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PubMed1995

The eighth Cct gene, Cctq, encoding the theta subunit of the cytosolic chaperonin containing TCP-1.

The chaperonin containing t-complex polypeptide 1 (TCP-1), as one of its subunits, CCT, is a cytosolic heterooligomeric molecular chaperone assisting in the folding of proteins in eukaryotic cytosol. We have isolated a Tcp-1-related 119-bp cDNA fragment from a human cDNA library by polymerase chain reaction, and cloned full-length mouse cDNAs orthologous to the human cDNA by hybridization. The nucleotide (nt) sequence of the longest mouse clone (1844 bp) shows an open reading frame (ORF) encoding a TCP-1-related polypeptide of 548 amino acids (aa) (59,562 Da). This gene is different from Tcp-1 and the six Tcp-1-related genes reported previously, Tcp-1 (Ccta), Cctb, Cctg, Cctd, Ccte, Cctz and Ccth, which encode subunits of CCT. The product of the novel gene was analysed using an antibody raised against the C terminus of the polypeptide deduced from the nt sequence. We found that this gene encodes a subunit of CCT (polypeptide S1; 62 kDa and pI 6.25 by two-dimensional gel analysis). We have named it Cctq, encoding the theta subunit of CCT (CCT theta). The aa sequence of CCT theta shows 23-29% identity to the other CCT subunits, alpha, beta, gamma, delta, epsilon, zeta and eta, and 29% identity to the archaebacterial chaperonin TF55. CCT theta also contains the motifs common to all the other subunits of CCT which are postulated to be involved in ATPase activity.

Amino Acid Sequence↗
PubMed1996

Molecular chaperones and the centrosome. A role for TCP-1 in microtubule nucleation.

Molecular chaperones play an important role in facilitating the proper maturation of many newly synthesized proteins. Here we provide evidence that molecular chaperones also participate in regulating the assembly of the microtubule cytoskeleton. Via indirect immunofluorescence analysis, both hsp 73 and TCP-1 localized within the centrosome in interphase and mitotic cells. These proteins, along with the centrosome-specific protein, pericentrin, were also present within an enriched preparation of centrosomes. Because the centrosome serves as an initiation site for microtubule growth, we examined the ability of cells to regrow their microtubule network in the presence of hsp 73 or TCP-1 specific antibodies. Purified tubulin and GTP were added to cells following the depolymerization and extraction of cellular microtubules. Microtubules were observed to nucleate off the centrosome using this system, even in the presence of anti-hsp 73 antibodies. Incubation with anti-TCP-1 antibodies, however, blocked microtubule regrowth off the centrosome. Similarly, anti-TCP-1 antibodies microinjected into living cells first treated with nocodazole also inhibited the regrowth of the microtubule network following removal of the microtubule poison. Our results complement earlier genetic studies in yeast implicating a role for TCP-1 in microtubule mediated processes, and may help to explain the previously reported mitotic and meiotic abnormalities associated with TCP-1 mutations.

Amino Acid Sequence↗
PubMed1997

TCP-11, the product of a mouse t-complex gene, plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction.

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH2) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH2, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Glu-ProNH2 inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function.

Acrosome↗
PubMed2004

Biocompatibility and osseointegration of beta-TCP: histomorphological and biomechanical studies in a weight-bearing sheep model.

The aim of the study was to investigate the biocompatibility, degradation, and biomechanical properties of beta-TCP (Cerasorb) in a weight-bearing sheep model. beta-TCP implant prototypes were implanted in the tibial head of adult merino sheep. After 6 and 12 months material explants were harvested for biomechanical, histological, and histomorphometrical analysis. Corresponding bone specimens of the intact bone of the contralateral leg were used as controls in the biomechanical test. Compression tests showed higher values for maximum fracture load, yield strength, and compression modulus after 6 and 12 months compared to control. Microscopically, the implants showed good osteoconduction and were incorporated into the bone; however, relevant amounts of beta-TCP were still present after 12 months. Histomorphological results revealed that beta-TCP had partially degraded between implantation and 6 months, but its share remained constant between 6 and 12 months. The bone volume fraction in the area of the implant (46% +/- 6.5%) was initially higher than in the corresponding bone area of the contralateral leg (31% +/- 9.6%), but after 12 months declined to 29% +/- 9.4% (control: 33% +/- 8.3%), while the share of beta-TCP remained constant at 36% +/- 12.2%. These findings were supported by microradiographic data. In conclusion, in a weight bearing implantation model beta-TCP showed good biocompatibility, osseointegration and beginning degradation, even though it was not further degraded between 6 and 12 months.

