Biomedical subjects

M Zhou

Publications and source records attributed to M Zhou.

At least 127 records · Page 7Linked to original sources

PubMed2000

A cDNA located on chromosome 7q32 shows loss of expression in epithelial cell line of nasopharyngeal carcinoma.

OBJECTIVE: To isolate and clone the tumor suppressor gene on chromosomal region 7q32 correlated with the carcinogenesis of human nasopharyngeal carcinoma (NPC). METHODS: The genotypes of polymorphic microsatellite markers on 7q32 in DNA from 24 biopsies of nasopharyngeal carcinoma and matched normal blood cells were identified. The expression levels of 20 expressed sequence tags (ESTs) on 7q32 between human nasopharyngeal carcinoma epithelial 1 (HNE1) and primary cultures of normal nasopharyngeal epithelial (PNNE) cells were compared using differential RT-PCR and Northern hybridization. The quantity of AA070437 DNA and mRNA was detected by differential PCR and differential RT-PCR, respectively. RESULTS: Loss of heterozygosity (LOH) was found in 25%-46% of NPC biopsies. AA070437 EST expression was down-regulated in HNE1 cell compared to PNNE cells. The down-regulation of AA070437 was found in 30.7% of NPC biopsies and allelic loss of AA070437 was observed in 29.1% of NPC biopsies. CONCLUSION: Our results show that AA070437 EST is negatively related with the occurrence of human NPC and may represent a candidate tumor suppressor gene of NPC on 7q32.

Chromosomes, Human, Pair 7↗

PubMed2000

Interaction of alcohol and the G to a substitution at the promoter region of the apolipoprotein AI gene in determining plasma apolipoprotein AI levels in Yi and Han Chinese.

OBJECTIVE: To investigate the influence on plasma lipid levels of alcohol and a common polymorphism in the human apolipoprotein AI gene promoter at a position 75 bp upstream of the transcription start site. METHODS: For this study, 742 healthy Yi and Han subjects all above 15 years old formed the total population which was divided into three groups: the Yi-farmer group, the Yi-emigrant group and the Han-resident group. All estimates of plasma lipids and apolipoproteins were performed using an auto-analyzer. Genetic DNA was prepared from the blood clots using the Triton X-100 lysis technique. Amplification of a 432 bp fragment of the apoAI gene promoter was performed using PCR followed by restriction digestion, electrophoresis and identification of the genotypes involved. RESULTS: The samples were divided on the basis of alcohol consumption: non-drinkers, 1-25 g/day, 26-75 g/day and > 75 g/day. Comparing the four alcohol consumption groups, plasma HDLC and apoAI levels were increased as the alcohol consumption increased with no evidence of threshold effects in the Yi-farmers and the Han people groups. A similar association was found in the Yi-emigrant group, but was not statistically significant. The frequencies of the A allele in the three populations were similar, and no significant difference of lipid and apolipoprotein levels was found between subjects with and without the A allele in the three populations. But, in Han and Yi-emigrant samples, the drinkers with the GG genotype had higher plasma HDLC and apoAI levels than non-drinkers with the same genotype, while the drinkers with the A allele had lower plasma HDLC and apoAI levels than drinkers without the A allele. Non-drinkers with the A allele had higher levels of apoAI than non-drinkers with GG genotypes. It was estimated that 18% of the variability of plasma apoAI level could be explained by the G to A polymorphism in non-drinkers of Yi-emigrants (F = 8.94, P < 0.01). CONCLUSIONS: The present data suggest that moderate alcohol consumption and the G to A substitution could lower the risk of coronary heart disease (CHD), but the beneficial effects of one will be negated by the other.

Adolescent↗

PubMed2000

[Influence of a G/A substitution at -75 bp promoter region of apolipoprotein A I gene and wine consumption on the plasma apolipoprotein A I levels in Yi and Han Chinese].

