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Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

Amino Acid Sequence↗

Cloning, structure and mRNA expression of human Cctg, which encodes the chaperonin subunit CCT gamma.

We describe the cloning, DNA sequence analysis and mRNA expression analysis of human Cctg (HsCctg), a gene that encodes the gamma-subunit of the eukaryotic cytosolic 'chaperonin-containing TCP-1' (CCT). Partial clones representing the 3' region of HsCctg cDNA were isolated from a human kidney cDNA library, and the missing 5' region was amplified directly from human kidney cDNA. The Cctg mRNA transcript is expressed in numerous human and mouse tissues and, like Tcp-1/Ccta, Cctg mRNA is expressed at higher levels in mouse testis when compared with kidney and brain. Southern-blot analysis has also revealed the Cctg gene to be highly conserved in mouse, rat, sheep and frog. The 1901 bp HsCctg cDNA has a coding region of 1635 bp and encodes a predicted 60 kDa protein (544 amino acids). The predicted HsCCT gamma amino acid sequence shares a high degree of sequence similarity with gamma-subunits from the mouse Mus musculus (98% similarity), the yeast Saccharomyces cerevisiae (75% similarity) and the protozoan Tetrahymena pyriformis (76% similarity) as well as with other members of the TF55/TCP-1 family, such as human TCP-1/CCT alpha (55% similarity) and TCP-20/CCT zeta (54% similarity). HsCCT gamma also shares conserved domains previously identified in the TF55/TCP-1 family of chaperonins and more distantly related chaperonins such as GroEL and Hsp60.

Adult↗

T-complex polypeptide-1 interacts with the erythrocyte cytoskeleton in response to elevated temperatures.

Chaperonins are double ring complexes composed of highly conserved 60-kDa protein subunits that are divided into two subgroups. Group II chaperonins are found in archaea and the cytoplasm of eukarya and are believed to function like other chaperonins as part of a protein folding system. We report here that human erythrocytes contain the group II chaperonin T-complex polypeptide 1 (TCP-1) and that this complex translocates from the cytoplasm to the cytoskeleton in response to heat treatment in the absence of overt cell damage. Identification as TCP-1 was determined by immunodetection for TCP-1alpha and corroborated by mass spectroscopy peptide sequencing. Direct visualization by immunofluorescence confirmed peripherally localized TCP-1 in response to heat treatment. Temperatures ranging from 37-50 degrees C were demonstrated to have distinct kinetic profiles of induced translocation. Heat-induced binding was shown by Triton shell analysis to be specifically associated with the cytoskeletal proteins. Furthermore, the binding was reversible following removal of the stimulatory condition. A stabilizing process is hypothesized based on the known interactions of chaperonins.

Chaperonin Containing TCP-1↗

Epstein-Barr virus-encoded nuclear protein EBNA-3 interacts with the epsilon-subunit of the T-complex protein 1 chaperonin complex.

OBJECTIVE: To find cellular proteins that associate with EBNA-3 (also called EBNA-3A), one of the Epstein-Barr virus (EBV)-encoded growth transformation-associated nuclear proteins. METHODS: Screening human cDNA libraries in the yeast two-hybrid system and performing an analysis of interaction in vitro as well as in cell lysates. RESULTS: EBNA-3 binds to the epsilon subunit of the chaperonin containing T-complex protein 1 (epsilon-TCP-1) in the yeast two-hybrid system. The cDNA clone isolated from a human lymphocyte library was found to encode the middle and C-terminal part of epsilon-TCP-1. The interaction was confirmed by showing that a GST fusion protein specifically precipitated EBNA-3 from CV1 cells infected with recombinant vaccinia virus expressing EBNA-3. The interacting region was mapped to the putative apical domain of epsilon-TCP-1. CONCLUSIONS: This study shows that large, virus-encoded transforming proteins such as EBNA-3 may receive help for their initial folding by chaperonin complexes. The recognition of the chaperonin complex likely occurs through specific interaction with one of the subunits. We suggest that nascent EBNA-3 may recognize the TCP-1 complex by interacting with the apical region of the epsilon subunit.