Animals↗
PubMed2006

Biodegradation process of alpha-TCP particles and new bone formation in a rabbit cranial defect model.

The purpose of the present study was to observe the biodegradation process of pure alpha-tricalcium phosphate (alpha-TCP) particles and to determine the efficacy of alpha-TCP as a space maintainer in a bone defect. We used 14 rabbits and prepared two cranial bone defects in each rabbit. One defect was left empty as a control, whereas the other was filled with alpha-TCP particles about 300 mum in diameter. Animals were sacrificed at 1 week, 4 weeks, and 8 weeks. The cranial bone was then embedded either in paraffin wax for the preparation of decalcified specimens, or in polyester resin for the preparation of nondecalcified specimens. All specimens were evaluated histologically and histomorphometrically. As a consequence of the degradation of alpha-TCP, a "reticulate structure" appeared in the particles at 1 week and new bone was observed in this structure at 8 weeks. The amount of new bone between the control and experimental groups was not significantly different at any of the time points. However, in the experimental group, new bone at the surface of alpha-TCP was evident even in the center of the defect whereas fibrous connective tissue was dominant in the control group. These results indicate that alpha-TCP is a degradable osteoconductive material that is able to act as a space maintainer for bone regeneration when applied to a bone defect. While there was no significant difference in total bone formation between the experimental and negative control groups, the space-maintaining and osteoconductive properties of the particles may result in more complete bone formation in longer-term studies.

Absorbable Implants↗
PubMed1991

Molecular structure of Tcp-10 genes from the t complex responder locus.

Male transmission ratio distortion (TRD) is a property of mouse t haplotypes which requires the t complex responder locus (Tcr). Tcr has been localized to a 70-160 kb region in t haplotypes. A candidate gene for the responder, called Tcp-10bt, has been cloned and is one member of a highly related gene family called Tcp-10 (formerly T66). Molecular evidence suggests that unique alternative splicing of the Tcp-10bt gene may be responsible for the mutant responder activity. Here we present the intron/exon structure of a representative Tcp-10 gene, and the characterization of alternative polyadenylation sites. The Tcp-10 genes contain 12 exons which span approximately 21 kb of DNA. At least six different polyadenylation sites are used, and none have a perfect consensus signal. This appears to be a common feature associated with testes-expressed transcripts. Since the gene we have analyzed is absent from many t haplotypes without apparent consequence, and no corresponding cDNAs have been isolated, it was speculated to be a pseudogene. However, no major sequence differences were found within the coding sequence to conclude that Tcp-10pst is a pseudogene.

Amino Acid Sequence↗
PubMed1985

Quantitative localization of [3H]TCP binding in rat brain by light microscopy autoradiography.

The anatomical localization of phencyclidine (PCP)/sigma-opiate receptors in rat brain was determined by quantitative light microscopy autoradiography using the new ligand N-(1-[2-thienyl]cyclohexyl) [3H]piperidine ([3H]TCP). TCP is a potent analog of PCP which possesses a higher affinity for PCP/sigma-opiate receptor than does PCP itself. The highest level of [3H]TCP binding was detected in the hippocampus. Intermediate levels were found in frontal cortex, striatum, amygdala and cerebellum. Specific [3H]TCP binding was undetectable in anterior commissure and corpus callosum. The distribution pattern of [3H]TCP binding sites is similar to the pattern obtained with [3H]PCP but more sharply defined. On the basis of its greater potency and specificity, [3H]TCP may prove superior to [3H]PCP as a molecular probe for the study of brain sigma opiate/phencyclidine receptors.

