AIMTo detect the expression of a novel protein, hemangiopoietin (HAPO), in the human fetal liver at the protein level.

METHODSMonoclonal antibodies (moAbs) against HAPO were produced by traditional hybridoma technique. Their affinities were calculated from the results of non-competitive ELISA and the antibodies were purified by protein G affinity chromatography. Expression of HAPO in the liver was detected by SDS-PAGE and Western blot.

RESULTSFive strains of moAbs were screened out in total, among which three were IgG1 and the other two were IgM. Their light trains were all belonged to kappa. The relative affinities of the three IgG1 form moAbs were 3.06 x 10(9) mol/L, 6.07 x 10(8) mol/L and 1.71 x 10(10) mol/L respectively. After purification, the purity of the moAbs could reach more than 99%. HAPO expression was detected at the protein level in the human fetal liver, and the apparent molecular weight of the nature HAPO was very close to but a little higher than our recombinant one.

CONCLUSIONHAPO was expressed in the human fetal liver at the protein level.

Antibodies, Monoclonal ; Blotting, Western ; Fetus ; Humans ; Immunoblotting ; Liver ; embryology ; metabolism ; Proteoglycans ; metabolism

OBJECTIVETo demonstrate the expression of proteolysis induce factor(PIF) in the gastrointestinal(GI) cancer cachexia patients and evaluate its role in cancer cachexia.

METHODSExamination of PIF was performed in urine samples from 28 GI cancer cachexia patients, 13 GI cancer patients without cachexia, and 12 weight loss patients with benign disease. PIF was added to the mice cultured C2C12 muscle cells, then the protein kinase B(Akt) phosphorylation and morphological change were measured.

RESULTSThe positive rate of PIF in urine of 28 cancer cachexia patients was 53.6%(15/28). In the other two groups, no positive result was detected. PIF could successfully induce Akt phosphorylation, cell atrophy, metamorphosis, and death. The peak of this phosphorylation could be detected after half an hour of the initiation of PIF at a concentration of 4 nmol/L.

CONCLUSIONSPIF is specifically and highly expressed in GI cancer cachexia patients' urine. PIF can induce cancer cachexia possibly by activating Akt phosphorylation and inducing downstream proteolysis.

Animals ; Cachexia ; etiology ; metabolism ; Cell Line ; Gastrointestinal Neoplasms ; complications ; Humans ; Mice ; Proteoglycans ; Proteolysis

Experimental findings have suggested that the metabolic activities of articular cartilage can be influenced by mechanical stimuli. Our recent mathematical analysis predicted that cyclic compressive loading may create periods of intermittent negative hydrostatic pressure within the cartilage extracellular matrix. Therefore, we hypothesize that intermittent negative hydrostatic pressure, created in the cartilage extracellular matrix during dynamic compression, has a stimulative effect on the biosynthesis of chondrocytes. In order to test this hypothesis, the present study developed a custom designed negative pressure generator to subject a monolayer culture of chondrocytes to an intermittent negative pressure. It was found that the intermittent negative pressure produced a 40% increase in proteoglycan and a l7% increase in non-collagenous protein synthesis during the pressurization period(p (0.05). The collagenous protein synthesis was not affected by the intermittent negative pressure regimen used in this study. After the intermittent negative pressurization, the metabolic activities of the chondrocytes returned to normal(control level). The intermittent negative pressure also produced an increase in the mRNA signals for aggrecan. Therefore, we conclude that intermittent negative pressure may be one of the major mechanical stimulators of chondrocytes in articular cartilage during dynamic compression.
Aggrecans ; Cartilage ; Cartilage, Articular ; Chondrocytes* ; Collagen ; Extracellular Matrix ; Hydrostatic Pressure* ; Metabolism* ; Proteoglycans ; RNA, Messenger

Formation of dermal collagen fiber is a complicated and sequential process with the progressive assembly of collagen. Collagen monomers form stepped and orderly protofibrils through longitudinal displacement. Subsequently, protofibrils or protofibrils and collagen are bonded by covalent bonds to form orderly lamellar structure of collagen fibers. Then collagen fibers are tightly wound into coarse collagen fiber bundles by covalent crosslinking. Decorin is a multifunctional small leucine-rich proteoglycan. It can prevent the aggregation of protofibrils by binding to the specific site of collagen with its core protein, and adjusting the spacing between the protofibrils with its glycosaminoglycan chain. Thus, by effecting the formation of collagen fibers with regulation of collagen assembly, decorin may help prevent scar formation and even promote regeneration.
Collagen ; Decorin ; metabolism ; Extracellular Matrix ; Extracellular Matrix Proteins ; metabolism ; pharmacology ; Fibrillar Collagens ; metabolism ; ultrastructure ; Fibrin ; metabolism ; Humans ; Microfibrils ; metabolism ; Proteoglycans ; metabolism ; pharmacology

OBJECTIVETo construct recombinant adenovirus expressing rat proteoglycan II (Ad-DCN) and to study its biological features.

