[MicroRNA-30a inhibits proliferation of hepatocellular carcinoma cells via targeted regulation of forkhead-box protein A1].

Objective: To investigate the expression of microRNA-30a (miR-30a) in hepatocellular carcinoma (HCC) and related molecular mechanisms in regulating HCC cell proliferation. Methods: A total of 30 pairs of HCC and adjacent tissue samples were collected, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of forkhead-box protein A1 (FOXA1). Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of HCC cells, luciferase reporter gene assay was performed to verify the target relationship between miR-30a and FOXA1, and MTT assay and Western blot were used to measure the proliferation of HepG2 cells and the protein expression of FOXA1 after miR-30a transfection. The t-test was used for comparison of data between two groups, and a one-way analysis of variance was used for comparison of data between multiple groups. P < 0.05 was considered statistically significant. Results: HCC tissue had significantly lower relative expression of miR-30a than adjacent tissue (1.049 ± 0.380 vs 1.982 ± 1.013, t = 3.985, P < 0.001). At 72 hours after miR-30a overexpression, there was a significant difference in the proliferative capacity of HepG2 cells between the blank control group, the miR-30a-NC group, and the miR-30a group (0.821 ± 0.006 vs 0.816 ± 0.013 vs 0.546 ± 0.020, F = 3.396, P < 0.05), suggesting that miR-30a overexpression significantly inhibited the proliferation of HepG2 cells. FOXA1 was a target gene of miR-30a and its protein expression was negatively regulated by miR-30a, and there was a significant difference in luciferase activity between wild-type and mutant FOXA1-3'UTR vectors (1.221 ± 0.024 vs 2.658 ± 0.031, F = 6.737, P < 0.05). In HepG2 cells, miR-30a overexpression significantly inhibited the protein expression of FOXA1, and there was a significant difference in the relative expression of FOXA1 between the blank control group, the miR-30a-NC group, and the miR-30a group (1.019 ± 0.016 vs 1.022 ± 0.017 vs 0.227 ± 0.021, F = 45.43, P < 0.05). Upregulating the protein expression of FOXA1 reversed the inhibitory effect of miR-30a on the proliferation of HepG2 cells, and there was a significant difference in the proliferative capacity of HepG2 cells between the miR-30a group and the miR-30a+FOXA1 group (0.524 ± 0.023 vs 0.843 ± 0.019, t = 2.507, P < 0.05). Conclusion: MiR-30a exerts its inhibitory effect on the proliferation of HCC cells by negatively regulating the expression of FOXA1.

Derivation and characterization of primordial germ cells from Guangxi yellow-feather chickens.

The Guangxi yellow-feather chicken is a very important breed used as a broiler in southern China, but the pure line is being threatened by continual introduction of foreign genetics into its breeding program to make it more marketable. In the current study, we isolated primordial germ cells (PGCs), a cell type committed to form sperm or eggs and that is responsible for passing genetic material from one generation to the next, from Guangxi yellow-feather chickens and cultured them in a cell-insert system. Three stable cell lines, all male, were established from 10 isolations. These cells proliferated and expressed germ cell-related markers such as SSEA-1 and EMA-1 after prolonged culture in vitro. After genetic modification, these PGCs retained significant potential to colonize the gonads and give rise to gametes when they were reintroduced into the vasculature of stage-15 HH embryos, confirming their germline cell characteristics. The ability to culture PGCs and preserve the genetics from this species would not only be of significant importance for biodiversity conservation, but also holds promise for use of these cells in breeding strategies in the future.

Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro.

1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including Ki, Ki', Vmax, and IC50 were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Kis of 13.9 and 38.6 µM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug-drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.

Role and diagnostic value of gene variants in assessing the risk of chronic obstructive pulmonary disease.

