A new contingent screening strategy increased detection rate of trisomy 21 in the first trimester.

BACKGROUND: Although the traditional contingent screening strategy is effective, there are still undetected low-risk trisomy 21. This study aims to define appropriate cut-off values of serum biochemical markers at low-risk and develop a strategy for sequential prenatal testing associated with first-trimester screening to increase the detection rate of trisomy 21. METHODS: This was a 9-year retrospective analysis of singleton pregnant women who underwent serum biochemical screening or combined first-trimester screening (CFTS) in the first trimester. For the low-risk group, the cut-off values of the serum biochemical markers were adjusted to determine the appropriate detection efficiency. Gravidas with abnormal serum biochemical markers at low-risk were advised to undergo further non-invasive prenatal screening (NIPS), whereas others continued with routine prenatal care. RESULTS: When cut-off values of free beta subunit of human chorionic gonadotropin (free ß-hCG) multiples of the median (MoM) or pregnancy-associated plasma protein A (PAPP-A) MoM were defined with ≥ 2.75 or ≤ 0.5, 7.72% (2,194/28,405) in the serum biochemical screening group and 12.36% (4,005/32,403) in CFTS group could be detected as abnormal results for further NIPS. Finally, 55.56% (5/9) and 85.71% (6/7) of trisomy 21 cases with false-negative results were detected, and the overall detection rate for trisomy 21 was improved by 10.64% (5/47) and 12.77% (6/47), respectively. CONCLUSIONS: The new contingent screening strategy can increase the detection rate of trisomy 21 compared with the traditional contingent screening strategy.

A Retrospective Analysis Of Different Contingent Screening Models For Fetal Down Syndrome In Southwestern China.

To discuss combinations of traditional screening and noninvasive prenatal screening (NIPS) and to compare which traditional screening is the most suitable first-line screening approach to NIPS, pregnant women were recruited in this retrospective observational study. Pregnant women underwent one of four traditional screening tests. The 9 contingent models were combined by high risk cut-offs of 1:50, 1:100, 1:270 and intermediate risk cut-offs of 1:1000, 1:1500, 1:2000. We analyzed cost and performance of various screening models with contingent screening of different risk cut-offs. Compared with other screening tests, combined first-trimester screening (CFTS) had the lowest proportion of high risk (≥1:270) with the highest detection rate (DR) (78.79%) and the lowest proportion of intermediate risk (1:271~1:1000). When intermediate risk was 1:51 ~1:1500, CFTS as first-line screening had the lowest cost with DR of 93.94%. Other screening tests as the first-line screening with intermediate risk of 1:51~1:1000 had the lowest cost, there DR were 90.91%, 84.62%, 91.67%, respectively. Our study demonstrated if only one traditional screening was allowed to screen pregnant women, CFTS was recommended as the first choice. According to local health and economic conditions, adopting appropriate traditional screening with suitable cut-offs as first-line screening will contributed to a cost-effective screening model.

Killer cell immunoglobulin-like receptor gene cluster predisposes to susceptibility to B-cell acute lymphoblastic leukemia in Chinese children.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. Killer cell immunoglobulin-like receptors (KIRs) are mainly expressed on natural killer (NK) cells and regulate killing of cancer cells. To investigate the possible association of KIR genes with B-ALL in Chinese children, we used polymerase chain reaction with sequence-specific primers (PCR-SSP) to determine the KIR genotypes of 137 B-ALL patients and 288 healthy children of Chinese Han origin. Herein we report no significant difference in the carrying frequency of individual KIR genes and haplotypes between patients and controls; however, individuals carrying C4Tx genotypes were more frequent in the B-ALL group compared with healthy controls (11.7% vs. 5.9%, P=0.038). In addition, the centromeric KIR gene cluster, KIR2DS2-2DL2-2DS3-2DL5, was significantly increased in the B-ALL group compared with healthy controls (13.9% vs. 7.3%, P=0.030). These data suggest that the C4Tx genotype and centromeric KIR gene cluster (KIR2DS2-2DL2-2DS3-2DL5) might predispose to susceptibility to B-ALL in Chinese children.

