Establishment and evaluation of in vitro galectin-3 inhibitor screening model / 药学学报

Galectin-3 (Gal-3) belongs to the galectin family and is specific in binding β-galactoside. Through its C-terminal domain, Gal-3 binds to the galactoside group of the glycosylated insulin receptor (IR) and inhibits IR signaling pathway, which leads to the insulin resistance. Thus, Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes. Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside. We expressed and purified human Gal-3 in Escherichia coli (E.coli), and labeled it with fluorescein isothiocyanate (FITC) in vitro. After incubating FITC labeled Gal-3 (Gal-3-FITC) with PANC-1 cells, which express glycosylated membrane protein, PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein. Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells, subsequently leads to the decrease of the fluorescent signal in PANC-1 cells. We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal. Further studies show this model is simple, stable, and repeatable with a Z' factor between 0.7 and 0.85. In sum, we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.

Preparation of SUN5-specific polyclonal antibody for detection of SUN5 expression in human germ cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a specific polyclonal antibody against full-length SUN5 for detecting the expression of SUN5 in human germ cells.</p><p><b>METHODS</b>Bioinformatic methods were used to compare the full-length SUN5 and its variant SUN5β, and a short peptide was designed based on the differential region to prepare SUN5 antibody. The prepared antibody was used to detect the expression of SUN5 in Ntera-2 cells and in human germ cells by Western blotting and immunofluorescence assay.</p><p><b>RESULTS</b>The short peptide was correctly synthesized and SUN5 antibody was obtained and purified. Western blotting showed that the prepared antibody was capable of recognizing full-length SUN5 in Ntera-2 cells, and SUN5 expression was localized on the nuclear membrane and in the cytoplasm as shown by immunofluorescence assay. Using this antibody, we detected SUN5 expression in the spermatocytes, round spermatids and sperms in human germ cells.</p><p><b>CONCLUSION</b>We successfully prepared SUN5-specific antibody. SUN5 is expressed in the spermatocytes, round spermatids and sperms in human germ cells, suggesting its important role in spermatogenesis.</p>

Surface modification and DNA-binding assessment of nano-hydroxyapatite / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the impact of surface modification on the DNA-binding ability of nano-hydroxyapatite (nHA).</p><p><b>METHODS</b>Chemical co-precipitation-hydrothermal synthesis was utilized to prepare the nHA particles, and polyethylenimine (PEI) was used for surface modification of the nHA. Transmission electron microscopic (TEM) observation and zeta potential detection of the nHA were carried out before and after surface modification. The abilities of the nanoparticles, at different pH values and different concentrations, for DNA-binding and DNA protection against nuclease digestion were assessed before and after surface modification by electrophoresis.</p><p><b>RESULTS</b>TEM observation showed a short rod-like morphology of PEI-modified nHA with uniform particle size and good dispersion; the nHA without the modification tended to aggregate with poor dispersion. With a positive zeta potential, the PEI-modified nHA showed an obviously enhanced ability of DNA binding at different pH values and concentrations, with strong capacity to protect the DNA against Dnase I digestion. At the concentration of 250 µg/ml and a pH value of 7.0, the nHA-PEI showed an optimal efficiency of DNA-binding and DNA protection.</p><p><b>CONCLUSION</b>nHA with surface modification by PEI can serve as an effective vector for DNA binding and transfer.</p>

Prokaryotic expression and purification of SPAG4L, a novel human testis gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.</p><p><b>METHODS</b>The fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.</p><p><b>RESULTS</b>The recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.</p><p><b>CONCLUSION</b>The recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.</p>

