Transgenic mice expressing Cre-recombinase specifically in M- or S-cone photoreceptors

PURPOSE: To establish lines of transgenic mice that express Cre-recombinase in M- or S-cone photoreceptors for generating cone photoreceptor-specific (conditional) mutants. METHODS: Five kilobases of 5' upstream sequence of the mouse red-green (M) opsin gene or 0.5 kb of the mouse blue (S) opsin gene was cloned into a Cre-expression plasmid. Transgenic mice were generated and characterized, and appropriate lines were established. The Cre-transgenic mice were crossed with ROSA26-lacZ mice (containing floxed beta-galactosidase gene) and analyzed to determine Cre-recombinase activity. RESULTS: Immunofluorescence study showed successful targeting of Cre-recombinase expression to cone photoreceptors. Double staining with anti-Cre antibody and anti-M- or anti-S-opsin antibody revealed specificity of Cre expression in M-opsin- and/or S-opsin-positive photoreceptors. Mating with ROSA26-lacZ mice demonstrated that Cre-recombinase was functionally active in M- or S-cones. CONCLUSIONS: Lines of transgenic mice that specifically express functional Cre-recombinase in M- or S-cones were established in this study. Because mutations in several widely expressed genes lead to photoreceptor degeneration, these transgenic mice should be valuable in generating conditional mutants to investigate the function of various genes specifically in cone photoreceptors.     

SsrA-mediated protein tagging in the presence of miscoding drugs and its physiological role in Escherichia coli

BACKGROUND: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging. RESULTS: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3' end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells. CONCLUSION: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.     

Behavior of HO-1-N-1, buccal mucosa carcinoma derived cells,on [Ca2+]i responses to stimulants

Buccal mucosa carcinoma-derived cell line, HO-1-N-1, epithelial-like cells, was obtained in order to investigate the characteristics of oral cancer cells and examine the [Ca2+]i responses to stimulants, such as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha ). Intracellular Ca2+ influx was observed by all stimulants that enhanced the [Ca2+]i response. However, intracellular Ca2+ release was not observed in response to growth factors. The [Ca2+]i response of BK (100 nM) was inhibited by 10 micro M of the BKB2 antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK, and HIST (1 mM) was completely inhibited by 100 nM of the H1 antagonist, (+)-chlorpheniramine, in the presence and absence of extracellular Ca2+ (1.5 mM).     

Symptomatic de novo arteriovenous malformation appearing 17 years after the resection of two other arteriovenous malformations in childhood: case report

OBJECTIVE AND IMPORTANCE: This report describes the first case of symptomatic de novo arteriovenous malformation (AVM) appearing ectopically after total resection of other AVMs. We discuss the growth phenomenon and the nature of AVMs. CLINICAL PRESENTATION: A 27-year-old woman with sudden headache and right-sided numbness was admitted to our hospital. Computed tomographic scans revealed a hemorrhage of the corpus callosum and the bilateral lateral ventricles. A cerebral angiogram demonstrated an AVM that was fed by the bilateral pericallosal arteries and drained into the inferior sagittal sinus. Seventeen years earlier, at the age of 10 years, the patient had undergone resection of two other AVMs. At that time, the newly presented AVM was not detected. This AVM had grown markedly and caused hemorrhage after 17 years. INTERVENTION: The AVM, which was located in the bilateral cingulate gyrus and the corpus callosum, was totally removed through a right frontal craniotomy. The patient was discharged without neurological deficits. CONCLUSION: Our findings suggest that patients who undergo complete resection of AVMs may sustain other de novo AVMs some years later. The growth of an AVM seems to be related to the patient's age at onset and the duration of the posttreatment period. We emphasize the importance of long-term follow-up in patients with cerebral AVMs treated during childhood.     

Trigeminal neuralgia: evaluation of neuralgic manifestation and site of neurovascular compression with 3D CISS MR imaging and MR angiography

PURPOSE: To evaluate three-dimensional (3D) constructive interference in steady-state (CISS) magnetic resonance (MR) imaging and MR angiography with multiplanar reconstruction (MPR) for detection of neurovascular compression (NVC) in patients with trigeminal neuralgia and to evaluate the relationship between clinical symptoms related to trigeminal branches and those related to the site of trigeminal nerve compression. MATERIALS AND METHODS: Fifty-four consecutive patients with trigeminal neuralgia were examined at 3D CISS imaging and MR angiography with a 1.5-T MR system. Original transverse and four reformatted images were used for image interpretation. Vascular contact with the trigeminal nerve at the root entry zone (REZ) was determined, and the nature of the involved vessels was identified. The position of the blood vessel compressing the nerve was classified into cranial, caudal, medial, or lateral sites. Statistical analysis was performed with the chi2 test or the Fisher exact test between two groups and with the chi2 test among more than two groups. RESULTS: In 12 of 15 patients who underwent surgery, the artery that was considered a responsible vessel at 3D CISS imaging and MR angiography was confirmed as such. In the other three patients, the vein was the responsible vessel, which was detected only at 3D CISS imaging. Sixteen (89%) of 18 patients with symptoms related to the maxillary division had NVC at the medial site of the REZ, while 16 (76%) of 21 patients with symptoms related to the mandibular division had NVC at the lateral site (P <.001, chi2 test). CONCLUSION: 3D CISS MR imaging with MPR is useful in the detection of NVC in patients with trigeminal neuralgia, compared with MR angiography. A close relationship was found between the region of neuralgic manifestation and the site of trigeminal nerve compression.     