Inhibition of Mitofusin-2 Promotes Cardiac Fibroblast Activation via the PERK/ATF4 Pathway and Reactive Oxygen Species.

Mitofusin-2 (Mfn2) is a key outer mitochondrial membrane protein, which maintains normal mitochondrial dynamics and function. However, its role in cardiac fibroblast activation remains poorly understood. In the present study, a rat model of transverse aortic constriction (TAC) was established to observe the cardiac fibroblast activation in vivo. TGF-ß1 treatment for 24 hours was used to induce cardiac fibroblast activation in vitro. As a result, the expression of Mfn2 decreased in the hypertrophic heart tissues and cardiac fibroblasts treated with TGF-ß1. siMfn2 and adenovirus were applied to mediate Mfn2 gene silencing and overexpression in cardiac fibroblasts to elucidate the relationship between Mfn2 and cardiac fibroblast activation, as well as the possible underlying mechanisms. Knockdown of Mfn2 further promoted TGF-ß1-induced cardiac fibroblast activation, while forced expression of Mfn2 attenuated this pathological reaction. The PERK/ATF4 pathway, one of the branches of endoplasmic reticulum (ER) stress, was identified to be involved in this process. Knockdown and overexpression of Mfn2 lead to aggravation or alleviation of the PERK/ATF4 pathway. Blocking this pathway by silencing ATF4 with siATF4 attenuated the pathological process. During the activation of cardiac fibroblasts, knockdown of Mfn2 also increased the production of reactive oxygen species (ROS), while ROS scavenger N-acetyl-l-cysteine (NAC) could attenuate the effect caused by knockdown of Mfn2. Our data suggested that inhibition of Mfn2 could promote cardiac fibroblast activation by activating the PERK/ATF4 signaling pathway and increasing the generation of ROS.

The iron-regulated transporter 1 plays an essential role in uptake, translocation and grain-loading of manganese, but not iron, in barley.

Transporters involved in manganese (Mn) uptake and intracellular Mn homeostasis in Arabidopsis and rice are well characterized, while much less is known for barley, which is particularly prone to Mn deficiency. In this study we have investigated the role of the iron-regulated transporter 1 (IRT1) for Mn uptake and translocation in barley plants. We employed an RNAi approach to reduce HvIRT1 expression to 5% of the wild-type level. This enabled characterization of the functional role of HvIRT1 by use of advanced imaging and phenotyping techniques applied to plants growing in hydroponics or soils with different Mn availability. Our results highlight the importance of HvIRT1 for the transport of Mn across the root endodermis into the stele. In the hvirt1-RNAi lines, a chlorotic phenotype with reduced shoot Mn concentration and impaired photosynthetic functionality was observed, especially under conditions with low Mn availability. We also document that HvIRT1 controlled the Mn distribution within the barley grain. Surprisingly, unlike other IRT1 orthologues, HvIRT1 played no significant role in iron uptake. We conclude that the barley IRT1 orthologue has a novel function with respect to ensuring sufficient shoot Mn concentrations. The preference of IRT1 for Mn instead of Fe is discussed in an evolutionary context.

[Expression of WT1 gene in children with acute myeloid leukemia].

