Animals ; Animals, Newborn ; Cells, Cultured ; Cyclic AMP ; metabolism ; Glucagon-Like Peptide 1 ; pharmacology ; Insulin ; secretion ; Islets of Langerhans ; drug effects ; secretion ; Peptide Fragments ; pharmacology ; RNA, Messenger ; genetics ; Rats

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.
Factor VIII ; genetics ; metabolism ; secretion ; Glycosylation ; HEK293 Cells ; Humans ; Inteins ; Mutation ; Peptide Fragments ; genetics ; metabolism ; secretion ; Protein Splicing ; Trans-Splicing ; Transfection

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

AIMTo determine whether serotonin, a major neurotransmitter in brain, can modulate the production of secretory beta-amyloid protein precursor (sAPP) by activation of serotonin 5-HT2C receptor.

METHODSThe hippocampal slices of rats were incubated with various concentrations of serotonin, M-110, or L-107. sAPP released into the incubation medium were assayed by Western blot analysis assay with monoclonal antibody 22C11 for 2 h.

RESULTSVarious concentrations of serotonin (1.0 x 10(-2) - 1.0 x 10(3) micromol x L(-1)), M-110, a serotonin 5-HT2C agonist (1.5 x 10(-6) - 1.5 x 10(3) micromol x L(-1)), showed positive effect on the production of sAPP while L-107, a serotonin 5-HT2C antagonist (1.0 x 10(-9) - 1.0 x 10(3) micromol x L(-1)), showed negative effect on the production of sAPP over controls.

CONCLUSIONSerotonin modulates production of secretory amyloid beta-protein precursor through serotonin 5-HT2C receptor in incubated rat hippocampal slices.

Amyloid beta-Protein Precursor ; secretion ; Animals ; Hippocampus ; metabolism ; In Vitro Techniques ; Male ; Peptide Fragments ; secretion ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT2C ; Serotonin ; pharmacology ; Serotonin 5-HT2 Receptor Agonists ; Serotonin 5-HT2 Receptor Antagonists

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

OBJECTIVETo investigate the effects of chitosan on the biological activities of the fibroblasts derived from different tissues.

METHODSThe biological activities of the fibroblasts derived from different tissues were evaluated with a MTT method for fibroblast proliferation, photic and electronic microscope for morphologic and subcellular structure, 3H-proline uptake method for collagen secretion and ELISA box for the secretion of TGF-beta 1, FGF-AB, and IL-8.

RESULTSThis study showed that the chitosan inhabited the proliferation of the fibroblasts and the secretion of the TGF-beta 1, FGF-AB and collagen of the fibroblasts with a dose-depended manner in the normal skin, hypertrophic scar and keloid groups, but it stimulated the IL-8. However, there were no significant differences among the three groups (P > 0.05).

CONCLUSIONThe chitosan could inhibit the growth, proliferation, biosynthesis and secretion of the fibroblasts, and it may be used to treat different scars.

Adolescent ; Adult ; Cell Division ; drug effects ; Chitin ; analogs & derivatives ; pharmacology ; Chitosan ; Dose-Response Relationship, Drug ; Female ; Fibroblast Growth Factors ; secretion ; Fibroblasts ; drug effects ; metabolism ; ultrastructure ; Hemostatics ; pharmacology ; Humans ; Interleukin-8 ; secretion ; Male ; Microscopy, Electron ; Peptide Fragments ; secretion ; Transforming Growth Factor beta ; secretion

The present study surveyed the prevalence of natural infection of the sheep esphagus muscle with sarcocysts of Sarcocystis ovicanis and examined induction of protective immunity using UV-attenuated sporocysts. The overall prevalence of natural infection of the sheep was 95%. Infectivity of the collected sarcocysts was confirmed by shedding of sporulated oocysts after feeding infected esophageal tissues to dogs. To induce protective immunity, lambs were immunized 3 times (once a week) with 1.5 x 10(4) sporocysts exposed to UV-light for 30 min (UV-30 group) or 60 (UV-60 group) min and then challenged with 1.5 x 10(4) normal sporocysts at the 3rd week post the 1st vaccination. These lambs showed high survival and less clinical signs of sarcocystosis than normal infected lambs. The attenuated sporocysts produced abnormal cysts; small in size and detached from the muscle fiber. These abnormalities were more obvious in UV-60 group than UV-30 group. Also, the IFN-gamma level and lymphocyte percentage were increased while the total leukocyte count was decreased in the UV-60 group compared with other groups. The high level of IFN-gamma may be an evidence for the induction of Th1 responses which may have protective effect against a challenge infection.
Animals ; Dogs ; Esophagus/parasitology ; Feces/parasitology ; Interferon-gamma/secretion ; Lymphocytes/immunology ; Oocysts/*immunology ; Peptide Fragments/secretion ; Prevalence ; Protozoan Vaccines/immunology ; Sarcocystis/cytology/*immunology/*radiation effects ; Sarcocystosis/epidemiology/immunology/prevention & control/*veterinary ; Severity of Illness Index ; Sheep/immunology/parasitology ; Sheep Diseases/immunology/*prevention & control ; Survival Analysis ; *Ultraviolet Rays ; Vaccines, Attenuated/immunology

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals ; Antibodies, Monoclonal/pharmacology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation ; Female ; Glucocorticoids/*pharmacology ; Herpes Simplex/*immunology ; Herpesvirus 1, Human/pathogenicity ; *Immunity, Cellular ; Interferon Type II/secretion ; *Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/metabolism ; Receptors, Interleukin-2/metabolism ; Receptors, Nerve Growth Factor/genetics/immunology/*metabolism ; Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/*immunology/metabolism/virology