Distribution of protocadherin 9 protein in the developing mouse nervous system.

Protocadherin 9 (Pcdh9) is a member of the protocadherin family, which includes many members involved in various phenomena, such as cell-cell adhesion, neural projection, and synapse formation. Here, we identified Pcdh9 protein in the mouse brain and examined its distribution during neural development. Pcdh9, with a molecular weight of approximately 180 kDa, was localized at cell-cell contact sites in COS-1 cells transfected with Pcdh9 cDNA. In cultured neurons, it was detected at the growth cone and at adhesion sites along neurites. In the E13.5 brain, prominent Pcdh9 immunoreactivity was detected in the dorsal thalamus along with other regions including the vestibulocochlear nerve. As development proceeded (E15.5-P1), Pcdh9 immunoreactivity became observable in various brain regions but was restricted to certain fiber tracts and brain nuclei. Interestingly, many Pcdh9-positive brain nuclei and fascicles belonged to the vestibular (e.g. vestibulocochlear nerve, vestibular nuclei, and the vestibulocerebellum) and oculomotor systems (medial longitudinal fascicles, oculomotor nucleus, trochlear nucleus, and interstitial nucleus of Cajal). In addition, we examined the distribution of Pcdh9 protein in the olfactory bulb, retina, spinal cord, and dorsal root ganglion. In these regions, Pcdh9 and OL-protocadherin proteins were differentially distributed, with the difference highlighted in the olfactory bulb, where they were enriched in different subsets of glomeruli. In the mature retina, Pcdh9 immunoreactivity was detected in distinct sublaminae of the inner and outer plexiform layers. In the dorsal root ganglion, only certain subsets of neurons showed Pcdh9 immunoreactivity. These results suggest that Pcdh9 might be involved in formation of specific neural circuits during neural development.

A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.

Retrospective analysis has shown that activating mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene are a predictor of response to gefitinib. We conducted a phase II trial to evaluate the efficacy and safety of gefitinib as first-line therapy for advanced non-small cell lung cancer (NSCLC) with EGFR mutations. Patients with stage IIIB or IV chemotherapy-naïve NSCLC with EGFR mutation were treated with 250 mg gefitinib daily. For mutational analysis, DNA was extracted from paraffin-embedded tissues and EGFR mutations were analysed by direct sequence of PCR products. Twenty (24%) of the 82 patients analysed had EGFR mutations (deletions in or near E746-A750, n=16; L858R, n=4). Sixteen patients were enrolled and treated with gefitinib. Twelve patients had objective response and response rate was 75% (95% CI, 48-93%). After a median follow-up of 12.7 months (range, 3.1-16.8 months), 10 patients demonstrated disease progression, with median progression-free survival of 8.9 months (95% CI, 6.7-11.1 months). The median overall survival time has not yet been reached. Most of the toxicities were mild. This study showed that gefitinib is very active and well tolerated as first-line therapy for advanced NSCLC with EGFR mutations.

Endobronchial ultrasonography with guide-sheath for peripheral pulmonary lesions.

The usefulness of endobronchial ultrasonography (EBUS) with guide-sheath (GS) as a guide for transbronchial biopsy (TBB) for diagnosing peripheral pulmonary lesions (PPL)s and for improving diagnostic accuracy was evaluated in this study. EBUS-GS-guided TBB was performed in 24 patients with 24 PPLs of < or =30 mm in diameter (average diameter=18.4 mm). A 20-MHz radial-type ultrasound probe, covered with GS was inserted via a working bronchoscope channel and advanced to the PPL in order to produce an EBUS image. The probe with the GS was confirmed to reach the lesion by EBUS imaging and X-ray fluoroscopy. When the lesion was not identified on the EBUS image, the probe was removed and a curette was used to lead the GS to the lesion. After localising the lesion, the probe was removed, and TBB and bronchial brushing were performed via the GS. Nineteen peripheral lesions (79.2%) were visualised by EBUS. All patients whose PPLs were visible on EBUS images subsequently underwent an EBUS-GS-guided diagnostic procedure. A total of 14 lesions (58.3%) were diagnosed. Even when restricted to PPLs <20 mm in diameter, the diagnostic sensitivity was 53%. In conclusion, endobronchial ultrasonography with guide sheath-guided transbronchial biopsy was feasible and effective for diagnosing peripheral pulmonary lesions.

[Clinical study of eight thymic carcinomas].

