[Value of CD, MPO, Ki-67 and C-MYC Positive Rate in the Pathological Tissues and C-MYC Gene of Patients with T-LBL/ALL for Predicting Prognosis].

OBJECTIVE: To explore the value of of CD, MPO, Ki-67, C-MYC positive rates in the pathological tissues and C-MYC gene of patients with T-LBL/ALL for predicting Prognosis. METHODS: Ninty cases of T-LBL/ALL patients in our hospital were selected and included in the T-LBL/ALL group, and 30 cases of lymphnode reactive hyperplasia were selected as control group. Immunohistochemical staining was used to detect the changes of CD, MPO, Ki-67 and C-MYC positive rate in 2 groups, and the changes of C-MYC gene were detected by fluorescence in situ hybridization. RESULTS: In 90 patients with T-LBL/ALL, there were CD1a+ 34 cases (37.8%), CD3+ 67 cases (74.4%), epsilon CD3+ 47 cases (52.2%), CD7+ 85 cases (94.4%), CD10+ 33 cases (36.7%), CD34+ 22 cases (24.4%), CD43+ 48 cases (53.3%), CD45RO+ 46 cases (51.1%), CD99+ 88 cases (97.8%), TDT+ 85 cases (94.4%); and CD23, CD20, and MPO all were negative; Ki-67>80% 47 cases (52.2% cases), Ki-67≤80%, 43 cases (47.8%). In 90 T-LBL/ALL patients, the positive rate of C-MYC (66.7%) was significantly higher than the control group (positive rate 0.0%) (P< 0.05); the Ki-67 index, mediastinal widening of T-LBL/ALL patients and the positive rate of C-MYC positively were correlated (P< 0.05). The overall survival rate (44.0%) of C-MYC negative patients was significantly higher than that of C-MYC positive patients (0.0%). The overall survival rate of C-MYC negative patients was significantly higher than that of C-MYC positive patients (P< 0.05).Ann Arbor staging, LDH, bone marrow involvement, mediastinal widening, Ki-67 positive index, and C-MYC protein expression of patients with T-LBL/ALL did not correlated with increased C-MYC gene breakage and copy number (P> 0.05). CONCLUSION: The overall survival rate of C-MYC positive patients decreases, which positively correlates with Ki-67 positive index and mediastinal width, suggesting that the prognosis of the patients with C-MYC protein expression is poorer.

4-phenylbutyric acid attenuates endoplasmic reticulum stress-mediated pancreatic ß-cell apoptosis in rats with streptozotocin-induced diabetes.

Endoplasmic reticulum stress (ERS) plays an important role in diabetes mellitus (DM), but the association between DM and ERS is unknown. We have previously shown that streptozotocin (STZ)-induced diabetes in rats is characterized by increased levels of ERS markers. Here, we tested whether the chemical chaperone 4-phenylbutyric acid (4-PBA) ameliorated ERS-associated apoptosis in pancreatic ß-cells in rats with STZ-induced diabetes. Male Sprague-Dawley rats were divided into 3 groups: control group, DM group, and DM model plus 4-PBA treatment group (4-PBA group). DM model rats were induced by injection of STZ (60 mg/kg) intraperitoneally, and 4-PBA was administered daily by gavage at a dose of 500 mg/kg body weight for 20 days. ß-cell apoptosis was higher in the DM group than in the control group. Moreover, the expression of caspase-3, Bax, and the ERS indicators Bip and CHOP was markedly elevated in the pancreas of rats in the DM group, whereas the expression of Bcl-2 was lower in these rats (P < 0.05). Blood glucose concentration in diabetic rats gradually decreased with 4-PBA treatment but remained higher at the end of the experiment compared to the concentration in control rats. Consistent with this, 4-PBA raised the fasting insulin level in diabetic rats; it also suppressed the expression of caspase-3, Bax, and ERS indicators but enhanced the expression of Bcl-2. In conclusion, 4-PBA protects pancreatic ß-cells from apoptosis in STZ-induced diabetes by attenuating the severity of ERS.

The role of Homer 1a in increasing locomotor activity and non-selective attention, and impairing learning and memory abilities.