OBJECTIVE: To study the influence of a common polymorphism of the human apolipoprotein A I gene promoter at a position of -75 bp upstream of the transcription start site and wine consumption on plasma lipid levels. METHODS: 742 healthy Yi and Han healthy subjects all above 15 years old to be the total population which was divided into three samples, namely, Yi-farmer sample, Yi-emigrant sample and Han-resident sample for this study. Estimations of plasma lipids and apolipoproteins were carried on through an auto-analyzer. Genetic DNA was prepared from the frozen blood clot using Triton x-100 lysis technique. Amplification of a 432 bp fragment of the apoA I gene promoter was performed via the PCR, followed by restriction digestion, electrophoresis and identification of the genotypes involved. Data analysis was done at last. RESULTS: Four groups of alcohol consumption were defined: the non-drinkers, 1 - 25 g alcohol intake/day, 26 - 75 g alcohol/day and > 75 g alcohol/day. A tendency of a mild persistent elevation of plasma HDL-C and apoA I was noticed corresponding to an increase of the amount of alcohol intake, and with no evidence of threshold effect observed in the samples of both the Yi-farmers and the Han people. Similar phenomenon was obtained in the sample of Yi-emigrants, but no statistical significance. The frequencies of the A allele of all 3 samples were similar. In Han and Yi-emigrant samples, the drinkers with genotypes of GG had a higher plasma HDL-C and apoA I level than that of the non-drinkers with the same genotypes. Drinkers with A allele had a lower plasma HDL-C and apoA I level than that of drinkers without A allele, and the non-drinkers with A allele had a higher levels of apoA I than of non-drinkers with genotypes of GG. It is estimated that 18% of the variability of plasma apoA I level could be explained by the G/A polymorphism in non-drinkers of Yi-emigrants (F = 8.94, P < 0.01). CONCLUSIONS: Current data suggest that a moderate alcohol consumption or a G to A substitution could make a lower incidence of CHD, but the beneficial effect of one will be negated by the other when both factors occur simultaneously. This finding is seemed valuable for a further study on the effect of the environmental factor or genetic factor in effecting the plasma apoA I level afterwards.

Adolescent↗

PubMed2000

[The expression of PCNA and NOR in carcinogenesis procession of hamster buccal pouch mucosa].

OBJECTIVE: To investigate the expression of proliferating cell nuclear antigen (PCNA) and nucleolar organizer regions (NOR) with the carcinogenesis model of hamster buccal pouch mucosa. METHODS: 48 Syrain hamsters with 6-8 weeks old and 70-80 g weight were selected. The material used for inducing cancer is 0.5% DMBA (7,12-dimethylbenzanthracene) in acetone. 0.5% DMBA was applied in the right buccal pouch of the hamster 3 times a week for 12 weeks. The control received no treatment. The time for collecting specimens was 3, 6, 9 and 12 weeks. The specimens were immediately fixed with 10% formalin, dyed with HE, and then two doctors major in histopathology evaluated with the WHO criterion (1986). The wax of immunohistochemical specimen was cut and placed on the APES pieces of glass, treated with 50 degrees C for 2 hours and preserved under the indoor temperature. The PCNA was examined with the LSAB technique of immunohistochemistry. The company called Zymed provided the MonAb. The positive control was a sample of human inflammatory hyperplastic lymphoma, and the negative was PBS replacing the MonAb. NOR count was determined in a method of silver nitrate staining. The test condition should be at 45 degrees C without light. RESULTS: 1. AgNOR was usually located in the basal-cell layer of normal epithelium, mainly in a single form. High dysplasia was mainly in an aggregated form, however, carcinoma in infiltration appeared to be in a mixed form. 2. PCNA expression was similar to that of the normal control in hyperplastic epithelia (3 weeks) at a moderate level in dysplasia epithelia (6-9 weeks). PCNA expressed highly in squamous cell carcinoma. The expression of the PCNA in line with malignant progress became stronger increasingly. The positive cell was perceivable in all epithelia of high dysplasia. CONCLUSION: PCNA has been significantly positive and correlative with AgNOR (r = 0.635, P < 0.001), however, the film of PCNA is much clearer and the practical value is greater.

Animals↗

PubMed2000

[Immunohistological observation of tonsillae in IgA nephropathy].

OBJECTIVE: To realize the relationship between IgA nephropathy (IgA-N) and immunological abnormality of tonsillae. METHOD: Thirty-one patients were diagnosed as IgA-N via renal puncture. Sixteen patients suffering from chronic tonsillitis as control. All resected tonsillae were labelled immunohistologically by ABC method. RESULT: The differences of expression of IgA, IgG in the tonsillae between above two groups were significantly obvious. The number of lymphocytes which can produce IgA increased in IgA-N tonsillae. CONCLUSION: The immune functions are abnormal in IgA-N patients. Tonsillectomy can prevent the antigen invasion, eliminate IgA originated from tonsillae, decrease the IgA immunocomplex in circulation, therefore decrease the IgA immunocomplex deposition in the basal membrane of glomeruli.

