The effect of dexamethasone (10(-5)M) and epinephrine (10(-6)M) on the biosynthesis of metallothionein (MT) in the perfused rat liver was investigated. MT synthesis was determined by measuring the incorporation of 14C-L-aspartic acid into liver MT fraction after the perfusion for five hours of isolated liver by artificial blood containing 14C-L-U-aspartic acid (0.2uci) with dexamethasone or epinephrine. MT was isolated by Sephadex G-75 column chromatography and DEAE Sephadex column chromatography. Incorporation of radioactive 14C into the MT fraction of perfused liver cytosol (9.0grams of liver) from dexamethasone treated, epinephrine treated and control groups were, respective1y, 0.72, 0.34 and 0.33% of total radioactivity infused. Total protein content in the MT fraction of liver perfused with dexamethasone and epinephrine were 0.80, 0.64mg/g liver compared to 0.52mg/g liver in the control. MT, a protein having a high content of cystein and metals is synthesized in the perfused rat liver and its induction is stimulated by dexamethasone, while epinephrine increased the accumulation of Zn in the MT fraction of the perfused rat liver. The present experiment confirms that MT synthesis and degradation are somewhat regulated by glucocorticoid hormone and epinephrine.
Animal ; Dexamethasone/pharmacology* ; Epinephrine/pharmacology* ; Female ; In Vitro ; Liver/drug effects ; Liver/metabolism* ; Metalloproteins/metabolism* ; Metallothionein/metabolism* ; Perfusion ; Rats ; Zinc/metabolism

Thlaspi caerulescens, the famous model plant of heavy-metal hyperaccumulator, can uptake and accumulate large amount of heavy metals in its above-ground part of the plants. However, the very low biomass in Thlaspi caerulescens makes this plant unfit for direct application in phytoremediation. In recent years, there are many reports about the physiological and molecular characterization of Thlaspi caerulescens under heavy metals stresses, including absorption, transport and intracellular detoxification processes (e.g., chelation and compartmentation). Research teams have conducted many studies of chelators in plants, such as organ acid, amino acid, phytochelatins, metallothioneins and nicotianamine, and so on. Several transport protein families, such as Zinc Regulated Protein, Cation Diffusion Facilitator, Natural Resistance and Macrophage Protein and Heavy Metal ATPase, play important role in short/long distance transport in the plant. In this review, we summarize the current knowledge of the physiological and molecular mechanisms of heavy metals accumulation in Thlaspi caerulescens, with particular emphasis on the roles of transporters and chelatins in modulating plant heave-metal-stress responses.
Absorption ; Azetidinecarboxylic Acid ; analogs & derivatives ; metabolism ; Biodegradation, Environmental ; Cation Transport Proteins ; genetics ; metabolism ; Metalloproteins ; genetics ; metabolism ; Metals, Heavy ; metabolism ; Phytochelatins ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Thlaspi ; genetics ; metabolism

OBJECTIVETo identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.

METHODSMaltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.

RESULTSMBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.

CONCLUSIONLMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.

Adaptor Proteins, Signal Transducing ; DNA-Binding Proteins ; genetics ; metabolism ; GATA1 Transcription Factor ; metabolism ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; genetics ; metabolism ; Periplasmic Binding Proteins ; Proto-Oncogene Proteins ; RNA Splicing ; Transcription Factors ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo evaluate the efficacy and safety of iron protein succinylate (IPS) oral solution in preventing and treating anemia of prematurity (AOP).

METHODSSixty premature infants less than 35 weeks of gestation were randomly divided into IPS (n=30) and polysaccharide iron complex (PIC) groups(n=30). Treatment began at two weeks after birth. The infants received IPS or PIC in addition to recombinant human erythropoietin. On days 14, 28, 42, and 60 after treatment, hemoglobin (Hb), red blood cell count(RBC), hematocrit (HCT), percentage of reticulocytes, serum iron, and serum ferritin were determined. Liver and renal functions were evaluated before and after treatment.

RESULTSThere were significant differences in the changing trends of RBC and HCT between the two groups (P<0.05). In the IPS group, RBC and HCT gradually decreased after birth, but began to rise gradually on days 28 and 42 of treatment; in the PIC group, RBC and HCT kept decreasing from birth to day 60 of treatment. On day 60 of treatment, the IPS group had significantly higher levels of Hb, RBC, HCT, serum iron, and serum ferritin than the PIC group (P<0.05). No notable adverse events occurred in either group.

CONCLUSIONSIPS oral solution has good efficacy and tolerability in preventing and treating AOP.