The current study aimed to investigate the possible role of Homer 1a in the etiology and pathogenesis of attention deficit hyperactivity disorder (ADHD). We divided 32 rats into four groups. The rats in the RNAi-MPH group were given the lentiviral vector containing Homer 1a-specific miRNA (Homer 1a-RNAi-LV) by intracerebroventricular injection, and 7 days later they were given three daily doses of methylphenidate (MPH) by intragastric gavage. The RNAi-SAL group was given Homer 1a-RNAi-LV and saline later. The NC-MPH group was given the negative control lentiviral vector (NC-LV) and MPH later. The NC-SAL group was given NC-LV and saline later. Rats that were given Homer 1a RNAi exhibited increased locomotor activity and non-selective attention, and impaired learning and memory abilities, which is in line with the behavioral findings of animal models of ADHD. However, MPH ameliorated these abnormal behaviors. All findings indicated that Homer 1a may play an important role in the etiology and pathogenesis of ADHD.

Increased locomotor activity and non-selective attention and impaired learning ability in SD rats after lentiviral vector-mediated RNA interference of Homer 1a in the brain.

Our previous studies found that Homer 1a, a scaffolding protein localized at the post-synaptic density (PSD) of glutamatergic excitatory synapses, is significantly down-regulated in the brain of spontaneous hypertensive rats (SHR), an animal model of attention deficit hyperactivity disorder (ADHD). Furthermore, a first-line treatment drug for ADHD, methylphenidate, can up-regulate the expression of Homer 1a. To investigate the possible role of Homer 1a in the etiology and pathogenesis of ADHD, a lentiviral vector containing miRNA specific for Homer 1a was constructed in this study. Intracerebroventricular injection of this vector into the brain of Sprague Dawley (SD) rats significantly decreased Homer 1a mRNA and protein expression levels. Compared to their negative controls, these rats displayed a range of abnormal behaviors, including increased locomotor activity and non-selective attention and impaired learning ability. Our results indicated that Homer 1a down-regulation results in deficits in control over behavioral output and learning similar to ADHD.

Homer expression in the hippocampus of an animal model of attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a pervasive neurobehavioral disorder. We previously demonstrated differential expression of some isoforms of Homer, a family of scaffolding proteins localized to the postsynaptic density of glutamatergic excitatory synapses, in the spontaneous hypertensive rat (SHR), which is the most frequently used animal model of ADHD. Since these changes were observed in the prefrontal cortex (PFC), a critical structure in ADHD, it was hypothesized that these Homer isoforms may play a role in ADHD. The present study aimed to extend these findings to the hippocampus, which has direct connections to the PFC and subserves attention and cognition, two functions that are disturbed in ADHD. Hippocampal mRNA and protein expression of several Homer isoforms were investigated in both SHR and control Wistar-Kyoto (WKY) rats using reverse transcription-polymerase chain reaction and Western blotting, respectively. Both mRNA and protein for Homer 1a and Homer 2a/b, but not Homer 1b/c, were expressed at significantly lower levels in the hippocampus of SHR compared to WKY rats. The effects of methylphenidate (MPH) on spatial learning and memory in SHRs were also examined using the Morris water maze and on hippocampal expression of Homer isoforms. MPH improved spatial learning and memory and up-regulated hippocampal expression of Homer 1a and Homer 2a/b, but not Homer 1b/c, in SHRs. The animal model of ADHD may have altered expression of Homer 1a and Homer 2a/b in the hippocampus, in addition to the PFC. Future studies will focus on elucidating the specific mechanisms of Homer 1a and Homer 2a/b in ADHD.

SP600125, an inhibitor of c-Jun NH2-terminal kinase, blocks expression of angiotensin II-induced monocyte chemoattractant protein-1 in human mesangial cells.

BACKGROUND: We investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, in the expression of angiotensin II (Ang II)-induced monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1), and in the production of fibronectin (FN), by human mesangial cells (HMCs). METHODS: JNK activation in cultured human mesangial cells was determined by Western blotting with an antibody against the phosphorylated Ser63 residue of c-Jun. Binding of the activator protein (AP-1) to the MCP-1 AP-1 motif was detected via the electrophoretic mobility shift assay (EMSA). The transient luciferase reporter was used to examine MCP-1 promoter activity; an RNase protection assay and ELISA were used respectively to detect the expression of MCP-1 mRNA and production of MCP-1, TGF-beta and FN. RESULTS: Anthra (1,9-cd) pyrazol-6(2H)-one (SP600125), a pharmacological inhibitor of JNK, almost completely abolished Ang II-induced Ser63 phosphorylation of c-Jun at concentrations of 5-20 micromol/L: JNK activity was reduced by 75% with 10 micromol/L SP600125, and by 90% with 20 micromol/L. Ang II increased AP-1 binding to the MCP-1 AP-1 motif in a time-dependent manner, as detected by EMSA, while SP600125 effectively blocked this increased AP-1 binding in a concentration-dependent manner. Treatment with 100 nmol/L Ang II led to a steady increase in MCP-1 mRNA expression, and to an enhanced production of MCP-1, TGF-beta and FN. These effects were blocked by SP60025 in a dose-dependent manner. SP600125 also reduced MCP-1 mRNA stability: the halflife of MCP-1 mRNA was approximately 5 hours in cells treated with Ang II only, but was reduced to 2 hours when treated with a combination of Ang II and SP600125. CONCLUSIONS: These results show that the JNK/AP-1 pathway is involved in the expression of MCP-1 and TGF-beta, and in extracellular matrix production. JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.

