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PubMed1994

A yeast TCP-1-like protein is required for actin function in vivo.

We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo.

Actins↗
PubMed2006

Differential recognition of citrate and a metal-citrate complex by the bacterial chemoreceptor Tcp.

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn(67), which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn(67) and allosteric switching.

Allosteric Regulation↗
PubMed1990

[3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in human frontal cortex: decreases in Alzheimer-type dementia.

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.

Alzheimer Disease↗
PubMed2004

An adaptive mechanism to guarantee the bandwidth fairness of TCP flows.

End-to-end TCP (transmission control protocol) congestion control can cause unfairness among multiple TCP connections with different RTT (Round Trip Time). The throughput of TCP connection is inversely proportional to its RTT. To resolve this problem, researchers have proposed many methods. The existing proposals for RTT-aware conditioner work well when congestion level is low. However, they over-protect long RTT flows and starve short RTT flows when congestion level is high. Due to this reason, an improved method based on adaptive thought is proposed. According to the congestion level of networks, the mechanism can adaptively adjust the degree of the protection to long RTT flows. Extensive simulation experiments showed that the proposed mechanism can guarantee the bandwidth fairness of TCP flows effectively and outperforms the existing methods.

Algorithms↗
PubMed1992

Tissue response to biphasic calcium phosphate ceramic with different ratios of HA/beta TCP in periodontal osseous defects.

The purpose of this study was to determine the optimal ratio of calcium hydroxyapatite (HA) to beta tricalcium phosphate (beta TCP) in a biphasic porous calcium phosphate (BCP) ceramic for effective repair of periodontal osseous defects. Defects were surgically produced in beagle dogs and made chronic for 4 months to simulate periodontal disease. Mucoperiosteal periodontal flaps were reflected, followed by osseous defect debridement and root planing. Specially prepared ceramic with different HA/beta TCP ratios were implanted into the prepared defects. The sites were allowed to heal for 6 months, animals were euthanized, and site-blocks were removed for histological study. During the follow-up phase, scaling and polishing were done once a month, and standardized probing attachment levels were recorded pre- and 6-months postoperatively. The Duncan's multiple range test showed that all the treatments produced statistically significant higher gain in probing attachment levels than the control group (0HA/0 beta TCP) (P < 0.05). Among the 7 "active" treatment groups, 2 (65/35 and 85/15) had significantly higher gain in probing attachment levels than those in 3 groups (50/50, 100/0, and 0/100) (P < 0.05). Histologically, higher HA ratio (but not 100% HA) showed accelerated new bone formation and new attachment levels. Based on histological results, the 85HA/15 beta TCP ratio appears to demonstrate greater gain in attachment level and bone regeneration in the treatment of periodontal osseous defects.

Alveolar Bone Loss↗
PubMed2000

Long-term sustained delivery of 3'-azido-2',3'-dideoxythymidine in vivo by means of HA and TCP delivery devices.

This study will attempt to determine the amount of circulating drug, when provided at sustained levels over an extended period of time, which provides efficacy and reduces the toxic side effects seen with the use of AZT in vivo. Two ceramic delivery systems were selected for this investigation namely tricalcium-phosphate (TCP) and hydroxyapatite (HA) ceramic implants. Three different dosages were selected (60, 90, 180 mg AZT). A total of 105 rats were used in this study and they were further divided into seven equal groups (n = 15 per group, 250-300 gm BW). Rats in groups I, II and III were implanted with TCP ceramic capsules containing 30, 60, and 90 mg AZT powder, respectively. Each rat in groups IV, V, and VI were implanted with HA capsules containing 30, 60, and 90 mg AZT powder, respectively. Rats in group VII were unimplanted and served as controls. The standard aseptic surgical techniques were performed in this investigation. Rats were anesthetized with a mixture of Zylazine/Ketamine, and their hind limbs shaved and scrubbed with providone iodine. The sterilized ceramics (gas/24 hours) were inserted under the skin using standard surgical techniques. After implantation, the site was sealed with wound clips, and the animals were injected with 0.1 ml of 200,000 units of Penicillin. At the end of 2, 4, and 6 weeks after implantation, five rats from each group were euthanized and the ceramic capsules, reproductive, and vital organs were removed and examined by following standard laboratory protocols. Results obtained from this study revealed that the rates of AZT released from TCP ceramic implants (30 mg = 2.38 +/- 0.23 ng/ml, 60 = 4.64 +/- 1.03 ng/ml, and 90 mg = 11.92 +/- 2.36 ng/ml serum AZT) were significantly higher than the rates of AZT released from HA ceramic implants (30 mg = 0.84 +/- 0.05 ng/ml, 60 = 2.40 +/- 0.83 ng/ml, and 90 mg = 6.41 +/- 1.24 ng/ml serum AZT). Data obtained from this investigation suggest that: (I) TCP and HA ceramic implants can be used effectively to deliver AZT in amounts capable of eliciting physiological responses in vivo, and (II) large fluctuations of AZT concentrations in the blood stream and tissues due to the conventional routes of administration could be eliminated by using ceramic sustained delivery system.

