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Functional Characterization of the Oat (Avena sativa L.) TCP Transcription Factor AsTCP38 Reveals Its Role in Low-Nitrogen Stress Tolerance.

Nitrogen limitation restricts plant growth, development, and yield in crops and forage species. Although TCP transcription factors are implicated in diverse abiotic-stress responses, the functions of most TCP genes in oat remain unclear. Here, we cloned and characterized the AsTCP38 gene, which is 1215 bp long and encodes a 405-amino-acid protein. The predicted protein contains a conserved TCP domain and shares its highest sequence similarity with Arabidopsis thaliana (A. thaliana) AtTCP15. The AsTCP38 protein localized to the nucleus, and promoter analysis identified cis-elements associated with light, hormone, and stress responses. We generated AsTCP38-overexpressing A. thaliana and wheat plants and screened an oat leaf yeast cDNA library for candidate interacting proteins. In these heterologous overexpression lines, AsTCP38 overexpression was associated with greater abscisic acid (ABA) sensitivity and improved seedling growth under low-nitrogen conditions. Changes in antioxidant-enzyme activities, nitrogen-metabolism-related enzyme activities, and endogenous hormone contents were also observed. Together, these findings suggest that AsTCP38 may participate in low-nitrogen responses and provide a basis for further functional studies in oat. Direct regulatory targets and the contribution of AsTCP38 to low-nitrogen adaptation in oat remain to be established.

Avena↗

Quantitative analysis of the cellular components of the fibrous tissue matrix surrounding ALCAP, HA, and TCP bioceramics using adult male rats as a model.

This investigation was specifically an attempt to correlate the thickness of this fibrous capsule and the various histological components that are seen in the fibrous capsule surrounding ALCAP, HA, and TCP ceramics at the S/C and I/P implantation sites in order to further elucidate the mechanisms of tissue-implant interaction. Eighteen male albino rats were randomly divided into three experimental groups (n = 5/group) and one control (n = 3). Animals in Groups I, II, and III were implanted with the ALCAP, HA, and TCP ceramics, respectively. Animals in Group IV were not implanted and acted as the control group. At 90 days post-implantation, the animals in all groups were euthanized and the fibrous tissue surrounding the ceramic devices and internal organs were harvested. After routine histological processing, sections of tissue was stained with hematoxylin and eosin and evaluated using light microscopy. Analysis of the data revealed the following: 1) The presence of macrophages, neutrophils, fibroblasts, vascularity, and thickness of the fibrous tissue matrix was found to be statistically different among the subcutaneously implanted ceramic groups (ANOVA, p < 0.01). 2) The presence of collagen (S/C) is not statistically different among the three ceramic groups (ANOVA, p = 0.76). 3) The presence of macrophages, neutrophils, fibroblasts, and collagen comprising the fibrous tissue surrounding the ALCAP, HA, and TCP ceramics (I/P), was found to be statistically different (ANOVA, p < 0.01). 4) Vascularity and thickness of the fibrous tissue surrounding the three I/P ceramics were not found to be statistically different among the three ceramic groups (ANOVA, p > 0.05).

Aluminum Compounds↗

[Periodontal regeneration by application of the porous beta-TCP/BMP artificial bone to Class II furcation defects].

OBJECTIVE: To study the regeneration of periodontal tissue after the application of beta-TCP/BMP artificial bone to class II furcation defects. METHODS: Periodontal defects were produced surgically around premolar teeth in 4 dogs. The flap surgery was performed and the root surfaces were curetted to remove the periodontal ligament and the cementum. A synthetic bioabsorble barrier manufactured from poly DL-lactide was used. The defects were divided into 3 groups (beta-TCP/BMP + PDLLA, PDLLA, control). 12 weeks after surgery, the animals were sacrificed and the teeth with their supporting periodontium were processed for light microscopic evaluation. RESULTS: The definitive evidences of regeneration were founded both in BMP/GTR group and GTR group, but the BMP/GTR group exhibited more new bone formation, cementum deposition occupying the majority of the furcation defects than the other two groups. CONCLUSION: It was concluded that the beta-TCP/BMP artificial bone can be used in GTR technique to promote periodontal regeneration.

Animals↗

[Experimental study on repairing rabbit femoral condyles defect by small intestinal submucosa and HA-TCP compositions at different ratios].