CYP isoforms involved in the metabolism of clarithromycin in vitro: comparison between the identification from disappearance rate and that from formation rate of metabolites

To clarify whether CYP2C19 is involved in the overall metabolism of clarithromycin (CAM) or not, in vitro studies using human liver microsomes and recombinant CYPs were performed by an approach based on the disappearance rate of parent compound from the incubation mixture. In addition, the results of disappearance rate were compared with those obtained from the formation rates of the major metabolites of CAM, 14-(R)-hydroxy-CAM and N-demethyl-CAM.The intrinsic clearance (CL(int)) values determined from the disappearance of CAM in nine different human liver microsomes were highly correlated with the testosterone 6beta-hydroxylation activity (r=0.957, p<0.001). The CL(int) of CAM was markedly reduced by selective inhibitors of CYP3A4 (ketoconazole and troleandomycin) and by polyclonal antibodies raised against CYP3A4/5 in human liver microsomes. Among the 11 isoforms of recombinant human CYP, only CYP3A4 revealed the metabolic activity for the disappearance of CAM. These results were fairly consistent with those obtained from the conventional approach based on the formation of major metabolites of CAM. Comparison of the kinetic parameters estimated from the disappearance rate of CAM and the formation rates of 14-(R)-hydroxy-CAM and N-demethyl-CAM indicates that N-demethylation and 14-(R)-hydroxylation account for 65% of CL(int) derived from the disappearance of CAM in human liver microsomes.The findings suggest that CYP3A4 plays a predominant role in the overall metabolic clearance of CAM as well as in the formation of 14-(R)-hydroxy-CAM and N-demethyl-CAM. CYP2C19 does not appear to be involved in the overall metabolism of CAM at least in human liver microsomes. A combination of the disappearance rate of a parent compound and the formation rate of metabolites appears to be a useful approach for estimating the percentage contribution of the formation of metabolites to the overall metabolic clearance of a parent compound in vitro.     

Gelatin particle indirect agglutination and enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis using Strongyloides venezuelensis antigen

Routine microscopical examination of stool specimens for diagnosis of strongyloidiasis is insensitive and serological methods using Strongyloides stercoralis antigen are at present not available for field studies. We evaluated 2 techniques, enzyme-linked immunosorbent assay (ELISA) and gelatin particle indirect agglutination (GPIA), using an antigen obtained from the rodent parasite, S. venezuelensis. Fifty-four Peruvian patients with different clinical forms of strongyloidiasis were studied: 12 asymptomatic, 31 symptomatic, and 11 hyperinfection cases. Our results demonstrate that both ELISA and GPIA using S. venezuelensis antigen are useful for diagnosis of strongyloidiasis, with sensitivities of 74.1% and 98.2%, respectively and a specificity of 100% for both techniques. We found that GPIA is a highly sensitive test for patients with suspected chronic infection and/or hyperinfection. In the hyperinfection cases, significantly lower concentrations of specific immunoglobulin antibodies and eosinophils (P < 0.001) were found compared with the asymptomatic and symptomatic cases.     

Caffeine diminishes cytotoxic effects of paclitaxel on a human lung adenocarcinoma cell line

This study was performed to investigate how caffeine modifies the cytotoxic effects of paclitaxel on a human lung carcinoma cell line. Caffeine doses up to 5mM had less effect on clonogenic survival. The cell killing effect, due to paclitaxel, increased in a dose-dependent manner up to 50 nM. For combined treatment with caffeine and paclitaxel, added caffeine reduced the cytotoxic effect of paclitaxel not only in dose-response but also in time-response curves. Caffeine combined with paclitaxel clearly suppressed cell proliferation in a dose-dependent manner. In the cell cycle analysis, caffeine alone caused early G1 accumulation, whereas paclitaxel alone caused an early increase in G2-M and a decrease in G1. As for the effect of caffeine on paclitaxel, caffeine suppressed the effect of paclitaxel on cell cycle distribution, where a dose-dependent early increase in G2-M and a decrease in G1 were not clear. We suggest that cell cycle modifying agents, such as caffeine, potentially diminish the cytotoxic activity of paclitaxel, and one should be careful when combining such agents.     

Validity of two-hour continuous hepatic arterial infusion chemotherapy with low-dose 5-FU for unresectable liver metastasis from colorectal cancer

The significance of hepatic arterial infusion chemotherapy for unresectable liver metastases from colorectal cancer was evaluated in 50 patients, who received either of the following regimens: 1 shot 5-FU + epirubicin + MMC (FAM group); hepatic arterial infusion of 5-FU for 2 hours + MMC (MF group); hepatic arterial infusion of 5-FU for 2 hours (5-FU group). There were no differences in the clinicopathological backgrounds of the patients among the groups. The mean survival time was 10.3 months, 16.0 months and 16.2 months in the FAM, MF and 5-FU groups. The effective percentages were 0%, 40% and 31% in the FAM, MF and 5-FU groups and the survival time of the effective cases was 18.1 months and 21.8 months in the MF and 5-FU groups. The MF group and 5-FU group showed significant improvement in prognosis. Concerning side effects, myelo-suppression and gastrointestinal toxicity appeared frequently in the MF group. In conclusion, 2-hour continuous hepatic arterial infusion with low-dose 5-FU for unresectable liver metastases from colorectal cancer may be helpful for improvement of prognosis.