Animals↗
PubMed1986

Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand, [3H]TCP.

Receptor binding sites for the phencyclidine (PCP) analogue, [3H]TCP, have been localized in the rat and guinea pig central nervous systems by in vitro autoradiography. Quantitation of [3H]TCP binding site densities in rat brain reveals highest levels in the forebrain, in particular the strata oriens and radiatum of the hippocampus, the molecular layer of the dentate gyrus and superficial layers of the cerebral cortex. Moderate levels of binding occur in the amygdala, thalamus, anterior olfactory nucleus, external plexiform layer of the olfactory bulb, olfactory tubercle, geniculate nuclei and deep layers of the cortex. Low levels of binding occur throughout most of the septum, diagonal band, hypothalamus, pons-medulla and cerebellum. Spinal cord grey matter also has low levels of binding. Excitotoxin lesions of the hippocampal formation, which destroy the pyramidal and granule cells, reduce the binding of [3H]TCP to strata radiatum and oriens and the molecular layer of the dentate gyrus by 60% suggesting that [3H]TCP labels intrinsic neurons in these regions. Residual binding is probably on afferent terminals. Ibotenic acid lesions of the caudate-putamen reduce [3H]TCP binding by 70%, indicating that binding sites are localized on intrinsic striatal neurons. 6-Hydroxydopamine lesions do not alter [3H]TCP binding levels in the caudate, suggesting the absence of binding sites on dopaminergic terminals in the caudate.

Animals↗
PubMed1990

A radiohistochemical measure of [3H]TCP binding to the activated NMDA-receptor-gated ion channel in rat brain.

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor is linked to an ion channel that is blocked by the phencyclidine analog N-(1-[thienyl]cyclohexyl)piperidine (TCP). Previous studies have shown that NMDA and glycine act together to increase the access of [3H]TCP to its binding site, presumably by increasing channel opening; NMDA/glycine-enhanced [3H]TCP binding performed under non-equilibrium conditions thereby serves as a dynamic molecular marker of channel activation. In this study we tested whether NMDA and glycine regulate [3H]TCP binding in slide-mounted brain sections. Striking activation of the NMDA ion channel was observed in neo- and allocortex; dentate gyrus and strata radiatum and oriens of CA1 and CA3 of hippocampal formation; and certain amygdaloid nuclei (basomedial, basolateral, and cortical). Other nuclei of the amygdala (central, posterolateral, and medial), basal ganglia, and numerous regions within the diencephalon, brainstem and cerebellum showed relatively little activation of the NMDA ion channel. Our demonstration of enriched NMDA/glycine-stimulated [3H]TCP binding in stratum radiatum of hippocampal region CA1 but not in cerebellar granule cell layer correlates with electrophysiologic studies that showed NMDA channel ion flux in CA1 but not in cerebellar granule cell layer in adult rats. These data demonstrate that NMDA and glycine regulation of [3H]TCP binding can be quantified with a radiohistochemical method to provide a regional measure of the activated NMDA-receptor-gated ion channel. This technique is a powerful tool for functional analysis of the NMDA receptor/channel complex in both physiologic and pathologic states.

Animals↗
PubMed1989

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes.

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.

Animals↗
PubMed1989

The binding of [3H]thienyl cyclohexylpiperidine ([3H]TCP) to the NMDA-phencyclidine receptor complex.

The high affinity binding of the phencyclidine derivative [3H]TCP to cortical membranes of the rat was investigated. In an extensively washed membrane preparation the binding of [3H]TCP was enhanced in the presence of L-glutamate and NMDA. The stimulation of the binding of [3H]TCP by L-glutamate was inhibited competitively by AP5 and non-competitively by MK801. The binding of [3H]TCP was also enhanced in the presence of glycine; this effect was insensitive to strychnine and inhibited non-competitively by AP5. Saturation experiments demonstrated that MK801 was a competitive inhibitor of the binding of [3H]TCP. These results suggest that [3H]TCP binds to a site similar to that which binds MK801; this site may be associated with the ion channel of the NMDA receptor.

