Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

酶消化结合组织块培养法用于颞下颌关节盘组织工程细胞扩增的初步研究

目的采用酶消化结合组织块培养法对山羊颞下颌关节（temporomandibular joint，TMJ）盘细胞进行体外培养和扩增，探索TMJ关节盘细胞体外培养及扩增的新方法。方法在无菌条件下，切取一月龄山羊TMJ关节盘，剪至1．0mm^3的碎块，用0．25％胰酶、0．01％I型胶原酶消化关节盘组织块，将消化好的组织块置入6孔板中培养。在倒置显微镜下连续观察细胞的形态变化及贴壁率，甲苯胺蓝染色、I型胶原免疫组化染色进行细胞鉴定，测定其生长曲线。结果原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞，7天贴壁细胞逐渐增多，第10天时细胞彼此相连，铺满平底，细胞以梭形为主，部分多角形。传代后12小时贴壁率达90％，大部分为多角形，4～5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒，胶原免疫纽化染色胞浆内可见棕黄色颗粒。结论酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力，可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。     

Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain

Summary The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture. Direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity were used to identify the capillary endothelial cells.Acknowledgments. We thank Dr J. Gripenberg for technical assistance. This research was supported by Finnish Cultural Foundation and carried out during the tenure of a fellowship provided for F. J. from the Finnish-Hungarian Cultural Exchange Program.     

Partial purification of an endogenous elicitor from suspension-cultured cells of red bean,Vigna angularis

Brief irradiation with a germicidal UV lamp of cells of red bean,Vigna angularis, cultured in suspension in a quartz flask caused the release into the culture medium of an endogenous substance with elicitor activity, as well as the accumulation of isoflavone glucoside stress metabolites in the cells. The active compound was fractionated using phenylalanine ammonia lyase (PAL)-inducing activity in fresh cells as a marker. The elicitor active principle appears to be a low molecular weight (<2000 MW) water-soluble acidic oligosaccharide.     

Histochemical localization of alkaline and acid phosphatase activities in the skin ofMystus (Mystus) vittatus Bl. (Siluriformes)

Summary An attempt has been made to localize alkaline and acid phosphatase activities in the skin ofMystus vittatus by using histochemical techniques. The alkaline phosphatase activity is found in metabolically active cells such as basal columnar cells, mucous cells and polygonal support cells. The acid phosphatase activity is intense in the outermost squamous support cells and in the basal columnar cells. These activities have been correlated with some physiological functions of the epidermis.Acknowledgment. We are thankful to P. Vishwanatham, Government College, Mhow, and Dr R.S. Shrivastava, Holkar Science College, Indore, for providing laboratory facilities and to the Council of Scientific and Industrial Research, New Delhi, for a fellowship for M.S.     

Effect of halogenmethylenebisphosphonates on bone cells in culture and on bone resorption in vivo

Dihalogenmethylenebisphosphonates increase alkaline phosphatase activity and fatty acid oxidation in calvaria cells in culture (Cl2MBP greater than Br2MBP approximately equal to F2MBP). The monohalogen ClMBP and the non-halogenated analogues are less active on phosphatase and inactive on or inhibitory towards fatty acid oxidation. The three dihalogenbisphosphonates and ClMBP inhibit bone resorption in vivo, Cl2MBP most strongly.     

Effect of epidermal growth factor on growth and maturation of fetal and neonatal rat small intestine in organ culture

Small intestinal explants from pre- and post-natal rats were incubated in an organ culture system in the absence and presence of epidermal growth factor (EGF). The rate of synthesis of small intestinal DNA and protein as well as the activity of lactase and alkaline phosphatase increased rapidly between 17 and 20-day gestational age, whereafter they declined. The maximal incorporation of 3H-thymidine and 14C-alanine into DNA and protein, respectively, was significantly stimulated by EGF (100 ng/ml). EGF had no effect on the activity of either lactase or alkaline phosphatase in the small intestinal explants.     