[Mechanisms of decorin inhibiting epithelial-to-mesenchymal transition induced by transforming growth factor beta1 in renal tubular epithelial cells].

OBJECTIVE: To investigate the mechanisms of decorin inhibiting epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor beta1 (TGF-beta1) in renal tubular epithelial cells. METHOD: HK-2 cells in vitro were divided into 4 groups: (1) negative control group; (2) decorin group, added with decorin 100 ng/ml ; (3) TGF-beta1 group, added with TGF-beta1 10 ng/ml; (4) decorin and TGF-beta1 group, added with decorin 100 ng/ml and TGF-beta1 10 ng/ml. The protein level of phosphor-ERK, phosphor-PI3K, phosphor-Smad(3) and beta-catenin was detected by Western blotting method. The snail mRNA level was tested by real time-PCR, while the lymphoid enhancer factor-1 (LEF-1) mRNA level was measured by RT-PCR. RESULTS: The snail (2.59 +/- 0.70:1.02 +/- 0.13) and LEF-1 mRNA (1.85 +/- 0.08:0.30 +/- 0.11) were significantly up-regulated, meanwhile the protein level of phosphor-ERK (1.11 +/- 0.09:0.47 +/- 0.07), phosphor-PI3K (14.79 +/- 1.02:2.48 +/- 0.06), phosphor-Smad(3) (0.95 +/- 0.02:0.08 +/- 0.01) and beta-catenin (1.46 +/- 0.20:0.49 +/- 0.05) were significantly increased in TGF-beta1 group compared to control group, while there were no statistically significant difference in all figures between control group and decorin group. The phosphor-ERK protein level (0.58 +/- 0.08) and the snail mRNA level (1.24 +/- 0.03) were significantly down-regulated in TGF-beta1 and decorin group compared to TGF-beta1 group, however there were no statistically significant differences in the level of phosphor-PI3K (15.84 +/- 1.64), phosphor-Smad(3) (0.90 +/- 0.04) and beta-catenin (1.42 +/- 0.09) between these two groups. CONCLUSION: Decorin inhibited EMT induced by TGF-beta1 which may be through blocking the ERK signal transduction pathway.

Microstructural, optical, and electrical properties of SnO thin films prepared on quartz via a two-step method.

A simple, cost-effective, two-step method was proposed for preparing single-phase SnO polycrystalline thin films on quartz. X-ray diffraction (XRD) analysis demonstrated that the annealed films were consisted of polycrystalline alpha-SnO phase without preferred orientation, and chemical composition analysis of the single phase in nature was analyzed using X-ray photoelectron spectroscopy (XPS). Transmittance spectra in UV-vis-IR range indicated that the average transmittance of both the as-deposited and the annealed SnO thin films was up to 70%. The optical band gap decreased from 3.20 to 2.77 eV after the annealing process, which was attributed to the crystalline size related quantum size effect. Photoluminescence (PL) spectrum of the annealed film showed only a weak peak at 585 nm, and no intrinsic optical transition emission was observed. Moreover, the p-type conductivity of SnO film was confirmed through Hall effect measurement, with Hall mobility of 1.4 cm(2) V(-1) s(-1) and hole concentration of 2.8 x 10(16) cm(-3).

Gene expression profiles in the prefrontal cortex of SHR rats by cDNA microarrays.

We have undertaken cDNA microarrays to identify differentially expressed genes in the prefrontal cortex (PFC) of spontaneously hypertensive-rat (SHR), a rodent model of attention deficit hyperactivity disorder (ADHD) versus control Wistar-Kyoto (WKY) rats. The analysis of the gene expression profiles indicated that 57 genes were up-regulated and 97 genes were down-regulated in the PFC of SHR. These predominately expressed genes included genes involved in neural development, immunity, transcription factor, monoamine neurotransmitter, metabolism, signal transduction, apoptosis and so on. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of ADHD.

