To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism

AIMTo investigate the antagonism of tetrandrine in acute renal injury induced by gentamicin in guinea pig.

METHODSThe experimental guinea pigs are divided into 4 groups by randomized blocks, they were group 1 (control), group 2 (tetrandrine), group 3 (gentamicin) and group 4 (gentamicin with tetrandrine). After 10 days, set apart urinary specimen for determination the activity of NAG, reserve renal specimen for observing the renal histology and measuring expression of actin and TGF-beta1.

RESULTSThe renal histology showed that the injury of group 3 was the most grievous, group 4 was lighter than group 3. In addition, the apoptosis was obviously less than group 3. The activity of urine of NAG in group 4 was lower than group 3. The levels of expression of actin and TGF-beta1 in group 4 were more than group 3.

CONCLUSIONWhen kidney is damaged by gentamicin, tetrandrine is able to reduce the activity of urinary NAG and inhibit the renal tubular epithelial cell apoptosis. Besides, tetrandrine can prevent breaking actin and evoke the endogenous transmission growth factor beta1 tune-up, the above results can indicate that tetrandrine has antagonism to acute renal injury induced by gentamicin in guinea pig.

Acetylglucosaminidase ; urine ; Actins ; metabolism ; Acute Kidney Injury ; chemically induced ; Animals ; Benzylisoquinolines ; pharmacology ; Drug Antagonism ; Drugs, Chinese Herbal ; pharmacology ; Gentamicins ; adverse effects ; Guinea Pigs ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVE: To learn the influence the gentamycin on C57BL/6J mice hear and cochlear hair cell ribbon synapses CaV1.3 calcium protein amount. To explore the relationship between hear loss and its dosage correlation change and significance. METHOD: The fixed amino glucoside to C57BL/6J mice was used to make abdominal cavity injection mold every day. The auditory brain-stem response ABR was used to measure the hear of mice in 7th, 14th, 28th after the injection. Immunofluorescence method was used to observe cochlear basement membrane of hair ribbon synapse CaV1.3 calcium channel proteins in the distribution and expression. Inner hair cells synaptic membrane was immune fluorescent tags with CtbP2 and CaV1. 3. RESULT: With the growth of the injected drugs, ABR threshold increased,but all the hair cells and shape had no obvious change. However the amount of hair rib bon synapse CaV1.3 calcium ion channel proteins in the expression had significant differences (P < 0.01). CaV1.3 calcium ion channel proteins increased slightly lower than normal at 7th day, significantly decreased at 14th day, had increased, increased quantity compare with 14th day, but at 28th day after intraperitoneal injection of gentamicin. CONCLUSION The increasing,decreasing and increasing trend of cochlear hair cells CaV1.3 proteins in the environment of amino glucoside drug toxicity showed that the increase of hair ribbon synapse CaV1.3 proteins may have a compensatory effect on the drug toxicity. With the increase of the drug toxicity effect, this kind of decompensated function could be the listening decline, which may be one of the mechanism of damage to hearing.
Animals ; Calcium Channels, L-Type ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; pharmacology ; Hair Cells, Auditory, Inner ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Proteomics

Though the pathogenic significance and the reservoir of Ewingella americana have not been clarified, this organism has caused several pathogenic infections, especially in immunocompromised patients. We report a pneumonia in a patient with chronic renal failure, who had chronic rejection of transplanted kidney. E. americana was identified to be the pathogen of pneumonia with clinical symptoms and signs and radiological examination. As soon as he was treated with ceftriaxone and isepamicin, clinical improvement was followed with no further growth of E. americana or other pathogenic isolates from sputum culture. This suggests to be the case of pneumonia caused by E. americana for the first time in the Korean literature.
Adult ; Anti-Bacterial Agents/pharmacology ; Ceftriaxone/pharmacology ; Enterobacteriaceae/*metabolism ; Enterobacteriaceae Infections/*complications ; Gentamicins/pharmacology ; Humans ; Kidney Failure, Chronic/*complications/*microbiology ; Kidney Transplantation ; Male ; Pneumonia/complications/*microbiology ; Sputum/metabolism ; Time Factors

AIMTo study the antagonistic action of acanthopanax senticosus injection (ASS) on gentamicin ototoxicity.

