[Complications of video-assisted thoracoscopic surgery].

We introduced video-assisted thoracoscopic surgery (VATS) for chest disorders in our institution in March, 1992. At first, many of the subjects' disorders were non-malignant diseases such as spontaneous pneumothorax, but later we started to perform this procedure for lung cancer and mediastinum neoplasm, with improved result over thoracoscopic surgical procedures. Now most of the chest disorders at our institution are treated with VATS. However, many kinds of complications due to manual techniques and instrument troubles surfaced during this period. Therefore, in this article we would like to describe the complications that we have experienced in our institution using VATS and discuss how we have attempted to deal with these complications.

Comparison of iridial pigmentation between latanoprost and isopropyl unoprostone: a long term prospective comparative study.

AIM: To compare incidence of iridial pigmentation prospectively induced by long term treatment with latanoprost and isopropyl unoprostone (hereafter, unoprostone) in Japanese patients with glaucoma. METHODS: Patients with glaucoma treated with prostaglandin (PG) related ophthalmic solutions were sequentially enrolled. Patients treated for more than 30 months with PG related ophthalmic solutions were subjected to analysis. The entry criteria were no history of intraocular surgery, laser iridotomy, and/or laser trabeculoplasty within 12 months before and after the enrolment; and no history of uveitis; no changes in antiglaucoma drugs within 6 months before and after the enrolment. Photographs of the irides were taken under the same conditions and three glaucoma specialists evaluated the iridial pigmentation with masking of patient information. The correlation of iridial pigmentation with the background factors and the reduction of intraocular pressure (IOP) before and after the treatment were investigated. RESULTS: 48 eyes in 48 patients satisfied the enrolment criteria (25 eyes in the latanoprost group, 23 eyes in the unoprostone group). At the end of the follow up period, iridial pigmentation was present in 15 patients (60.0%) in the latanoprost group and seven patients (30.4%) in the unoprostone group. The correlation between development of iridial pigmentation and age, sex, concurrent use of other ophthalmic solutions, and IOP reduction was not significant. CONCLUSIONS: The incidence of iridial pigmentation induced by latanoprost or unoprostone is high in the case of long term treatment. Iridial pigmentation did not affect PG related ophthalmic solution induced IOP reduction.

Iridial pigmentation induced by latanoprost ophthalmic solution in Japanese glaucoma patients.

PURPOSE: To investigate the incidence of iridial pigmentation induced by latanoprost ophthalmic solution in Japanese glaucoma patients by a prospective and observer-masked study. PATIENTS AND METHODS: Sixty-nine eyes of 69 glaucoma patients were included. Patients who had undergone intraocular surgery, laser trabeculoplasty, and laser iridotomy within 12 months before enrollment, and patients with history of uveitis and any changes in antiglaucoma drugs within 6 months before enrollment were excluded. Iridial photographs were taken by one examiner under the same conditions at 1, 3, and 6 months after the initiation of latanoprost treatment. Three glaucoma specialists, masked of patient information, independently assessed the iridial pigmentation. Cases with iridial pigmentation diagnosed by three specialists were categorized as showing a definite increase in iridial pigmentation. RESULTS: A definite increase in iridial pigmentation occurred in 3.5%, 9.7%, and 35.0% of eyes within 1, 3, and 6 months of treatment, respectively. Age, gender, or concomitantly used eyedrops did not significantly influence the incidence of iridial pigmentation within 6 months of instillation. A reduction of intraocular pressure by latanoprost did not differ significantly between patients with and without iridial pigmentation. CONCLUSION: The incidence of iridial pigmentation by latanoprost ophthalmic solution in Japanese patients was higher than previously reported values in pigmented races.

Cystoid macular edema associated with topical latanoprost in glaucomatous eyes with a normally functioning blood-ocular barrier.