METHODSRat DCN gene was inserted into an E1 and E3-substituted adenovirus shuttle plasmid. And this shuttle plasmid including DCN was recombined with AdEasy-1 in BJ5183-AD-1 electroporation competent cells to form recombinant adenovirus-DCN plasmid, which was further transfected into Ad293 cells to passage adenovirus. The control recombinant adenovirus, Ad-LacZ, was also constructed in the same way. After plaque forming trail and adjusted with PBS, Ad-DCN and Ad-LacZ were obtained with titer 1x10(9) pfu x ml(-1). PCR, Western blot and MTT analysis were used to detect the expression of DCN or the bioactivity of expressed DCN.

RESULTDCN was detected in Ad-DCN infected CHO cells by PCR and Western blot, but not in Ad-LacZ infected CHO cells. MTT analysis results showed that the supernatant from the culture of Ad-DCN infected CHO cells could abrogate the inhibitive effect of TGFbeta1 on proliferation of CCL-64 cells. The proliferation rate of TGFbeta1 + Ad-DCN treated cells was significantly higher than that of TGFbeta1 + Ad-lacz or TGFbeta1 treated cells [(0.5252 +/-0.04 compared with 0.2826 +/-0.02 or 0.2918 +/-0.02) OD, P <0.05] and lower than that of control cells [(0.9332 +/-0.08) OD, P <0.05].

CONCLUSIONThe constructed recombinant adenovirus can express biologically active decorin.

Adenoviridae ; genetics ; metabolism ; Animals ; Genetic Vectors ; Male ; Proteoglycans ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic

OBJECTIVETo observe the impacts of thermosensitive moxibustion (TSM) on the expressions of nitric oxide (NO), typeⅠdisintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), typeⅡcollagen and proteoglycan (PG) in the rabbit models of knee osteoarthritis (KOA) and explore the mechanism of TSM on KOA.

METHODSA total of 42 Japanese long-eared male rabbits were divided into a blank group (6 rabbits), a model group (6 rabbits), a moxibustion group (24 rabbits) and a sham-operation group (6 rabbits) according to the random number table. In the blank group, the rabbits were fed normally. In the model and moxibustion groups, the papain injection was given to establish KOA models. The rabbits in the sham-operation group were treated with the intracavity injection of 0.9% NaCl solution. The rabbits were forced to move for 30 min every day, continuously for 15 days during modeling. At the end of modeling, in the moxibustion group, moxibusiton was applied at "Dubi" (ST 35), once a day, 40 min each time, for 14 days totally. According to the temperature changes during moxibustion, the rabbits were divided into a TSM group and a non-TSM group. 6 rabbits were collected randomly from the two groups. The usual feeding was given in the blank group, the model group and the sham-operation group, without any intervention. The body mass and behavioristics changes were observed in each group. At the end of treatment, the nitrate reduction method was adopted to determine NO expression in the serum. The real-time PCR was adopted to determine the expressions of ADAMTS-4, typeⅡcollagen and PG in the cartilage.

RESULTS① After modeling, compared with the blank group, the body mass was all reduced and the Lequesne MG score was increased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). After intervention, compared with the blank group, the body mass was decreased and the Lequesne MG score was increased in the model and sham-operation groups (<0.05, <0.01). Compared with the model group, the body mass was increased and the lequesne MG score was decreased in the TSM, non-TSM, and sham-operation groups (<0.05, <0.01). Compared with the non-TSM group, the body mass in the TSM group was increased remarkably (<0.05), but the difference in Lequesne MG score was not statistically significant (>0.05). ② After intervention, compared with the blank group, the expressions of NO and ADAMTS-4 were all increased and the expressions of typeⅡcollagen and PG were decreased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the model group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the non-TSM group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group after intervention (all <0.05).

CONCLUSIONThe thermosensitive moxibustion alleviates the inflammatory reactions and protects the joint cartilage through inhibiting the expressions of NO and ADAMTS-4 to achieve the effects in the treatment of KOA.

ADAMTS4 Protein ; metabolism ; Animals ; Cartilage ; metabolism ; Collagen Type III ; metabolism ; Male ; Moxibustion ; Nitric Oxide ; blood ; Osteoarthritis, Knee ; therapy ; Proteoglycans ; metabolism ; Rabbits ; Random Allocation

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the feasibility of using cartilage link protein of hyaluronic acid (HA-CLP) for defining the tumor boundary in a mouse model of lung carcinoma.