METHODSGuinea pigs were randomly divided into control group, GM group, ASS group, and ASS + GM group. The changes of hearing threshold, cochlear morphology, expression of caspases-3 were determined by ABR, TEM, and Western blot, respectively.

RESULTSThe ABR threshold in GM group increased markedly. There was no significant difference in ABR threshold between ASS group and control group, but the ABR threshold in ASS group was much lower than that both in GM group and ASS + GM group. Severely injured hair cells with morphological characteristics of apoptosis were found under TEM in GM group, and the hair cells were less injured in ASS + GM group. The results of Western blot showed that the expression of caspase-3 increased markedly in GM group, but it increased slightly in ASS + GM group.

CONCLUSIONASS may antagonize the GM ototoxicity by inhibiting the expression of caspases-3.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Eleutherococcus ; Gentamicins ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; cytology ; drug effects ; Plant Extracts ; pharmacology

OBJECTIVETo study the protective effect of peperphentonamine hydrochloride (PPTA) against gentamicin-induced cochlear damage and its mechanism to inhibit cell apoptosis.

METHODSGuinea pigs with normal hearing were randomized into control, gentamicin, and PPTA treatment groups, and the guinea pigs models of gentamicin-induced cochlear damage received intraperitoneal injection of PPTA. The changes of hearing of the guinea pigs were evaluated with auditory brainstem response (ABR) test, and the protein expression of caspase-3 in the cochlear tissue was detected using Western blotting. TUNEL staining, scanning and transmission electron microscopy were performed to observe the morphological changes of the cochlea.

RESULTSThe threshold in ABR in PPTA treatment group was significantly higher than that in the control group (P<0.05) but significantly lower than that in gentamicin group. Western blotting showed a significantly increased caspase-3 expression in gentamicin group (P<0.001); caspase-3 expression in PPTA group was obviously higher than that in the control group but much lower than that in gentamicin group (P<0.001). TUNEL assay and electron microscopy revealed serious damages of the hair cells in gentamicin group with numerous apoptotic cells in the organ of Corti, stria vascularis and spiral ganglion, and such cochlear damages were obviously alleviated in PPTA group.

CONCLUSIONPPTA can protect against gentamicin-induced cochlear damage in guinea pigs by decreasing the protein expression of caspase-3 to inhibit cell apoptosis.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cochlea ; drug effects ; pathology ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; adverse effects ; Guinea Pigs

OBJECTIVETo explore the relationship of gentamicin-induced cochlear damage with autophagy-related protein LC3, beclin1, Na(+-)K(+-)2Cl(-) cotransporter (NKCC1) mRNA and endothelin-1 (ET-1), and investigate the protective mechanism of PPTA against gentamicin-induced cochlear damage.

METHODSSixty guinea pigs were randomly divided into control group (with saline and artificial perilymph injections), model group (with gentamicin and artificial perilymph injections), concurrent treatment group (with gentamicin and PPTA injections), model control group (with artificial perilymph injection 7 days after gentamicin injection) and delayed treatment group (with PPTA injection 7 days after gentamicin injection). Saline and gentamicin (160 mg/kg) were injected intraperitoneally, and artificial perilymph and PPTA were injected into the otocysts on a daily basis for 7 consecutive days. Hearing impairment of the guinea pigs was analyzed with ABR, and the protein expressions of beclin1 and LC3 in cochlear tissue were tested. The expression of NKCC1 mRNA was detected with RT-PCR, and the expression of ET-1 was detected immunohistochemically.

RESULTSThe ABR thresholds in the model group and model control group were similar (P>0.05) , but significantly higher than those in the other 3 groups (P<0.05); the threshold was significantly lower in concurrent treatment group than in delayed treatment group (P<0.05). Compared with those in the other 4 groups, the expressions of LC3 II, beclin1, and NKCC1 mRNA were significantly increased in the model group (P<0.05); and those in delayed treatment group were significantly lower than those in the model control group (P<0.05). The expressions of ET-1 in the Corti organ, striavascularis and spiral ganglion were significantly higher in the model group but significantly lower in the control group than those in the other 4 groups; ET-1 expression was significantly lower in delayed treatment group than in the model control group.

