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Distribution of beta-N-acetylglucosaminidase, hyaluronoglucosaminidase and acrosin in buffalo and goat spermatozoa.

The distribution of beta-N-acetylglucosaminidase, hyaluronoglucosaminidase and acrosin in buffalo and goat sperm acrosomes was studied. The three hydrolases were found to occur in soluble and bound forms. In the bound form, they were associated with the denuded sperm and were maximally solubilized at pH 3.0. The possible role of beta-N-acetylglucosaminidase in fertilization is discussed.

Acetylglucosaminidase

Histochemical characterization of mucosubstances in bone and soft tissue-tumors.

The present investigation endeavors to characterize the mucosubstance content of 170 myxoid and chondromatous tumors and chordomas by histochemical methods. The results obtained using the critical electrolyte concentration (CEC) method as introduced by Scott and co-workers23,24 were compared with those obtained by staining with alcian blue and toluidine blue at different pH's with and without pretreatment with bovine testicular hyaluronidase. Tissues known biochemically to contain different heteroglycans were used as controls: synovial fluid and cock's comb (hyaluronic acid) stained with alcian blue up to a MgCl2 concentration of 0.1 M; fetal cartilage (chondroitin 4- and 6-sulphate) pulposus with notochordal remnants (keratan sulphate) up 10 1.0 M. The staining reaction of intramuscular myxoma and myxoid liposarcoma corresponded to that of synovial fluid and cock's comb (containing hyaluronic acid). Benign chondromatous tumors (osteochondroma, enchondroma, extraskeletal chondroma, chondromatosis in bursae, synovia, and tendon) as well as well-differentiated chondrosarcomas had a similar staining reaction to that of adult cartilage (containing keratan sulphate). However, the intensity of the reaction was lower in these tumors than in the adult cartilage, indicating that the keratan sulphate content of the tumors is lower. Most of the moderately well-differentiated chondrosarcomas, the poorly differentiated chondrosarcomas, and pulmonary metastases of chondrosarcoma, as well as mesenchymal chondrosarcoma and extra-skeletal chondrosarcoma possessed the same staining properties as fetal cartilage, known to contain chondroitin 4- and 6-sulphate but not keratan sulphate. A few of the moderately well-differentiated chondrosarcomas stained up to a MgCl2 concentration of 1.0 M. Three cases of poorly differentiated chondrosarcomas stained with alcian blue up to 0.35-0.45 M in the lowest differentiated areas, indicating the presence of sulphated heteroglycans, as chondroitin 4- and 6-sulphate. Most chordomas possessed the same staining properties as fetal cartilage; however, a few chordomas stained in the same way as notochordal remnants of nucleus pulposus (containing keratan sulphate), which are thought to be the origin of these tumors. The results of staining of the tumors in the present series with the Scott technique corresponds well with toluidine blue and alcian blue at different pH's with and without pretreatment of the sections with testicular hyaluronidase. Since bone and soft tissue tumors may contain varying mucosubstances depending on the tissue of origin and on differentiation, histochemical investigation of the heteroglycan content of these tumors may be a valuable diagnostic aid.

Adult

Urinary glycosaminoglycan patterns in angiosarcoma of the liver.

Glycosaminoglycans extracted from 24-hour urine specimens from patients with hepatic angiosarcoma and from normal/controls were separated as cetylpyridinium complexes into "hyaluronic acid," "chondroitin sulfate," and "heparin" fractions, then further separated and characterized by anion-exchange chromatography and hyaluronidase susceptibility. The chromatographic pattern of the urinary chondroitin sulfate fraction in patients with angiosarcoma of the liver differed from those of controls in that there was a relative increase in the total amount of uronic acid in a hyaluronidase-resistant fraction and a decrease in a fraction susceptible to hyaluronidase digestion. These changes appeared to become more pronounced with advancing disease. Chromatographic patterns and determinations of hyaluronidase susceptibility indicated that the resistant fraction was heparan sulfate and that the susceptible fraction was chondroitin-4-sulfate and/or chondroitin-6-sulfate.

Chondroitin Sulfates

The isolation of single cells from the avian salt gland.

Collagenase, hyaluronidase, and trypsin were used to isolate single cells from the avian salt gland. Three cell types were distinquishable in the resultant cell suspension: peripheral, intermediate, and principal cells. The fine structure of these cells is described and related to the morphology of the intact gland.

Animals

Enzyme treatment of spinal cord transected rats.

Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols of Matinian and Andreasian's two most successful treatment regimens, we repeated their experiments using United States produced pure hyaluronidase and trypsin or trypsin and elastase. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiated proline by regenerated corticospinal axons. None of the treated animals differed from control animals treated only with the enzyme vehicle. No animals walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.