Animals↗
PubMed2002

Effect of TCP sintering temperatures on MRC-5 fibroblast proliferation and viability.

Calcium/Phosphate ceramics have recently been used as orthopaedic implants, drug delivery systems, and models for studies on bone remodeling. Although their use has been very successful, problems such as aseptic loosening, coagulation, fibrous tissue formation, and inflammation have been noted. The objective of the study was to investigate the effects of tri-calcium Phosphate (TCP) ceramics sintered at different temperatures on the proliferation of MRC-5 fibroblasts. This was accomplished by light microscopy morphologic analysis, measurements of MDA (a measure of cell damage) and total protein, and cell counts. The experimental design consisted of four experimental groups and one control group. 96 TCP ceramics were divided into 4 groups of 24, with each group being assigned a different sintering temperature of 800, 1000, 1200 or 1400 degrees celcius. Each temperature group was then further divided into 8 ceramics per phase of 24, 48 and 72 hours. All data were analyzed using Sigma Stat software. The data collected from this study provided useful information pertaining to the optimum manufacturing conditions that produce high proliferation rates with minimal cellular damage. At 72 hours (the phase simulating chronic conditions), cell counts and MDA analysis showed that the cells incubated with the TCP sintered at 1200 degrees C had the greatest proliferation and least damage when compared to the other groups. Morphological, MDA and protein analysis showed the cells incubated with the 1400 degrees C TCP capsules to have undergone lysis or other extreme cellular alterations. Overall, the data collected revealed a marked alteration in cellular function and morphology over the range of sintering temperatures that were tested.

Biocompatible Materials↗
PubMed1990

Sequence and structural homology between a mouse T-complex protein TCP-1 and the 'chaperonin' family of bacterial (GroEL, 60-65 kDa heat shock antigen) and eukaryotic proteins.

A mammalian cytoplasmic protein TCP-1, encoded by a gene within the mouse t-complex, has been found to exhibit highly significant (p much less than 0.00001) sequence homology to the 'chaperonin' family of bacterial and eukaryotic proteins (viz. groEL protein of E. coli, rubisco subunit binding protein of plant chloroplasts, yeast hsp58 and mammalian P1 proteins and 60-65 kDa mycobacterial antigen). With the introduction of few gaps, the amino acid sequence of TCP-1 shows between 60-63% similarity (17-20% identical residues and 42-45% conserved substitutions) throughout its length to various chaperonin proteins, indicating a common evolutionary origin. The sequence data also suggest that in contrast to the endosymbiotic origin of mitochondrial and chloroplast chaperonins, the cytoplasmic TCP-1 may have directly descended from the common universal ancestor via eukaryotic lineage. The observed similarity between TCP-1 and the 60-65 kDa bacterial 'common antigen' is also of importance from the viewpoint of immune/autoimmune response.

Amino Acid Sequence↗
PubMed1996

The effect of HA, TCP and ALCAP bioceramic capsules on the viability of human monocyte and monocyte derived macrophages.

The relationship between various bioceramics used in surgical implantation and inflammatory cellular response has not been fully elucidated. The objective of this study was to investigate the effect of various biomedical ceramics such as tricalcium phosphate (TCP), hydroxyapatite (HA), and aluminum-calcium-phosphorous oxide (ALCAP) on the adherence and viability of human monocyte and monocyte derived macrophages in vitro. The monocytes were isolated from human peripheral blood and seeded at a density of 5 x 10(5) cells/well according to standard laboratory procedures. Cells were considered macrophages after remaining in culture for 24 hours. Cells were then plated in each microtiter well loaded with ceramic capsules (HA, TCP and ALCAP) and buffered control. At the end of 1, 2, 3, and 7 days the viability and cell number of monocyte or monocyte derived macrophages were determined using an established assay. Cell number was determined in control wells with known amounts of cell number, a standard curve was generated by plotting absorbance units versus cell number. Biochemical analysis was performed on the aliquots obtained from the experimental and control wells at the end of each phase of the investigation. The data from this experiment suggest that: (I) monocytes and macrophages are capable of adhering to the surface of HA, TCP and ALCAP in an in vitro environment for over a 7 day period. (II) Long term incubation of ceramic capsules with macrophages revealed that the cells experienced gradual disassociation phenomenon with a greater number of cell detachment seen in the ALCAP contained wells. (III) SEM analysis of representative capsules demonstrated that there is an increase in the number of micropores on the surface of the materials after contacting a cellular environment. This observation suggest that the material surface has been modified (TCP > HA = ALCAP). (IV) Biochemical analysis of aliquots at the end of each phase showed a significant change (P < 0.05) in the activity of catalase and superoxide dismutase (SOD). Information obtained from this study provided new insights on the interrelationship between bioceramics and the possible cell response during chronic inflammation at the site of implantation.