Chaperonin Containing TCP-1↗

Bone-bonding behavior of plasma-sprayed coatings of BioglassR, AW-glass ceramic, and tricalcium phosphate on titanium alloy.

The bone-bonding behavior of three kinds of bioactive ceramics coated on titanium alloy by the plasma-spray technique was investigated. Titanium alloy (Ti-6A1-4V) coated with BioglassR (45S5), apatite-wollastonite containing glass ceramic (AW), or beta-tricalcium phosphate (TCP) was prepared, and rectangular specimens were implanted into the tibial bones of mature male rabbits, which were sacrificed 8 or 24 weeks after implantation. The tibiae containing the implants were dissected out and subjected to detachment tests to measure the failure load. The bone-implant interface was investigated by Giemsa surface staining, contact microradiography, and scanning electron microscopy-electron probe microanalysis (SEM-EPMA). Eight weeks after implantation, the failure loads for implants coated with BioglassR, AW, and TCP were 1.04 +/- 0.94, 2.03 +/- 1.17, and 3.91 +/- 1.51 kg, respectively, and 24 weeks after implantation, the respective failure loads were 2.72 +/- 1.33, 2.39 +/- 1.30, and 4.23 +/- 1.34 kg. Failure loads of AW- and TCP-coated implants did not increase significantly with time. After the detachment test, breakage of the coating layer was observed. Bioactive ceramics can act as stimulants that induce bonding between bone and metal implants. However, failure load of metal implants coated with the bioactive ceramics was lower than that of bulk AW or TCP. It appears impossible to obtain a higher failure load using a bioactive-ceramic coating on titanium alloy. Histologically, the coating layer was found to become detached from the metal implant and the bone tissue bonded to the coating layer. SEM-EPMA observation revealed breakage of the coating layer, although bonding between bone and the coating layer was evident. A Ca-P-rich layer was observed at the interface between bone and the AW coating, and a Ca-P-rich and a Si-rich layer were observed at the interface between bone and the BioglassR coating. For clinical application, it would seem better to use coated metal implants for short-term implantation. However, there is a possibility of breakage of the coating layer because of both dissolution of the bioactive ceramic and mechanical weakness at the interface between the coating layer and the metal implant.

Alloys↗

Biomechanical characterization of a biodegradable calcium phosphate hydraulic cement: a comparison with porous biphasic calcium phosphate ceramics.

Biomechanical properties of a biodegradable calcium phosphate hydraulic cement (CPHC) were tested with rabbits. The cement was composed of beta-tricalcium phosphate (beta-TCP), monocalcium phosphate monohydrate (MCPM), and calcium sulfate hemihydrate (CSH), beta-TCP-MCPM-CSH cement. Cylinders of 4.7 mm in diameter and 10 mm in length were put into bone cavities created in the distal epiphysis of femurs in rabbits. Cylinders of the same size of porous biphasic calcium phosphate ceramics (BCPC, 75% hydroxyapatite and 25% beta-TCP) were implanted as references. Two, 4, 12, and 16 weeks after the operation, the rabbits were sacrificed. Histomorphometry showed that the cement was resorbed, leaving only 7.67 +/- 1.81% of bone cavity after 12 weeks. Newly formed bone occupied 34.59 +/- 4.00% of the cavity. Cylindrical bone-material composites were cut out with a small dental burr. Compressive force was applied to the specimens and compressive strength, elastic modulus, and toughness were calculated. The same tests were performed on cylinders of normal bone from the same site, which served as controls. The compressive strength and the toughness of the cement-bone composite were higher than those of normal bone and porous ceramics 12 weeks after the operation (p < 0.05). At 16 weeks the compressive strength and the toughness returned to the normal bone values. The elastic modulus of the porous ceramic-bone composite was higher than the normal bone at 4, 12, and 16 weeks after surgery (p < 0.05). We found that the beta-TCP-MCPM-CSH cement is replaced by new bone and that the cement-new bone composite has similar or better mechanical properties than normal bone within 16 weeks. This study suggests the usefulness of a particular cement for filling bone defects or for temporary fixation of orthopedic implants.