Animals

Lidase treatment of spinal cord transected rats.

Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols outlined by Matinian and Andreasian, we repeated a portion of their experiment using a Lidase preparation manufactured in the USSR. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiate proline by regenerated corticospinal axons. The only difference between treated and control animals was that the mean total body weight of the treated animals increased more than that of controls (p less than 0.05). No animal walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.

Animals

Enhancement of urate solubility by connective tissue. I. Effect of proteoglycan aggregates and buffer cation.

Proteoglycan aggregates (PGC) facilitated dissolution of 2 1/2 times more sodium urate crystals than nonaggregated proteoglycans. This effect of aggregates on urate solubility was abolished by digestion of the aggregates with hyaluronic acid beta1 leads to 3 hydrolase. PGC, however, did not sustain urate concentrations in supersaturated solutions. Potassium urate was severalfold more soluble than sodium urate. In vivo, where they exist predominantly as their sodium salts in the extracellular connective tissue, proteoglycans may not markedly influence the solubility of sodium urate.

Animals

A fetuin-like antigen from human nephroblastoma.

An antigen was detected in pooled human nephroblastomas using antiserum prepared in rabbits against an ethylemediaminetetra acetic acid (EDTA) extract of the tumors. This antigen was not found in normal human plasma or kidney extracts, and was not related to the ABO or Forssman blood groups. The antigen was detected in extracts of cultured nephroblastoma cells, but was not present in extracts of normal human fetal kidney cell cultures. The antigen is believed to be present at the cell surface, as cell viability was not significantly lowered during the extraction procedure. A reaction of complete identity was demonstrated by Ouchterlony double diffusion experiments with this antigen and purified bovine fetuin. The antigen was not found in extracts of human fetal spleen, thymus or kidney, nor in human fetal serum. Furthermore, the antigen does not possess determinants in common with the human alpha-fetoprotein of hepatomas, nor was it detected in human renal clear cell carcinoma. Initial characterization of the antigen showed it to be nondialysable, not sedimentable at 100,000 times g for 2 h, stable to repeated freeze-thawing and to incubation at 56 degrees C for 1 h, and water soluble over a wide pH range. The antigen was susceptible to digestion with pronase and trypsin and possibly hyaluronidase, but not to ribonuclease or neuraminidase. The protein portion is therefore of major importance to the structural integrity of this antigen. The relationship between this antigen and other abnormal materials reported previously in nephroblastoma patients is being studied.

Adolescent

Markers to distinguish normal and neoplastic mammary epithelial cells in vitro: comparison of saturation density, morphology and concanavalin A reactivity.

Normal and premalignant mouse mammary epithelial cells can be prepared in high yields by collagenase dissociation of minced glands followed by a brief, differential centrifugation to remove contaminating fibroblasts and fat cells. The major difficulties in preparing pure cultures in quantity are 1) incomplete dissociation of gland material, and 2) cell death during enzymatic digestion. These problems are eliminated by careful selection of collagenases for dissociation. Normal and premalignant mammary epithelial cells are morphologically indistinguishable from malignant mouse mammary epithelial cells in primary monolayer cultures. In addition, the growth rates and saturation densities achieved by normal mammary epithelial cells are indistinguishable from those of malignant mammary epithelial cells in primary culture. In both cases, a monolayer of cells is preserved with no evidence of focal overgrowth. Malignant adenocarcinoma mammary cells can however be distinguished from normal mammary epithelial cells by virtue of differences in their surface interactions with concanavalin A. A hemadsorption assay using Con-A-coated erythrocytes was the most sensitive indicator for these differences. In hemadsorption assays malignant mammary epithelial cells were half-maximally reactive with 2.5 mug/ml concanavalin A, while normal cells were completely unreactive even at concanavalin A concentrations five-times higher. Premalignant mammary epithelial cells were as reactive as malignant mammary epithelial cells in the hemadsorption assays. Hemadsorption of malignant cells was observed in primary and secondary cultures of epithelium as well as in cell lines. Malignant cells forming mammary adenocarcinomas were as highly reactive as malignant cells forming scirrhous carcinomas. Malignant cells not releasing mammary tumor virus (MuMTV) were as reactive as cells releasing that virus. Adsorption of concanavalin-A-coated erythrocytes to normal mammary epithelial cells could be induced by brief treatment of cell monolayers with hyaluronidase. Exposure of active sites was not affected with either trypsin or collagenase. Our results show that while the growth of malignant cells does not serve to distinguish them from normal cells in monolayer culture, surface changes do exist which can be identified by differences in concanavalin A reactivity. Since the earliest transformants identifiable in vivo (premalignant) have undergone conversion of the surface marker, concanavalin-A-mediated hemadsorption provides a sensitive measure for mammary epithelial cell transformants in vitro.