Eight patients with thymic carcinoma were surgically treated at Sapporo City General Hospital from September 1990 to January 2002. The histological subtypes of thymic carcinoma were squamous cell in 5, undifferentiated in 2, and small cell in one. After median sternotomy, 5 patients had total resection of the tumor, and 3 had incomplete resection due to extensive mediastinal invasion involving the main pulmonary artery in the A-P window. Of 3 cases with intra pericardial malignant effusion, one patient survived 7 years without recurrence of the tumor after the exploratory operation followed by mediastinal irradiation without chemotherapy. In this patient, existence of the malignant cells in the pericardial effusion was not associated with poor outcome.

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells.

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.

Effects of Batatasin III and Its Analogs on Gibberellic Acid-Dependent α-Amylase Induction in Embryoless Barley Seeds and on Cress Growth.

The effects of batatasin III and its analogs on gibberellic acid (GA3)-dependent α-amylase induction in embryoless barley seeds and on cress root-growth were examined. Batatasin III was most effective and caused 68% inhibition of α-amylase induction at 4×10(-4) M, but its potency was low compared with that of abscisic acid. In the cress test, p-hydroxybibenzyl had high activity.

The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage.

The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif. The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein. The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE. Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells. Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources. The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters. In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure. Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities. These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation.

Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. II. Defective DNA repair synthesis by xeroderma pigmentosum cell extracts.

We have constructed a cell-free DNA repair system with UV-irradiated SV40 minichromosomes, as described in the accompanying paper (Sugasawa, K, Masutani, C., and Hanaoka, F. (1993) J. Biol. Chem 268, 9098-9104). In this study, we examined DNA repair synthesis by cell extracts from seven xeroderma pigmentosum (XP) complementation groups, A through G. DNA repair synthesis by XP cell extracts was lower than that with repair-proficient human 293 cell extract and did not increase to the level with the latter on increase in the amount of cell extract or the incubation time. The defects of XP cell extracts were complemented by addition of extracts from cells of different complementation groups, indicating that defective proteins in XP-A through G cells are directly involved in DNA repair. Addition of T4 endonuclease V, which is reported to complement defects of XP cells, stimulated DNA repair synthesis by the 293 cell extract, and also complemented the defects of all XP cell extracts. The XPAC gene product was shown to be involved in DNA repair synthesis using anti-xpac serum and xpac protein produced in Escherichia coli. Anti-xpac serum inhibited DNA repair synthesis by the 293 cell extract and xpac protein reversed the inhibition. Furthermore, xpac protein complemented the defects of extracts of two lines of XP-A cells (XP2OSSV and XP12ROSV) but had no effect on the reactions of extracts from cells of other complementation groups. These findings are consistent with previous results obtained in experiments with cells, indicating that our system is useful for analyzing the mechanisms of DNA excision repair in mammalian cells.

Identification and characterization of xpac protein, the gene product of the human XPAC (xeroderma pigmentosum group A complementing) gene.

We have cloned human xeroderma pigmentosum group A complementing (XPAC) cDNA that encodes a "zinc finger" protein with a predicted size of 31 kDa. To detect the xpac protein in cells, we raised antibody against a recombinant human xpac protein. Using this antibody, we identified the xpac protein in the nucleus of cells. In normal human cells, 40- and 38-kDa proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A reduced amount of the smaller protein was detected in XP 39OSSV cells, which show low UV sensitivity, and no xpac proteins were detected in XP 2OSSV cells, which show high UV sensitivity. These levels of xpac proteins in xeroderma pigmentosum cells were determinants of heterogeneity of the DNA repair defect in group A xeroderma pigmentosum. Synthesis of the xpac protein did not increase after UV irradiation.

Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells.

Microinjection of cell extracts prepared from both human placenta and HeLa cells into xeroderma pigmentosum (XP) cells of complementation group A restores unscheduled DNA synthesis (UDS) in these cells after UV irradiation [de Jonge, A., Vermeulen, W., Klein, B. & Hoeijmakers, J. (1983) EMBO J. 2, 637-641]. These cells also showed normal resistance to UV irradiation. The half-life of the factors in the cell extracts corresponding to the UDS activity (factor A) was 14 hr in XP cells of group A, and the maximal level of UDS was exerted 2 hr after microinjection. The factors were sensitive to protease treatment but not to RNase treatment and were found to be approximately equal to 160 and approximately equal to 90 kDa by gel filtration. These two fractions of the factor(s) acted specifically in XP cells of complementation group A among complementation groups A, B, C, D, F, G, and probably E and H.