Adolescent↗

PubMed2000

[The development of a brain surgery path planning system].

Based on the patient's CT/MRI Section images, the brain surgery path Planning system makes surgeon freely draw out the interesting regions through the use of feasible interactive drawing contour function. And by fast judgement, the system is able to give out the risky value of the path. Finally, the system would directly show the surgery path in 3-D display.

Brain↗

PubMed1999

Localization of the ATP-sensitive potassium channel subunit (Kir6. 1/uK(ATP)-1) in rat brain.

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.

Adenosine Triphosphate↗

PubMed1999

Developmental changes in the expression of Shaker- and Shab-related K(+) channels in neurons of the rat trigeminal ganglion.

We have investigated properties of voltage-gated K(+) channels in neurons of the pre- and postnatal rat trigeminal ganglion (TG). To correlate functional data with information on gene expression of Shaker- and Shab-related channels in these pseudo-unipolar neurons, the patch-clamp technique was combined with the single-cell reverse transcription-polymerase chain reaction (RT-PCR). A majority (80%) of prenatal TG neurons possessed only sustained delayed rectifier currents with half-maximal current inactivation at -30 mV. In the postnatal cells, steady-state inactivation of sustained currents occurred at more negative voltages (half-maximal inactivation at -58 mV). About 65% of the postnatal cells displayed a transient outward component in addition to the sustained currents. With increasing age, the sensitivity of sustained currents to 4-aminopyridine (4-AP) decreased significantly. The Shaker channel toxins, alpha-dendrotoxin and agitoxin-2 (50 and 10 nM), were much less effective. Discrimination between both stages with tetraethylammonium chloride (5 mM) was not possible since the currents were reduced generally by about 50%. After recording, the cell content was harvested and single-cell RT-PCR was performed to compare K(+) current properties and mRNA expression within the same cell. Most cells simultaneously expressed several different Shaker- and Shab-like transcripts. At postnatal day 14, the frequency of cells carrying transcripts encoding Kv1.1 decreased. Detailed analysis revealed a higher 4-AP sensitivity of TG neurons expressing Kv1.1 transcripts.

4-Aminopyridine↗

PubMed1999

An important von Hippel-Lindau tumor suppressor domain mediates Sp1-binding and self-association.

VHL is the causative gene for both von Hippel-Lindau (VHL) disease and sporadic clear-cell renal cancer. We showed earlier that VHL downregulates vascular endothelial growth factor transcription by directly binding and inhibiting the transcriptional activator Sp1. We have now mapped the VHL Sp1-binding domain to amino acids 96-122. The 96-122 domain is disproportionately affected by substitution mutations, which interfere with the VHL-Sp1 interaction. Deletion of the 96-122 domain prevents VHL effects on Sp1 DNA binding and on VHL target gene expression, indicating the domain contributes importantly to VHL tumor suppressor activity. Nevertheless, prevention of the VHL-Sp1 interaction only partially abrogates VHL's transcriptional repressor activity, supporting the existence of VHL transcriptional effectors in addition to Sp1. VHL also directly interacts with the Sp1 zinc fingers and self-associates via the 96-122 domain, which furthermore suggest the domain may bind other metalloproteins and contribute to VHL dominant-negative effects.

Amino Acid Substitution↗

PubMed1999

Gamma-heregulin: a fusion gene of DOC-4 and neuregulin-1 derived from a chromosome translocation.

gamma-Heregulin was identified as an isoform resulting from alternate splicing of the neuregulin-1 gene, after cloning of its cDNA from the MDA-MB-175 breast cancer cell line. gamma-Heregulin was shown to promote growth of cultured MDA-MB-175 cells resulting from activation of its cognate ErbB tyrosine kinase reporters. We show here that gamma-heregulin is transcribed from a fusion gene resulting from a chromosome translocation in MDA-MB-175 cells. The fusion chromosome is described as dic(8:11)(8qter-->8p12::11q13-->11pter). As a result, the 5' end of the gamma-heregulin gene is derived from the stress-induced gene, DOC-4 (11q13), while the 3' end is from the neuregulin-1 gene (8p12). Thus, expression of gamma-heregulin is under the control of the DOC-4 promoter. By contrast with MDA-MB-175 cells, RT-PCR failed to detect a gamma-heregulin transcript in either E9.5 to E13.5 embryonic mouse tissues, adult mouse tissues or other human tumour cell lines. We conclude, therefore, that gamma-heregulin is not a native isoform of the neuregulin-1 gene, but a novel growth factor that may contribute to tumour cell proliferation.