Animals↗

Hydrophobicity, surface tension, and zeta potential measurements of glass-reinforced hydroxyapatite composites.

Wettability and zeta potential studies were performed to characterize the hydrophobicity, surface tension, and surface charge of P2O5-glass-reinforced hydroxyapatite composites. Quantitative phase analysis was performed by the Rietveld method using GSAS software applied to X-ray diffractograms. Surface charge was assessed by zeta potential measurements. Protein adsorption studies were performed using vitronectin. Contact angles and surface tensions variation with time were determined by the sessile and pendent drop techniques, respectively, using ADSA-P software. The highest (-18.1 mV) and lowest (-28.7 mV) values of zeta potential were found for hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), respectively, with composite materials presenting values in between. All studied bioceramic materials showed similar solid surface tension. For HA and beta-TCP, solid surface tensions of 46.7 and 45.3 mJ/m2, respectively, were obtained, while composites presented intermediate surface tension values. The dispersive component of surface tension was the predominant one for all materials studied. Adhesion work values between the vitronectin solution and HA and beta-TCP were found to be 79.8 and 88.0 mJ/m2, respectively, while the 4.0 wt % glass composites showed slightly lower values than the 2.5 wt % ones. The presence of beta-TCP influenced surface charge, hydrophobicity, and protein adsorption of the glass-reinforced HA composites, and therefore indirectly affected cell-biomaterial interactions.

Adsorption↗

Bone growth on and resorption of calcium phosphate coatings obtained by pulsed laser deposition.

Three different calcium phosphate coatings of crystalline hydroxyapatite (HA), alpha- and beta-tricalcium phosphate (alpha+beta-TCP), or amorphous calcium phosphate (ACP) obtained by pulsed laser deposition on Ti-6Al-4V were incubated in a potentially osteogenic primary cell culture (rat bone marrow) in order to evaluate the amount and mode of mineralized bone matrix formation after 2 weeks with special emphasis on the type of interfacial structure that was created. Evaluation techniques included fluorescence labeling and scanning electron microscopy. The resistance to cellular resorption by osteoclasts was also studied. Bone matrix delaminated from the ACP coatings, while it remained on the HA and the alpha+beta-TCP coatings even after fracturing. A cementlike line was seen as the immediate contiguous interface with the nondegrading dense HA surface and with the surface of the remaining porous beta-TCP coating. Highly dense and crystalline HA coatings do not dissolve but are capable of establishing a strong bond with the bone matrix grown on top. Chemical and mechanical bonding were considered in this case. Cellular resorption was practically not observed on the HA coatings, but it was observed on the alpha+beta-TCP coatings. Resorption took place as dissolution that was due to the acidic microenvironment.

Alloys↗

Dissolution of dense carbonate apatite subcutaneously implanted in Wistar rats.

Hydroxyapatite (HA) is used as a biomaterial in orthopedic applications because it is similar in composition to bone mineral; however, carbonate apatite (CHA) is closer in chemical composition to bone mineral because bone mineral contains significant amounts of carbonate, yet there have been few reports comparing biological responses to HA and CHA. It is generally agreed that bone forms a bond of some kind to HA, and there is conflicting evidence as to whether HA is resorbed in vivo or not. However, comparative reports generally agree that beta-tricalcium phosphate (beta-TCP) is removed faster than HA from an implant site by an undetermined combination of resorption and/or dissolution. The work reported here attempts to provide the first directly comparable subcutaneous dissolution data for dense sintered HA, beta-TCP, and CHA in rats. The weight losses of HA, beta-TCP, and 3.2 wt % CHA were approximately 60, 520, and 320 mg m(-2) day(-1), respectively. Histological sections did not show any evidence of giant cells, and all of the samples were encapsulated with fibrous tissue. beta-TCP and (to a lesser degree) CHA were found to be resistant to dissolution around the edges of the sample. An X-ray analysis did not indicate that any phase transformation had occurred in the dissolution resistant region.