Meta-analyses have revealed many positive associations between gene variants and susceptibility to chronic obstructive pulmonary disease (COPD). However, some of those positive results may be false positives. Therefore, we investigated the genetic polymorphisms associated with COPD risk and determined their diagnostic value. We extracted the odds ratio (OR) and 95% confidence interval for each polymorphism from published meta-analyses concerning gene variants and COPD susceptibility in October 2014, subsequently we calculated false-positive report probabilities (FPRPs) for statistically significant associations (P value < 0.05). We determined the diagnostic value of the true positive polymorphisms of COPD using the Meta-DiSc software. Twenty-five gene polymorphisms were significantly associated with COPD risk. The FPRP test results were as follows: 1) when the prior probability was 0.001 and the OR was 1.5, ADAM33 rs612709, CHRNA3/5 rs1051730, CHRNA3/5 rs8034191, CHRNA3/5 rs16969968, and TGFB1 rs1800470 were truly associated with COPD risk (FPRP < 0.2); 2) when the prior probability was 0.000001 and the OR was 1.5, all the variants except TGFB1 rs1800470 remained noteworthy; and 3) when the probability was 0.000001 and the OR was 1.2, ADAM33 rs612709 and CHRNA3/5 rs1051730 remained true positives. Unfortunately, the results of the diagnostic accuracy meta-analyses suggested that none of the variants had high value for COPD diagnosis.

Long-term efficacy of miglustat in paediatric patients with Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) is a rare inherited neurovisceral disease characterized by progressive neurological manifestations. Oral miglustat was first approved for the treatment of children and adults with NP-C in Europe in 2009. There are still relatively few published data on the long-term efficacy and safety of miglustat in patients with NP-C in clinical practice. We report the effects of up to 6 years of treatment with miglustat 100 mg t.i.d. in five children. Overall, 3/5 patients displayed progressive dysphagia before starting miglustat, and 4/5 showed marked cognitive and/or motor impairment. The mean age at treatment start was 11.6 years, and the median (range) duration of therapy so far is 4 (4.1 to 6.1) years. No treatment dose alterations were required, but therapy was interrupted for 1-3 months at least once in all patients due to supply issues. Swallowing function was stabilised during miglustat therapy, with no significant increase in Han dysphagia scale or aspiration-penetration index scores among four evaluable patients (p > 0.05). Scores on the mini-mental state examination indicated an improvement in cognitive function during the first 3-6 months of miglustat therapy, followed by stabilisation up to 5 years. Ambulatory function remained stable for at least the first 2 years of treatment in most patients, but there was a trend towards deterioration thereafter, possibly related to treatment interruptions. The safety/tolerability profile of miglustat was similar to previous clinical studies, although reports of gastrointestinal disturbances were rare. Overall, miglustat appeared to stabilise key parameters of neurological disease progression.

Treatment of Niemann-Pick disease type C in two children with miglustat: initial responses and maintenance of effects over 1 year.

Niemann-Pick disease type C (NP-C) is a lipid storage disorder characterized by the accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system of certain cells in the central nervous system (CNS) and visceral organs. Clinical symptoms include progressive neurological deterioration and visceral organomegaly. Miglustat, a small iminosugar molecule approved for the treatment of Gaucher disease, reversibly inhibits glucosylceramide synthase, which catalyses the first committed step in glycosphingolipid synthesis. The physicochemical properties of miglustat allow it to cross the blood-brain barrier and suggest possible benefits in lysosomal storage diseases affecting the CNS. Here, we present findings in two children with NP-C, aged 14 years (patient 1) and 9 years (patient 2), treated with miglustat for 1 year. Before treatment, patient 1 presented with severe difficulties in swallowing and walking, and patient 2 with problems mostly affecting communication and social interaction. Videofluoroscopic studies in patient 1 demonstrated a substantial improvement in swallowing by month 6 of treatment, and ambulation index measurements indicated improved walking. Mini Mental-State Examination (MMSE) assessments in patient 2 showed cognitive improvement by month 6, which was sustained up to month 12. Liver/spleen volume and plasma chitotriosidase activities were stabilized in both cases. There was no weight loss during treatment. Patient 1 experienced severe but self-limiting paresthesia, which was not associated with peripheral neuropathy. We conclude that miglustat can provide therapeutic benefits in CNS symptoms and allows stabilization of systemic disease in childhood-onset NP-C. Further follow-up is crucial to determine the long-term maintenance of these effects.

Paraganglioma manifesting as shock: a case report.

