Biomedical subjects

X Zhou

Publications and source records attributed to X Zhou.

At least 325 records · Page 18Linked to original sources

PubMed1999

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Amino Acid Sequence↗

PubMed1999

High-performance liquid chromatography determination of residue levels on chicken carcasses treated with cetylpyridinium chloride.

Cetylpyridinium chloride (CPC) has been found to be effective in reducing contamination of chicken carcasses from a variety of microorganisms, including Escherichia coli O157:H7, Salmonella typhimurium, Campylobacter jejuni, Aeromonas hydrophila, Listeria monocytogenes, and Staphylococcus aureus. A procedure has been developed to determine residue levels on chicken carcasses after CPC treatment. For the analysis, chicken carcasses were extracted with 95% ethanol. The CPC concentration in the extract was measured by high-performance liquid chromatography (HPLC) with ultraviolet detection using dodecylpyridinium chloride (DPC) as an internal standard. The method was validated in the concentration range of 3-200 microg/ml CPC in ethanolic extract. This assay is rapid, precise, and accurate.

Animals↗

PubMed1999

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.

Affinity Labels↗

PubMed1999

Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization.

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Little is known regarding K19 regulation or function, and the only other type I keratin that has been studied in terms of regulation is keratin 18 (K18). We characterized K19 phosphorylation as a handle to study its function. In vivo, serine is the major phosphorylated residue, and phosphopeptide mapping of 32PO4-labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation. Mutation of Ser-10 or Ser-35 followed by transfection confirmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35 --> Ala K19 showed a filament assembly defect as compared with normal or with Ser-10 --> Ala K19. Comparison of K18 and K19 phosphorylation features in interphase cells showed that both are phosphorylated primarily at a single site, preferentially in the soluble versus the insoluble keratin fractions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results demonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphorylations share some features but also have distinct properties that suggest different regulation of type I keratins within the same cells.

Animals↗

PubMed1999

[Genetic linkage of polymorphisms of type I angiotensin II receptor gene to Chinese Han hypertension]

OBJECTIVE: To observe the CA repeat polymorphism of the type I angiotensin II receptor gene on Chinese Han people, and evaluate the genetic linking to hypertension. METHODS: The CA repeat polymorphism at 3'-flanking region of AT1R gene was studied by amplified fragment length polymorphism (Amp-FLP ) in Chinese Han people from Shandong and Xi'an. RESULTS: There were 9 alleles ranging from 130bp to 146bp in all subjects. The allele frequencies were 0.01 to 0.38, and allele A4 was more frequent in the control. The heterozygosity of CA repeat locus was 0.789, the polymorphic information content, 0.746. There was no significant difference in distribution of CA repeat polymorphism genotype between different regions in same ethnic (P>0.05), but the allele frequency of CA repeat polymorphism in hypertensive group was significantly different from that of control (P<0.05). There was no detectable distortion from Hardy-Weinberg equilibrium in both groups. CONCLUSION: The results suggest that CA repeat polymorphism of AT1R gene be associated with Chinese Han hypertensive people.

Journal Article↗

PubMed1999

Role of the 20-hydroxyl group in camptothecin binding by the topoisomerase I-DNA binary complex.

Recent findings concerning the structure of the covalent binary complex formed by DNA topisomerase I and its DNA substrate, as well as the nature of interactions with inhibitors that bind reversibly to this binary complex, have led to two proposed models for the binding of the prototype inhibitor camptothecin to the DNA-topisomerase I binary complex. While these models differ in many regards, they both suggest the involvement of the 20-OH group of camptothecin in a donor hydrogen bond with an enzyme side chain functional group. Presently, five analogues of camptothecin that differ only at C-20 have been evaluated for their ability to bind to the topoisomerase I-DNA binary complex and thereby inhibit enzyme function. Both 20-chloro- and 20-bromocamptothecin bound as well to the enzyme-DNA binary complex as 20-aminoCPT despite the absence of a substituent at C-20 capable of contributing a donor hydrogen bond.

Base Sequence↗

PubMed1999

Increased generation of CD8+ T cell clones in p53 mutant mice.

Very few cultured CD8+ T cell clones can normally be obtained from a single mouse and maintained in long-term culture. To improve the yield, we immunized p53 mutant mice with peptides of Sendai virus (FAPGNYPAL) and influenza virus (ASNENMETM) origin. Substantially more clones could be derived from p53-/- mice than from similarly treated wild-type mice (p53+/+); an intermediate yield was obtained from heterozygous mice (p53+/-). CTL lines or clones from p53-/- mice exhibited greater proliferative activity and resistance to gamma-irradiation than those from p53+/+ mice, and were cytolytically potent.