PURPOSE: To study prospectively using optical coherence tomography (OCT) whether topical latanoprost induces retinal disorders, such as cystoid macular edema, in patients with glaucoma and a normally functioning blood-ocular barrier. METHODS: Sixty-eight eyes of 38 patients with glaucoma and no history of intraocular surgery, uveitis, or laser trabeculoplasty were studied. Before initiation of latanoprost treatment and after 1, 3, and 6 months of treatment, OCT images were taken, and the following tests were performed: visual acuity examination, fundus ophthalmoscopy, intraocular pressure measurement, and fundus color photography. To evaluate retinal thickness in the fovea accurately. OCT scanning was repeated six times, and the smallest value was used as the retinal thickness in the fovea. RESULTS: Latanoprost ophthalmic solution did not influence retinal thickness in the fovea at any investigated time points compared with the time before instillation, and no changes were observed in visual acuity, ophthalmoscopic findings, and fundus photographs. The intraocular pressure was reduced significantly at all investigated time points compared with the time before instillation. CONCLUSIONS: It is unlikely that topical latanoprost induces retinal disorders, such as cystoid macular edema, in glaucomatous eyes with a normally functioning blood-ocular barrier.

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida.

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.

Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress.

Expression of the MRP1 gene encoding the GS-X pump and of the gamma-GCSh gene encoding the heavy (catalytic) subunit of the gamma-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like gamma-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1, 4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably gamma-GCSh-transfected cell lines down-regulated endogenous MRP1 and gamma-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and gamma-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and gamma-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and gamma-GCSh expression.

Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues.

Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples. Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump. We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996). In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA. Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861. In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed. Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities. Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

Application of synchrotron radiation to ultrafast spectroscopy.

A novel application of synchrotron radiation to ultrafast optical spectroscopy is demonstrated. The application is based on the short coherence time of broadband synchrotron radiation and employs a conventional interferometer. From a detailed study of the coherence of synchrotron radiation, it is shown that the coherent interference between two synchrotron radiation beams, split from a single beam, can provide ultimate time resolution down to a few femtoseconds. Experimental results of ultrafast spectroscopy using broadband synchrotron radiation are presented; these include free-induction decay and photon echoes in the visible and ultraviolet regions.

Requirement for two conserved cysteine residues in the Ada protein of Escherichia coli for transactivation of the ada promoter.

Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids. Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E. coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity. Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent. This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter. Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents. An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine. This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter. Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter. We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.

Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.

Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis.

8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues. Human cells contain enzyme activity, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA. We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence. We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence. The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity. When the human cDNA was expressed in E. coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity. In such cells, the frequency of spontaneous mutation was greatly reduced. We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals.

Regulatory elements for expression of the alkA gene in response to alkylating agents.

Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base changes within this sequence led to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promoter. There are considerable differences between the mechanisms of action of Ada protein on the promoters of alkA and ada, even though the expression of both genes is positively regulated by Ada protein.

An altered DNA sequence encompassing the ras gene of Harvey murine sarcoma virus.

The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and assigned the coding region of a transforming protein, p21, to the sequence. Examination of nucleotide sequence, taken together with the result of analysis of the ras mRNAs (1), has revealed that p21 is encoded from a continuous coding region starting with the 5' proximal initiation codon but not a processed protein. However, there were found several differences between the sequence published by Dhar et. al. (2) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacement of glycine by alanine at position 122 of p21. The evidences are presented with some of actual gel autoradiographs.

The specific, diverse effects of purine and pyrimidine nucleoside-5'-di(tri)-3'-diphosphates on the eucaryote translation system in vitro.

All the eight 5'-di(tri)-3'-diphosphates of four common ribonucleosides were prepared by the enzymic pyrophosphoryl transfer catalysed by Streptomyces adephospholyticus ATP:nucleotide pyrophosphokinase (E.C.2.7.6.4) form dATP to the respective 5'-phosphates, and their effects on the translation of mRNAs by a wheat germ system in vitro were studied. (p) ppPupp decreased the total 14C-leucine incorporation directed by a rat liver mRNA whereas (p) ppPypp did not. With a silkworm pupa ovary mRNA, distinctly reverase results were obtained. Gel electrophoretic profiles of the translation products disclosed the mRNA species-specific stimulatory or inhibitory effects for each of the polyphosphates tested.