Prefrontal cortex Homer expression in an animal model of attention-deficit/hyperactivity disorder.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a pervasive neurobehavioral disorder affecting approximately 5% of children and adolescents and 3% of adults, and the prefrontal cortex (PFC) may play the most critical role in the expression of ADHD. Converging previous studies indicate a potential role of Homer--a scaffolding protein family localized at the postsynaptic density (PSD) of glutamatergic excitatory synapses--in behavioral pathologies associated with neuropsychiatric disorders. Accordingly, we speculate that these Homer isoforms might contribute to the etiology and development of ADHD. METHOD: We investigated the differential mRNA and protein expressions of several Homer isoforms in the PFC of the spontaneous hypertensive rat/Wistar-Kyoto rats (SHR/WKY), the most frequently used animal model of ADHD, using RT-PCR and western blotting. Furthermore, we examined the effects of methylphenidate (MPH) exposure on the behaviors and the expression of different Homer isoforms in the PFC of SHR, using Làt maze, RT-PCR and western blotting, respectively. RESULTS: Homer 1a and Homer 2a/b, but not Homer 1b/c, were expressed at a significantly lower levels in the PFC of SHR compared with WKY. MPH exposure decreased the locomotor activity and non-selective attention of SHR, and it up regulated the expression of Homer 1a and Homer 2a/b, but not Homer 1b/c, in the PFC of SHR. CONCLUSION: It is plausible that Homer 1a and Homer 2a/b may be involved in the etiology and pathogenesis of ADHD. Future work will focus on elucidating the specific mechanisms of Homer 1a and Homer 2a/b in ADHD.

[The effects of core proteoglycan on the expressions of I and III collagen in human renal tubular epithelial cell induced by TGFbeta1 in vitro].

AIM: To explore the roles of core proteoglycan and TGFbeta1 on the expressions of I and III collagen in human renal tubular epithelial cell line(HK-2) in vitro. METHODS: Confluent HK-2 cells were exposed to TGFbeta1 and core proteoglycan for up to 48 h. The cells were divided into four groups. Group (1), negative control group; group(2), single 10 microg/L TGFbeta1 treated group; group (3), 10 microg/L TGFbeta1+10 microg/L core proteoglycan group; group (4), 10 microg/L TGFbeta1+100 microg/L core proteoglycan group. Morphologic characterization of HK-2 cells was shown by invertmicroscope; Precise amounts of I and III collagen mRNA were measured by RT-PCR. RESULTS: After 48 h, morphology of (1) group cells had no changes, most cells were normal shape; (2) group cells took great changes, most cells converted into spindle shape, like fibroblast, (3) and (4) groups, spindle shape cells reduced significantly. In contrast to (1) group, the expressions of I collagen in (2) group from mRNA significant increased by 27.86-fold. The expressions of III collagen increased by 21.83-fold. Comparing (3) and (4) groups to(2) group, the expressions of I collagen from mRNA effectively decreased 36.39% and 53.36%. III collagen expressions increased 26.35% and 47.96%èP<0.05érespectively. But, neither (3) group nor (4) group alone could regulate I and III collagen mRNA to normal levels. CONCLUSION: Core proteoglycan can inhibit the expressions of I and III collagen in HK-2 cells induced by TGFbeta1 in vitro. Possibly, suggest core proteoglycan contribute to the regulation of renal fibrosis.

[Effects of core proteoglycan on the transdifferentiation of human renal tubular epithelial cell induced by transforming growth factor-beta1 in vitro].

OBJECTIVE: To study the effects of core proteoglycan on the transdifferentiation of human renal tubular epithelial cell induced by transforming growth factor beta1 (TGF-beta1) in vitro. METHOD: The cultured HK-2 cells were divided into six groups: A. negative control group; B. 10 ng/ml TGF-beta1 group; C. 10 ng/ml core proteoglycan treated group; D. 100 ng/ml core proteoglycan treated group; E. 10 ng/ml TGF-beta1 + 10 ng/ml core proteoglycan group; F. 10 ng/ml TGF-beta1 + 100 ng/ml core proteoglycan group. The changes in configuration of HK-2 cells were inspected 48 hours after adding the stimulating factor. At the same time, changes in mRNA of keratin, alpha-smooth muscle actin, vimentin were analyzed. RESULTS: Compared with group A, group B showed great changes in the morphology of cells, most cells converted into spindle shape, like fibroblast; groups E and F, especially group F showed significantly reduced spindle shape cells. Compared with group A, groups C and D had no significant changes in morphology of cells Compared with 10 ng/ml TGF-beta1 group and negative control, the mRNA expression of alpha-smooth muscle actin and vimentin had significant increase, but that of keratin reduced (P < 0.05). However, after combined treatment with TGF-beta1 and core proteoglycan, alpha-smooth muscle actin and vimentin expression were reduced significantly, while expression of keratin was up-regulated. Single core proteoglycan treated group and negative control group had no dramatic differences (P > 0.05). CONCLUSION: TGF-beta1 can induce the transdifferentiation of human renal tubular epithelial cell and core proteoglycan has some inhibitory effect on transdifferentiation of human renal tubular epithelial cell induced by TGF-beta1 in vitro.