Animals↗

Evaluation of calcium phosphates and experimental calcium phosphate bone cements using osteogenic cultures.

In this study, rat bone marrow cells (RBM) were used to evaluate two biodegradable calcium phosphate bone cements and bioactive calcium phosphate ceramics. The substances investigated were: two novel calcium phosphate cements, Biocement F and Biocement H, tricalcium phosphate (TCP), surface-modified alpha-tricalcium phosphate [TCP (s)] and a rapid resorbable calcium phosphate ceramic consisting of CaKPO(4) (sample code R5). RBM cells were cultured on disc-shaped test substrates for 14 days. The culture medium was changed daily and also examined for calcium, phosphate, and potassium concentrations. Specimens were evaluated using light microscopy, and morphometry of the cell-covered substrate surface, scanning electron microscopy, and energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. Areas of mineralization were identified by tetracyline labeling. Except for R 5, rat bone-marrow cells attached and grew on all substrate surfaces. Of the different calcium phosphate materials tested, TCP and TCP (s) facilitated osteoblast growth and extracellular matrix elaboration to the highest degree, followed by Biocements H and F. The inhibition of cell growth encountered with R 5 seems to be related to its high phosphate and potassium ion release.

Absorbable Implants↗

Preparation and compressive strength of alpha-tricalcium phosphate/gelatin gel composite cement.

A novel alpha-tricalcium phosphate (TCP) and gelatin gel composite cement was prepared, and the effects of gelatin content, liquid/powder ratio, setting time, and additives (rod-like hydroxyapatite and CaTiO3 particles) on the microstructure and compressive strength of the setting product were investigated. Addition of gelatin gel to alpha-TCP cement resulted in the formation of a porous solid possessing pores of 20-100 microm in diameter whose pore diameter increased with increasing gelatin gel content. The compressive strength of alpha-TCP cement after 1 week increased from 9.0 to 14.1 MPa with increasing gelatin gel content up to 5 wt % and thereafter decreased. The compressive strength of the cement containing 5 wt % gelatin gel increased with time up to 35 MPa after 1 month whereas without gelatin gel it was approximately 20 MPa. Dispersion of 5 wt % of rod-like hydroxyapatite and CaTiO3 powders with alpha-TCP cement containing 5 wt % gelatin gel increased the compressive strength after 1 week from 14.1 to 31.3 and 34.8 MPa, respectively.

Bone Cements↗

Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering.

Chitosan scaffolds reinforced by beta-tricalcium phosphate (beta-TCP) and calcium phosphate invert glass were fabricated with a low-cost, bioclean freeze-drying technique via thermally induced phase separation. The microstructure, mechanical performance, biodegradation, and bioactivity of the scaffolds were studied. The composite scaffolds were macroporous, and the pore structures of the scaffolds with beta-TCP and the glass appeared very different. Both the compressive modulus and yield strength of the scaffolds were greatly improved, and reinforced microstructures were achieved. The bioactivity tests showed a continuous decrease in both Ca and P concentrations of a simulated body fluid (SBF) after the scaffolds with beta-TCP were immersed in the SBF for more than 20 h, which suggests that an apatite layer might be formed on the scaffolds. However, the same was not observed for the pure chitosan scaffolds or the scaffolds incorporated with the glass. This was further confirmed by micrographs from scanning electron microscopy. This study suggests that the desirable pore structure, biodegradation rate, and bioactivity of the composite scaffolds might be achieved through controlling the ratio of chitosan and calcium phosphates or beta-TCP and the glass.

Biocompatible Materials↗

Dendritic architecture of rat somatosensory thalamocortical projection neurons.