Animals↗

PubMed1999

Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by tissue iron deposition secondary to excessive dietary iron absorption. We recently reported that HFE, the protein defective in HH, was physically associated with the transferrin receptor (TfR) in duodenal crypt cells and proposed that mutations in HFE attenuate the uptake of transferrin-bound iron from plasma by duodenal crypt cells, leading to up-regulation of transporters for dietary iron. Here, we tested the hypothesis that HFE-/- mice have increased duodenal expression of the divalent metal transporter (DMT1). By 4 weeks of age, the HFE-/- mice demonstrated iron loading when compared with HFE+/+ littermates, with elevated transferrin saturations (68.4% vs. 49.8%) and elevated liver iron concentrations (985 micrograms vs. 381 micrograms). By using Northern blot analyses, we quantitated duodenal expression of both classes of DMT1 transcripts: one containing an iron responsive element (IRE), called DMT1(IRE), and one containing no IRE, called DMT1(non-IRE). The positive control for DMT1 up-regulation was a murine model of dietary iron deficiency that demonstrated greatly increased levels of duodenal DMT1(IRE) mRNA. HFE-/- mice also demonstrated an increase in duodenal DMT1(IRE) mRNA (average 7.7-fold), despite their elevated transferrin saturation and hepatic iron content. Duodenal expression of DMT1(non-IRE) was not increased, nor was hepatic expression of DMT1 increased. These data support the model for HH in which HFE mutations lead to inappropriately low crypt cell iron, with resultant stabilization of DMT1(IRE) mRNA, up-regulation of DMT1, and increased absorption of dietary iron.

Animals↗

PubMed1999

Potassium uptake through the TOK1 K+ channel in the budding yeast.

The current through TOK1 (YKC1), the outward-rectifying K+ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K+ auxotrophy of a mutant missing the two K+ transporters, TRK1 and TRK2. This trk1Delta trk2Delta double mutant hyperexpressing the TOK1 transgene had a higher internal K+ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K+ outward current activating at membrane potential (Vm) above the K+ equilibrium potential (EK+), a small inward current was consistently observed when the Vm was slightly below EK+. The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba2+ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K+ channel can take up K+ into yeast cells, at least under certain conditions.

Fungal Proteins↗

PubMed1999

Central peptidergic neurons are hyperactive during collateral sprouting and inhibition of activity suppresses sprouting.

Little is known regarding the effect of chronic changes in neuronal activity on the extent of collateral sprouting by identified CNS neurons. We have investigated the relationship between activity and sprouting in oxytocin (OT) and vasopressin (VP) neurons of the hypothalamic magnocellular neurosecretory system (MNS). Uninjured MNS neurons undergo a robust collateral-sprouting response that restores the axon population of the neural lobe (NL) after a lesion of the contralateral MNS (). Simultaneously, lesioned rats develop chronic urinary hyperosmolality indicative of heightened neurosecretory activity. We therefore tested the hypothesis that sprouting MNS neurons are hyperactive by measuring changes in cell and nuclear diameters, OT and VP mRNA pools, and axonal cytochrome oxidase activity (COX). Each of these measures was significantly elevated during the period of most rapid axonal growth between 1 and 4 weeks after the lesion, confirming that both OT and VP neurons are hyperactive while undergoing collateral sprouting. In a second study the hypothesis that chronic inhibition of neuronal activity would interfere with the sprouting response was tested. Chronic hyponatremia (CH) was induced 3 d before the hypothalamic lesion and sustained for 4 weeks to suppress neurosecretory activity. CH abolished the lesion-induced increases in OT and VP mRNA pools and virtually eliminated measurable COX activity in MNS terminals. Counts of the total number of axon profiles in the NL revealed that CH also prevented axonal sprouting from occurring. These results are consistent with the hypothesis that increased neuronal activity is required for denervation-induced collateral sprouting to occur in the MNS.

Animals↗

PubMed1999

COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.

The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis.

Apoptosis↗

PubMed1999

pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.

Binding Sites↗

PubMed1999

Expression of the avian Na,K-ATPase subunits in Dictyostelium discoideum.

This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na, K-ATPase subunits. However, Dictyostelium expressed avian Na, K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the alpha1- and beta1-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian beta-subunit alone but only expressed the alpha1-subunit when the beta1-subunit was co-expressed. Subunit assembly occurred in cells expressing both alpha1- and beta1-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na, K-ATPase or free beta-subunit to the plasma membrane.

Animals↗

PubMed1999

Giant pandas discriminate individual differences in conspecific scent.

