Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.
Child ; Humans ; Brain/pathology* ; Cations/metabolism* ; Chlorides/metabolism* ; Epilepsy/metabolism* ; Malformations of Cortical Development, Group I/metabolism* ; Solute Carrier Family 12, Member 2/metabolism* ; Symporters/metabolism*

Administration, Oral ; Glucose Transporter Type 2 ; metabolism ; Humans ; Intestinal Absorption ; physiology ; Organic Anion Transporters ; metabolism ; Peptide Transporter 1 ; Pharmacokinetics ; Sodium-Glucose Transporter 1 ; metabolism ; Symporters ; metabolism

Human Sodium/Iodide symporter gene cDNA was amplified from thyroid tissue of the patient suffering from Graves disease by RT-PCR, and T/A cloned into pGEM-TEasy-NIS for sequencing, subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene, and then forwarded to homologous recombinant in the bacteria BJ5183 that already contained AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the 293 cells to obtain recombinant adenovirus. The results showed that the recombinant AdNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 2. 5 - 3 x 10(9) efu/ml. So, the recombinant adenovirus vector carrying hNIS was successfully constructed, thus providing a basis for researches on 131I therapy in nonthyroid carcinoma.
Adenoviridae ; genetics ; metabolism ; DNA, Complementary ; genetics ; Genetic Vectors ; genetics ; metabolism ; Graves Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Symporters ; biosynthesis ; genetics

This study sought to probe the feasibility of instituting a radioiodide treatment for androgen-independent prostate cancer by adenovirus transfer of the hNIS gene. A recombinant adenovirus, Ad-CMV-NIS, that expressed the NIS gene under the control of cytomegalovirus (CMV) promoter was constructed. In vitro, after infection with Ad-CMV-NIS,PC-3 prostate cancer cells exhibited an uptake of perchlorate-sensitive iodide, approximately 120 times higher than that exhibited by negative control Ad-CMV-GFP-infected cells. The half-time of efflux was 26.6 min. Clonogenic assays demonstrated that Ad-CMV-NIS-infected cancer cells were selectively killed by exposure to 131I. In vivo, Ad-CMV-NIS infected tumors showed significant radioiodine accumulation (16.30 +/- 8.72)% ID/g at 2h postinjection) with an effective half-life of 5.4h. The tumor could be clearly visualized by 131I scintigraphy. These data indicate that infection with Ad-CMV-NIS is an efficient way to induce radioiodide uptake in vitro and in vivo, thus suggesting that NIS-based gene therapy has the potential for use in androgen-independent prostate cancer.
Adenoviridae ; genetics ; metabolism ; Genetic Therapy ; methods ; Humans ; Iodine Radioisotopes ; therapeutic use ; Male ; Prostatic Neoplasms ; genetics ; metabolism ; radiotherapy ; Symporters ; genetics ; Transfection ; methods

Case-Control Studies ; Child, Preschool ; Congenital Hypothyroidism ; DNA ; genetics ; Female ; Humans ; Hypothyroidism ; genetics ; Infant ; Iodine ; metabolism ; Male ; Mutation ; Polymerase Chain Reaction ; Sodium ; metabolism ; Symporters ; genetics

Radioiodine therapy of carcinoma could be mediated by transferring the genes which participate in the process of iodine metabolism in thyroid. The correlative genes are sodium/iodine symporter gene, thyroid peroxidase gene and the specific thyroid transcription factors, and others. The objective gene can specifically express in carcinoma by inserting the tissue-specific promoter/enhancer upstream of them, so radioiodine could be used to treat varied carcinomas. The radioiodine uptake in carcinoma cells was obviously increased and the radioiodine therapy of carcinoma was effective after those genes had expressed in carcinoma cells. The main problem was that the effective half-time of radioiodine in cells was too short to produce the ideal effect of radioiodine therapy. Moreover, 211At and 188Re could be transferred by sodium/iodine symporter and they could be used to treat the carcinoma that is capable of radioiodine uptake.
Animals ; Gene Expression Regulation, Neoplastic ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Iodine Radioisotopes ; therapeutic use ; Neoplasms ; genetics ; radiotherapy ; Symporters ; genetics ; metabolism

BACKGROUND AND OBJECTIVELung cancer harms people's health or even lives severely. Especially, the therapy of non-small cell lung cancer (NSCLC) has not been obviously improved for many years. The aim of this study is to transfer the human sodium/iodide symporter (hNIS) and the human thyroperoxidase (hTPO) genes into H460 lung cancer cell line, and to study the uptake ability of iodide after co-transfected hTPO and hNIS gene in cell lines.

METHODSThrough cloning, recombination, packaging and amplifying, the recombinant adenosine virus (AdTPO) was constructed. Then the protein expression of AdTPO was tested by Western blot. After transfected hNIS gene into human lung cancer cell line H460 through liposome, stably expressing hNIS gene cell lines (hNIS-H460) selected by G418 antibiotics was determined as hNIS-H460 group. Using AdTPO, hTPO gene was transducted into hNIS-H460, as AdTPO-hNIS-H460 group. H460 cell without hNIS gene was applied as control group (H460). Then, we investigated the 125I uptake assay of the above cells.

RESULTSWe were successful in co-transfecting hNIS and hTPO gene into human lung cell lines H460, and were obtained hNIS and hTPO gene lung cancer cell lines (hNIS-H460 and AdTPO-hNIS-H460). In AdTPO-hNIS-H460, hNIS-H460 and H460, the uptake ability of 125I was (59 637.67 +/- 1 281.13), (48 622.17 +/- 2 242.28) and (1 440.17 +/- 372.86) counts x min(-1). The uptake ability of 125I was 41 fold higher in AdTPO-hNIS-H460 than in blank control H460 (P < 0.01), and 34 fold higher in hNIS-460 than in blank control H460 (P < 0.01), and 1.2 fold higher in AdTPO-hNIS-H460 than in hNIS-H460 (P < 0.01).

