Autophagic flux is essential for the downregulation of <Emphasis Type="Italic">D</Emphasis>-dopachrome tautomerase by atractylenolide I to ameliorate intestinal adenoma formation

Colorectal cancer is generally believed to progress through an adenoma - carcinoma sequence. Adenomatous polyposis coli (APC) mutations serve as the initiating event in adenoma formation. The Apc^Min/+ mouse harbors a mutation in the APC gene, which is similar or identical to the mutation found in individuals with familial adenomatous polyposis and 70% of all sporadic CRC cases. Autophagy is a constitutive process required for proper cellular homeostasis. However, its role in intestinal adenoma formation is still controversial. Atractylenolide I (AT1) is a sesquiterpenoid that possesses various clinically relevant properties such as anti-tumor and anti-inflammatory activities. The role of AT1 on adenoma formation was tested in Apc^Min/+ mice and its underlying mechanism in regulating autophagy was documented. D-dopachrome tautomerase (D-DT) was identified as a potential target of AT1 by an proteomics-based approach. The effects of p53 modification on autophgic flux was monitored in p53^?/? and p53^+/+ HCT116 cells. Small interfering RNA was used to investigate the function of Atg7 and D-DT on autophagy programme induce by AT1. AT1 effectively reduced the formation of adenoma and downregulated the tumorigenic proteins in Apc^Min/+ mice. Importantly, AT1 stimulated autophagic flux through downregulating acetylation of p53. Activation of Sirt1 by AT1 was essential for the deacetylation of p53 and downregulation of D-DT. The lowered expression of COX-2 and β-catenin by AT1 were partly recovered by Atg7 knockdown. AT1 activates autophagy machinery to downregulate D-DT and reduce intestinal adenoma formation. This discovery provides evidence in vivo and in vitro that inducing autophagy by natural products maybe a potential therapy to ameliorate colorectal adenoma formation.     

Impact of Sur1 gene inactivation on the morphology of mouse pancreatic endocrine tissue

In congenital hyperinsulinism of infancy (CHI), the loss of K-ATP channels (composed of Kir6.2 and SUR1 subunits) in β cells induces permanent insulin secretion and severe hypoglycaemia. By contrast, Sur1 ^?/? mice do not present such defects. We have investigated the impact of Sur1 gene inactivation on mouse islet cell morphology, structure and basic physiology. Pancreata were collected from young, adult and old wild-type (WT) and Sur1 ^?/? mice. After immunostaining for hormone, the total endocrine tissue, cell proportion, cell size and intra-insular distribution, hormone content and Glut-2 expression were quantified by morphometry. Basic physiological parameters were also measured. In young Sur1 ^?/? mice, the total endocrine tissue and proportion of β cells were higher (P<0.05) than in WT mice, whereas the proportion of δ cells was lower (P<0.01). In old Sur1 ^?/? mice, α cells were frequently located in the central regions of islets (unlike WT islets) and their proportion was increased (P<0.05). Glut-2 protein and mRNA levels were lower in old Sur1 ^?/? islets (P<0.02). Insulinaemia, fasting insulin and glucagon contents were equivalent in both groups of pancreata. Thus, the islets of Sur1 ^?/? mice present morphological modifications that have not been described in CHI and that might reflect an adaptive mechanism controlling insulin secretion in these mice.     

Analytical modeling of the mechanics of early invasion of a merozoite into a human erythrocyte

In this study, we used a continuum model based on contact mechanics to understand the mechanics of merozoite invasion into human erythrocytes. This model allows us to evaluate the indentation force and work as well as the contact pressure between the merozoite and erythrocyte for an early stage of invasion (γ = 10%). The model predicted an indentation force of 1.3e ^?11N and an indentation work of 1e ^?18J. The present analytical model can be considered as a useful tool not only for investigations in mechanobiology and biomechanics but also to explore novel therapeutic targets for malaria and other parasite infections.     

The role of interspecies differences in the ratio of choline-containing phospholipid fractions of rodent erythrocytes in response of these cells to the effect of membranotropic compounds

In in vitro experiments, interspecies differences were revealed in the erythrocyte responses in varied rodent species—laboratory mice (Mus musculus L.), tundra voles (Microtus oeconomus Pall.) and bank voles (Myodes glareolus Pall.)—to the effect of chemical agents able to interact with membrane lipids and disrupt the membrane structure (detergent Triton X-100, oxidative stress inductor AAPH, antioxidant ionol or BHT, uranyl ion). It was hypothesized that these differences are due to physicochemical peculiarities of the erythrocyte membrane structure, specifically, the ratio of choline-containing fractions of phospholipids (phosphatidylcholine and sphingomyelin). The use of blood erythrocytes as an in vitro experimental model to study the mechanisms of toxicity as well as antioxidant and membrane-protective properties of compounds of different nature was shown to imply the choice of an adequate source of erythrocytes in view of considerable speciesdependent structural specificity of the lipid component of mammalian erythrocyte membranes.     

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm^-/- and p53^-/- consistently develop thymic lymphoma early in life ^1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols ^1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain ^1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm^-/- [FVB/N-Atm^tm1Led/J] ² and p53^-/- [129/S6-Trp53^tm1Tyj/J] ⁵ mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.Download video file.^{(97M, mov)}     

Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC^1311/+; orthologous to human APC^1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC^1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5′-flanking, coding, and 3′ untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC^1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC^1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC^1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p?= 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.     