Paraganglioma is a rare neuroendocrine tumor in children that rarely manifests as shock. We describe the case of a 12-year-old girl with paraganglioma who developed impaired cardiac function, pulmonary edema, and shock at the time of admission. Her blood pressure stabilized after intravenous normal saline rescue and dopamine treatment. However, hypertension was noted thereafter. After a series of examinations, paraganglioma was diagnosed and excision of the tumor was performed. After surgery, blood pressure stabilized and her cardiac function had fully recovered at 4 months' follow-up.

Congenital intracranial teratoma.

Congenital intracranial teratoma is a rare disease. A fetus with a congenital intracranial teratoma presenting with a disproportionately enlarged head at 27 weeks gestation is presented. Prenatal ultrasonography and fetal magnetic resonance imaging demonstrate a huge, heterogenous intracranial mass in the left supratentorial region, with the left cerebral hemisphere being compressed and flattened. The infant died of respiratory failure within 24 hours of birth at 28 weeks gestation. On postmortem examination the histologic report revealed an immature teratoma. Fetal MRI is helpful in the prenatal diagnosis and evaluation of intracranial tumor.

Power Doppler ultrasound imaging in neonatal cerebral venous sinus thrombosis.

Neonatal sinus thrombosis is a rare occurrence in sick neonates. Because of its nonspecific manifestations, the incidence is underestimated. This disease may not be demonstrated by conventional color Doppler and is diagnosed by computed tomography or magnetic resonance imaging. The authors report a neonate with neonatal sinus thrombosis diagnosed by power Doppler and suggest that the technique may be used as a less expensive and more available screening and follow-up method in high-risk neonates.

Physical mapping of RFLP markers on four chromosome arms in maize using terminal deficiencies.

Terminal deficiencies (TDs) generated by the r-X1 deletion system in maize were used to physically map RFLP markers on the short arm of chromosome 2 (2S) and the long arm of chromosome 6 (6L), chromosome 8 (8L), and chromosome 10 (10L). Five TDs on 2S, 8 on 6L, 10 on 8L, and 20 on 10L were isolated using the recessive morphological markers lg1, py1, j1(gl18), and sr2, respectively, for selection. Two exceptional TDs on 2S and 8L also have a second breakpoint on the long arm of chromosome 2 (2L) and 8L, respectively. The physical mapping of RFLP probes in relation to TD breakpoints was done by Southern hybridization. The five TDs on 2S divide chromosome 2 into four regions, all of which are distinguishable by RFLP markers. Likewise, three remaining chromosome arms are divided by TDs into RFLP-marked regions: 8 TDs divide 6L into five regions, 10 TDs divided 8L into seven regions, and 20 TDs divide 10L into three regions. The linear order of the physical map of 6L and 8L is consistent with that of the genetic maps, but that of 2L and 10L is not. Four groups of markers on 2S as well as 2L, and two on 10L are in reverse order in the physical map compared with the genetic maps. Other intriguing results are that breakpoints of TDs on 6L and 8L are distributed throughout the selected region, but most of those on 2L and 10L cluster in a region near the centromere: a single TD arose after fertilization.

Magnetic resonance imaging in patients with hemodialysis-related arthropathy.

From April 1993 to December 1993, we prospectively studied the knees of 15 patients (mean age, 48 +/- 11 yr) receiving long-term hemodialysis (mean duration, 9 +/- 4 yr) using magnetic resonance imaging (MRI) techniques including, T1 weighted spin-echo, multiplanar gradient recalled, and postcontrast T1 with chemical shift-selective, fat-saturation pulse sequences. Analysis of these images revealed that the three findings most indicative of hemodialysis-related arthropathy were intramedullary, cortical and soft tissue lesions. Knee pain was significantly correlated with the presence of soft tissue lesions. Cortical lesions were usually associated with soft tissue lesions. Inflammatory changes adjacent to soft tissue lesions were demonstrated in postcontrast studies in all patients with soft tissue lesions. Increases in water content in those lesions appeared to increase the signal intensity. Our results indicate that MRI is useful in demonstrating the extent of hemodialysis-related arthropathy involvement, especially in hemodialysis patients suffering from knee pain.