OBJECTIVE: To study an optimal ratio of small intestinal submucosa (SIS) and (hydroxyapatite-tricalcium phosphate, HA-TCP, SIS/HA-TCP) compositions according to the effect of SIS/HA-TCP compositions with different ratios on repairing rabbit femoral condyle defect. METHODS: Thirty-six rabbits were made into bone defect models of 6 mm in diameter and 10 mm in depth in both sides of femoral condyles. Three different ratios of SIS/HA-TCP compositions (w/w: 1, 0. 5, 0. 25) were implanted into rabbit femoral condyle defect. After 2, 4, 8 and 12 weeks of operation, the repair effect was observed grossly. The histological evaluations were performed by histological scoring system and computer imaging analysis system. RESULTS: The amount of new bone formation in SIS/HA-TCP(0. 5) group was more than that in SIS/HA-TCP(1) and SIS/HA-TCP(0. 25) groups. Histological observation: In SIS/HA-TCP (1) group, few new bone formation was seen and bone defect was repaired in the 12th week. In SIS/HA-TCP (0.5) group, immature woven bone was found in the defect in the 2nd week; more immature woven bone appeared and formed trabeculae in the 4th week; the regenerated bone was vigorously growing into the interspaces of the implanted materials in the 8th week; the implanted materials was basically replaced by bony structure and the lamellar bone appeared in the 12th week. The results of SIS/HA-TCP (0. 25) group were similar to that of SIS/HA-TCP(0. 5) group. The histological scoring was higher in SIS/HA-TCP (0.5) and SIS/HA-TCP (0.25) groups than that in SIS/HA-TCP (1) group (P<0.05) in the 2nd, 4th, 8th, and 12th weeks. The scoring was higher in SIS/HA-TCP (0.5) group than that in SIS/HA-TCP (0.25) group in the 2nd and 12th weeks (P< 0.05). In new bone formation and the degradation of HA-TCP, SIS/HA-TCP (0.5) and SIS/HA-TCPC (0.25) groups were superior to SIS/HA-TCP (1) group (P<0.05), SIS/HA-TCP (0.5) group was superior to SIS/HA-TCP (0.25) group (P<0.05). CONCLUSION: SIS/ HA-TCP (0.5) has better effects of repairing bone defect and it can be used as a reference ratio in constructing bone scaffolds.

Animals↗

Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs.

Beta tricalcium phosphate (TCP) was employed as a nonimmunogenic biodegradable delivery system for bovine bone morphogenetic protein (BMP). A BMP/TCP composite was implanted in adult dogs with skull trephine defects of a critical size of 1.4 cm that would otherwise remain unhealed in the lifetime of the individual. BMP/TCP implants induced 91%-100% incorporation by deposits of new bone. In comparison, control implants of TCP impregnated with bovine serum albumin (BSA/TCP) induced 0%-8% incorporation, or only marginal host bed reactive bone formation. The retention of unabsorbed TCP in the host bone four months after implantation suggests that further research should be encouraged to obtain a formulation of sintered calcium phosphate that could be resorbed more rapidly and in the process more completely replaced by bone.

Animals↗

Expression of the Tcp-1 locus of the mouse during early embryogenesis.

The synthesis of proteins by preimplantation mouse embryos was studied by two-dimensional (2D) gel electrophoresis of proteins labelled with [35S]methionine. The product of the Tcp-1 locus of the mouse, p63/6.9, is synthesized by morula and blastocyst stage embryos, but is not detectable in 2-cell embryos. These results indicate that maternal expression (oocyte-specific) of this gene is not occurring and that this gene may be developmentally regulated. Blastocysts which are allowed to implant in vitro have rates of protein synthesis that permit fluorographs of single embryos to be performed. These embryos can be unambiguously identified for their t-complex genotype. Quantitation of the amounts of the Tcp-1a and Tcp-1b allelic products in morula embryos suggested delayed expression of the paternal allele.

Animals↗

Comparative examinations of 99mTc-DMS preparations obtained by labelling dimercaptosuccinate kits with different formulations. II. Comparison of chemical and biological characteristics of TcP-5 and MPI kits.