Administration, Oral ; Anemia, Neonatal ; blood ; drug therapy ; prevention & control ; Erythrocyte Count ; Female ; Hematinics ; therapeutic use ; Hematocrit ; Humans ; Infant, Premature ; Iron ; metabolism ; Male ; Metalloproteins ; adverse effects ; therapeutic use ; Solutions ; Succinates ; adverse effects ; therapeutic use

Functional proteins designed de novo have potential application in chemical engineering, agriculture and healthcare. Metal binding sites are commonly used to incorporate functions. Based on a de novo designed protein DS119 with a βαβ structure, we have computationally engineered zinc binding sites into it using a home-made searching program. Seven out of the eight designed sequences tested were shown to bind Zn(2+) with micromolar affinity, and one of them bound Zn(2+) with 1:1 stoichiometry. This is the first time that metalloproteins with an α, β mixed structure have been designed from scratch.
Amino Acid Sequence ; Binding Sites ; Computer Simulation ; Escherichia coli ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Kinetics ; Magnetic Resonance Spectroscopy ; Metalloproteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Engineering ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Zinc ; chemistry ; metabolism

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo study the immunophenotype and prognostic significance of primary gastrointestinal diffuse large B-cell lymphoma, with reference to Hans, Choi and Tally algorithms.

METHODSThe clinicopathologic features and follow-up data in 90 cases of primary gastrointestinal diffuse large B-cell lymphoma were analyzed by Kaplan-Meier method, Log-rank test and Cox regression model. Immunohistochemistry was carried out using EliVision and EnVision methods for CD20, CD3ε, CD10, bcl-6, MUM-1, CD5, bcl-2, GCET1, FOXP1, LMO2, BLIMP1 and Ki-67.

RESULTSThe age of patients ranged from 27 to 83 years (mean = 58 years). The male-to-female ratio was 1.31:1. Amongst the 90 cases studied, 64.4% (58/90) involved the stomach and 35.6% (32/90) involved the intestine. The immunohistochemical findings were as follows: 100% positivity for CD20, 0% for CD3ε and CD5, 17.8% (16/90) for CD10, 75.6% (68/90) for bcl-6, 52.2% (47/90) for MUM-1 (cut off was 30%), 43.3% (39/90) for MUM-1 (cut off was 80%), 50.0% (45/90) for GCET1, 45.6% (41/90) for FOXP1, 23.3% (21/90) for LMO2, 42.2% (38/90) for bcl-2 and 8.9% (8/90) for BLIMP1. The Ki-67 index ranged from 20% to 95% (median = 80%). According to Hans algorithm, 51.1% of the cases belonged to germinal center B-cell (GCB) subtype and 48.9% belonged to non-GCB subtype. In contrast, Choi algorithm classified 55.6% cases as GCB subtype and 44.4% as activated B-cell (ABC) subtype. According to Tally algorithm, 34.4% were of GCB subtype and 65.6% of non-GCB subtype. Most of the patients (67.8%, 61/90) received chemotherapy and 68.9% (62/90) underwent surgical resection. The overall 2, 3 and 5-year survival rates were 58.5%, 52.8% and 49.8%, respectively. The overall 2, 3 and 5-year survival rates in the CHOP therapy group were 68.5%, 61.2% and 52.9%, respectively.

CONCLUSIONSThere is no significant difference in ratio between the GCB and non-GCB/ABC subtypes by Hans and Choi algorithms. The non-GCB subtype seems to be more prevalent according to Tally algorithm. Although there is no significant difference in survival between GCB and non-GCB/ABC subtypes by the 3 algorithms, GCB subtype tends to show a better survival. In univariate analysis, LDH level, international prognostic index, chemotherapy, surgical resection, B symptoms, number of involved sites and clinical stage are found to have prognostic significance. In multivariate analysis, Choi algorithm, Tally algorithm, chemotherapy, surgical resection, LDH level and clinical stage are independent prognostic factors.

Adaptor Proteins, Signal Transducing ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD20 ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; therapeutic use ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; therapeutic use ; Female ; Forkhead Transcription Factors ; metabolism ; Germinal Center ; pathology ; Humans ; Immunophenotyping ; Interferon Regulatory Factors ; metabolism ; Intestinal Neoplasms ; classification ; drug therapy ; metabolism ; pathology ; surgery ; Kaplan-Meier Estimate ; LIM Domain Proteins ; Lymphoma, Large B-Cell, Diffuse ; classification ; drug therapy ; metabolism ; pathology ; surgery ; Male ; Metalloproteins ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neprilysin ; metabolism ; Prednisone ; therapeutic use ; Proportional Hazards Models ; Proto-Oncogene Proteins ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; Repressor Proteins ; metabolism ; Serpins ; metabolism ; Stomach Neoplasms ; classification ; drug therapy ; metabolism ; pathology ; surgery ; Survival Rate ; Vincristine ; therapeutic use