Amino Acids↗
PubMed1989

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes.

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.

Acrosome↗
PubMed1990

Developmental study of [3H]TCP and [3H]glycine binding sites in the rat hippocampus.

The postnatal development of [3H]thienylphencyclidine ([3H]TCP) sites in rat hippocampus has been studied autoradiographically and with membrane preparations. [3H]TCP binding increased progressively from birth to adulthood; this is due to a change in the maximal number of sites (Bmax) but not in the affinity (Kd). A different developmental pattern was found for strychnine-insensitive [3H]glycine binding which also increased after birth, but reached adult levels earlier than [3H]TCP binding. The ontogenesis of TCP or glycine sites also differed from that previously described for N-methyl-D-aspartate (NMDA) sites in the hippocampus. In neonatal, as in adult hippocampus, [3H]TCP binding was enhanced by NMDA or glycine and reduced by Mg2+. We suggest that TCP sites are functionally coupled to the NMDA receptor-ion channel complex in developing as in mature hippocampus, but that there are developmental changes in the receptor channel complex.

Aging↗
PubMed2003

Effects of alpha-TCP and TetCP on MC3T3-E1 proliferation, differentiation and mineralization.

Calcium phosphates with high solubility in water such as alpha-tricalcium phosphate (alpha-TCP) and tetracalcium phosphate (TetCP) have received considerable attention as components of bone-substitution materials. However, the osteoblast response to these materials has not yet been clearly understood. This study examined the effects of alpha-TCP and TetCP on osteoblast proliferation, differentiation and mineralization in the culture system of MC3T3-E1 cells. Cells were cultured in a differentiation medium with or without alpha-TCP or TetCP at 1 or 10 microM, and the number of cells attached to the culture plates was determined. To examine osteoblast differentiation, the alkaline phosphatase (ALP) activity was measured and the expression of osteoblastic markers analyzed by RT-PCR. In addition, mineralization was evaluated by staining the calcium deposit with Alizalin red. Culture in the presence of alpha-TCP or TetCP showed no significant influence on cell proliferation. ALP activities of the cells were enhanced by both calcium phosphates for 3d and the expression of type I collagen was promoted at 12h and 1d after incubation. Enhancement of bone-like tissue formation by the addition of alpha-TCP or TetCP at 10 microM was observed after 7d incubation and thereafter. The results of the present study indicate that alpha-TCP and TetCP promote osteogenesis by increasing collagen synthesis and calcification of the extra-cellular matrix.

3T3 Cells↗
PubMed1997

Solid state synthesis and thermal stability of HAP and HAP - beta-TCP composite ceramic powders.

Powders of pure beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HAP) and a biphasic composite mixture of HAP+beta-TCP were prepared by solid state reaction between two commercially available calcium-based precursors namely, tricalcium phosphate (TCP) and calcium hydroxide (Ca(OH)2). These reactants mixed in the molar ratios ranging from 3 : 0 to 3 : 4 (designated T0 to T4) in deionized water, milled and slip-cast into discs were heat treated in the temperature range of 600 degrees C to 1250 degrees C. The products formed were characterized by X-ray diffraction (XRD) and i.r. spectroscopic techniques for identification of phases formed and functional groups present in them. While tricalcium phosphate and calcium hydroxide taken in the molar ratio of 3 : 2 and 3 : 3 resulted in pure HAP when heat treated at 1000 degrees C for 8 h, the 3 : 1 and 3 : 1.5 molar ratio compositions resulted in a biphasic mixture of HAP+beta-TCP for similar heat treatments. Heat treatment of 3 : 4 molar ratio composition of tricalcium phosphate and calcium hydroxide at 1000 degrees C yielded HAP with free CaO as the secondary phase. Products of heat treatment at higher temperatures (1150 and 1250 degrees C) for even shorter duration (2 h) while not differing from the products obtained from T0 and T2 cases at 1000 degrees C (pure beta-TCP and pure HAP), change in the case of T1, T1.5, T3 and T4 to products with lesser percentages of HAP containing beta-TCP (in the case of T1 and T1.5) or CaO (in the case of T3 and T4) as the secondary phase.