High-resolution computed tomography in pulmonary lymphangio(leio)myomatosis and pulmonary tuberous sclerosis.

We report two cases where high-resolution computed tomography (HRCT) assisted in the diagnosis of a patient with lymphangioleiomyomatosis and a patient with pulmonary tuberous sclerosis. HRCT was used in both cases where the chest radiographs and conventional computed tomographic scans appeared relatively normal but the conditions were strongly suspected.

[Gene cloning, expression of fusion protein TGF alpha-PE 40 and its inhibition activity on cancer cell growth].

Recombinant plasmid pYX382 and pYX3825 were constructed by fusing the cDNA encoding transforming growth factor type alpha (TGF alpha) to Pseudomonas exotoxin gene (PE) in which the cell recognition domain was deleted. The chimeric proteins produced by host E. Coli cells BL21 transformed by plasmid pYX382 and pYX3825 are termed TGF alpha-PE 40 which reacts with antibody against TGF alpha or antibody against PE in immunoblotting to show a 46 kd protein band reflecting the fusion of 56 kD TGF alpha peptide and 40 kD truncated Pseudomonas exotoxin molecule. An additional signal sequence OmpA was inserted into upstream region of TGF alpha cDNA in plasmid pYX3825 resulting in the partly secreting of expression product into medium and periplasm of the cells. TGF alpha-PE 40 was purified from medium by MONO Q ion exchange column and TSK 250 gel filtration column attached to Pharmacia EPLC system. The TGF alpha-PE 40 molecules showed a very strong activities inhibiting the protein synthesis and killing the cancer cells overexpressing EGF receptor on the cell surface.

[Analysis of EGFR gene transcription in rat embryo and adult tissues].

EGFR gene transcription in rat embryo and adult tissues were analysed by RNA Northern and Dot hybridization using 2.4 Kb human EGFR cDNA pE 7 as probe. In Northern hybridization (Fig. 1), the normal rat liver RNA showed a major 5.6 Kb RNA band which was a common EGFR mRNA present in human normal and cancer cells. For the comparison of EGFR gene transcription in embryo of different stages and different tissues, the density scanning data of dot hybridization signals are shown as bars in accompanying figures. The results indicated that the highest level of EGFR transcription was seen in rat embryo at 10th to 12th day during neonatal development (Fig. 2 and Fig. 3). In adult rat tissues, EGFR gene was commonly expressed with some variation in different tissues, for example, much more expressed in kidney than in liver, heart, brain and spleen, and moderately expressed in testis and lung (Fig. 4). The EGFR gene transcription could be induced in liver by hepatectomy. Fig. 5 showed the increase of EGFR gene transcripts at the 2-8 th hour of liver regeneration and the decrease after the 8th hour, even reaching a value lower than that of normal liver after the 24th hour. The results suggest that EGFR gene transcripts are commonly present in a wide range of normal tissues and may be involved in the control of proliferation and differentiation in embryo and adult tissues.

[EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells].

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.

A study on Chinese phenylalanine hydroxylase gene restriction site polymorphism.

Human phenylalanine hydroxylase (PAH) cDNA was applied as a hybridization probe to analyzing the following 8 restriction fragment length polymorphisms (RFLP) in the PAH genes of 80 normal and 28 phenylketonuric Chinese patients: BglII, 3.6 kb/1.7 kb; EcoRI, 17 kb/11 kb; EcoRV, 30 kb/25 kb; HindIII, 4.2 kb/4.0 kb; MspIa, 23 kb/19 kb; MspIb, 4.0 kb/2.2 kb; PvuIIa, 19 kb/6.0 kb and PvuIIb, 11.5 kb/9.1 kb. The frequencies of the above RFLP in normal Chinese and PKU patients are: 0.13, 0.83, 0.77, 0.81, 0.12, 0.04, 0.70, 0.10 and 0.12, 0.93, 0.89, 0.81, 0.04, 0, 0.69, 0.04, respectively. This study reveals significant differences between the frequencies of individual RFLPs when these values are compared with those of the Caucasians. Finally, the detection of RFLPs in the PAH gene in the Chinese population will permit prenatal diagnosis of PKU in China.