OBJECTIVE: To study WT1 gene expression in children with acute myeloid leukemia (AML) and its possible correlations to clinical outcomes. METHODS: Bone marrow samples were collected from 45 children with AML (excluding acute promyelocytic leukemia, AML-M3) at different time points of AML treatment and follow-up. WT1 gene expression levels in bone marrow mononuclear cells were assayed by real-time fluorescence quantitative PCR. The correlation between WT1 expression and prognosis was retrospectively analyzed. RESULTS: The WT1 expression level in AML children with bone marrow blast cell percentage of >60% was significantly higher than in those with bone marrow blast cell percentage of ≤ 60% (p<0.05). The lower WT1 expression level was documented in children with AML-M2 compared with in children with other non-M2 subtypes (p<0.05). WT1 expression level in patients in complete remission was significantly lower than that in patients at diagnosis or relapse (p<0.01). The 2-year disease-free survival (DFS) in patients with higher WT1 expression was significantly lower than in those with lower WT1 expression at the end of induction chemotherapy (p<0.05). The 2-year overall survival (OS) and DFS in patients with ≥1 log WT1 reduction range were significantly higher than those with <1 log reduction of WT1 expression level at the end of induction chemotherapy (p<0.05). WT1 expression levels tended to rise 2-3 months prior to bone marrow relapse. CONCLUSIONS: WT1 expression level is closely correlated prognosis in children with AML. Dynamic monitoring of WT1 expression level is of great clinical importance in terms of individualized management, prognosis evaluation and relapse prediction.

[Expression of CD20 in B-cell precursor acute lymphoblastic leukemia and its correlation with clinical outcomes].

OBJECTIVE: To determine whether expression of CD20 is associated with clinical outcomes of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: 271 newly diagnosed childhood BCP-ALL during January 2009 to May 2013 were enrolled in this study. The patients were treated in line with the Chinese Childhood Leukemia Group ALL 2008 protocol (CCLG-ALL 2008). The clinical feature, early therapeutic response and clinical outcomes of the patients with a CD20 positive (CD20+ BCP) expression were compared with those with a CD20 negative (CD20- BCP) expression. RESULTS: CD20- BCP accounted for 45.76% (124 cases) of all participants. There were no significant differences between CD20- BCP and CD20- BCP patients in gender distribution, age, WBC counts when diagnosis was made, proportion of prednisone poor responders, and distribution of risk categories (P > 0.05). Patients of 10 years or older comprised 25.81% and 14.29% of CD20+ BCP and CD20- BCP patients, respectively (P = 0.017). Pro-B and pre-B cases accounted for 43.55% and 59.86% of CD20- BCP patients respectively, compared with 56.45 and 40.14% in CD20- BCP patients (P = 0.007). CD20+ BCP patients had 12.20% Philadelphia positive ALL and 6.50% BCP-ALL with TEL-AML1 fusion gene, compared with 4.86% (P = 0.03) and 18.06% (P = 0.005) in those of CD20 BCP. No significant differences were found between the two groups of patients in 15-day (77.50% vs. 74.13%, P = 0.525) and 33-day (95.04% vs. 95.83%, P = 0.757) complete remission rates. No significant differences (P > 0.05) were found in predicted 4-year event-free survival CEFS (78.00% +/- 4.96%) vs. (79.05% +/- 5.40%)) and predicted 4-year overall survival (OS (83.01% +/- 6.13%) vs. (93.64% +/- 2.46%)) between the two groups of patients either. CONCLUSION: CD20 positivity was not found to be associated with worse prognosis of children with BCP-ALL. More studies are needed to validate the correlation between CD20 and unfavorable outcomes in BCP-ALL.

Association analysis of four candidate genetic variants with sporadic amyotrophic lateral sclerosis in a Chinese population.

Recently, four single nucleotide polymorphisms (SNPs), including rs2814707 in the 9p21, rs12608932 in the UNC13A gene, rs13048019 in the TIMA1 gene, and rs2228576 in the SCNN1A gene have been reported to be associated with the risk for developing amyotrophic lateral sclerosis (ALS) in Caucasian population. However, this association is not consistent among different studies and yet to be tested in ALS patients in Mainland China. This study included 397 sporadic ALS (SALS) patients and 287 unrelated Chinese healthy controls from Southwest China. Four SNPs listed above were genotyped by using Sequenom's iPLEX assay. No significant differences in the genotype distributions or minor allele frequencies in all SNPs were found between ALS group and control group, between the spinal-onset group and bulbar-onset group, and between the early-onset group and the late-onset group. Our results suggest that these SNPs are unlikely to be common cause of SALS in Chinese population.

GDF15-mediated upregulation of ferroportin plays a key role in the development of hyperferritinemia in children with hemophagocytic lymphohistiocytosis.