Base Sequence↗

PubMed1999

Macrophage colony-stimulating factor reduces tert-butyl hydroperoxide induced oxidative injury to monocytes/macrophages.

The transformation of macrophages into foam cells is an important event in the development of atherosclerosis, and the oxidative injury caused by oxidized low density lipoprotein (Ox-LDL) plays an essential role in that process. It has been proved that macrophage colony-stimulating factor (M-CSF) could prevent the progression of atherosclerosis in Watanabe heritable hypercholesterolemic (WHHL) rabbits. We proposed that the anti-atherogenic effect of M-CSF was partly associated with its protective effect on monocyte-derived macrophages from Ox-LDL induced oxidative injury. In order to prove this, we investigated the effect of M-CSF on the oxidative injury caused by tert-butyl hydroperoxide (tbOOH) to mouse peritoneal macrophages and U937/J774 cell lines. The results showed that M-CSF could protect mouse peritoneal macrophages from oxidative injury (presented by cell morphology and cell survival rate); L929 cell-conditioned medium (L929-CM) had the same effect as M-CSF; and anti-M-CSF monoclonal antibody could mostly block the protective effect of L929-CM on macrophages. L929-CM was proved to be also able to decrease the impact of plasma membrane fluidity in U937 and J774 cells treated with tbOOH. Incubation with tbOOH caused DNA fragmentation in U937 cells. The presence of L929-CM greatly reduced the number of apoptotic U937 cells characterized by DNA fragmentation. From these results, we concluded that M-CSF could protect monocytes/macrophages from oxidative injury. It may be one of the mechanisms which explain the anti-atherogenic effect of exogenous M-CSF in WHHL rabbits.

Animals↗

PubMed1999

An active site of transforming growth factor-beta(1) for growth inhibition and stimulation.

Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulator. It inhibits growth of many cell types, including epithelial cells, but stimulates growth of others (e.g. fibroblasts). The active site on the TGF-beta molecule, which mediates its growth regulatory activity, has not been defined. Here, we show that antibody to a TGF-beta(1) peptide containing the motif WSLD (52nd to 55th amino acid residues) completely blocked both (125)I-TGF-beta(1) binding to TGF-beta receptors and TGF-beta(1)-induced growth inhibition in mink lung epithelial cells. Site-directed mutagenesis analysis revealed that the replacement of Trp(52) and Asp(55) by alanine residues diminished the growth inhibitory activity of TGF-beta(1) by approximately 90%. Finally, while wild-type TGF-beta(1) was able to stimulate growth of transfected NIH 3T3 cells, the double mutant TGF-beta(1) W52A/D55A was much less active. These results support the hypothesis that the WSLD motif is an active site of TGF-beta(1), which is important for growth inhibition of epithelial cells and growth stimulation of fibroblasts.

Amino Acid Sequence↗

PubMed1999

Serum choline activates mutant acetylcholine receptors that cause slow channel congenital myasthenic syndromes.

We have found that mutant acetylcholine receptor channels (AChRs) that cause slow-channel congenital myasthenic syndromes are activated by serum and that the high frequency of openings in serum is reduced by treatment with choline oxidase. Thus, slow-channel congenital myasthenic syndrome AChRs at the neuromuscular junction are likely to be activated both by steady exposure to serum choline and by transient exposure to synaptically released transmitter. Single-channel kinetic analyses indicate that the increased response to choline is caused by a reduced intrinsic stability of the closed channel. The results suggest that a mutation that destabilizes the inactive conformation of the AChR, together with the sustained exposure of endplates to serum choline, results in continuous channel activity that contributes to the pathophysiology of the disease.

Animals↗

PubMed1999

Is gut the major source of proinflammatory cytokine release during polymicrobial sepsis?