The chemical and biological characteristics of two dimercaptosuccinate kits were examined and compared: TcP-5 (produced at the Boris Kidric Institute - Vinca) in the freeze-dried form and MPI - the aqueous solution of tin dimercaptosuccinate. The radiochemical composition of the injection solutions was examined by various methods and the method of ascending paper chromatography in 50% aqueous methanol was found as satisfactory. Both preparations has similar compositions. Both preparations showed maximum renal concentrations 3-4 hrs after injection. Liver uptake of MPI was higher than that of TcP-5. The bench-life of both preparations was compared by measuring organ distribution. TcP-5 had a bench-life at least six hrs with a kidney/liver uptake ratio from 5.60 to 8.52, whereas MPI had a satisfactory bench-life of less than half an hour after labelling; thereafter, hepatic concentration rose and the kidney/liver uptake ratio dropped to 1.64.

Animals↗

[Experimental study on bone formation induced by porous HA-beta-TCP bioceramics].

Three types of HA-beta-TCP active bioceramics which have various porosity and porous diameters were implanted into femurs and muscles of dogs. Histological and biomechanical tests proved that the porous HA-beta-TCP could make the active stage of bone formation moved up in bones and induce new bone formation in muscles. The pores were easily filled with new bones in samples which had smaller porous diameter than samples having larger porous diameter. The final strength after accomplishment of repair process was closely related with porosity. In samples with lower porosity, the proportion of organic bone and the strength were also lower, but in samples with higher porosity the proportion of organic bone and the strength were also higher. Up to the fourth month postoperatively, the strength of HA-beta-TCP with proper porous diameter and porosity which had been implanted into bones was as high as that of organic bone.

Animals↗

Characterization of haloperidol and trifluperidol as subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonists using [3H]TCP and [3H]ifenprodil binding in rat brain membranes.

[3H]TCP and [3H]ifenprodil binding to N-methyl-D-aspartate (NMDA) receptors in rat forebrain membranes was used to compare the inhibition of haloperidol and trifluperidol with that of ifenprodil and eliprodil. In the [3H]TCP binding assay, inhibition curves of ifenprodil, eliprodil, haloperidol and trifluperidol revealed two affinity states in the presence of glutamate, glycine and spermidine. The potency of these agents to inhibit the high-affinity fraction of the binding agreed with the results of other studies investigating their potency to block glutamate-induced current at recombinant NR1a/NR2B NMDA receptors expressed in Xenopus oocytes. These agents also inhibited [3H]ifenprodil binding in a biphasic manner, whether in the absence or the presence of either the sigma site ligand GBR-12909 or spermidine. Spermidine reduced the fraction of high-affinity sites labeled with [3H]ifenprodil. The only alteration in the affinity was a decrease in the IC50 value of haloperidol to inhibit the high-affinity fraction of [3H]ifenprodil binding. GBR-12909 also reduced the fraction of [3H]ifenprodil sites inhibited by these compounds with high affinity, with no change in the affinity for either fraction. These data suggest that spermidine is neither a competitive antagonist at the fraction of the binding inhibited by these agents with high affinity, nor is this fraction of the binding to sigma sites. Haloperidol and trifluperidol represent a new class of agent that interacts at a site that is labeled by [3H]ifenprodil as well as [3H]TCP in rat brain membranes and that closely reflects ifenprodil's voltage-independent site on the recombinant NR1a/NR2B subtype of the NMDA receptor.

Animals↗

A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg).

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA(Arg3) (AGC) gene.

Amino Acid Sequence↗

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1.

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the zeta subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of Mr 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTzeta-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.

Amino Acid Sequence↗

Pleiotropic mutation in a tendril TCP gene underlies the yield-enhancing multiple-flowering trait in summer squash (Cucurbita pepo).

Crop yield is a focal point in plant breeding. Regulation of lateral budding through apical dominance was a central target of crop domestication, directly affecting crop production. The young fruits of Cucurbita pepo, summer squash, are produced on plants characterized by apical dominance and differentiation of a single flower bud per leaf axil. A single recessive mutation, mf, results in differentiation of more than one flower per leaf axil, thereby directly increasing production because of the continual day-to-day harvest of the summer squash crop. Positional cloning of the Cucurbita pepo mf (Cpmf) gene denoted a frameshift mutation in a TCP transcription factor, Cp4.1LG13g07780, as causative for the increase in axillary flowering. Cpmf is an ortholog of a tendril-development TCP gene in other cucurbits, and likewise, the recessive allele of Cpmf is associated with distorted tendril development. Gene function is context dependent, and we propose that multiple flowering is a unique pleiotropic attribute of mutation in a tendril-development gene of C. pepo. Characterization of a C. pepo collection confirmed a significant association of the Cpmf mutation with multiple flowering and showed that the mutant allele is absent in ancestral C. pepo and one of its two cultivated subspecies. The beneficial mutation occurred and was selected after the domestication of the other subspecies, during its cultivation for young fruit production. We demonstrate the discovery of a causative yield-increasing sequence variant and its practical utilization in breeding. Our findings provide a molecular target for creation of high-yielding, multiple-flowering summer squash cultivars through marker-assisted breeding or precise genome editing.

