Specimens of human articular cartilage obtained at replacement surgery were divided into three groups, i.e. weight-bearing and non-weight-bearing portions of normal control cartilage, and osteoarthritic cartilage. Fractions of 2 M guanidine hydrochloride-cartilage extracts obtained by Sephadex G-75, chromatography contained serine proteinases which had the ability to degrade gelatin and proteoglycan aggregate. The serine proteinase activity of the weight-bearing control cartilage was two times higher than that of the non-weight-bearing control cartilage. Moreover, the activity of osteoarthritic cartilage was about 80 times higher than that of non-weight-bearing control cartilage. Fractions of control cartilage extracts on. Sephadex G-75 chromatography contained inhibitory activity against trypsin and collagenase with M, of 14,000 and 36,000, respectively. The trypsin inhibitor level in osteoarthritic cartilage was lower than in control cartilage. The level of collagenase inhibitor in control cartilage was higher in non-weight-bearing portions than in weight-bearing portions. However, no significant collagenase inhibitor activity was observed in osteoarthritic cartilage. On sodium dodiecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by zymography, the cartilage serine proteinases had M, of 67,000 and 22,000. The data suggest that the increased level of destructive proteinases acting in the presence of the decreased level of inhibitors may lead to the extracellular matrix degradation in osteoarthritic cartilage.
A human bladder carcinoma cell line HTB9, which produces colony-stimulating factor (CSF), was grown on artificial capillaries in a hollow fiber culture unit. The extracapillary fluid was collected every 2 days, and the levels of CSF and other components of the fluid were evaluated. The level of CSF produced by HTB9 cells in the fluid was sufficiently high and production of CSF was detected for as long as 40 days without a cell passage.
A highly sensitive sandwich enzyme immunoassay for parvalbumin has been developed. The amount of parvalbumin was measured by the sandwich enzyme immunoassay using anti-parvalbumin IgG-coated polystyrene balls and anti-parvalbumin Fab’-horseradish peroxidase conjugate. The minimum detection limit was 30 pg parvalbumin per assay tube. The present enzyme immunoassay was about 30-fold more sensitive than the previously described radioimmunoassay in which the detection limit was 1 ng/tube. The assay showed no cross-reactivity with calmodulin and S-100. The coefficients of variation in within-run and between-run were 6.8-8.9% and 7.0-9.6%, respectively. Concentrations of parvalbumin in various rat tissues were determined by this assay system. High concentrations of the protein were observed in fast skeletal muscles such as gastrocnemius and tibialis anterior. On the other hand, the concentration in soleus muscle was only about two hundredths that in fast skeletal muscles. Parvalbumin was also present in the heart, lung and kidney. The results suggest that parvalbumin may also play an important role in the regulation of Ca2+ ions in various tissues other than skeletal muscles.
A low molecular weight factor in chicken and rabbit steroid-free sera (LMS) can induce in vitro maturation of intrafollicular oocytes of the fish Oryzias latipes by a two-step incubation procedure, but not that of follicular cell-free oocytes. In vitro maturation of intrafollicular oocytes induced by gonadotropin and 17α,20β-didihydroxy-4-pregnen-3-one (DHP) was reversibly inhibited by LMS. The inhibitory, as well as the inductive effect, of the rabbit LMS was biphasic and dosedependent, with an optimum concentration at 0.5 mg/ml. Cyclic AMP production was also stimulated by a similar concentration of LMS. Addition of LMS to the culture medium enhanced secretion of estradiol-17β and DHP by granulosa cells. Further incubation after removal of LMS from culture medium resulted in increased secretion of DHP and decreased secretion of estradiol-17β. The present findings suggest that LMS is available as a tool for investigation of the mechanism of maturation of medaka oocytes.
