The amyloid structure of mouse RIPK3 (receptor interacting protein kinase 3) in cell necroptosis.

RIPK3 amyloid complex plays crucial roles during TNF-induced necroptosis and in response to immune defense in both human and mouse. Here, we have structurally characterized mouse RIPK3 homogeneous self-assembly using solid-state NMR, revealing a well-ordered N-shaped amyloid core structure featured with 3 parallel in-register ß-sheets. This structure differs from previously published human RIPK1/RIPK3 hetero-amyloid complex structure, which adopted a serpentine fold. Functional studies indicate both RIPK1-RIPK3 binding and RIPK3 amyloid formation are essential but not sufficient for TNF-induced necroptosis. The structural integrity of RIPK3 fibril with three ß-strands is necessary for signaling. Molecular dynamics simulations with a mouse RIPK1/RIPK3 model indicate that the hetero-amyloid is less stable when adopting the RIPK3 fibril conformation, suggesting a structural transformation of RIPK3 from RIPK1-RIPK3 binding to RIPK3 amyloid formation. This structural transformation would provide the missing link connecting RIPK1-RIPK3 binding to RIPK3 homo-oligomer formation in the signal transduction.

[Association between left ventricular twist/untwist and diastolic dysfunction of high cardiovascular risk population in the community].

OBJECTIVE: To assess the association between left ventricular (LV) twist and untwist with the severity of diastolic dysfunction of high cardiovascular risk population in the community. METHODS: This cross-sectional survey was performed in high cardiovascular risk people with normal left ventricular (LV) ejection fraction in an urban community of Beijing (n = 620). Normal LV diastolic function was defined in 305 subjects, mild diastolic dysfunction in 266 subjects and moderate/severe diastolic dysfunction in 49 subjects. Peak LV twist, peak twist velocity, peak untwist velocity and untwist rate were measured in apical and basal short-axis images using speckle tracking echocardiography. RESULTS: Peak LV twist was similar among subjects with normal diastolic function, mild diastolic dysfunction and moderate/severe diastolic dysfunction. Peak twist velocity [(129.3 ± 45.3)°/s vs. (118.0 ± 36.2)°/s] and untwist velocity [(-132.9 ± 50.4) °/s vs. (-121.2 ± 41.4)°/s] were significantly higher in mild diastolic dysfunction group than in normal diastolic function group (all P < 0.01) and similar between normal diastolic function and moderate/severe diastolic dysfunction group (P > 0.05). Untwist rate of moderate/severe diastolic dysfunction decreased significantly than that of normal diastolic function [(41.9 ± 32.9)°/s vs. (57.7 ± 36.2) °/s, P < 0.01] and mild diastolic dysfunction group [(41.9 ± 32.9)°/s vs. (60.9 ± 39.9) °/s, P < 0.01]. CONCLUSIONS: Twist and untwist parameters are increased/preserved in population with normal systolic function and mild diastolic dysfunction and "normalized" or reduced in those with advanced diastolic dysfunction. The maintaining (if not increasing) of LV twist in early diastolic dysfunction might serve as a compensatory mechanism in case of reduced myocardial relaxation in these subjects.

Detection of the rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil using a ligase detection reaction assay.

BACKGROUND: Adefovir dipivoxil (ADV) is effective for treatment of chronic hepatitis B virus (HBV) infection, but long-time ADV therapy leads to drug resistance because of HBV reverse transcriptase mutations. We developed a sensitive and specific method for detecting the rtA181V/T and rtN236T mutations associated with ADV resistance in chronic hepatitis B patients, based on a ligase detection reaction (LDR). METHODS: HBV templates were amplified by polymerase chain reaction (PCR), followed by LDR and electrophoresis on a sequencer. The assay was evaluated using 165 serum samples, and plasmid controls. RESULTS: In a mixture of wild-type and mutant plasmids, the assay could detect mutant plasmid at 1%. Complete concordance between the PCR-LDR assay and sequencing analysis was observed for 141 of 148 samples (95.3%) at codon 181, and 143 of 148 samples (96.6%) at codon 236. Discordant results were confirmed to be consistent with the PCR-LDR assay by subclone sequencing. Seventeen samples could not be detected by both of the methods due to low HBV DNA levels. CONCLUSIONS: The PCR-LDR assay can sensitively and specifically detect the rtA181V/T and rtN236T mutations, and may be used for monitoring ADV resistance in patients infected with HBV.

Undifferentiated embryonal sarcoma of liver in an old female: case report and review of the literature.