CONCLUSIONThe uptake ability of 125I could increase by co-transfected hNIS and hTPO genes into human lung cancer cell lines H460.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Iodide Peroxidase ; genetics ; Iodine Radioisotopes ; pharmacokinetics ; therapeutic use ; Lung Neoplasms ; metabolism ; therapy ; Symporters ; genetics ; Transfection

OBJECTIVETo explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment.

METHODSTo tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus. The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by (125)I uptake assay, (125)I influx-course and (125)I-efflux-course. A glioma model was established with inoculation of the U251 cells in nude mice, and the inhibiting effect of (131)I on the tumor growth was tested in the mouse models.

RESULTSThe hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus. The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene. The uptake of (125)I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells. The tumor volume of the mice after (131)I treatment was significantly decreased in comparison with that before treatment.

CONCLUSIONRadioactive (131)I treatment after HNIS-based gene transfer can be enhanced and effectively inhibit the tumor growth in nude mice.

Adenoviridae ; genetics ; Animals ; Brain Neoplasms ; pathology ; therapy ; Genetic Therapy ; Glioma ; pathology ; therapy ; Humans ; In Vitro Techniques ; Iodides ; Iodine Radioisotopes ; metabolism ; therapeutic use ; Mice, Nude ; Symporters ; genetics ; Transfection

OBJECTIVETo study changes in the expression levels of parvalbumin (PV), glutamate decarboxylase 67 (GAD67) and K+-Cl- cotransporter 2 (KCC2) in the brain tissue of rats with schizophrenia (SZ) induced by dizocilpine (MK-801), and to investigate the mechanism involving gamma-aminobutyric acid (GABA) by which NMDA receptor blocker induces SZ in the perinatal period.

METHODSThirty-six neonatal male Sprague-Dawley rats were randomly assigned to two batches on postnatal day 6. Each batch was divided into normal control (treated by 0.9% normal saline), SZ-development model (treated by subcutaneous injection of 0.1 mg/kg MK-801 on postnatal days 7-10; bid), and SZ-chronic medication model groups (treated by intraperitoneal injection of 0.2 mg/kg MK-801 on postnatal days 47-60; qd). On postnatal day 63, the brain tissue of the first batch of rats was obtained and then fixed with paraform for histological sections; expression levels of PV and GAD67 in the medial prefrontal cortex (mPFC) and hippocampus CA1 were measured by immunohistochemistry. Simultaneously, the second batch of rats was sacrificed and the mPFC and hippocampus were obtained and homogenized; expression levels of KCC2 in the mPFC and hippocampus were measured by Western blot.

RESULTSExpression levels of PV and GAD67 in the mPFC and hippocampus CA1 were significantly lower in the SZ-development and chronic medication model groups than in the normal control group (P<0.05). Expression levels of KCC2 in the mPFC and hippocampus were significantly lower in the SZ-development model group than in the SZ-chronic medication model and normal control groups (P<0.05).

CONCLUSIONSThe expression changes of PV and GAD67 in SZ can be simulated using the SZ development model induced by MK-801, which might affect the development of the GABA system in the PFC and hippocampus by downregulating KCC2 expression.

Animals ; CA1 Region, Hippocampal ; chemistry ; Dizocilpine Maleate ; pharmacology ; Glutamate Decarboxylase ; analysis ; Immunohistochemistry ; Male ; Parvalbumins ; analysis ; Prefrontal Cortex ; chemistry ; Rats ; Rats, Sprague-Dawley ; Schizophrenia ; etiology ; metabolism ; Symporters ; analysis

The present study was done to determine whether endogenous nitric oxide (NO) plays a role in the regulation of sodium transporters in the kidney. Male Sprague-Dawley rats were treated with NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/L drinking water) for 4 weeks. Control rats were supplied with tap water without drugs. Expression of Na, K-ATPase, type 3 Na/H exchanger (NHE3), Na/K/2Cl cotransporter (BSC1), and thiazide-sensitive Na/Cl cotransporter (TSC) proteins was determined in the kidney by Western blot analysis. Catalytic activity of Na,K-ATPase was also determined. The treatment with L-NAME significantly and steadily increased the systemic blood pressure. Total and fractional excretion of urinary sodium decreased significantly, while creatinine clearance remained unaltered. Neither plasma renin activity nor aldosterone concentration was significantly altered. The alpha1 subunit expression and the catalytic activity of Na, K-ATPase were increased in the kidney. The expression of NHE3, BSC1 and TSC was also increased significantly. These results suggest that endogenously-derived NO exerts a tonic inhibitory effect on the expression of sodium transporters, including Na, K-ATPase, NHE3, BSC1, and TSC, in the kidney.
Animals ; Blotting, Western ; Carrier Proteins/*biosynthesis ; Enzyme Inhibitors/pharmacology ; Kidney/drug effects/metabolism ; Male ; NG-Nitroarginine Methyl Ester/*pharmacology ; Na(+)-K(+)-Exchanging ATPase/biosynthesis ; Nitric Oxide Synthase/*antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Drug/biosynthesis ; Sodium/*metabolism ; Sodium Chloride Symporters/biosynthesis ; Sodium-Hydrogen Antiporter/biosynthesis ; Sodium-Potassium-Chloride Symporters/biosynthesis