Effect of epidermal growth factor on growth and maturation of fetal and neonatal rat small intestine in organ culture

Summary Small intestinal explants from pre- and post-natal rats were incubated in an organ culture system in the absence and presence of epidermal growth factor (EGF). The rate of synthesis of small intestinal DNA and protein as well as the activity of lactase and alkaline phosphatase increased rapidly between 17 and 20-day gestational age, whereafter they declined. The maximal incorporation of³H-thymidine and¹⁴C-alanine into DNA and protein, respectively, was significantly stimulated by EGF (100 ng/ml). EGF had no effect on the activity of either lactase or alkaline phosphatase in the small intestinal explants.Acknowledgment. The project was supported by grants from the Veterans Administration Research Service. The authors wish to thank Dr. M. C. Geokas, Chief, Department of Medicine, Veterans Administration Medical Center, Martinez, CA, for providing us with excellent laboratory facilities and for his encouragement in this study.     

Histochemical response of alkaline phosphatase activity during the healing of cutaneous wounds in a cat-fish

Summary The activity of alkaline phosphatase in the various tissue components of the regenerating skin of a cat-fish has been studied. A marked increase in alkaline phosphatase activity in the cells of migrating epithelium has been correlated with their highly active state. High alkaline phosphatase in the basal cells after 2 days has been found to have played an important role in cell multiplication and differention. The functional significance of this enzyme is discussed in relation to the granulation tissue formation.     

Über DPNH-Diaphorase und alkalische Phosphatase bei Präimplantationsstadien des Goldhamsters (Mesocricetus auratus Waterh.)

Summary The activity of the DPNH-diaphorase and the alkaline phosphatase were examined in golden-hamster eggs prior to fixation. The reactions for DPNH-diaphorase as well as for alkaline phosphatase were found to be positive already in the undivided egg and in early cleavage stages. The reaction products of both enzyme determinations showed a histophotometrically measurable polar distribution in the cytoplasm, in the one-cell stage as well as in the individual blastomeres of the examined early cleavage stages.     

Die alkalische Phosphatase-Aktivität des Ratten-serums nach anaphylaktoidem Schock

Summary At seven different intervals after the elicitation of anaphylactoid shock (compound 48/80), determinations of alkaline phosphatase (AP) activity were carried out in rat serum. No significant increase could be detected. These findings exclude extrarenal factors as the cause for increased AP-activity in urine following anaphylactoid shock.     

Alkaline phosphatase isozymes in cultured human cancer cells

Alkaline phosphatase, an ubiquitous enzyme is known to exist in several isozymic forms. At least three different isozymes have now been identified in humans. Alkaline phosphatase isozymes are among the substances synthesized ectopically by a variety of human tumors and many continuous cell lines derived from different cancers have retained the capacity to produce these membrane-located glycoproteins. This paper reviews the identification of alkaline phosphatase isozymes in cultured tumor cells and relates these findings with recent developments concerning these cell membrane located glycoproteins.     

Alkaline phosphatase isozymes in cultured human cancer cells

Summary Alkaline phosphatase, an ubiquitous enzyme is known to exist in several isozymic forms. At least three different isozymes have now been identified in humans. Alkaline phosphatase isozymes are among the substances synthesized ectopically by a variety of human tumors and many continuous cell lines derived from different cancers have retained the capacity to produce these membrane-located glycoproteins. This paper reviews the identification of alkaline phosphatase isozymes in cultured tumor cells and relates these finding with recent developments concerning these cell membrane located glycoproteins.     

Differential developmental pattern of acid and alkaline phytase and phosphatase activities in rat intestine

Summary Rat intestine was found to show a distinct acid phytase activity (pH optimum 4.7) in addition to that of an alkaline phytase (pH optimum 8.0). The phytase and phosphatase activities were found to differ in their developmental pattern and responded differentially to some inhibitors. Thus the two activities seem to be due to two independent enzymes and are not the activity of a nonspecific phosphatase as has been suspected formerly.     

Differential developmental pattern of acid and alkaline phytase and phosphatase activities in rat intestine.

Rat intestine was found to show a distinct acid phytase activity (pH optimum 4.7) in addition to that of an alkaline phytase (pH optimum 8.0). The phytase and phosphatase activities were found to differ in their developmental pattern and responded differentially to some inhibitors. Thus the two activities seem to be due to two independent enzymes and are not the activity of a nonspecific phosphatase as has been suspected formerly.     

