondronectin interacts with proteoglycan and then the complex interacts with the collagen substrate and with the cell surface to promote cell adhesion. Further evidence for a direct interaction of chondronectin with the glycosaminoglycan portion of the proteoglycan is the selective binding of chondronectin to dextran-Sepharose, dextran having been shown to inhibit attachment to an extent similar to that of chondroitin sulfate.

Chondronectin: physical and chemical properties.

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.

Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.

The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

Laminin detection in normal and retinitis pigmentosa human retina.

Epiretinal membrane formation in the posterior pole is an almost constant feature of eyes with retinitis pigmentosa. Using a modified direct immunofluorescence reaction, we demonstrated a strong reaction with antilaminin in a band-like pattern on the inner retinal surface of 3 eyes with retinitis pigmentosa. There were also strongly-laminin-positive pigmented and unpigmented cells scattered throughout the degenerated retinas. This pattern was in sharp contrast to that seen in normal retinas, where the linear band of reaction at the internal limiting membrane was faint and accompanied by a zone of reaction at the level of the outer limiting membrane. Quantitation of laminin by ELISA revealed a six- to ten-fold increase (per mg protein) in retinitis pigmentosa retinas, as compared with normal. Since both glial and retinal pigmented epithelial cells can synthesize and deposit laminin, both may participate in the gliosis that occurs during the course of retinitis pigmentosa.

Altered synthesis of Bruch's membrane proteoglycans associated with dominant retinitis pigmentosa.

Proteoglycans, extracellular matrix molecules that have been shown to have filtration properties in some tissues, make up a significant proportion of the structural macromolecules of Bruch's membrane. Bruch's membrane may provide a selective filtration barrier between the choriocapillaris and the pigmented epithelium (PE) and outer retina. In this paper, we compare the proteoglycans extracted from metabolically-labeled normal and retinitis pigmentosa (RP) Bruch's membranes. Isolated RP Bruch's membrane proteoglycans were larger than those from normal donor eyes when chromatographed on a column of Sepharose CL-4B. In addition to the increased size, there was also a dramatic increase in the proportion of heparan sulfate proteoglycan being synthesized in RP. Considering the structural and filtration properties of proteoglycans, alterations such as these could affect the functioning of Bruch's membrane and, possibly, the PE and the outer retina.

Identification of a bone matrix-derived chemotactic factor.

When demineralized bone matrix powder is implanted subcutaneously in the rat, the early responses involve the appearance and proliferation of mesenchymal cells at the site of implantation, followed by cartilage and bone formation. The ability of cells to migrate to the implant suggests that chemotaxis may be a critical event in this process. Therefore, using the modified Boyden chamber assay, we tested extracts of demineralized bone matrix for chemotactic activity. We have identified and partially purified, on molecular sieve chromatography, a heat labile and trypsin-sensitive protein (Mr = 60,000-70,000) that is a potent chemoattractant for mouse calvaria, osteoblast-like cells (MMB-1), but not for monocytes (putative osteoclast precursors). These findings suggest that chemotactic protein(s) have a significant role in the recruitment of osteoprogenitor cells to a site of bone repair.

Immunofluorescence localization of fibronectin in chondrosarcoma cartilage matrix.

In this study, we have compared the extracellular matrix components and the in vitro adhesion characteristics of normal rat epiphysial chondrocytes with those from the Swarm rat chondrosarcoma, which has many of the biochemical characteristics of normal cartilage. With the use of immunofluorescence techniques, tissue slices and chondrocytes in culture were tested for the presence of collagen types I and II, cartilage-characteristic proteoglycan, and fibronectin. Both normal and tumor matrix contained type II collagen and cartilage proteoglycan, but only the tumor matrix contained fibronectin. In culture, tumor-derived chondrocytes continued to accumulate fibronectin in their matrix, even after deposition of type II collagen and proteoglycans, while normal chondrocytes did not. When the attachment characteristics of both types of chondrocytes were compared, tumor chondrocytes required fibronectin for attachment, while normal chondrocytes used another attachment factor that had been identified previously as chondronectin. These studies suggest that, although biochemically similar to normal chondrocytes, tumor chondrocytes are no longer able to express the regulatory mechanisms for fibronectin accumulation.