This study examines dendrites from physiologically characterized and intracellularly labelled thalamocortical projection (TCP) neurons from the rat ventrobasal complex (VB) and posterior nucleus (POm). The goals were to provide quantitative descriptions of TCP neuron dendrites, examine underlying design principles of dendritic morphology, and determine correlations between dendritic size parameters. Forty-four dendrites from seven VB neurons and 21 dendrites from three POm TCP neurons that responded to low-threshold mechanical stimuli were reconstructed and quantitatively analyzed at the light microscopic level. The dendritic architecture of the neurons was remarkably similar in most parameters studied, including the percentage of dichotomous branching, contribution of terminal branches to total dendritic length, and branching symmetry. There was a positive correlation between stem dendrite diameter and the length of the entire dendrite arbor, making it possible to estimate the total length of a dendritic arbor by measuring the stem dendrite diameter. The correlations of the VB and POm dendrites had different slopes. The path distance (the distance from the soma to a dendritic end point) of individual dendrites showed only a small variation with large differences in the total dendritic length of an arbor. The constant diameter of distal dendrites shows that dendrite diameter is a poor predictor of synaptic location on the dendritic tree. Although the morphology of neurons and their individual dendrites varied considerably in overall size and qualitative appearance, when examined qualitatively, many aspects of dendritic structure were similar within and between groups. We suggest that the rat somatosensory TCP neurons have a stereotyped dendritic architecture and present data which provide a base for future comparative, developmental, and plasticity studies.

Animals↗

The human homologue of the mouse t-complex gene, TCP1, is located on chromosome 6 but is not near the HLA region.

Southern blot analysis indicates that there are four sequences in the human genome related to the mouse t-complex gene Tcp-1. All four genes were cloned and partial sequencing showed that one of them was a functional gene, and the other three were pseudogenes. The human sequences were all approximately 90% related to each other and 82-89% related to the mouse Tcp-1a sequence. Human TCP1 cDNA clones from both fibrosarcoma and B cell lines confirmed that there was a single expressed gene. mRNA transcripts of different sizes were accounted for by two different polyadenylation signals. The human TCP1 gene shared some amino acid substitutions with the mouse t-complex allele (Tcp-1a) which were not found in Tcp-1b. The functional human TCP1 gene was mapped, using a panel of somatic cell hybrids, as well as in situ analysis, to the long arm of chromosome 6 at 6q23-qter and thus is not closely linked to the HLA complex on the short arm. For this reason and others it is unlikely that there is a human equivalent of the mouse t-complex.

Amino Acid Sequence↗

Resorbability and solubility of zinc-containing tricalcium phosphate.

Using zinc-containing tricalcium phosphate (ZnTCP) as the zinc carrier for zinc-releasing calcium phosphate ceramic implants promoted bone formation around the implants. Because no quantitative information was available on the equilibrium solubility and resorbability of ZnTCP, in vitro equilibrium solubility and in vivo resorbability of ZnTCP were determined and compared quantitatively in this study. The solubility of ZnTCP decreased with increasing zinc content. The negative logarithm of the solubility product (K(sp)) of ZnTCP was expressed as pK(sp) = 28.686 + 1.7414C - 0.42239C(2) + 0.063911C(3) - 0.0051037C(4) + 0.0001595C(5) in air, where C is the zinc content in ZnTCP (mol %). The solubility of ZnTCP containing a nontoxic level of zinc (<0.63 wt %) decreased to 52-92% of the solubility of pure tricalcium phosphate (TCP) in the pH range 5.0-7.4. However, the in vivo resorbed volume of ZnTCP containing the same amount of zinc was much lower than that expected from the in vitro solubility, becoming as low as 26-20% of that of TCP. Cellular resorption of TCP is substantially a process of dissolution in a fluid with an acidic pH that is maintained by the activities of cells. Therefore, the reduction of the resorbability of ZnTCP could be attributable principally to its lowered cellular activation property relative to that associated with pure TCP.

Algorithms↗

Enhanced ingrowth of porous-coated CoCr implants plasma-sprayed with tricalcium phosphate.