Effect of different glucose concentrations on the expressions of insig-1 and insig-2 mRNA during the differentiation of 3T3-L1 cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the effect of different concentrations of glucose on the differentiation of 3T3-L(1) and the expression of insig-1 and insig-2 mRNA, and to explore the effect of insulin-induced gene in the differentiation and formation of adipocytes and lipogenesis.@*METHODS@#The 3T3-L(1) cells were induced to differentiate in high glucose concentration (25 mol/L G.S), low glucose concentration (5.5 mol/L G.S), and mannitol (19.5 mol/L Mannitol +5.5 mol/L G.S), respectively. The differentiation of 3T3-L(1) cells was examined by oil red "O" straining, and the expression of insig-1,insig-2 mRNA and AP2 mRNA was examined by RT-PCR and in situ hybridization.@*RESULTS@#With the differentiation of 3T3-L(1) cells, the expression of insig-1 and insig-2 mRNA was gradually up-regulated. The expression of insig-1 and insig-2 mRNA significantly increased while AP(2) mRNA decreased in the low glucose concentration inducing group and mannitol inducing group. In the high glucose concentration inducing group, the cell differentiation was poor (P<0.05). There was no difference between the low glucose concentration and the mannitol group in the differentiation of 3T3-L(1) cells, and in the expression of insig-1 and insig-2 and AP(2) mRNA.@*CONCLUSION@#Different concentrations of glucose may influence the cell differentiation and the low glucose concentration promotes insig-1 and insig-2 gene expression, which may lead to the inhibition of the differentiation and lipogenesis of preadipocytes.

Expression of PDCD5 in multiple myeloma and its relation with BCL-2 / 中南大学学报(医学版)

OBJECTIVE@#To determine the expression of apoptosis related gene PDCD5 in multiple myeloma (MM), and to analyze the relation between PDCD5 and BCL-2.@*METHODS@#The expressions of PDCD5 and BCL-2 protein and mRNA were determined by immunohistochemical staining method, flow cytometry (FCM) and reverse transcription polymerase chain reaction (RT-PCR) method in bone marrow mononuclear cells. We also analyzed the relation between PDCD5 and BCL-2.@*RESULTS@#Immunohistochemical staining showed that PDCD5 protein positive cell percentage, staining intensity index (SII) of PDCD5 protein, BCL-2 protein positive cell percentage, and SII of BCL-2 protein were (34.75 +/- 6.49)%, (281.16 +/- 75.33), (29.97 +/- 5.57)%, and (224.94 +/- 57.72) in the MM group and (52.98 +/- 5.84)%, (462.84 +/- 39.77), (5.56 +/- 1.95)%, and (27.84 +/- 9.75) in the control group (all P < 0.05). Results of FCM showed that PDCD5 protein positive percentage and mean fluorescence intensity of PDCD5 were (78.11 +/- 21.63)% and (61.73 +/- 11.04) in the MM group and (89.46 +/- 9.98)% and (353.04 +/- 123.26) in the control group (all P < 0.05). RT-PCR showed that relative expression of PDCD5 and BCL-2 mRNA were (0.33 +/ -0.07) and (0.33 +/- 0.08) in the MM group and (0.53 +/- 0.05) and (0.12 +/- 0.02) in the control group (all P < 0.05). The positive cell percentage of PDCD5 and BCL-2 protein was negative correlation (r = -0.86, P < 0.05); the expression of PDCD5 and BCL-2 mRNA was the same status (r = -0.90, P < 0.05).@*CONCLUSION@#The expressions of PDCD5 protein and mRNA in MM patients are down-regulated, but the expressions of BCL-2 protein and mRNA are up-regulated. The mRNA and protein expression of PDCD5 and BCL-2 has negative correlation.