Animals

The effects of hyalurodinase upon tumor formation in BALB/c mice painted with 7,12-dimethylbenz-(a)anthracene.

Experiments were performed to investigate the effects of hyaluronidase on chemical carcinogenesis. Two experiments were carried out using BALB/c mice. In the first experiment the mice were divided into three groups, viz. (1) painted with 7,12-dimethylbenz(a)anthracene (DMBA), (2) injected with hyaluronidase and painted with DMBA and (3) injected with saline and painted with DMBA. In the second experiment the mice were divided into three groups: (1) painted with DMBA, (2) injected with hyaluronidase and painted with DMBA and (3) injected with heat-inactivated hyaluronidase and painted with DMBA. The tumor incidence and size of tumors were significantly lower in the group treated with hyaluronidase than in the other groups. The latent period was increased. The mitotic index of the skin adjacent to the tumors at the end of the experiment was decreased. These studies show that hyaluronidase can act as an anticarcinogenic agent.

9,10-Dimethyl-1,2-benzanthracene

An effect of accumulated matrix on sulfation among cells in a cartilage colony: an autoradiographic study.

In this report an autoradiographic approach is used to compare synthetic activities of cells within differentiated cartilage colonies. While amino acid incorporation is umiform throughout the colony, H-3-uridine is incorporated more actively by cells having little matrix, cells which are typically in the peripheral regions of a colony. On the other hand S-35-O4 is incorporated most actively by cells in the colony centers. This difference in sulfation appears to occur independently of the mitotic state of the cells, since it is apparent in both growing and near-stationary cultures. Instead, there is a correlation between the accumulation of extracellular matrix and more active levels of sulfation. In support of the idea that matrix creates a microenvironment more favorable to chondrogenesis is the observation that a brief treatment with hyaluronidase, which removes about 60% of the S-35-O4 from prelabeled cultures, depresses isolation of labeled glycosaminoglycans. The possible role of extracellular matrices in altering the expression of differentiated functions by creating a more favorable microenvironment is considered.

Amino Acids

Studies on the interaction of concanavalin A with glycoproteins.

Lectins (phytohaemagglutinin) are known to have the unique property of binding with certain specific sugars, polysaccharides and glycoproteins. Although the kinetics of interaction between lectins and sugar have been extensively studied, the binding characteristics of the lectins with various glycoproteins are not well understood. In this laboratory a systematic study has been initiated in relation to the interaction of lectins with glycoproteins. Concanavalin A is known to bind alpha-glucosides, mannosides and biopolymers having these sugar configurations. A galactose binding protein from caster bean has been purified to homogeneity and was found to contain mannose. This lectin was used as the source of glycoprotein for studying its interaction with concanavalin A. This study showed that the interaction is temperature dependent and the dissociation is time and alpha-methyl glucoside concentration dependent. This has led to speculate a model for cell-lectin interaction. Using concanavalin A it has been shown that all the lysosomal enzymes from brain studied were glycoprotein in nature. Moreover, using Sepharose-bound concanavalin A it has been possible to devise a method by which these lysosomal enzymes could be purified considerably. With the knowledge that the interaction between lectin and glycoprotein is not only dependent on the specific sugar present in the glycoprotein, but also on the nature of the glycoprotein it was possible to develop a novel method for immobilizing various glycoprotein enzymes, such as arylsulphatase A, hyaluronidase and glucose oxidase.

Animals

Development of the basal lamina and extracellular materials in the early chick embryo.

Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.

Animals

[Electron microscopic investigations of vitreous collagen after treating the vitreous with liquefying substances (author's transl)].

By electron microscopic studies collagenase, hyaluronidase, HCl, ascorbic acid, and iron ions have been found to attack the collagen fibers of bovine vitreous. Because of the possible role of ascorbic acid in collagen synthesis and the ability of ascorbic acid to degrade hyaluronic acid and collagen we suggest that the ascorbic acid of the vitreous essentially participates in construction and metabolism of the vitreous body.

Animals

[Observations of the surface of the articular cartilage after treatment with hyaluronidase (author's transl)].

1. The articular surfaces of the femoral head of rats have been studied by scanning electron microscopy after in vitro treatment with hyaluronidase. 2. The cartilage surface has been digested mildly by the buffer medium (control). 3. The matrix of the cartilage has selectively been removed by the hyaluronidase. 4. Chondrocytes have been seen on the surface after treatment with hyaluronidase for a longer period. 5. We discuss the result of enzymatic digestion.

Animals