Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northernblot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

A plasmid-mediated metallo-␤-lactamase gene was cloned from a carbapenem-resistant Serratia marcescens strain, AK9373. The metallo-␤-lactamase gene was identical to the bla IMP , and it was located in the space between an integrase-like gene and an aac(6)-Ib-like gene. The deduced amino acid sequence for the putative integrase gene showed considerable identity (60.9%) to that of the Escherichia coli integrase reported. Sequences similar to the GTTRRRY and an atypical 59-base element containing a 67-bp inverted repeat sequence, which were peculiar to the integrase-dependent recombination, were also conserved in the flanking regions of the bla IMP gene. These findings imply that the metallo-␤-lactamase gene in S. marcescens AK9373 is carried by a novel integron-like element that is mediated by a transferable large plasmid.Carbapenems, such as imipenem, are potent agents for chemotherapy in infectious diseases caused by gram-negative bacteria including the family Enterobacteriaceae, since they are quite stable to hydrolysis by ␤-lactamases produced by these organisms (3, 4, 9, 10, 12). However, several clinical isolates of Bacteroides fragilis, Aeromonas hydrophila, and Pseudomonas aeruginosa were reported to be resistant to these agents because of production of metallo-␤-lactamases (7,13,15,19) belonging to Ambler's class B (1). Recently, we also isolated an imipenem-resistant Serratia marcescens strain, TN9106, and characterized a novel enterobacterial metallo-␤-lactamase, IMP-1, produced by this strain (14). Transfer of carbapenem resistance was observed in some strains of B. fragilis (5) and P. aeruginosa (19), though genetic characterization has not been done yet. In the present study, we investigated the structural features of an element carrying the metallo-␤-lactamase gene mediated by a transferable large plasmid harbored by an imipenem-resistant S. marcescens strain, AK9373 (11).(This study was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 4 to 7 October 1994 [2].) Cloning of the imipenem resistance gene. S. marcescensAK9373 showing high resistance to various broad-spectrum ␤-lactams including imipenem was isolated from a patient with a urinary tract infection at a general hospital in Anjyo, Japan, in 1993 (11). The total DNA of this strain was prepared and digested with BamHI; then, the resultant fragments were ligated in plasmid vector pMK16 (14). Escherichia coli HB101 was transformed with these recombinant plasmids, and several colonies grown on Luria-Bertani agar plates supplemented with 8 g of ceftazidime per ml were isolated (14). A 9-kb insert was generally found among the recombinant plasmids harbored by these ceftazidime-resistant transformants. The restriction sites of several endonucleases in the recombinant plasmid pSMB731 were determined as shown in Fig. 1, and the imipenem resistance gene was localized near the SmaI site by deletion analysis. Sequence analyses and identification of ORFs. The BamHISacI fragment was subcloned int...

Recent studies have reported increased podoplanin expression by cancer cells and stromal cells, but little is known about its expression and biological significance in adenocarcinoma of the lung. We examined podoplanin expression by both cancer cells and stromal cells in 177 consecutive lung adenocarcinoma cases and analyzed relations between podoplanin expression and both clinicopathological factors and outcome. Podoplanin expression was observed on the apical membrane of the cancer cells in only 9 of the 177 (5.1%) cases. By contrast, cancer-associated fibroblasts (CAFs) were found to express podoplanin in 54 cases (30.5%). Podoplanin (1) CAFs were found only in invasive adenocarcinoma and none were found in noninvasive adenocarcinoma. Conventional prognostic factors were significantly correlated with podoplanin expression by CAFs. The univariate analyses and log-rank test showed that podoplanin expression was significantly associated with shorter survival time (p < 0.001 and p < 0.001, respectively). We divided the cases into 3 groups according grade based on the proportion of CAFs expressing podoplanin [a grade 0 group (n 5 123), a grade 1 group (n 5 36) and a grade 2 group (n 5 18)]. The result showed that conventional prognostic factors were significantly correlated with the grade of podoplanin expression by CAFs. Furthermore, the grade 2 group tended to have a shorter survival time than the grade 1 group (p 5 0.092). The results of this study highlight the importance of podoplanin expression by CAFs and provide new insights into the biology of the cancer microenvironment in adenocarcinoma of the lung.

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Many seed storage proteins, including monomeric 2S albumin and polymeric prolamin, contain conserved sequences in three separate regions, termed A, B, and C, which contain the consensus motifs LxxC, CCxQL, and PxxC, respectively. Protein-sorting mechanisms in rice (Oryza sativa) endosperm were studied with a green fluorescent protein (GFP) fused to different segments of rice a-globulin, a monomeric, ABC-containing storage protein. The whole ABC region together with GFP was efficiently transported to protein storage vacuoles (type II protein bodies [PB-II]) in the endosperm cells and sequestered in the matrix that surrounds the crystalloids. Peptide Gln-23 to Ser-43 in the A region was sufficient to guide GFP to PB-II. However, GFP fused with the AB or B region accumulated in prolamin protein bodies. Substitution mutations in the CCxQL motif in the B region significantly altered protein localization in the endosperm cells. Furthermore, protein extracts containing these substituted proteins had increased amounts of the endoplasmic reticulum (ER) chaperons BiP (for binding protein), protein disulfide isomerase, and calnexin as a part of protein complexes that were insoluble in a detergent buffer. These results suggest that the ER chaperons and disulfide bonds formed at the dicysteine residues in CCxQL play critical roles in sorting fused proteins in the endosperm cells.

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To explore the significance of phosphatidylinositol-3-kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild-type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over-expressed wild-type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage-independent growth activity and migration activity of immortalized airway epithelium 16HBE14o-cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild-type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.