BACKGROUND: Growth differentiation factor 15 (GDF15), a divergent TGFß superfamily, has recently been implicated in the modulation of iron homeostasis, acting as an upstream negative regulator of hepcidin, the key iron regulatory hormone produced primarily by hepatocytes. However, little is known about possible roles that GDF15 might play in the regulation of iron homeostasis and development of hyperferritinemia in children with hemophagocytic lymphohistiocytosis (HLH). PROCEDURES: We compared serum GDF15 level and mRNA expressions of GDF15 and key molecules of iron metabolism, and made correlations between their expressions in children with HLH and control children. RESULTS: Serum GDF15 level was remarkably higher in HLH group than that in controls, with median serum concentration of 1,700 and 260 pg/ml, respectively (P < 0.001). In addition, GDF15 mRNA was significantly upregulated but independent of hypoxia-inducible factor-mediated oxygen signaling pathway. More importantly, GDF15 induction was positively correlated to upregulation of ferroportin, the only cellular iron exporter, and to upregulation of ferritin heavy chain. CONCLUSIONS: Our study suggests that GDF15 induction helps suppress further activation of macrophages in stressful physiologic states as HLH, and is intimately implicated in the development of hyperferritinemia by modulating the hepcidin-ferroportin axis, resulting in enhanced ferroportin-mediated iron efflux.

Suppression of inflammation by tumor-derived exosomes: a kind of natural liposome packaged with multifunctional proteins.

Exosomes are small-membrane vesicles secreted by hematopoietic and malignant epithelial cells as well as trophoblasts. The composition of cancerous exosomes has been proven to play pivotal roles in the maintenance of the microenvironment that is beneficial for the progression of cancer, such as Fas-ligand-triggered lymphocyte apoptosis. We supposed that the immunosuppressive effect of cancerous exosomes might be helpful in the treatment of diseases characterized by overactivation of the immune system and subsequent tissue injury. The aim of this study was to evaluate the protective effect of tumor-derived exosomes in the mice model of lipopolysaccharide (LPS)-induced inflammation. Tetrazolium (MTT) and DNA electrophoresis were used to measure the cytotoxicity of exosomes on lymphocytes. Pathologic observation of tissue sections, serologic analysis of aspartate aminotransferase/alanine aminotransferase (AST/ALT), and urinary analysis of protein were used to assess the protection effect of exosomes in LPS-induced multiorgan damage. In vitro outcomes of MTT and DNA electrophoresis showed the cytotoxicity of exosomes on lymphocytes. Together with the alleviation of organ damages evaluated by urine protein, serum AST/ALT, and pathologic analysis, we confirmed the possibility that pretreatment of mice with exosomes, produced by H22 hepatic tumor cells, resulted in protection against LPS-induced tissue damage, which is caused by overactivation of the immune system and inflammation response. This therapeutic strategy will raise an interesting way to search new therapeutics in pairs of diseases with complementarities in etiology and pathology, namely, a strategy of taking advantage of the mutual complementarities between diseases.

Interleukin 17 induces up-regulation of chemokine and cytokine expression via activation of the nuclear factor κB and extracellular signal-regulated kinase 1/2 pathways in gynecologic cancer cell lines.

