The Linkage Region in the Polypeptide of Pig Costal Cartilage Proteoglycan

Proteoglycan from pig costal cartilage and fragments obtained by proteolytic digestion were characterized by equilibrium ultracentrifugation and amino acid analysis.

The proteoglycan extractable in 4 M guanidinium chloride yielded, after proteolytic digestion with trypsin and chymotrypsin, a chondroitin sulfate peptide containing four chains of polysaccharide. The unextractable residue yielded chondroitin sulfate peptide containing only two chains. The amino acid composition indicated a fairly uniform spacing between all four chains with an average of eight amino acid residues between the serine residues involved in linkage.

Following the alkaline sulfite elimination-addition reaction, free peptide was isolated and found to contain one unsubstituted serine residue for every two linked glycosidically. Glycine and glutamic acid were the only two amino acids sufficiently abundant to be part of an invariant sequence near to serine residues destined to be glycosy-lated. The linkage region of the polypeptide also contains some substituted serine residues which do not carry a full chondroitin sulfate chain.     

Decorin Expression, Straw-like Structure, and Differentiation of Human Costal Cartilage

Costal cartilage is much understudied compared with the load-bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken and pigeon chests, respectively. A lack of understanding of the ultrastructural and molecular biology of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. This study analyzed the structure of marginal human costal cartilage (ribs 6-10) through scanning electron and atomic force microscopes and identified the presence of straw-like structures running longitudinally. We also demonstrated that chondrocytes tend to occur singly or as doublets and that centrally located cells produce high levels of aggrecan compared with more peripherally located cells measured using immunohistochemistry. Gene expression from mRNA extracted from cartilage showed high levels of decorin expression, likely associated with the large, complex tubular structures running through this cartilage type. COL2A1, ACAN, and TIMP1 also showed higher levels of expression compared with ACTB. Analysis of gene expression ratios demonstrate that costal cartilage is under differentiated compared with published ratios for articular cartilage, likely due to the vastly different biomechanical environments of each cartilage type. Further studies need to establish whether findings described here from the costal margins are significantly different than the cartilage of the "true ribs" and how these values change with age.     

Regenerative Potential of Mandibular Condyle Cartilage and Bone Cells Compared to Costal Cartilage Cells When Seeded in Novel Gelatin Based Hydrogels

The field of temporomandibular joint (TMJ) condyle regeneration is hampered by a limited understanding of the phenotype and regeneration potential of cells in mandibular condyle cartilage. It has been shown that chondrocytes derived from hyaline and costal cartilage exhibit a greater chondro-regenerative potential in vitro than those from mandibular condylar cartilage. However, our recent in vivo studies suggest that mandibular condyle cartilage cells do have the potential for cartilage regeneration in osteochondral defects, but that bone regeneration is inadequate. The objective of this study was to determine the regeneration potential of cartilage and bone cells from goat mandibular condyles in two different photocrosslinkable hydrogel systems, PGH and methacrylated gelatin, compared to the well-studied costal chondrocytes. PGH is composed of methacrylated poly(ethylene glycol), gelatin, and heparin. Histology, biochemistry and unconfined compression testing was performed after 4 weeks of culture. For bone derived cells, histology showed that PGH inhibited mineralization, while gelatin supported it. For chondrocytes, costal chondrocytes had robust glycosaminoglycan (GAG) deposition in both PGH and gelatin, and compression properties on par with native condylar cartilage in gelatin. However, they showed signs of hypertrophy in gelatin but not PGH. Conversely, mandibular condyle cartilage chondrocytes only had high GAG deposition in gelatin but not in PGH. These appeared to remain dormant in PGH. These results show that mandibular condyle cartilage cells do have innate regeneration potential but that they are more sensitive to hydrogel material than costal cartilage cells.

     

Light and Electron Microscope Immunolocalization of Alpha-fetoprotein in Rat Liver Cells in vivo and in vitro

In neonatal rat liver, AFP is localized only in typical hepatocytes. Their lobular distribution changes throughout the neonatal period. Some AFP+ cells also contain albumin. Less than 5% of AFP+ cells incorporate ³H-thymidine (2-hour pulse). Injections of dexamethasone suppress AFP positivity but not albumin positivity nor ³H-thymidine incorporation. AFP is also localized in typical hepatocytes in newborn rat isolated liver cells or liver explants in culture. During the early phase of AFP induction in rats, by mDAB feeding, AFP is detected in cells smaller than the normal hepatocytes and preferentially situated in periportal areas of the liver.
Electron microscope immunoperoxidase localizations in newborn rat liver show that AFP is present on bound ribosomes, in the lumina of rough and smooth endoplasmic reticulum, and in the Golgi apparatus. Direct AFP measurement on isolated organelles confirmed this distribution. It indicates a synthesis and secretion pattern similar to that of albumin.     

