Novel protease bound with chromatins in normal and tumorous tissues of rats

A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma? Rhodamine sarcoma≥ AH 130≥ thymus> spleen? kidney≥ liver? brain.     

Detecting metabolic differences in fetal and adult sheep adipose and skeletal muscle tissues

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two‐photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.     

Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types

We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.     

Distinctive fibroblastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content

Summary The glycosaminoglycan (GAG) content of rabbit skin, oral mucosa, and cultured [³H]-glucosamine-labeled dermal and submucosal fibroblasts was compared. Skin contained predominantly dermatan sulfate (DS) and a small amount of hyaluronic acid (HA), whereas mucosa contained primarily keratan sulfate (KS) and smaller quantities of HA and DS. Culture medium from dermal and submucosal fibroblasts contained GAGs co-electrophoresing with DS, HA, and chondroitin sulfate (CS), although the relative proportions of these GAG differed. CS isolated from dermal and mucosal fibroblast culture medium co-electrophoresed with chondroitin 4-sulfate (C4-S) on cellulose acetate, whereas dermal medium CS was resistant to digestion by chondroitinase ABC, and mucosal medium CS was chondroitinase ABC-susceptible. The pericellular matrix of dermal fibroblasts contained primarily DS and C4-S/C6-S, as confirmed by chondroitinase ABC digestion; the corresponding fraction of mucosal fibroblasts contained HS and a GAG co-electrophoresing with a C6-S standard, yet resistant to digestion by chondroitinase ABC. Thus the GAG content of dermal and mucosal fibroblasts differed both qualitatively in terms of the type of GAG secreted into the culture medium and pericellular matrix, and quantitatively, in terms of the relative proportions of these GAGs in both fractions. These differences support the concept of distinctive fibroblastic subpopulations in skin and mucosal tissue, inasmuch as the cells were subjected to identical culturing conditions. This work was supported by research grant 15878 (C.N.B.) from the Shriners Hospitals for Crippled Children and DE 07803 (C.N.B.) from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown.

Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.     

Stem cells in adult tissues

In recent years the concept of a stem cell has evolved to encompass the hypotheses that stem cells exist within many adult tissues, and that a common 'interchangeable' progenitor cell may exist within the bone marrow capable of regenerating and repairing tissues throughout the body. As more knowledge is gained about stem cells, their potential roles in disease processes, including the development and progression of cancer, have moved to the forefront. The underlying hypothesis of this review is that cell fate is determined by a combination of intrinsic and extrinsic factors; growth and differentiation are regulated through intracellular integration of a multitude of signals initiated by internal and external stimuli. The development of successful stem cell based therapies may depend on experimental approaches that consider both the intrinsic and extrinsic factors that control cell fate.     

Lymphocyte entry into inflammatory tissues in vivo. Qualitative differences of high endothelial venule-like vessels in sponge matrix allografts vs isografts

Sponge matrix allografts and isografts become extensively encapsulated and neovascularized after s.c. implantation. Sponge allografts acquire alloantigen-reactive T lymphocytes, whereas sponge isografts fail to do so, even though these T cells are continuously circulating in the peripheral blood. We have investigated the possibility that the vascular endothelia regulates lymphocytic accumulation in sponge matrix implants. In normal lymph nodes, specialized high endothelial venules (HEV) regulate lymphocyte extravasation from the blood. We have now identified HEV-like vessels in sponge matrix allografts. These vessels are operationally defined as "HEV-like" in that they react with mAb MECA 325 which identifies murine HEV, and bind lymphocytes in ex vivo adhesion assays. In contrast, sponge isografts contain MECA 325 reactive vessels that are significantly smaller than those found in allografts. Further, vessels of sponge isografts do not readily bind lymphocytes in ex vivo adhesion assays. Immunohistologic analysis also revealed that the small MECA 325+ vessels present in sponge isografts are consistently found in close proximity to nerve bundles. Although this MECA 325 reactive vessel-nerve bundle association is also observed in sponge allografts, large MECA 325 reactive vessels are widely distributed in allografts. Our data suggest that small, poorly adhesive MECA 325 reactive vessels develop in sponge isografts and allografts, possibly under the influence of local nerve tissue. These vessels respond to regional alloimmune responses by developing into the larger HEV-like vessels capable of binding lymphocytes in sponge allografts. The value of this experimental system as an in vivo model to evaluate mechanisms involved in neovascularization and endothelial differentiation is discussed.     

Changes in DNase and DNA-polymerase activities in different tissues of chick with age.

The presence of acid and alkaline DNases in nuclei of chick brain cells has been demonstrated. The activities of these two DNases along with those of DNA-polymerases were assessed in chicken tissues with known varied cell proliferative capacities (eg., spleen, kidney and brain) at different ages. The results indicate that the acid and alkaline DNases are probably the 'house keeping' enzymes with a constitutive role in DNA repair process, the former with a DNA-repair process that is linked to cell proliferation (DNA synthesis) while the latter with the basal DNA-repair operations that must go on at all times without any regard to the cell division process. Chicken brain, unlike that of rat, possesses significant levels of aphidicolin sensitive DNA-polymerase(s) that are considered to be more replication oriented enzymes suggesting that the replication potential of adult and old avian brain cells may be different from that of a mammalian brain.