Cucurbita↗

Integrated analysis of ATAC-seq and RNA-seq reveals the TCP-ARF molecular module related to pathogenic process of phytoplasma infection in Paulownia fortunei.

BACKGROUND: Witches&#x2019; broom is an important disease of the Paulownia fortunei. Understanding the pathogenesis of witches&#x2019; broom is a prerequisite for its prevention and control. Phytoplasma is the pathogen of Paulownia witches&#x2019; broom. RESULTS: We investigated the changes in chromatin accessibility before and after phytoplasma infection in Paulownia fortunei by analyzing the DNA accessibility (ATAC-seq). In phytoplasma-infected P. fortunei (PFI) compared to healthy samples (PF), the closed regions of chromatin(1187 regions) were three times more than the open regions (352 regions). Fifty one percent of the accessible chromatin regions were overlapped with either H3K27ac or H3K9ac peaks. The closed regions were enriched in the conserved motif TGGGC[CT] that is recognized by the TCP transcription factor family. The closed regions in PFI are intersected with ARF family gene locus. The gene PfARF3 was verified to interact with the PfTCP23 transcription factor. The PfTCP23 was predicted to be interacted with the effector pawb44 in the pathogen of phytoplasma. CONCLUSIONS: The phytoplasma infection in P. fortunei is involved in the chromatin changes of the DNA accessibility and histone modification. The binding regions of TCP23 were found to be changed mostly in the accessibility between PFI and PF. The TCP-ARF module was found to be the possible regulatory module inducing the crinkled leaf trait.

RNA-Seq↗

Comparative study of bone cements prepared with either HA or alpha-TCP and functionalized methacrylates.

The properties of bone cements prepared with both hydroxyapatite (HA) and alpha-tricalcium phosphate (alpha-TCP) and methacrylates containing acidic or basic groups are the main interest of this article. The presence of methacrylic acid or diethyl amino ethyl methacrylate as comonomers in the bone cement and both ceramic types as filler were found not to affect the amount of residual monomer, which was generally less than 4.5 wt%. In contrast, setting times, maximum temperature, and glass transition temperature were found to be composition dependent. For samples with acidic comonomer, a faster setting time, a higher maximum temperature, and higher glass transition temperatures were observed compared to those with the basic comonomer. The presence of the fillers slightly increased the setting time but did not affect the other parameters. The mechanical properties of the filled bone cements depended mainly on composition and type of testing. Both HA or alpha-TCP filled systems fulfilled the minimum compressive strength required for bone cement application, although a significantly lower value was observed for the alkaline comonomer systems. The minimum bending strength was not satisfied by any of these formulations. The tensile and shear strength of these composites ranged from 20 to 37.9 and from 18 to 27 MPa, respectively. In all cases it was higher for bone cements containing methacrylic acid. The results of this study suggest that the properties of dry unfilled bone cements prepared with MAA are comparable to CMW 3 in mechanical terms but inferior in their setting properties.

Bone Cements↗

TGF-beta1-enhanced TCP-coated sensate scaffolds can detect bone bonding.