CONCLUSIONPPTA offers protection against gantamicin-induced cochlear damage in guinea pigs by inhibiting cell autophagy and suppressing of NKCC1 and ET-1 expressions. Early intervention with PPTA produces better therapeutic effect, suggesting that gantamicin causes irreversible injury of the auditory cells.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; metabolism ; Beclin-1 ; Cochlea ; drug effects ; Endothelin-1 ; metabolism ; Gentamicins ; adverse effects ; Guinea Pigs ; Hearing Loss ; chemically induced ; prevention & control ; Microtubule-Associated Proteins ; metabolism ; Solute Carrier Family 12, Member 2 ; metabolism

OBJECTIVETo study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16(INK4a) plays a role in tumor suppression.

METHODSA conditional targeting vector with 2.0 kb EcoR I/Xba I fragment as short arm and 5.9 kb SpeI/NotI fragment as long arm was built. Of the 2 direct locus crossing- over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16(INK4a) exon 1a and the other at 1633 bp downstream of the initiate code. Both exon 1a and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, t he targeting vector was introduced into ES cells through electroporation.

RESULTSTwenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization.

CONCLUSIONTargeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.

Animals ; Anti-Bacterial Agents ; pharmacology ; Antiviral Agents ; pharmacology ; Base Sequence ; Cell Division ; drug effects ; genetics ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Drug Resistance ; genetics ; Embryo, Mammalian ; cytology ; drug effects ; metabolism ; Exons ; genetics ; Ganciclovir ; pharmacology ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Mice ; Molecular Sequence Data ; Recombination, Genetic ; Stem Cells ; cytology ; drug effects ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection

To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N1 to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.
Antineoplastic Agents ; pharmacology ; Base Sequence ; Dose-Response Relationship, Drug ; Drug Screening Assays, Antitumor ; methods ; Enhancer Elements, Genetic ; genetics ; Gene Expression ; drug effects ; Gentamicins ; pharmacology ; Green Fluorescent Proteins ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Lentinan ; pharmacology ; Microscopy, Fluorescence ; Molecular Sequence Data ; Oligonucleotides ; genetics ; Proline ; analogs & derivatives ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thiocarbamates ; pharmacology ; Transfection

PURPOSE: Since November 2006, imipenem-resistant Acinetobacter baumannii isolates have increased in Kyung Hee University Hospital in Seoul, Korea. The purpose of this study was to determine the genetic basis and molecular epidemiology of outbreak isolates. MATERIALS AND METHODS: Forty-nine non-repetitive isolates of the 734 IRAB strains were investigated in order to determine their characteristics. The modified Hodge and the ethylenediaminetetraacetic acid (EDTA)-disk synergy test were performed for the screening of carbapenemase and metallo-beta-lactamase production. Multiplex polymerase chain reaction (PCR) assays were performed for the detection of genes encoding for OXA-23-like, OXA-24-like, OXA-58-like and OXA-51-like carbapenemase. Pulsed-field gel electrophoresis (PFGE) was performed for strain identification. RESULTS: All isolates showed 100% resistance to ciprofloxacin and gentamicin, 97.9% resistance to cefepime, piperacillin/tazobactam, aztreonam, ceftazidime and piperacillin, 93.9% resistance to tobramycin and 57.1% resistance to amikacin. All of the 49 isolates (100%) showed positive results in the modified Hodge test and negative results in the EDTA-disk synergy test. They all (100%) possessed the encoding gene for an intrinsic OXA-51-like carbapenemase and an acquired OXA-23-like carbapenemase in the multiplex PCR assay. PFGE patterns revealed that all isolates were clonally related from A1 to A14. CONCLUSION: It is concluded that all of the 49 IRAB isolates acquired resistance to imipenem by producing OXA-23 carbapenemase and they might have originated from a common source.
Acinetobacter Infections/epidemiology/*microbiology ; Acinetobacter baumannii/*drug effects/genetics ; Anti-Bacterial Agents/*pharmacology ; Cephalosporins/pharmacology ; Ciprofloxacin/pharmacology ; Disease Outbreaks ; Drug Resistance, Multiple, Bacterial/genetics/physiology ; Electrophoresis, Gel, Pulsed-Field ; Gentamicins/pharmacology ; Humans ; Imipenem/*pharmacology ; Korea/epidemiology ; Microbial Sensitivity Tests ; beta-Lactamases/genetics/*metabolism ; beta-Lactams/*pharmacology