The functions and mechanisms of chemical communication are well established for many species of small mammal amenable to investigation, but systematic research is virtually absent for large, rare species. The present study marks the first systematic investigation of chemical communication in the endangered giant panda, Ailuropoda melanoleuca. Using a habituation-discrimination paradigm, we sought to determine whether giant pandas can discriminate between scents from different individuals on the basis of chemical cues afforded by female urine and male anogenital gland secretions. Pandas habituated to successive presentations of scent stimuli from a single individual, as evidenced by diminished responsiveness across five habituation trials. In the discrimination test for male anogenital gland secretions, subjects of both sexes investigated a new individual's scent significantly more than a familiar scent to which they were previously habituated. Neither sex significantly discriminated novel from habituated female urine, although a strong tendency to differentiate the stimuli was evident. We also found that the sex of the subject affected patterns of response. Females showed a significant preference for male anogenital gland secretions, whereas males investigated both types of scent stimuli equally. In general, male subjects were more responsive than females to conspecific scent, especially female urine. This study provides clear evidence that giant pandas distinguish individual odours emanating from male anogenital secretions, and implies that these odours contain individually distinctive chemical signatures. These results have implications that can be applied to the conservation and management of this species. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗

PubMed1999

Evidence that IGF-I acts as an autocrine/paracrine growth factor in the magnocellular neurosecretory system: neuronal synthesis and induction of axonal sprouting.

The ability of mature oxytocinergic (OT) and vasopressinergic (VP) neurons of the magnocellular neurosecretory system (MNS) to undergo axonal growth implies that one or more growth factors may be active in the adult MNS, yet little is known regarding their possible identity. One such potential factor is insulin-like growth factor I (IGF-I). We have examined the expression of IGF-I mRNA and IGF-I-immunoreactivity (IGF-I-ir) in the mature MNS and have also determined the in vivo response of OT and VP neurons to hypothalamic implants of IGF-I. In situ hybridization revealed moderate labeling of IGF-I mRNA in both the supraoptic (SON) and the paraventricular (PVN) nuclei of adult male rats. RT-PCR analysis confirmed the presence of authentic IGF-I mRNA in extracts of the basal hypothalamus. Faint IGF-I-ir was detected in scattered magnocellular neurons within both the PVN and the SON of normal rats, but IGF-I-ir was much more intense and the majority of MNS neurons including those in the accessory nuclei were immunoreactive in sections from rats given colchicine, as were some parvocellular neurons in the PVN. Confocal microscopy revealed that IGF-I-ir was present in both OT and VP neurons, but VP neurons contained the most intense IGF-I-ir. Finally, a dramatic growth response of OT but not of VP fibers was observed following implantation of polymer rods containing IGF-I into the hypothalamus. A dense OT fiber plexus grew along the cannula track and OT fibers invaded the leptomeninges ventral to the SON and encircled the rostral cerebral artery. To our knowledge this is the first demonstration of axonal sprouting by mature OT neurons in response to an identified growth factor and the first direct demonstration of sprouting in response to exogenous IGF-I in the adult CNS. These findings suggest that IGF-I is synthesized and transported by adult MNS neurons where it may act as an autocrine and/or paracrine growth factor.

Animals↗

PubMed1999

Posttranscriptional destabilization of the bradykinin B1 receptor messenger RNA: cloning and functional characterization of the 3'-untranslated region.

We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3'-untranslated region (3'-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFalpha, phorbol ester, bradykinin, and desArg10-kallidin contain the same 3'-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3'-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3'-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression.

3' Untranslated Regions↗

PubMed1999

Opening of Rice Floret in Rapid Response to Methyl Jasmonate.

Effects of methyl jasmonate (MeJA) on rice floret opening were investigated in seven cultivars or hybrid combinations covering various variety types. Intact or excised panicles, judged to have florets just before anthesis, were soaked in 4 x 10(-5) - 4 x 10(-3)M MeJA solutions for 2 min at different temperatures. The results indicated that MeJA significantly induced opening of rice florets within about 30 min, with the most rapid induction occurring just 6 min after treatment. Numbers of induced opening florets are correlated with MeJA concentrations. Higher concentrations of MeJA induced more florets. pH values had no influence on MeJA effect, but MeJA required less time and induced more florets at 34 degrees C than at 25 degrees C. As far as we know, this is the first evidence that floret opening is induced by plant hormones. CO(2) evolution from panicles was also increased by MeJA treatment. Field experiments revealed that perfect flowering synchrony between the cytoplasmic male sterile (CMS) and restorer lines in hybrid seed production could be obtained by spraying MeJA solution on CMS line plants at the rate of 25 mg/m(2). As a result, many more hybrid seeds were harvested.

Journal Article↗

PubMed1999

Nonrandom cytogenetic alterations in hepatocellular carcinoma from transgenic mice overexpressing c-Myc and transforming growth factor-alpha in the liver.

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-alpha double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4'-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene and myc transgene site of integration. Moreover, its early occurrence indicates that this is a primary and relevant alteration to the initiation of the neoplastic process. In addition, the concordance between the breakpoints observed during the early dysplastic stage of hepatocarcinogenesis and the stable deletions of chromosomes 1, 4, 6, 7, 9, and 12 in the tumors provides evidence for preferential site of genetic changes in hepatocarcinogenesis.

Age Factors↗