The clinical characteristics of undifferentiated (embryonal) sarcoma of the liver (UESL) were investigated and the best treatment modalities were recommended. Both histology and immuno-histochemistry demonstrated the cellular features of this peculiar tumor. The tumor size was 12 cm multiply 9 cm multiply 8 cm in the right liver lobe. The patient underwent surgical resection of the tumor. The postoperative recovery was uneventful and she died eight months after diagnosis. The tumor showed mixed spindle and polygonal cells within the myxoid matrix. Some tumor cells contained eosinophilic hyaline globules that were positive for resistant diastase. Immunohistochemistry showed positive vimentin. Stellate and spindle cells were positively stained with alpha-1-antichymotrypsin (AACT) and CD68. This case indicates that UESL is not obviously differentiated in old-aged adults.

Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.

AIM: To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS: Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS: Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 (33%), 41 (33%) and 45 (36%) samples, and genotype C in 76 (60%), 76 (60%) and 81 (64%) samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real-time PCR was the rapidest and cheapest among the three assays. CONCLUSION: Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

[Clinical significance of AFP-L3 variants determined by micro centrifugal column].

OBJECTIVE: To evaluate the clinical significance of AFP-L3 in patients with hepatocellular carcinoma. METHODS: Serum AFP-L3 variants were separated by micro centrifugal column, and detected by chemiluminescence. RESULTS: AFP and AFP-L3 levels were higher in patients with hepatocellular carcinoma than those in patients with chronic hepatitis (P<0.001); as a diagnostic target, the sensitivity and specificity of AFP-L3 were 72.3 percent and 97.2 percent, respectively. Eight patients with hepatitis have higher AFP-L3, but none of them were found with carcinoma by CT three months later. CONCLUSION: AFP-L3 is very useful in the diagnosis of patients with hepatocellular carcinoma.

Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor.

Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo(r)), replaced the alpha-lactalbumin gene in a 210kb human alpha-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.

[Expression of goat beta-casein gene targeting vector in mammary gland cell].

The study of mammary gland bioreactor is in the ascendant. In order to generate transgenic goats of well-controlled expression of exogenic genes, we constructed a human lactoferrin (hLF) gene targeting vector containing promoter, exon 1, intron1 and some of exon 2 (about 6.1 kb fragment) and exon 6 approximately 9 (about 3.3 kb fragment) of the goat beta-casein gene as well as hLF minigene, neo gene inserted into them and tk gene ligated to the 3' end of the construct. The 9.4 kb goat genomic sequences as homologous arms were initially amplified by PCR with local goat tissue DNA. The expression vector was named pBC-tk-neo-hlf. Then the recombinant plasmid pBC-tk-neo-hlf containing hLF minigene was transfected into mice mammary tumor cell line C127 by liposome, cell clones were selected with G418. After proliferating, the transfected cells were induced with insulin, luteotropic hormone and hydrocortisone. The result of Western-blotting analysis showed that the transfected cells can secrete hLF protein, and the recombinant protein expressed in cultured cell supernatant has the similar molecular weight as the native protein. The expression level detected by ELISA was 0.21 microg/mL. This result indicated that the targeting vector could efficiently direct the expression of hLF in mammary cells,and it confirmed the validity of the constructed vector. At the same time, C127 cell line proved to be useful for evaluating the regulation of a foreign gene expression in mammary gland specific expression vector.

[Flow cytometric detection and significance of four cyclins in esophageal cancer].

OBJECTIVE: To study the expression and significance of cyclin E, cyclin D1, CDK4 and p27 protein in esophageal squamous cell cancer (ESCC) and their correlation with tumor differentiation and lymph node metastasis. METHODS: Expressions of cyclin E, cyclin D1, CDK4 and p27 protein in 65 patients with ESCC were quantitatively detected by flow cytometry. RESULTS: The expressions of cyclin E, cyclin D1, CDK4 in poorly-differentiated ESCC were higher than those in well-differentiated ESCC (P = 0.0275, 0.0001, 0.0174). The expression of p27 in poorly-differentiated ESCC was lower than that in well-differentiated ESCC (P = 0.0042). There was positive correlation between cyclin E and cyclin D1, cyclin D1 and CDK4, but negative correlation between cyclin D1 and p27. The expressions of all four proteins were not correlated with lymph node metastasis. CONCLUSION: The expressions of cyclin E, cyclin D1, CDK4 and p27 are closely related to tumor differentiation of ESCC. An imbalance between positive and negative control of cell cycling might be critical in the carcinogenesis of esophageal squamous cell cancer.