Aluminum Oxide↗
PubMed2004

A subclass of myosin XI is associated with mitochondria, plastids, and the molecular chaperone subunit TCP-1alpha in maize.

The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1alpha (TCP-1alpha) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1alpha isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1alpha co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1alpha for regulating folding or providing protection against denaturation.

Actins↗
PubMed1996

Analysis of chaperonin-containing TCP-1 subunits in the human keratinocyte two-dimensional protein database: further characterisation of antibodies to individual subunits.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two-dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP-1, also known as CCT alpha, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP-1, and a number of epitope-related proteins were identified.

Animals↗
PubMed1998

A Dictyostelium discoideum homologue to Tcp-1 is essential for growth and development.

Tcp-1 (t-complex polypeptide 1 gene) was first identified in the mouse as relevant for tail-less and embryonic lethal phenotypes. Since then, its homologous sequences have been isolated in several other species, and the yeast Tcp-1 has been shown to encode a molecular chaperon for actin and tubulin. In a random sample of genes expressed in the gamete of Dictyostelium discoideum (Dd), we encountered a sequence containg the TCP1 motifs. The complete ORF of the gene (DdTcp-1) showed more than 60% similarity to TCP-1 of several organisms, including human. DdTcp-1 was found to be expressed in both sexually mature and immature cells at the growth phase. Although the sexual process itself was not affected, antisense interference of this gene resulted in severe retardation of cell growth, leading to the complete cessation of division. In addition, the antisense transformants stopped asexual development at the finger stage. These results suggest an important function of DdTcp-1 in growth and development of this organism.

Amino Acid Sequence↗
PubMed1999

Healing of segmental bone defects in rats induced by a beta-TCP-MCPM cement combined with rhBMP-2.

A beta-tricalcium phosphate-monocalcium phosphate monohydrate (beta-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 x 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 microg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 microg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for beta-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 microg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the beta-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects.

Animals↗
PubMed1996

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Animals↗
PubMed1992

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

Adenosine Triphosphatases↗
PubMed1991

Plasma-sprayed coatings of tetracalciumphosphate, hydroxyl-apatite, and alpha-TCP on titanium alloy: an interface study.

In order to study the interaction of calcium phosphate coatings with bone tissue, coated titanium cylinders with a standard size were implanted in dog femora. Coatings were made by plasma spraying powders of hydroxylapatite, beta-whitlockite, and tetracalciumphosphate particles. The plasma spraying process turns beta-whitlockite into alpha-TCP. Bone bonding and bone formation were evaluated by mechanical push-out tests and histological observations. Hydroxylapatite and tetracalciumphosphate coatings show an interface strength after 3 months of implantation of 34.3 +/- 6.5 MPa and 26.8 +/- 3.9 MPa, respectively, while alpha-TCP and blanco titanium lead to an interface strength of 10.0 +/- 3.5 MPa and 9.7 +/- 1.3 MPa, respectively. Histological examinations revealed that hydroxylapatite and tetracalciumphosphate give rise to an excellent bone formation, while alpha-TCP and blanco titanium evoked remodeling and less bone contact.

Alloys↗
PubMed1983

[3H]TCP: a new tool with high affinity for the PCP receptor in rat brain.

PCP binding sites have previously been demonstrated in the central nervous system with [3H]PCP. We now describe the binding properties to rat brain membranes of [3H]TCP, a PCP derivative. It is very advantageous to use [3H]TCP instead of [3H]PCP for the 3 following reasons: (i) it has a better affinity (Kd = 7.4 nM) for PCP binding sites than PCP itself; (ii) it dissociates slowly from its binding sites (t 1/2 = 20 min); (iii) the non-specific binding component obtained with [3H]TCP is much lower than that found with [3H]PCP.

Animals↗
PubMed1992

TCP enhances the survival of human fetal spinal cord cells in culture.

Dissociated cell cultures were prepared from human spinal cords of 7-10-week-old fetuses. After 10 weeks progressive neuronal necrosis was observed in controls whereas N/1-(2-thienyl)cyclohexyl/piperidine (TCP) enhanced the survival time of the cells. After 21 weeks the number of gamma-aminobutyric acid (GABA)ergic and neuron specific enolase (NSE)-stained neurons was higher in the TCP-treated cultures than in controls. TCP appears to be a promising drug for long term survival of neurons.

Biomarkers↗