Circadian variations in the plasma insulin response to food intake, and effects of adrenalectomy and treatment with corticosterone were examined in rats which had been fed during a restricted time of day (2000-2400) for 2 weeks. When rats were refed at 2000 after starvation, the plasma insulin level increased to a much higher level than when they were refed at 0800. In adrenalectomized rats, the plasma insulin response to refeeding was much attenuated at 2000 and its clock-time difference was abolished. When corticosterone was administered to adrenalectomized rats immediately before refeeding, the clock-time difference in the postprandial insulin response was restored, with a greater response at 2000. From these results, together with the previous finding that the plasma corticosterone level was higher at the scheduled feeding time (2000), it was concluded that the circadian difference in the postprandial insulin response was attributable to the circadian adrenocortical rhythm.
The present study deals with immunohistochemical localization of S-100 protein in the blood vessels of the pig and cattle. Immunoreactivity for S-100 protein was selectively seen in the endothelial cells of arteries, but not in those of veins. The immunoreactivity was also found in the endothelial cells of blood capillaries; the endothelial cells of continuous (non-fenestrated) type showed a moderate to intense immunoreactivity, while those of fenestrated type were immunonegative. Immunostaining using antisera specific to α- or β-subunit of S-100 protein revealed that the endothelial immunoreactivity was due to the β-subunit. The present study suggests that S-100 protein in the endothelial cells may be involved in the mechanism of transcytosis like in fat cells which also contains S-100 protein.
In the organ culture of rat anterior pituitary fragments, microvillous cells and ciliated cells rapidly covered the surface of the explant. The cilia showed the 9+2 fibril configuration. Both types of surface cells lacked secretory granules. The rough endoplasmic reticulum and the Golgi apparatus were poorly developed in these cells, while bundles of filaments were frequently observed. The two types of cells formed junctional complexes between adjacent cells. Some surface cells of the explant were immunoreactive against anti-S-100 protein serum. These characteristics resemble those of folliculo-stellate and marginal cells found in the pituitary in situ. Microvillous and ciliated cells covered the glandular cells with their thin cytoplasmic projections. The basement membrane was observed on the basal side of the surface cells. It is suggested that the surface cells originate from inside the explant, and support the glandular cells.
Cross-reactivity of six novel somatostatin (SRIF) analogs has been examined by SRIF-specific radioimmunoassays using anti-SRIF sera OAL-272 or OAL-283. The analogs examined possessed different chain length in which the S-S linkage was replaced by a methylene bridge of α-amino suberic acid (D-Asu). In the assay using antiserum OAL-272, the labeled SRIF failed to displace any of the six analogs, whereas in the assay with antiserum OAL-283, five of the analogs with the exception of the shortest analog (Phe-D-Trp-Lys-Thr-D-Asu) were bound very weakly (0.01-0.06% vs. SRIF). The results indicate that antisera specific to each of the analogs will be required for assays of these analogs in biological fluids.
Human aorta specimens with no atherosclerotic lesions were obtained at autopsy from 126 individuals, and analyzed for the content of the cross-linking amino acid, histidinoalanine. The histidinoalanine content increased with an increase in age, although marked variability was seen in old age.
Homology of the P0 protein to the immunoglobulin-cell adhesion molecule superfamily was examined by comparing their amino acid sequences. The predicted secondary structure of the amino-terminal domain of P0 protein was suggested to be similar to all immunoglobulin domains consisting of two β-sheets with antiparallel β-strands. The amino-terminal domain including two cysteine residues at 21 and 98 showed considerable sequence homology with the variable domains of immunoglobulins. By a homology search using a computor program, significant homologies were found between the amino-terminal domain of P0 protein and the members of immunoglobulin superfamily including cell adhesion molecules such as the neural cell adhesion molecule and the myelin-associated glycoprotein. We concluded that P0 protein is a member of the immunoglobulin-cell adhesion molecule superfamily and may be involved in the primordial immunoglobulin domain. The amino-terminal domain of P0 protein is located on the extracellular side and may function as an adhesion molecule to form the myelin intraperiod line.