catena-Poly[[diaqua-zinc(II)]-µ-piper-azine-1,4-diacetato-κN,O:N,O].

The asymmetric unit of the title compound, [Zn(C(8)H(12)N(2)O(4))(H(2)O)(2)](n), contains a Zn(II) ion residing on an inversion center, half of a centrosymmetric piperazine-1,4-diacetate ligand (L) and a water mol-ecule. The Zn(II) ion is trans-coordinated by two N,O-bidentate L ligands and by two water mol-ecules in a distorted octa-hedral geometry. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link polymeric chains into a three-dimensional supra-molecular structure.

[Oxalylbis(aza-nedi-yl)]bis-{[amino-(2-pyrid-yl)methyl-ene]ammonium}.

The title compound, C(14)H(16)N(8)O(2) (2+)·2ClO(4) (-), was prepared by reaction of bis-[amino-(2-pyrid-yl)methyl-ene]oxalohydrazide with perchloric acid. The mol-ecular symmetry is C(i) and thus the asymmetric unit comprises one half-mol-ecule. The dihedral angle between the aromatic ring and the plane of the oxamide group is 70.8 (3)°. The perchlorate anions and the cations are connected by inter-molecular N-H⋯O hydrogen bonds.

trans-Diaqua-bis[(E)-3-(dimethyl-amino)-1-(2-pyrid-yl)prop-2-en-1-one-κN,O]cobalt(II) dinitrate dihydrate.

In the title compound, [Co(C(10)H(12)N(2)O)(2)(H(2)O)(2)](NO(3))(2)·2H(2)O, the Co(II) ion, located on an inversion center, is trans-coordinated by two N,O-bidentate chelating (E)-3-(dimethyl-amino)-1-(2-pyrid-yl)prop-2-en-1-one ligands and by two water mol-ecules in a slightly distorted octa-hedral geometry. Inter-molecular O-H⋯O hydrogen bonds link the cations, anions and water mol-ecules into layers parallel to the ac plane. The crystal packing also exhibits weak inter-molecular C-H⋯O hydrogen bonds.

(5,7,7,12,14,14-Hexamethyl-1,4,8,11-tetra-azacyclo-tetra-deca-4,11-diene-κN,N,N,N)(thio-cyanato-κS)nickel(II) perchlorate monohydrate.

In the title compound, [Ni(SCN)(C(16)H(32)N(4))]ClO(4)·H(2)O, the Ni(II) ion is coordinated by the four N atoms of the tetra-azacyclo-tetra-deca-4,11-diene macrocyclic ligand and by the S atom of a thio-cyanate anion. The perchlorate anion is rotationally disordered around one Cl-O bond between two orientations; the occupancies refined to 0.61 (4) and 0.39 (4). Inter-molecular O-H⋯N, N-H⋯O and N-H⋯N hydrogen bonds link two cations, two anions and two solvent water mol-ecules into a centrosymmetric cluster. The crystal packing is further stabilized by weak inter-molecular C-H⋯O hydrogen bonds.

(E)-3-Dimethyl-amino-1-(2-thien-yl)prop-2-en-1-one.

The mol-ecular skeleton of the title mol-ecule, C(9)H(11)NOS, is essentially planar: the thio-phene ring is inclined to the mean plane of the rest non-H atoms by 2.92 (3)°. The crystal packing exhibits no significantly short inter-molecular contacts.

[Infiltration of DCs and CD4+ T cells in muscle after DNA immunization].

OBJECTIVE: In order to get a better understanding of the mechanism of DNA initiating specific cytotoxicity T lymphocyte (CTL) immune response, we observed the local infiltration in injection site at different time after DNA immunization and further studied the characters of infiltration cells. METHODS: The local infiltration in injection sites was observed by HE staining at 6 h, 12 h and 1-6 days after DNA immunization. Infiltration cells were further studied with the mice dendritic cells marker CD205, T lymphocyte marker CD4 and CD8 by immunohistological methods. RESULTS: phAFP injection into mouse muscle led to a local infiltration. Infiltrates reached peak at 3-6 days after injection and appeared in several discrete clusters within the muscles. The predominant cell types found in infiltrates were macrophage-like cells and lymphocytes. Immunohistological staining revealed CD205+ and CD4+ cells. CD4+ cells could be detected in any of the injected muscles. CD205+ cells were only seen at 24 h to day 3, appearing later and disappearing earlier than CD4+ cells. However, CD8+ immunostaining positivity could not be found. CONCLUSION: Our findings indicate that the character of local infiltration after DNA immunization is different from the inflammation of injection. The recruitment of CD205+ to the injection site suggests that DCs and Th cells play an important role in DNA immunization.