Journal Article↗
PubMed1996

Ethanol-induced inhibition of [3H]thienylcyclohexylpiperidine (TCP) binding to NMDA receptors in brain synaptic membranes and to a purified protein complex.

N-Methyl-D-aspartate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol-insensitive, but dizocilpine (MK-801)-sensitive, binding of N-[1-(2-[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration-dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by L-Glu when L-Glu was present at concentrations < 100 microM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of L-Glu (200-400 microM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has L-Glu-, Gly-, and [3H]TCP-binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either L-Glu or Gly. Activation constants (Kact) for L-Glu and Gly acting on the purified complex were 12 and 28 microM, respectively. Ethanol had no significant effect on the Kact of L-Glu but caused an increase in Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both L-Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.

Animals↗
PubMed2002

[The Transverse Coloplasty Pouch (TCP)--function and subjective judgement in comparison with the surgically more complicated J-Pouch].

The present study shows the comparison of function and subjective judgement between two pouch-constructions after TME as treatment for rectal cancer. In 61 cases we constructed a J-Pouch, in 25 cases a transverse coloplasty pouch (TCP). The sex distribution was nearly the same. The influence of additional treatment like neoadjuvant radiotherapy or adjuvant chemo-radiatio was considered. Patients were asked about stool frequency, -consistency, continence, quality of life etc. 9 patients were not alive at follow up. 54 patients (70 %) could be interviewed and examined. 6 months after operation patients with a J-Pouch had a daily stool frequency of 4 times versus 6 times with a TCP. 19 % of patients with a J-Pouch suffered from incontinence problems compared to 33 % with a TCP. Obstipation was only a problem after J-Pouch-construction (12 %). The question on a "good" quality of life was positively answered by 56 % of patients in the J-pouch-group versus 39 % in the TCP-group. 17 % after J-pouch-construction versus 6 % after TCP-construction judged the quality of life as "bad". The results of this study show the TCP being a good alternative pouch-construction instead of the surgically more complicated J-pouch after TME.

Aged↗
PubMed2006

[Effect of beta-TCP containing the bone-growth promoting compound, TAK-778, on the vertex bone of rat--change of volmetric bone formation corresponding to the containing volume of TAK-778].

PURPOSE: This study was conducted to histomorphologically determine the quantity of bone formation induced by TAK-778, a 3-benzothiepin derivative, (Takeda Pharmaceutical Co.Ltd.), in various amounts, using b-tricalcium phosphate granules (beta-TCP; OLYMPUS Corp.) as a carrier for the osteogenetic agent. METHODS: Ten-week-old female SD rats were used. An incision was made over the parietal region of the head. The cranial periosteum was ablated and a titanium tube was fixed with an adhesive resin cement to the central part of the head, through which a mixture of TAK-778 and beta-TCP was implanted under various conditions. Tissue specimens were prepared at 4, 8, and 16 weeks after the implantation for histomorphological examination, and the proportion of new bone formation was compared at fixed time points using the NIH imaging software. The amount of new bone formation was examined every week after the implantation of TAK-778 at various doses, and the mean values were compared using Fisher's PSLD test (P< 0.05). RESULTS: The histomorphological observations revealed new bone formation in all the groups, irrespective of the amount and the duration of implantation of TAK-778. A comparative study revealed that the amount of new bone formation was the largest at 16 weeks following the implantation of a mixture of beta-TCP and 100 mg of TAK-778. CONCLUSIONS: 1. The present study confirmed the acceleration of new bone formation soon after TAK-778 implantation. 2. The results suggested that the action of TAK-778 could be maintained over time if the agent was used in combination with beta-TCP. 3. The time-course of bone formation differed depending on the proportion of TAK-778 and beta-TCP in the mixture used. 4. TAK-778 at the dose of 10 mg or 50 mg was more effective than that at the dose of 100 mg for the early formation of new bone. These results indicate that TAK-778 accelerates the formation of new bone and that beta-TCP is a useful carrier for TAK-778.

Animals↗