Phosphoprotein phosphatase activity of bovine intestinal alkaline phosphatase

The phosphoprotein phosphatase activity of a commercial preparation of bovine intestinal alkaline phosphatase (EC 3.1.3.1) was examined using phosvitin and dentine phosphoprotein as substrates. Over 90% and 70% of the phosphorus from dentine phosphoprotein and phosvitin were hydrolyzed in 2 h. The optimum pH of the enzyme for the dephosphorylation of phosvitin and dentine phosphoprotein was nearly 6. No protein phosphatase activity was observed when the alkaline phosphatases from bovine liver and pulp were investigated.     

Slice cultures of cerebellar,hippocampal and hypothalamic tissue

Summary Cerebellar, hippocampal and hypothalamic slices prepared from newborn and 7-day-old rats were cultured by means of the roller-tube technique. Identification of cells was made easier by the fact that at least part of the characteristic cytoarchitecture of the tissue was preserved in vitro. Cerebellar Purkinje cells and neurones of the deep cerebellar nuclei were recognized on the basis of their size, their location within the culture and their dendritic arborization. Pyramidal cells of all hippocampal subfields displayed their characteristic basal and apical dendritic trees with numerous spinous processes. Hippocampal granule cells gave rise to a monopolar dendritic arbor; their axons terminated in the dentate hilus and CA3 region. Golgi-like immuniperoxidase staining allowed localization of groups of neurophysin-positive neurones in slices prepared from the anterior hypothalamus. These neurones, bilaterally bordering the third ventricle, usually displayed a simple dendritic arborization and fine beaded axons.—Cultivation of brain slices prepared from young rats offers particular advantages in that the cultured cells are organized in an organotypic monolayer and individual living neurones may be directly visualized.Acknowledgments. I would like to thank Dr J. Zimmer and Dr M. Sofroniew for their valuable help with the Timms and immunoperoxidase staining techniques and Mr R. Schlichter and L. Wohlfart for illustrating the culture procedure. The excellent technical assistance of Ms L. Rietschin and E. Hoffmann is gratefully acknowledged.     

Retroviral-mediated gene therapy for the treatment of citrullinemia. Transfer and expression of argininosuccinate synthetase in human hematopoietic cells

Citrullinemia is a recessive genetic disease caused by a deficiency in argininosuccinate synthetase (AS). Retroviruses were used to transduce the human AS gene into cultured human cells. Using amphotropic viruses with high titer (>10⁶ cfu/ml), we were able to correct the defect in cultured fibroblasts from citrullinemic patients. Retroviral transduction of the human AS gene into human bone marrow cells was also studied. Co-cultivation was used to infect the cells and up to 80% of progenitor cells were found to be carrying and expressing the AS retrovirus after infection. When the infected cells were kept in culture, integration and expression of the retrovirus was observed. Retroviral sequences were present and expressed in the cultured bone marrow-derived cells for up to 10 weeks.     

Cellular and molecular mechanism of low-turnover osteopenia in the klotho-deficient mouse

The mouse homozygous for a disruption of the klotho locus (KL-/- or klotho mouse) exhibited multiple pathological conditions resembling human aging. We observed osteopenia in KL-/- mice with a low bone turnover, in which the decrease in bone formation exceeded the decrease in bone resorption and resulted in net bone loss. This pathophysiology resembles closely that of senile osteoporosis in humans. Osteoblastic cells from KL-/- mice proliferated normally in vitro; however, they showed much lower alkaline phosphatase activity and mineralized matrix formation than those from control mice. Cultured osteoclastic cells from KL-/- mice had normal resorbing activity and survival rate, but the differentiation of osteoclastic cells from their precursors was significantly disturbed: in the co-culture of osteoblastic cells and osteoclast precursor cells, the formation of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells was extremely poor only when osteoclast precursor cells originated from KL-/- mice independently of the origin of the osteoblastic cells. In addition, we found that osteoprotegerin a secreted factor which inhibits osteoclastogenesis, was up-regulated in KL-/- mice. We conclude that a defect in klotho gene expression leads to the independent impairment of osteoblast and osteoclast differentiation, which can be a cause of low-turnover osteoporosis.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Summary Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25–2.0 Mrads is optimal for cell growth.