The tumor suppressor p53 protein is a transcription factor that plays a central role in the cellular response to DNA damage, and it can cause either G1 arrest or apoptosis. Recently, it was shown to induce the tumor suppressor p21Waf1/Cip1/Sdi1 (p21), which inhibits cyclin-CDK complex kinase activity. Although the etiology of idiopathic pulmonary fibrosis (IPF) is still uncertain, it is postulated that IPF begins with an initial inflammatory lesion localized to the alveolus and progresses on to chronic inflammation with alveolitis. We examined whether p53 and p21 are upregulated in association with chronic DNA damage in the bronchial and alveolar epithelial cells in patients with IPF in an attempt to repair the injury. We performed in situ detection of DNA strand breaks or apoptosis (TUNEL) in the tissues as well as immunohistochemistry (IHC) for p53 and p21. Positive signals by TUNEL were detected mainly in the bronchiolar and alveolar epithelial cells in 10 of 14 lung specimens from patients with IPF. On the other hand, no positive signal by TUNEL was detected in normal lung parenchyma or in specimens of pulmonary emphysema. The IHC demonstrated that p53 and p21 were expressed especially in hyperplastic bronchial and alveolar epithelial cells of lung tissues from all patients with IPF, except five specimens for p21. These results are consistent with those obtained by TUNEL. In normal lung parenchyma and specimens of pulmonary emphysema, p53 and p21 were not detected except in scattered alveolar macrophages and in the epithelial cells within localized fibrotic regions. These results suggest that p53 and p21 are upregulated in association with chronic DNA damage, resulting in either G1 arrest or apoptosis so that the DNA damage can be repaired in IPF. We speculate that chronic DNA damage and repair may lead to mutation of the p53 gene and tumorigenesis in IPF.

ADC seems to be a promising tool for monitoring the early response to or predicting prognosis after chemotherapy of NSCLC.

. Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases. Vacuolar Hϩ -pyrophosphatase (V-PPase) 1 belongs to the fourth class of electrogenic proton pump in addition to the P-, F-, and V-type ATPases. The proton pumping reaction couples with the hydrolysis of PP i . V-PPase acidifies vacuoles together with vacuolar H ϩ -ATPase in the plant cell and actively exports protons from the cytosol in the bacterial plasma membrane (1-3). V-PPase has the simplest structure among the proton pumps except for bacteriorhodopsin, a light-driven proton pump. The molecular mass calculated from the cDNA sequences range from 80 to 81 kDa for V-PPases of land plants and algae (for a review, see Refs. 3 and 4), while V-PPases in photosynthetic bacteria Rhodospirillum rubrum (5) and archaebacteria Pyrobaculum aerophilum (6) are relatively small. The simplicity of the enzyme structure and its substrate is an advantage to analyze the structure-function relationship. The enzyme activity is stimulated by K ϩ at relatively high concentrations. Also, Mg 2ϩ is essential to form a Mg-PP i complex and to keep the active conformation of V-PPase (1, 7, 8). Ca There is a putative substrate-binding motif of DXXXXXXXKXE in the cytoplasmic loop (1, 11). This was supported by immunochemical study with an antibody specific to this sequence (DVGADLVGKVE) (12). This sequence is common among VPPases not only from land plants but also from Chara corallina (10), Acetabularia acetabulum (13), R. rubrum (5), Thermotoga maritima (GenBank™ accession number AE001702), and P. aerophilum (6). Studies using substrate analogs, such as aminomethylenebisphosphonate, have also provided information on the catalytic domain (14 -16). Furthermore, the N-ethylmaleimide-binding cysteine residue (Cys 634 ) (17) and the N,NЈ-dicyclohexylcarbodiimde-binding residues (Glu 305 and Asp 504 ) (18) have been identified by a combination of site-directed mutagenesis of Arabidopsis V-PPase and heterologous expression in yeast.The aim of this study is to clarify the substrate-binding site of V-PPase by the method of site-directed and random mutagenesis. We prepared a line of constructs, in which charged residues in a putative substrate-binding site were replaced, expressed in Saccharomyces cerevisiae, and then examined for enzymatic properties. V-PPase has been proposed to have three conserved regions (3, 10). In addition to a putative PP i -binding site in the first conserved region, we investigated the two acidic motifs in the first and third conserved regions. Each aspartic acid residue in the two acidic regions was substituted and examined for enzymatic properties. Here, the functional roles of these residues on the substrate hydrolysis, binding of free Mg 2ϩ , and a coupling reaction between PP i hydrolysis and proton transport were examined. The similarity of the conserved functional motifs of V-PPase with the P-type ATPase is also discussed.

No abstract

The tonoplast K ؉ membrane transport system plays a crucial role in maintaining K ؉ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K ؉ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ϳ2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K ؉ over Na ؉ . NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca 2؉ , but a cytosolic concentration of 45 M Ca 2؉ resulted in a 2-fold increase in the amplitude of the K ؉ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K ؉ currents. These results show that NtTPK1 is a novel tonoplast K ؉ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.Plants take up potassium (K ϩ ) from the soil and plant cells accumulate K ϩ to regulate the membrane potential and turgor pressure. The cytoplasmic K ϩ concentration is tightly controlled at ϳ100 mM (1). Vacuoles are major subcellular reservoirs for controlling K ϩ homeostasis in plant cells (1). During cell expansion, for instance during stomata opening or cell growth, tonoplast transport system moves K ϩ into the vacuole, whereas, under conditions of salinity stress, K ϩ is replaced by Na ϩ (2-5).Several kinds of genes encoding K ϩ channels and K ϩ transporters have been identified in the Arabidopsis thaliana genome, and their function and tissue and cellular distribution have been extensively studied. They consist of two families, the Shaker-type channels, with six hydrophobic transmembrane domains and a single pore domain, and the two-pore K ϩ channel (TPK) 2 family, with four transmembrane and two pore domains. Six different genes encoding TPK-type channels are present in A. thaliana. AtTPK4 is targeted to the plasma membrane (6), while the other five, AtTPK1, AtTPK2, AtTPK3, AtTPK5, and AtKCO3, are localized in the vacuolar membrane (7). AtTPK1 and AtTPK4 have been functionally characterized. AtTPK4 shows a voltage-independent K ϩ profile in Xenopus laevis ooctyes and in yeast, and the K ϩ current is inhibited by extracellular Ca 2ϩ and reduced by shifting the cytosolic pH from 7.5 to 6.3, but is not affected by the external pH (6). AtTPK1 has different properties to AtTPK4 (7,8). In the yeast and plant ...