Although studies have shown that the gut is capable of being a cytokine-producing organ and that the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 are upregulated following the onset of sepsis, it remains unknown whether the gut is indeed the major source of the increased cytokine production under such conditions. To determine this, male rats were subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) or sham operation followed by the administration of normal saline solution subcutaneously (i.e., fluid resuscitation). Systemic and portal blood samples were taken simultaneously at 2, 5, 10, or 20 h after CLP or sham operation. Plasma levels of TNF-alpha, IL-1beta, and IL-6 were determined using an enzyme-linked immunosorbent assay. In additional animals, the small intestine was harvested at 10 h after CLP or sham operation and examined for TNF-alpha, IL-1beta, and IL-6 gene expression by RT-PCR. The results indicate that the levels of TNF-alpha, IL-1beta, and IL-6 in both systemic and portal blood samples were significantly elevated during sepsis with the exception that the increase in IL-1beta was not significant at 2 h after CLP. However, there were no significant differences in the levels of those proinflammatory cytokines between systemic and portal blood at any points after the onset of sepsis. Moreover, there were no significant alterations in the proinflammatory cytokine gene expression in the small intestine at 10 h after CLP. Since the levels of TNF-alpha, IL-1beta, and IL-6 were not significantly increased in portal blood as compared to systemic blood and since there was no upregulation of gene expression for these cytokines, it appears that organs other than the gut are responsible for the upregulated proinflammatory cytokines during polymicrobial sepsis.

Animals↗

PubMed1999

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.

Adenosine Triphosphate↗

PubMed1999

A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics.

1. During routine sequencing of our mouse muscle alpha subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. 2. PCR amplification of genomic DNA confirmed that the BALB/C mouse alpha subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. 3. In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. 4. Kinetic modelling of alphaV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine ( approximately 160 microM in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of approximately 1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. 6. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, alphaG153S, was re-examined. The properties of this mutant were similar with a V or an A at position alpha433.

Algorithms↗

PubMed1999

In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme.

The influence of sequence context on the ability of DNA polymerase to bypass sites of base loss was addressed using an in vitro selection system. Oligonucleotides containing either an aldehydic abasic site or tetrahydrofuran surrounded by four randomized bases on both the 5' and 3' sides were used as templates for synthesis by phage T4 DNA polymerase holoenzyme proficient or deficient in the 3'-->5' proofreading exonuclease activity. Successful bypass products were purified, subcloned and the sequences of approximately 100 subclones were determined for each of the four polymerase/lesion combinations tested. Between 7 and 19 % of the bypass products contained deletions of one to three nucleotides in the randomized region. In bypass products not containing deletions, biases for and against certain nucleotides were readily noticeable across the entire randomized region. Template strands from successful bypass products of abasic sites had a high frequency of T in most of the randomized positions, while those from bypass products of tetrahydrofuran had a high frequency of G at the positions immediately to the 3' and 5' side of the lesion. Consensus sequences were shared by successful bypass products of the same lesion but not between bypass products of the two lesions. The consensus sequence for efficient bypass of tetrahydrofuran was over-represented in several frames relative to the lesion. T4 DNA polymerase inserted A opposite abasic sites 63 % of the time in the presence of proofreading and 79 % of the time in its absence, followed by G>T>C, while the insertion of A opposite tetrahydrofuran ranged between 93 % and 100 % in the presence and absence of proofreading, respectively. Finally, sequence context influenced the choice of nucleotide inserted opposite abasic sites and consensus sequences which favored the incorporation of nucleotides other than A were defined.

Bacteriophage T4↗

PubMed1999

Structural basis of the tight binding of pyridoxal 5'-phosphate to a low molecular weight protein tyrosine phosphatase.

Pyridoxal 5'-phosphate (PLP) binds tightly to bovine low Mr protein tyrosine phosphatase (BPTP), but it is a very poor substrate for the enzyme. The structural basis of this tight binding of PLP is examined here by a variety of methods. Binding constants of a number of PLP analogues were measured with wild-type BPTP, and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The tight binding of PLP (Ki = 7.6 microM) causes a downfield shift of the His-72 Cepsilon1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR spectroscopy with an MLEV-17 spectral editing scheme was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. UV-visible spectrophotometric titration also supported this conclusion. The binding constant of PLP to a C17A mutant was similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is "essential" for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. It is concluded that Asp-129 binds to the pyridinium nitrogen of PLP and that this renders Asp-129 effectively unavailable to serve its essential catalytic role as a general acid. The interactions described here should be useful in the design of specific inhibitors of this and related phosphotyrosyl protein phosphatases.

Animals↗