[Effect of insulin-like growth factor I on the transdifferentiation and collagen synthesis of human renal tubular epithelial cells].

AIM: To investigate the role of insulin-like growth factor I(IGF-I) in transdifferentiation and collagen synthesis of human renal tubular epithelial cell line HK2 in vitro. METHODS: Cultured HK2 cells were divided into two groups: (1) control group; (2) IGF-I-treated group(25, 50, 100, 200 microg/L, respectively). The cell morphological changes were traced with inverted microscope, and the expression of alpha-smooth muscle actin (alpha-SMA), collagen I alpha and collagen III alpha mRNA was detected by RT-PCR. Concentration of collagen I secreted into the culture supernatant was determined by ELISA. RESULTS: In HK2 cells, IGF-I stimulated the morphological oval-to-fusiform transdifferentiation of the cells, and upregulated alpha-SMA, collagen I alpha and collagen III alpha mRNA expression(P<0.05). Secreted collagen I level was also up-regulate by IGF-I in the concentration of 100 and 200 microg/L, respectively. CONCLUSION: IGF-I can promote the transdifferentiation of human renal tubular epithelial cells and stimulate the synthesis of collagen.

Prolonged exposure to resistin inhibits glucose uptake in rat skeletal muscles.

AIM: To assess the effects and mechanisms of the action of resistin on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells. METHODS: Rat myoblasts (L6) were cultured and differentiated into myotubes followed by stimulation with single commercial resistin (130 ng/mL, 0-24 h) or cultured supernatant from 293-T cells transfected with resistin-expressing vectors (130 ng/mL, 0-24 h). Liquid scintillation counting was used to quantitate [3H] 2-deoxyglucose uptake. The translocation of insulin-sensitive glucose transporters GLUT4 and GLUT1, synaptosomal-associated protein 23 (SNAP23) and GLUT protein content, as well as the tyrosine phosphorylation status and protein content of insulin receptor substrate (IRS)-1, were assessed by Western blotting. RESULTS: Treatment of L6 myotubes with single resistin or cultured supernatant containing recombinant resistin reduced basal and insulin-stimulated 2-deoxyglucose uptake and impaired insulin-stimulated GLUT4 translocation. While SNAP23 protein content was decreased, no effects were noted in GLUT4 or GLUT1 protein content. Resistin also diminished insulin-stimulated IRS-1 tyrosine phosphorylation levels without affecting its protein content. The effects of recombinant resistin from 293-T cells transfected with resistin-expressing vectors were greater than that of single resistin treatment. CONCLUSION: Resistin regulated IRS-1 function and decreased GLUT4 translocation and glucose uptake in response to insulin. The downregulated expression of SNAP23 may have been partly attributed to the decrease of glucose uptake by resistin treatment. These observations highlight the potential role of resistin in the pathophysiology of type 2 diabetes related to obesity.

Resistin-binding peptide antagonizes role of resistin on white adipose tissue.

AIM: To investigate the direct effects of resistin and resistin-binding peptide (RBP) on lipid metabolism and endocrine function in adipose tissue. METHODS: Rat white adipose tissue was cultured in vitro and incubated for 24 h with 30 ng/mL recombinant rat resistin protein (rResistin) or combined with RBP of varying concentrations(1x10(-12) mol/L, 1x10(-10) mol/L, 1x10(-8) mol/L). Free fatty acids (FFA) released into medium was measured by a colorimetric kit. The levels of protein secretion and mRNA expression of TNF-alpha and adiponectin were detected by ELISA kit and RT-PCR respectively. RESULTS: The levels of FFA released into medium were significantly increased after 24 h of exposure to rResistin, but significantly decreased after RBP was applied, although there was no difference between the 3 concentrations. The protein level and gene expression of TNF-alpha in adipose tissue were significantly increased after 24 h of exposure to rResistin, but only obviously decreased after incubated with 1x10(-8) mol/L RBP. The levels of protein secretion and mRNA expression of adiponectin in adipose tissue were significantly decreased after 24 h of exposure to rResistin, but increased after incubated with RBP with the higher concentrations. CONCLUSION: RBP can effectively antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue.