3 polymorphisms of gene GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship among 3 polymorphisms of GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness(PTR).@*METHODS@#The 26th exon, the 30th exon and the 21st intron of gene GP IIb in 110 individuals were amplified by polymerase chain reaction (PCR), and the PCR products were analyzed with single-strand conformation polymorphism(SSCP) and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok I enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion.@*RESULTS@#There were polymorphisms of gene GP IIb in the 26th, 30th exon and the 21st intron, and the mutations were: T changed into G in 13,959 th of the 26th exon; C changed into T in 16,997 th of the 30th exon; the 9 bps deletion occurred in 11,996-12,004 th of the 21st intron. The 3 polymorphisms had synchronization in the individuals. The results of Fok I enzyme indicated that the frequency of HPA-3a was 83.6% (92/110)and 81.9%(119/147), and that of HPA-3b was 16.4%(18/110) and 19.1%(28/147) in the normal individuals and the patients respectively. There was no significant difference between the patients and normal individuals (P>0.05). After the platelet transfusion, the antibodies of all the cases of the homotype platelet transfusion were negative, but the antibodies in 2 cases of the control group were positive, and there was antibody to HPA-3a in one of the antibodies positive cases.@*CONCLUSION@#(1)There is close linkage among the polymorphisms of gene GP IIb, which is T->G in 13 959 th of the 26th exon, C->T in 16,997 th of the 30th exon, and the 9 bps deletion in 11,996-12,004 th in the 21st intron. (2)The gene frequency of HPA-3a/3b is similar in the normal individuals and patients with hematologic diseases. (3) HPA-3 system may be one of the reasons for PTR in Chinese.

Potential risk of porcine endogenous retrovirus cross-species transmission in neonatal pig islets under xenotransplanted condition / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the potential risk of porcine endogenous retrovirus (PERV) cross-species transmission xenotransplanted with microencapsulated neonatal pig islets (NPIs).@*METHODS@#Ten dogs were randomly divided into an experiment group and a control group. The experiment group was transplanted with microencapsulated NPIs, and the control group was transplanted with non-microencapsulated NPIs. Glucose tolerance test (GTT) was performed to evaluate the function of microencapsulated NPIs after the transplantation; immunity histochemistry was used to detect the microencapsulated NPIs in the liver of dogs which had been transplanted after 28 days; PCR and RT-PCR were performed to detect PERV and pig mitochondrial (mt) DNA in the blood samples obtained from recipients at various time points after the transplantation.@*RESULTS@#The level of serum special porcine C peptide increased significantly after the injection of glucose for 15 approximately 30 min in dogs which were transplanted with the micro-encapsulated NPIs over 2 weeks, while special porcine C peptide could not be detected in the control group. Immunity histochemistry showed that a few microencapsulated NPIs were still alive in the liver of the dog, and the liver was not damaged. PCR and RT-PCR showed that pig mt DNA and PERV could not be detected in the experiment group 1 approximately 28 days after the transplantation, while very weak expression of that in the control could be detected in the first 4 days and disappeared 10 days after the transplantation.@*CONCLUSION@#Microencapsulated NPIs can survive and have biological function in dogs. There is no evidence of PERV replication, suggesting that the xenotransplantation with microencapsulated NPIs can prevent PERV effectively, and may have great value.

Porcine endogenous retrovirus in Daweizi pigs in Hunan / 中南大学学报(医学版)

OBJECTIVE@#To detect porcine endogenous retrovirus (PERV) in Daweizi pigs and to provide basic parameters of evaluating the biological safety for xenotransplantation from pigs to humans.@*METHODS@#Ear tissues from 42 individuals were randomly collected from a Daweizi pig population. PCR and RT-PCR were performed to detect PERV proviral DNA and mRNA respectively. Finally, env-A, env-B, and env-C were amplified, sequenced, and analyzed using the BLAST software in National Center for Biotechnology Information.@*RESULTS@#PERV proviral DNA and mRNA could be detected in the 42 individuals by PCR and RT-PCR, respectively. env-A, env-B and env-C were detected in all the individuals. Compared with other pig species (AY288779, DQ011794 and AY534304), there was 1 and 8 bp differences in the sequences of env-A and env-C, while no difference in env-B.@*CONCLUSION@#PERV exists and has transcriptive activity in Daweizi pigs. The predominate subtype is PERV-ABC. Env genes are firstly cloned and sequenced in Daweizi pigs and there are polymorphism in the breed. As to the biological safety, the breed was not suitable as a donor in xenotransplantation.