Tricalcium phosphate (TCP) is an osteo-conductive bioceramic which, when applied to a porous-coated prosthesis, may enhance osseous ingrowth and mechanical stability. TCP plasma-sprayed and unsprayed porous-coated tibial intramedullary rods were bilaterally implanted in seven adult rabbits. All rabbits were killed at 12 weeks. Pull-out tests were performed on 4 rabbits while all were evaluated histologically for osseous response and adverse tissue reaction. TCP-sprayed implants showed significantly greater osseous ingrowth in comparison to unsprayed implants. Neither implant type exhibited adverse tissue reactions. Average pull-out strengths were 69 lb for treated rods and 72 lb for controls (p greater than 0.05); quality of fit for all pull-out specimens except one was deemed poor. We conclude that plasma-sprayed TCP enhances osseous ingrowth into porous-coated devices. However, our data further suggest that enhanced ingrowth may not always lead to enhanced fixation.

Alloys↗

Apatite formation on three kinds of bioactive material at an early stage in vivo: a comparative study by transmission electron microscopy.

Apatite formation on the surface of three kinds of bioactive material at an early stage after implantation in bone was studied using transmission electron microscopy (TEM). The materials were apatite- and wollastonite-containing glass-ceramic (A-W GC) as a surface-active glass-ceramic, dense sintered hydroxyapatite (HA) as a surface-active ceramic, and dense sintered beta-tricalcium phosphate (beta-TCP) as a resorbable ceramic. Particles of these materials, ranging from 100-300 microns in diameter, were implanted into rat tibiae, and specimens were prepared at 3, 7, 10, and 14 days after implantation. For A-W GC, dissolution of the glassy and probably wollastonite phase was observed in the surface region on and after the third day, and a collagen-free thin apatite layer on the surface of the material was evident on and after the seventh day. This apatite layer was observed before the mineralization of the surrounding bone matrix and was sometimes evident even where the material bordered on the bone marrow. On and after the tenth day, the surrounding bone matrix calcified and A-W GC-bone bonding through an apatite layer was completed. For HA, a mineralized collagen-free layer was observed on the surface of the ceramic on and after the tenth day. This layer was always present near calcifying bone and it was difficult to distinguish from immature bone. For beta-TCP, such a surface mineralized layer was rarely evident, even just before bone-ceramic contact, and finally the bone bonded to beta-TCP directly. Cell-mediated degradation of beta-TCP was frequently observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Osteoclastic resorption of calcium phosphates is potentiated in postosteogenic culture conditions.

Cell-mediated resorption of densely sintered hydroxyapatite (HA1250), tricalcium phosphate (TCP), and 600 degrees or 900 degrees C calcined hydroxyapatite (HA600 and HA900, respectively), was investigated by using two culture systems. The first was an osteoclastic cell culture, and the second was a two-stage culture that was composed of a bonelike tissue formation on the substrata in the first stage and its subsequent resorption by osteoclasts in the second stage. Neither of the materials showed resorption or surface alterations in the osteoclastic cell culture, except for some limited phagocytotic activity on HA600 and HA900. In the two-stage culture, production of mineralized extracellular matrix was only observed on HA1250 and TCP, and its subsequent resorption by osteoclastlike cells was evident. Small and occasionally larger tartrate-resistant acid phosphatase positive cells produced 20-150 microns diameter resorption pits in both the mineralized extracellular matrix on HA1250 and TCP and the surfaces of HA600 and HA900. Resorption of the mineralized extracellular matrix on TCP also resulted in degradation of the underlying ceramic surface, mainly initiating from intergrain boundaries, whereas the surface of HA1250 remained unaltered. The results of this study clearly demonstrate that osteoclastic resorption of calcium phosphates is potentiated in postosteogenic culture conditions. A possible role for bone matrix constituents in cell-mediated resorption is hypothesized, whereas the occurrence of resorption seems to be mainly governed by the combined effects of material characteristics such as grain size and crystal structure.

Acid Phosphatase↗