METHODSLung carcinoma was induced in KM mice by chemical carcinogenesis. HA-CLP separated from bovine cartilage and purified by affinity chromatography was labeled with (125)I for autoradiography. Immunohistochemical analysis and Western blotting were used to examine the efficiency of HA-CLP in defining the boundaries of the lung tumors.

RESULTSWith autoradiography, the clearest image of lung cancer was obtained at 2 h. With immunohistochemical method, the tumor boundary was the most clearly displayed at 2 h when the strongest signals of HA-CLP was detected; Western blotting also showed the clearest bands of HA-CLP at 2 h.

CONCLUSIONHA-CLP has the immunogenicity of HABP, and can efficiently indicate lung tumor boundary in autoradiography and immunohistochemistry.

Animals ; Autoradiography ; methods ; Extracellular Matrix Proteins ; metabolism ; pharmacology ; Female ; Hyaluronic Acid ; metabolism ; Immunohistochemistry ; Iodine Radioisotopes ; Lung Neoplasms ; radiotherapy ; Male ; Mice ; Proteoglycans ; metabolism ; pharmacology ; Radiotherapy, Image-Guided ; methods

OBJECTIVETo evaluate the biological effect on the synthesis of the extracellular matrix (ECM) in the cultivation of adult degenerative nucleus pulposus cells using the stiring microcarrier system in vitro.

METHODSThirty-four specimens were collected after intervertebral fusion operations of the patients with intervertebral disc herniation diseases from September 2005 to May 2009. The specimens were then randomly allocated into 2 groups for in vitro cultivation: monolayer culture group and microcarrier culture group. On the exponential phase, SP-ABC immunohistochemical staining and Western blot quantitative analysis were conducted in the two groups to detect the collagen type I and II. Proteoglycan contents of two groups in different growth phases were detected with (35)S-sulfate incorporation assay.

RESULTThe expressions of collagen type I and II in microcarrier culture group were significantly higher than those in monolayer culture group: SP-ABC immunohistochemical staining (collagen type I: 32.5 ± 4.4 vs. 15.2 ± 1.2, t = 2.871, P < 0.01; collagen type II: 43.6 ± 4.1 vs. 23.1 ± 2.2, t = 2.375, P < 0.05); Western blot quantitative analysis (collagen type I: 0.62 ± 0.08 vs. 0.50 ± 0.06, t = 3.327, P < 0.01; collagen type II: 1.46 ± 0.08 vs. 0.86 ± 0.04, t = 2.453, P < 0.05). Nucleus pulposus cells cultivated in stiring microcarrier system showed significantly increased proteoglycan synthesis than monolayer culture group does on both exponential phase and stationary phase (exponential phase: 34 821 ± 312 vs. 21 046 ± 673, t = 2.134, P < 0.05; stationary phase: 45 134 ± 175 vs. 32 193 ± 713, t = 2.801, P < 0.01).

CONCLUSIONSThe expression of collagen type I, II and proteoglycan of adult degenerative nucleus pulposus cells are positive regulated by the stiring microcarrier system, which can be used in the mass amplification of the adult degenerative nucleus pulposus cells.

Adult ; Aged ; Cell Culture Techniques ; Collagen ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Humans ; Intervertebral Disc ; cytology ; Male ; Middle Aged ; Proteoglycans ; metabolism ; Random Allocation ; Young Adult

Deficits in the clearance of amyloid β protein (Aβ) by the peripheral system play a critical role in the pathogenesis of sporadic Alzheimer's disease (AD). Impaired uptake of Aβ by dysfunctional monocytes is deemed to be one of the major mechanisms underlying deficient peripheral Aβ clearance in AD. In the current study, flow cytometry and biochemical and behavioral techniques were applied to investigate the effects of polysaccharide krestin (PSK) on AD-related pathology in vitro and in vivo. We found that PSK, widely used in therapy for various cancers, has the potential to enhance Aβ uptake and intracellular processing by human monocytes in vitro. After administration of PSK by intraperitoneal injection, APP/PS1 mice performed better in behavioral tests, along with reduced Aβ deposition, neuroinflammation, neuronal loss, and tau hyperphosphorylation. These results suggest that PSK holds promise as a preventive agent for AD by strengthening the Aβ clearance by blood monocytes and alleviating AD-like pathology.
Alzheimer Disease/pathology* ; Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Cognition ; Disease Models, Animal ; Mice ; Mice, Transgenic ; Monocytes/pathology* ; Polysaccharides/therapeutic use* ; Proteoglycans