Rat anterior pituitary cells in vitro can partly reconstruct in vivo topographic affinities

Hormone-producing cells in the rat anterior pituitary gland are not randomly distributed; rather, there are specific topographic affinities among five cell types (Noda et al., Acta Histochem. Cytochem. 2001;34:313-319). In this study we reconstructed these affinities, at least partially, in primary monolayer culture. Pituitary cells collected from adult male rats were enzymatically dispersed and cultured for 72 hr at a density of 1 x 10(5) cells/cm(2). We double-immunostained cells using antibodies against hormones, and then used confocal laser microscopy to examine the ability of the cells to attach to each other. We also statistically analyzed the affinity of all combinations of the five types of hormone-producing cells. We observed clusters by electron microscopy to identify junctional complexes between the cells. Confocal laser microscopy indicated that the features and attachment patterns of hormone-producing cells in vivo were similar to those in vitro. Statistical analyses revealed that the rates at which the five types of hormone-producing cells attached to growth hormone (GH)-, prolactin (PRL), and luteinizing hormone (LH)-producing cells were unequal, which suggests there are specific topographic affinities. The specific rates of adrenocorticotropic hormone (ACTH)-producing cell attachment to GH cells, LH to PRL cells, and PRL to LH cells were high, whereas that of PRL attachment to PRL cells was low. In addition, the rates correlated with the data from our previous in vivo study. Ultrastructural observations revealed few junctional complexes between hormone-producing cells. These results indicate that anterior pituitary hormone-producing cells can attach to specific types of cells by means of specific and/or nonspecific adhesion factors, and can reconstruct the topographic nature of the pituitary gland.     

A Critical Role of Activin A in Maturation of Mouse Peritoneal Macrophages in vitro and in vivo

Activin A, a multifunctional factor of the transforming growth factor-beta （TGF-β） superfamily, is mainly produced by microglia and macrophages, and its anti-inflammatory and pro-inflammatory activities are both related to macrophage functions. However the direct effect of activin A on the rest macrophages in vivo remains unclear. In the present study, the results showed that activin A not only increased NO and IL-1β release, but also promoted phagocytic abilities of mouse peritoneal macrophages in vitro and in vivo, whereas it did not influence MHC Ⅰ and MHC Ⅱ expression. Moreover, we found that activin A significantly upregulated the expressions of CD14 and CD68, markers of mature macrophages, on the surface of macrophages in vitro and in vivo. These data suggest that activin A can induce primary macrophage maturation in vitro and in vivo, but may not trigger the acquired immune response via regulating expression of MHC molecules involved in presentation of antigen.     

Elevated Levels of Annexin I Protein in vitro and in vivo in Rat and Human Mammary Adenocarcinoma

Annexin I is a phospholipid and actin binding protein which may play a role in signal transduction to the cytoskeleton. Previous work reported the differential expression of annexin I mRNA among rat adenocarcinoma cell lines of various metastatic potential (MTLn3, MTLn2, MTC.4: highest to lowest, respectively) (Pencil et al. 1993, Breast Cancer Res Treat, 25, 165–74). This relationship has been extended to the protein level in in vitro cultures using Western blotting and flow cytometry. Annexin I protein levels in MTLn3 cells are 3- to 5-fold higher than in MTC.4 cells. The weakly metastatic cell line MTLn2 shows levels 1.5- to 2.5-fold higher than MTC.4. In vivo tumors were produced by injecting 1 × 10⁶ cells into mammary fat pads of syngeneic rats and necropsies were performed 40 days later. Semiquantitative immunohistochemical color image analysis was performed using a polyclonal rat annexin I specific antibody. Annexin I protein expression was highest in lung metastases of MTLn3, at 8-fold the levels observed in the MTC.4 primary tumors. MTLn3 cells in the primary tumor had an annexin I specific optical density 3-fold higher than that of cells in the MTC.4 primary tumor. MTLn2 primary tumors had an annexin I specific optical density 1.5- fold higher than MTC.4. A proportion of human mammary adenocarcinomas also have positive annexin I immunoreactivity, often with more uniform annexin I staining in the lymph node metastases. These results suggest that there may be survival advantages for nascent metastatic cells with high annexin I levels This revised version was published online in August 2006 with corrections to the Cover Date.     

大小鼠血液流变学11项指标的分析测定

本实验测定鼠类11项血液流变学正常值,并在不同鼠种、鼠龄、性别和取血方式间进行比较。结果提示:实验动物的血液流变学值受鼠种,鼠龄、性别及取血方式的影响,因此在同一实验中应保持上述几方面的一致。本实验特别提出取血方式对血液流变学测定结果的影响。并认为心脏取血由于简便、采血量多、较少受其它因素影响,因而在血液流变学实验中是一个较好的取血方法。     

Metabolism of Fracture Callus of Rat in vitro: II. Incorporation of 35S-Sulphate, 3H-Proline and 32P-Phosphate

The incorporation rate of ³⁵S-sulphate into the glycosaminoglycans of callus in vitro was at the highest on the 7th day after the fracture corresponding with the proliferation of cartilage. Protein synthesis per callus occurred at the maximal rate 4 days later (during the transition of cartilaginous to ossifying callus) as judged from the radioactivities of ³H-hydroxyproline and ³H-proline in collagen and other proteins. Radioactive phosphate accumulated maximally into the cartilaginous matrix on the 7th day and into the osseous matrix on the 21st day after the fracture. No differences in activities were detected between heated and intact slices indicating non-enzymatic uptake of phosphate ions from the medium.     

Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo

Human, rat, and mouse studies have demonstrated the existence of a population of adipose-derived adult stem (ADAS) cells that can undergo multilineage differentiation in vitro. However, it remains unclear whether these cells maintain their multilineage potential in vivo. The aim of this study was to examine the in vitro and in vivo characteristics and behavior of a potential population of murine ADAS (muADAS) cells isolated from the visceral fat of the abdominal cavity of C57BL/10J mice. We used flow cytometry to examine the cells' expression of CD29, CD31, CD45, CD34, CD44, CD144, CD146, Flk1, and Sca-1. The isolated cell population was CD45 negative, which precludes contamination by hematopoietic cells, but was partially positive for Sca-1 and CD34: 2 stem-cell markers. After induction in conditioned medium, the muADAS cells gained the ability to undergo adipogenic, osteogenic, chondrogenic, myogenic, and hematopoietic differentiation in vitro. The muADAS cells readily differentiated to form bone and cartilage in vivo for up to 24 weeks, but their ability to regenerate muscle or reconstitute bone marrow was found to be limited.     

Biosynthesis of Acetylcholine in Different Brain Regions in vivo Following Alternative Methods of Sacrifice by Microwave Irradiation

Endogenous acetylcholine and biotransformation of tritium-labelled choline (³H-CH) were studied in mouse brain regions following different methods of sacrifice, viz. dislocation of the spine (7 min until enzymes inactivated), whole body microwave irradiation (7 s) and irradiation of the head (0.25 s). The brain temperature was measured in different locations 10 to 60 s after irradiation. The slope of the temperature time curves indicated a brain temperature of about 85–90°C at the termination of exposure to both types of irradiation. Acetylcholinesterase (AChE) and choline acetyltransferase (CAT) were practically completely inactivated when measured one to two min after sacrifice. For turnover studies, mice were killed 1, 5, 10 or 20 min after i.v. injection of 15 nmol of ³H-Ch. The brains were dissected into 6 regions, extracted and analysed. No significant difference (except in cortex) in the amount of endogenous ACh was found when whole body irradiation was used in comparison to dislocation of the spine. However, the amount of ³H-acetylcholine CH-ACh) was much higher in the stri-atum, hippocampus and cortex, in particular. With the shorter inactivation time (head irradiation) endogenous ACh was markedly increased in the striatum, cortex, medulla, oblongata and midbrain. However, there was no further increase in the radioactive ACh. The difference regarding the post-mortem sensitivity of endogenous and radioactive ACh does not seem to have been due to methodological artifacts but rather suggests that they are handled differently by the brain tissue. Plots of the specific radioactivity (SA) of Ch and ACh us. time indicated fairly distinct precursor-product relationship in the different regions, when the animals were sacrificed by irradiation of the head.     

Expression of Keratinocyte Growth Factor and Its Receptor in Rat Tracheal Cartilage: Possible Involvement in Wound Healing of the Damaged Cartilage

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.     

Synthesis of Brain-Specific Acidic Proteins in Rat and Mouse Cerebral Slices

Optimal conditions for incorporation of radioactive amino acids into protein by slices of brain have been established. Protein synthesis continued for at least 4 h at 35°. The highest incorporation of amino acids into protein was obtained with 20–50 mg wet weight per ml incubated at pH 7.1. After incubation of 500 mg of slices acidic proteins were separated by saltfractionation and electrophoresis on 14% polyacrylamide gel. The most acidic band was shown to contain S-100 protein. Identification was solubility of the protein in 100% (NH₄)₂SO₄, electrophoretic mobility at pH 8.9, precipitation with specific antibodies, and 2-dimen-sional electrophoresis in polyacrylamide gel. The protein band was insoluble in 50 % methanol and contained between 0.06 and 0.2% of the radioactivity present in the total soluble proteins; while other well separated acidic proteins contained about 10 times the radioactivity of S-100 protein.     

Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth.     

大鼠和小鼠胚胎后肾移植的形态学研究

目的观察不同胚龄的大鼠和小鼠的后肾植入同种异体远交系成年宿主体内后的生长变化,探讨胚胎后肾移植的最佳胚龄及血管起源。方法应用光、电镜及免疫组织化学技术观察不同胚龄的后肾移植后,肾脏各部的发育情况及后肾内CD31+阳性细胞的分布。结果离体后肾移植10天后成熟的肾小体形成,胚龄16天的大鼠和胚龄13天的小鼠后肾移植10天后,排斥反应轻微;胚龄20天的大鼠后肾和胚龄14、16天的小鼠后肾移植10天后,出现明显排斥反应,且随着胚龄的增长排斥反应逐渐加重。CD31+阳性细胞分布在肾小体毛细血管内皮及部分皮质中的间质细胞。结论胚龄16天的大鼠和胚龄13天的小鼠后肾中均无成熟肾小体出现,是移植的最佳时间;后肾移植物内的毛细血管袢是由移植物的间质细胞分化而来的。