OBJECTIVES: Previous studies have revealed that interleukin 17 (IL-17) contributes to pathological processes in many solid tumors. However, the roles of IL-17 in gynecologic cancer still remain elusive, hindering the deep understanding of gynecologic tumorigenesis. METHODS: In the present study, to delineate the functional roles of IL-17 in gynecologic cancer, IL-17 stimulation was introduced in cell lines of 3 gynecologic cancers, and IL-17-induced expression of chemokines and cytokines and possible signaling pathways were investigated. RESULTS: Our results showed that in HEC-1-B (human endometrial cancer) cells, IL-17 stimulation induced mRNA level increases of CCL2, CCL5, CCL20, CXCL2, and IL-8. Similar treatment in HeLa cells caused increases in the mRNA levels of CCL2, CXCL2, IL-6, and IL-8, and in SKOV3 cells, mRNA levels of CCL2, CCL20, CXCL1, CXCL2, IL-6, and IL-8 increased. The increases in mRNA levels induced by IL-17 were dose- and time-dependent. Furthermore, with the addition of the NF-κB (nuclear factor κ-light-chain-enhancer of activated B) and extracellular signal-regulated kinase inhibitors pyrrolidine dithiocarbamate and PD98059, the IL-17-induced CCL2 mRNA level was significantly compromised. IL-17 stimulation also activated phosphorylation of IκBα and extracellular signal-regulated kinase 1/2 in a time-dependent manner. CONCLUSION: These results demonstrated that IL-17 may regulate chemokines and cytokines in gynecologic cancers.

[TfR2 mRNA expression in bone marrow mononuclear cells of children with hyperplastic anemia and its implications].

The aim of this study was to investigate the expression of transferrin receptor 2 (TfR2) mRNA in bone marrow mononuclear cells (BMMNC) of children with hyperplastic anemia (HA), to analyze the correlation of TfR2 mRNA expression level with Hb level, bone marrow erythroid hyperplasia, iron status in body and underlying diseases, and to evaluate the role of TfR2 in erythroid hemopoiesis and the useful value in diagnosis of HA. The experiment was divided into 2 groups: test group, in which 40 patients with HA were enrolled, and control group in which 10 patients without erythroid disorders and hematological malignancies confirmed by bone marrow examination were enrolled. The bone marrow samples of patients in mentioned above 2 groups were collected, the TfR2 mRNA expression in BMMNC of patients with HA was detected by fluorescence-quantitative PCR, the correlation of HA with bone marrow erythroid hyperplasia, iron status of body and underlying diseases was analyzed. The results showed that the relative level of TfR2 mRNA expression in HA patients was significantly higher than that in control patients. The TfR2 mRNA expression level negatively correlated with Hb level in peripheral blood (r = -0.715), while it positively correlated with ratio of bone marrow erythroblasts (r = 0.533). It is concluded that TfR2 mRNA expression in HA patients increases and closely correlates with hyperplasia status of bone marrow and anemia level in peripheral blood.

N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1.

AMMONIUM TRANSPORTER (AMT) proteins are conserved in all domains of life and mediate the transport of ammonium or ammonia across cell membranes. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized. High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato. However, in the paralogue LeAMT1;3, trimeric complexes were not detected. LeAMT1;3 differs from the other AMTs by an unusually short N-terminus, suggesting a role for the N-terminus in oligomer stability. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. Swapping of the N-terminus between LeAMT1;1 and LeAMT1;3 showed that sequences in the N-terminus of LeAMT1;1 are necessary and sufficient for stabilization of the interaction among the subunits. Two N-terminal cysteine residues are highly conserved among AMT1 transporters in plants but are lacking in LeAMT1;3. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.

Destructive pulmonary staphylococcal infection in a boy with hyper-IgE syndrome: a novel mutation in the signal transducer and activator of transcription 3 (STAT3) gene (p.Y657S).

UNLABELLED: Hyper IgE syndrome (HIES) is a rare primary immunodeficiency disorder, characterized by eczema, recurrent skin and lung infections, and significantly elevated serum IgE level. It was previously diagnosed based on clinical manifestations and laboratory markers that were not specific to the disease. Recent studies have demonstrated that mutations in signal transducer and activator of transcription 3 (STAT3) cause the autosomal dominant or sporadic HIES, which make the disease definitively characterized at molecular level. Here, we reported a 3-year old Chinese boy with neonatal-onset rash and multiple serious Staphylococcus aureus infections including recurrent skin abscesses, liver abscess, sepsis, and destructive pulmonary infection (pneumonia, multiple pulmonary abscesses, pyopneumothorax, and finally, pneumatocele). Genetic study revealed a heterozygous mutation in exon 21 of STAT3 gene (g.66583 A > C, c.1970A > C) in the boy, which resulted in a substitution of tyrosine at the amino acid position 657 to serine (p.Y657S) in the Src homology 2 (SH2) domain of STAT3. Functional prediction with bioinformatics programs of the Sorting Intolerant from Tolerant (SIFT) and the Polymorphism Phenotyping (PolyPhen) reported "deleterious (SIFT score 0.02)" and "probably damaging (PSIC score difference 2.94)" values, respectively. Further study of family members revealed that neither his parents, nor his twin brother carried the mutation, indicating the mutation was likely to occur de novo in our patient. CONCLUSION: The mutation,p.Y657S,in SH2 domain of STAT3 is a disease-causing mutation in the boy with HIES.