Identification of differentially expressed genes in omental adipose tissues of obese patients by suppression subtractive hybridization.

To identify differentially expressed genes between obese individuals and normal control, we have undertaken suppression subtractive hybridization (SSH). Omental adipose tissues were obtained via abdominal surgery for appendicitis in both 13 obese subjects [BMI (body mass index) >30 kg/m2] and 13 normal subjects (BMI >18 and <25 kg/m2). Following SSH, about one thousand clones were sequenced and found to derive from 426 different genes. These predominately expressed genes included genes involved in lipid metabolism, cytokines, signal transduction, GLUT4 translocation, cell cycle and growth, cytoskeleton, and others. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of obesity.

[Transdifferentiation and collagen-synthesis effects of exogenous connective tissue growth factor on renal tubular epithelial cells in vitro].

OBJECTIVE: To investigate the effects of recombinal human connective tissue growth factor (rhCTGF) stimulation on epithelial-myofibroblast transdifferentiation (EMT) and collagen-synthesis in human renal tubular epithelial cell line (HK2) in vitro. METHODS: The cultured HK2 cells were stimulated with rhCTGF of 5 ng/mL. The morphological changes were observed under an inverted microscope. The cells were collected at 0, 3, 6, 12, 24 and 48 hrs after rhCTGF stimulation. The expression of E-cadherin,alpha-smooth muscle actin (alpha-SMA), collagen Ialpha1 (Col Ialpha1) and collagen IValpha1 (Col IValpha1) mRNAs were detected by RT-PCR. RESULTS: rhCTGF stimulation changed the HK2 cell appearance from oval to fusiformdown-regulated the E-cadherin mRNA expression and up-regulated alpha-SMA mRNA expression, but had no effects the Col Ialpha1 and Col IValpha1 mRNA expression. CONCLUSIONS: Exogenous CTGF can mediate the EMT but has no collagen-synthesis effects on HK2 cells.

A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation.

BACKGROUND: Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor. METHODS: Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide. RESULTS: Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation. CONCLUSION: RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.

[The cloning of human Smac gene and its pro-apoptotic effect on Burkitt's lymphoma cells].

OBJECTIVE: Second mitochondria-derived activator of caspase (Smac) is a recently identified, novel pro-apoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac promotes activation of caspases by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The objective of the study was to examine the pro-apoptotic effect of human Smac gene on Burkitt's lymphoma Raji cells. METHODS: The full length cDNA of human Smac gene was amplified by reverse transcription-PCR from total RNA of HEK-293 cells. The PCR product was ligated with linearized vector pGEM-T-easy supplied in the TA cloning kit and sequenced. The correct cDNA of full length Smac was subcloned into eukaryocytic expression vector pcDNA3.1/myc-his and transfected into human Burkitt's lymphoma cell line Raji by lipofectamine-mediated transfection. The expression of full length Smac was determined by Western blot. Morphological observation was done with the laser scanning confocal microscope by double staining the Raji cells with Hoechest 33,258 and propidium iodide. Flow cytometry was used to evaluate apoptosis. Relative caspase-3 activity was determined by colorimetric assay. RESULTS: Recombinant eukaryocytic expression vector pcDNA3.1/Smac, which contained full length Smac, was successfully constructed. After pcDNA 3.1/Smac was transfected into human Burkitt's lymphoma Raji cell line for 24 hours, Raji cells showed apparent apoptosis with a percentage of (43.7 +/- 2.5)%, which was higher than that of non-transfected group and free vector-transfected group (P < 0.05). Compared with non-transfected group (0.136 +/- 0.036) and free vector-transfected group (0.138 +/- 0.026), the relative caspase-3 activity of Raji cells transfected by pcDNA3.1/Smac (0.936 +/- 0.041) was significantly enhanced (P < 0.05). CONCLUSION: Transfection and expression of human Smac gene could significantly induce apoptosis of human Burkitt's lymphoma Raji cells. The mechanism is associated with the increase of caspase-3 activity.

[Suppressive effects of par-4 antisense oligodeoxynucleotide on up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects].

OBJECTIVE: To investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects. METHODS: Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye. RESULTS: Par-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01). CONCLUSIONS: par-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.