[Construction and expression of anti-human AFP ScFv gene in BL-21 (DE3) E. coli].

OBJECTIVE: To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity. METHODS: VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test. RESULTS: The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%. CONCLUSION: We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.

[Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1].

OBJECTIVE: To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli. METHODS: The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test. RESULTS: It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1. CONCLUSION: VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

[Construction and expression of human alpha-fetoprotein recombinant plasmid].

OBJECTIVE: To construct AFP-expressing plasmid and study the expression in eukaryotic cells. METHODS: Total RNA was isolated from fetal liver tissue. Full-length human AFP cDNA was obtained by RT-PCR amplification and then recombined into eukaryotic expression plasmid pcDNA3.1. The AFP sequence of the recombinant plasmid phAFP was determined. The AFP expressions in CHO transfected with phAFP and in muscle after injection phAFP were investigated by immunohistological methods. RESULTS: The restriction endonuclease mapping of recombinant plasmid showed 1.8 kb fragment as well as full-length human AFP cDNA, and the sequence is consistent with that from GenBank. The phAFP was successfully expressed in CHO and in muscle tissues. CONCLUSION: These results suggested that AFP can be used as a target gene of DNA vaccine in heptocellular carcinoma therapy.

[Study on the TIL and NK of IL-2 injected via pelvic retroperitoneal space in gynecological cancer patient].

OBJECTIVE: To verify the feasibility and effect of biotherapy instituted via pelvic retroperitoneal space on gynecological cancer. METHODS: Injecting IL-2 (and/or) 5-Fu through a tube installed in the pelvic retroperitoneal space. Counting the subpopulation of T cell and NK of lymph-nodes of pelvis after the drugs being by FCM. RESULTS: The numbers of CD3+, CD4+, CD8+, CD25+ and NK cells in treatment group were significantly higher than those in the control group. And the numbers of these cells in the IL-2 + 5-Fu group were significantly higher than those in the 5-Fu group. The CD25+ and NK cell numbers in the IL-2 group were significantly higher than those in the 5-Fu group (P < 0.05). CONCLUSION: The IL-2 injected via pelvic retroperitoneal space can promote the activity, development and infiltrating of T cell and NK cell in the tumor tissue.

[The effect of basic fibroblast growth factor in ovarian cancer growth and angiogenesis].

OBJECTIVE: To investigate whether basic fibroblast growth factor (bFGF) can induce the proliferation, invasion and angiogenesis of ovarian cancer or not. METHODS: Human ovarian cancer cell lines SKOV(3) 1 x 10(4)/ml were plated in 24-well dishes, bFGF at 5, 10,15 and 20 ng/ml was added and crystal violet staining was given daily for 8 days, cell numbers were counted by determining OD490. SKOV(3) cells were plated in the center of 50% extra cellular matrix gel, bFGF at 5 and 10 ng/ml was added and the migration distance of cells was measured daily. SKOV(3) 5 x 10(7)/ml were transplanted to BALB/c nude mice subcutaneous. One week later, bFGF, bFGF-MAb or 0.9% nature sodium was injected subcutaneously surrounding the tumor twice a week. Eight weeks later, the experiment ended and the volume of the tumors were measured. Intratumoral microvessel density (MVD) was measured by immunohistochemistry staining for factor VIII. RESULTS: bFGF at 0-10 ng/ml could stimulate the proliferation of SKOV(3) concentration dependently (P<0.05). On the fifth day, the cell proliferation in 10 ng/ml group was 121% above control. bFGF could stimulate the invasion of SKOV(3) concentration dependently (P<0.05). On the seventh day, the migration distance in 5 ng/ml group was 1.16 cm and 153% above control, and that in 10 ng/ml group was 1.86 cm and 245% above control. The average volume of transplanted tumors and MVD in bFGF group were 180% and 146% above control respectively those in bFGF-MAb group were 63.7% and 62.8% above control respectively. CONCLUSION: bFGF can stimulate proliferation, invasion and angiogenesis of ovarian cancer markedly; bFGF-MAb can inhibit the angiogenesis and growth of ovarian cancer.