[Expression of mitochondrial ferritin in K562 leukemic cell during ATRA-induced cell differentiation].

OBJECTIVE: To investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during ATRA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules. METHODS: K562 cells cultured with or without ATRA (1 micromol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward granulocyte lineage was confirmed by microscopic study (Wright's staining) and flowcytometry. Expression levels of MtF, TfR1 and Fn were evaluated with semiquantitative RT-PCR, while K562 cells cultured without ATRA as control. RESULTS: Over 21.2% of K562 cells demonstrated features of granulocyte, and the expression of CD13 on cell surface increased significantly at day 5 with ATRA treatment (P < 0.05, compared with control). K562 cells could express a certain level of MtF before ATRA-induced differentiation. With increase of ATRA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 86.5% and 79.2% of that before ATRA treatment. While Fn mRNA expression increased to 1.21 folds of that before ATRA treatment. CONCLUSION: MtF expression is downregulated during ATRA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.

[Expression of transferrin receptor 2 in mononuclear cells from children with acute leukemia].

OBJECTIVE: To study the expression of TFR2 mRNA in mononuclear cells of bone marrow and peripheral blood from children with acute lymphoblastic leukemia, and to explore possible correlations between TFR2 expression and a panel of prognostic/risk factors. METHODS: Bone marrow or peripheral mononuclear cells were isolated from 56 children with newly diagnosed acute lymphoblastic leukemia (ALL) and 15 normal children as control. TFR2 mRNA expression level in mononuclear cell was determined by real-time fluorescence quantitative RT-PCR. RESULTS: Relative expression level of TFR2 mRNA in prednisone good responders (median: 0.0848) was significantly higher than that in prednisone poor responders (median: 0.0126) (P = 0.038). The medians of TFR2 mRNA expression levels in low-risk, moderate-risk and high-risk ALL were 0.1677, 0.0728 and 0.0125 respectively (P = 0.003). The medians of TFR2 mRNA expression levels in three ALL groups with initial WBC counts less than 50 x 10(9)/L, from 50 x 10(9)/L to 100 x 10(9)/L and more than 100 x 10(9)/L were 0.0974, 0.0294 and 0.0078 (P = 0. 013). In addition, the relative TFR2 mRNA level in B-lineage ALL was significantly higher than that in T-ALL, with medians of 0.0636 and 0.0065 respectively (P = 0.004). Ranked correlation analysis revealed that TFR2 expression was negatively correlated to initial white blood cell count, percentage of blast cells and absolute numbers of blasts in peripheral blood, with ranked correlation coefficients of -0.398, -0.307 and 0.421 respectively (correponding P values were 0.003, 0.022 and 0.001). CONCLUSION: TFR2 might be a novel prognostic factor for ALL.

[Isolation of multipotent mesenchymal stem cells from the tissue of umbilical cord for osteoblasts and adipocytes differentiation].