The role of the P2X7 receptor in murine cutaneous leishmaniasis: aspects of inflammation and parasite control

Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The immunopathology of leishmaniasis caused by L. amazonensis infection is dependent on the pathogenic role of effector CD4⁺ T cells. Purinergic signalling has been implicated in resistance to infection by different intracellular parasites. In this study, we evaluated the role of the P2X7 receptor in modulating the immune response and susceptibility to infection by L. amazonensis. We found that P2X7-deficient mice are more susceptible to L. amazonensis infection than wild-type (WT) mice. P2X7 deletion resulted in increased lesion size and parasite load. Our histological analysis showed an increase in cell infiltration in infected footpads of P2X7-deficient mice. Analysis of the cytokine profile in footpad homogenates showed increased levels of IFN-γ and decreased TGF-β production in P2X7-deficient mice, suggesting an exaggerated pro-inflammatory response. In addition, we observed that CD4⁺ and CD8⁺ T cells from infected P2X7-deficient mice exhibit a higher proliferative capacity than infected WT mice. These data suggest that P2X7 receptor plays a key role in parasite control by regulating T effector cells and inflammation during L. amazonensis infection.     

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon <Emphasis Type="Italic">Tyr-Cre</Emphasis>-Mediated Deletion of the Autophagy Gene <Emphasis Type="Italic">Atg7</Emphasis>

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7^f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7^f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7^f/f Tyr-Cre mice relative to Atg7^f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7^f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7^f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.     

Impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in Africa: case studies of South Africa,Zambia and Ethiopia

Background

Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of Plasmodium -infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of Plasmodium -infected erythrocytes, development of parasitaemia and thus the course of malaria.

Methods

Human erythrocytes were infected in vitro with Plasmodium falciparum (P. falciparum) (strain BinH) in the absence and presence of azathioprine (0.001 – 10 μM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with Plasmodium berghei (P. berghei) ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection.

Results

In vitro infection of human erythrocytes with P. falciparum increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (≥ 1 μM) and in vitro parasitaemia (≥ 1 μM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of P. berghei -infected mice (from 0% to 77% 22 days after infection).

Conclusion

Azathioprine inhibits intraerythrocytic growth of P. falciparum, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.     

Mutant <Emphasis Type="Italic">TP53</Emphasis> G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

Background

TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53?/?.

Results

MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.

Conclusions

These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

     

Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-¹³C]glucose and [1,2-¹³C]acetate injection with ex vivo ¹³C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-¹³C]glutamate and [4-¹³C]glutamine, and percent enrichment in [4-¹³C]glutamate were detected in the α-syn KO mice. [1,2-¹³C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.     

Spontaneous recombinase activity of Cre–ERT2 in vivo

Inducible Cre–ERT recombinase technology is widely used for gene targeting studies. The second generation of inducible Cre–ERT recombinase, hemizygous B6.129S-Tg(UBC-cre/ERT2)1Ejb/J (hereafter abbreviated as Cre–ERT2), a fusion of a mutated estrogen receptor and Cre recombinase, was engineered to be more efficient and specific than the original Cre–ERT. The putative mechanism of selective Cre-mediated recombination is Cre sequestration in the cytoplasm in the basal state with translocation to the nucleus only in the presence of tamoxifen. We utilized both a reporter mouse (B6.129 (Cg)-Gt(ROSA)26Sor ^{tm4(ACTB-tdTomato,-EGFP)Luo} /J) and endothelin converting enzyme-1 floxed transgenic mouse line to evaluate Cre–ERT2 activity. We observed spontaneous Cre activity in both settings. Unintended Cre activity is a confounding factor that has a potentially large impact on data interpretation. Thus, it is important to consider background Cre activity in experimental design.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).     

Inactivation of bone morphogenetic protein 1 (<Emphasis Type="Italic">Bmp1</Emphasis>) and tolloid-like 1 (<Emphasis Type="Italic">Tll1</Emphasis>) in cells expressing type I collagen leads to dental and periodontal defects in mice

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the β-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the β-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 ^flox/flox and Tll1 ^flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3 kb Col1a1-Cre mice with the Bmp1 ^flox/flox and Tll1 ^flox/flox mice, we further generated Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.     

Association of <Emphasis Type="Italic">p</Emphasis>53 codon 72 polymorphism and survival of North Indian lung cancer patients treated with platinum-based chemotherapy

p53 helps in maintaining genomic stability by undergoing cellular arrest, DNA repair or cellular apoptosis during DNA damage. So, as to find the association of p53Arg ⁷² Pro towards lung carcinogenesis and overall survival of North Indian lung cancer patients, single nucleotide polymorphic variant (rs1042522) was analyzed. 840 subjects including 420 cases and 420 controls were recruited and genotyped using PCR-RFLP technique for p53Arg ⁷² Pro polymorphic site. Association was analyzed using adjusted odds ratio along with its confidence intervals (95?% CI) and p value predicted from logistic regression whereas overall survival for lung cancer patients was obtained using Kaplan–Meir and Cox regression model for different parameters to obtain hazard ratio and survival time with statistical significance (log-rank p value). None of the variant genotypes for p53Arg ⁷² Pro showed any association towards lung cancer risk or any specific histological subtype. Lung cancer subjects with Pro/Pro genotype had better median survival time as compared to Arg/Pro genotype (10 months; HR?=?0.65; 95?% CI?=?0.45–0.95; p?=?0.03). Furthermore, female lung cancer patients with Arg/Pro (HR?=?0.08; 95?% CI?=?0.02–0.34; p?=?0.0005) and Pro/Pro (HR?=?0.21; 95?% CI?=?0.06–0.67; p?=?0.008) genotypes showed a better overall survival and hence a better prognosis as compared to males. Our data also reveals that lung cancer patients with ECOG scores between 0 and 1 and carrying the Pro/Pro had better chances of survival. p53 codon 72 polymorphism could play a role as a prognostic marker in lung cancer patients.