Porous polybutylene terephthalate (PBT) scaffold systems were tested as orthopedic implants to determine whether these scaffolds could be used to detect strain transfer following bone growth into the scaffold. Three types of scaffold systems were tested: porous PBT scaffolds, porous PBT scaffolds with a thin beta-tricalcium phosphate coating (LC-PBT), and porous PBT scaffolds with the TCP coating vacuum packed into the scaffold pores (VI-PBT). In addition, the effect of applying TGF-beta1 to scaffolds as an enhancement was examined. The scaffolds were placed onto the femora of rats and left in vivo for 4 months. The amount of bone ingrowth and the strain transfer through various scaffolds was evaluated by using scanning electron microscopy, histology, histomorphometry, and cantilever bend testing. The VI-PBT scaffold showed the highest and most consistent degree of mechanical interaction between bone and scaffold, providing strain transfers of 68.5% (+/-20.6) and 79.2% (+/-8.7) of control scaffolds in tension and compression, respectively. The strain transfer through the VI-PBT scaffold decreased to 29.1% (+/-24.3) and 30.4% (+/-25.8) in tension and compression when used with TGF-beta1. TGF-beta1 enhancement increased the strain transfer through LC-PBT scaffolds in compression from 9.4% (+/-8.7) to 49.7% (+/-31.0). The significant changes in mechanical strain transfer through LC-PBT and VI-PBT scaffolds correlated with changes in bone ingrowth fraction, which was increased by 39.6% in LC-PBT scaffolds and was decreased 21.3% in VI-PBT scaffolds after TGF-beta1 enhancement. Overall, the results indicate that strain transfer through TCP-coated PBT scaffolds correlate with bone ingrowth after implantation, making these instrumented scaffolds useful for monitoring bone growth by monitoring strain transfer.

Animals↗

Regional changes in [3H]D-aspartate and [3H]TCP binding sites in Alzheimer's disease brains.

The specific binding of [3H]D-aspartate, a marker for the presynaptic glutamate uptake site, and [3H]N-(1-[2-Thienyl]cyclohexyl)-piperidine [( 3H]TCP), a high affinity ligand for the N-methyl-D-aspartate (NMDA)-associated phencyclidine binding site, was measured in homogenates of brain from normal subjects and from subjects with neuropathologically confirmed Alzheimer's disease. Alzheimer's disease was associated with a reduction in [3H]D-aspartate binding density in temporal cortex and caudate nucleus. By contrast, a reduction in the receptor density for [3H]TCP binding was only recorded in the frontal cortex. Thus, glutamate-containing nerve terminals are severely reduced in Alzheimer's disease, whilst the postsynaptic NMDA-phencyclidine receptor complex is much less affected. These findings have implications for theories of glutamate neurotoxicity in Alzheimer's disease.

Aged↗

Equilibrium analysis of [3H]TCP binding: effects of glycine, magnesium and N-methyl-D-aspartate agonists.

It has been reported that glutamate can increase the binding of [3H]TCP to phencyclidine (PCP) receptors by an action on receptors which are selective for N-methyl-D-aspartate (NMDA). Recently this laboratory has reported that glycine and magnesium can amplify this effect of NMDA agonists in well-washed, lysed cortical membranes. Here we report that maximally effective concentrations of glutamate (10 microM), NMDA (300 microM), MgCl2 (300 microM) and glycine (10 microM) increase the affinity of the PCP receptor for [3H]TCP by approximately 4-fold in the absence of any change in the density of PCP receptors. However, in combination with glutamate, magnesium had the further effect of increasing the Bmax by about 75%. Finally, a synaptosomal P2 preparation, which had not been washed to minimize the concentration of endogenous effectors had a Bmax value similar to the well-washed preparation, but had a KD value 8-fold lower. These data indicate that the primary effect of NMDA agonists, glycine, and low concentrations of magnesium ions is to convert the PCP receptor from a low-affinity to a high-affinity state. These data are discussed in relation to the functional regulation of the NMDA ionophore.

Animals↗

Isolation and identification of a peptide from rat brain which inhibits [3H]TCP binding.

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue [3H]TCP was utilized to screen for inhibitors of binding in extracts of rat brain. 2. Extracts were prepared from rat cortex and hippocampus by methods employing aqueous acid or acidified methanol. Samples were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The most active fraction was further purified by high performance-size exclusion chromatography. 3. Size exclusion chromatography revealed two zones of activity, corresponding to mol. wts of 4000-8000 Da and 1000-2000 Da. Final purification of the lower molecular weight material was achieved by RP-HPLC. 4. Two well-separated peaks were shown to be homogeneous. Their amino acid sequences were determined by automated Edman degradation and data base searching identified these two peaks as the undecapeptide Substance P and its oxidized counterpart (Substance P sulfoxide). 5. Comparative HPLC of synthetic Substance P, or its sulfoxide, as well as spectral analysis confirmed the identity of the isolated peptides. 6. Synthetic Substance P inhibits specific [3H]TCP binding in the radioreceptor assay.

Amino Acid Sequence↗