OBJECTIVE: To explore the methods and conditions for isolating and proliferating multipotent mesenchymal stem cells (MSCs) from the tissue of umbilical cord, with an aim to induce osteogenic and adipogenic differentiation in vitro. METHOD: The cord was dissected along the long axis, with vessels pulled away and then sutured into a "loop". Collagenase solution was filled into the loop. Suspended cells were collected from the loop suspension after 6-8 hours and centrifuged. The cells were finally cultured in polystyrene dishes. The single cell-derived colonies were obtained and tested for their immunophenotype and osteoblast and lipoblast differentiations. RESULT: Adherent cells were obtained from the tissue of umbilical cord, which proliferated and formed single cell-derived colonies. The colonies presented matrix cells immunophenotype and differentiated into osteoblasts that produced mineralized matrices, which were stained by alizarin red and alkaline phosphatase. The colonies also differentiated into adipocytes that accumulated lipid vacuoles, which were demonstrated by the morphology and oil red stains. CONCLUSION: MSCs can be isolate from the tissue of umbilical cords and proliferate in vitro. The proliferated colonies show matrix cell immunophenotypes and can differentiate into osteoblasts and adipocytes.

[Expression of mitochondrial ferritin in K562 leukemic cell during TPA-induced cell differentiation].

Mitochondrial ferritin (MtF), a new player in iron metabolism, first identified in 2001, is highly homologous to ferritin both structurally and functionally. Preliminary studies have suggested that MtF might play very important roles in the regulation of mitochondrial iron homeostasis. Leukemic cells, just like other malignant cells, demand more iron for their greater proliferation potential. However, little is known about what roles MtF might play in leukemic cell iron metabolism and cell proliferation. The aim of this study was to investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during TPA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules. K562 cells cultured with or without TPA (16 nmol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward monocyte lineage was confirmed by microscopic study (Wright's staining) and flow cytometry. Semiquantitative RT-PCR was performed to determine mRNA expression, with house-keeping gene beta-actin as control reference. This study revealed that over 95% of K562 cells showed morphological features of monocyte/macrophage, and the expression of CD64 on cell surface increased significantly at day 5 with TPA treatment. K562 cells could express a certain level of MtF before TPA-induced differentiation. With increase of TPA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 50.3% and 68.2% of that before TPA treatment. While Fn mRNA expression increased to 1.97 folds of that before TPA treatment. It is concluded that MtF expression is downregulated during TPA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.

Nitrogen-dependent posttranscriptional regulation of the ammonium transporter AtAMT1;1.

Ammonium transporter (AMT) proteins of the AMT family mediate the transport of ammonium across plasma membranes. To investigate whether AMTs are regulated at the posttranscriptional level, a gene construct consisting of the cauliflower mosaic virus 35S promoter driving the Arabidopsis (Arabidopsis thaliana) AMT1;1 gene was introduced into tobacco (Nicotiana tabacum). Ectopic expression of AtAMT1;1 in transgenic tobacco lines led to high transcript levels and protein levels at the plasma membrane and translated into an approximately 30% increase in root uptake capacity for 15N-labeled ammonium in hydroponically grown transgenic plants. When ammonium was supplied as the major nitrogen (N) form but at limiting amounts to soil-grown plants, transgenic lines overexpressing AtAMT1;1 did not show enhanced growth or N acquisition relative to wild-type plants. Surprisingly, steady-state transcript levels of AtAMT1;1 accumulated to higher levels in N-deficient roots and shoots of transgenic tobacco plants in spite of expression being controlled by the constitutive 35S promoter. Moreover, steady-state transcript levels were decreased after addition of ammonium or nitrate in N-deficient roots, suggesting a role for N availability in regulating AtAMT1;1 transcript abundance. Nitrogen deficiency-dependent accumulation of AtAMT1;1 mRNA was also observed in 35S:AtAMT1;1-transformed Arabidopsis shoots but not in roots. Evidence for a regulatory role of the 3'-untranslated region of AtAMT1;1 alone in N-dependent transcript accumulation was not found. However, transcript levels of AtAMT1;3 did not accumulate in a N-dependent manner, even though the same T-DNA insertion line atamt1;1-1 was used for 35S:AtAMT1;3 expression. These results show that the accumulation of AtAMT1;1 transcripts is regulated in a N- and organ-dependent manner and suggest mRNA turnover as an additional mechanism for the regulation of AtAMT1;1 in response to the N nutritional status of plants.