Identification and Characterization of a Novel aac(6′)-Iag Associated with the bla IMP-1–Integron in a Multidrug-Resistant Pseudomonas aeruginosa

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6′)-Iag, bla _IMP-1, a truncated form of bla _IMP-1, and a truncated form of aac(6′)-Iag. The aac(6′)-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6′)-Iz. Recombinant AAC(6′)-Iag protein showed aminoglycoside 6′-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6′)-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla _IMP-1 and aac(6′)-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.     

Synthesis of 3,6-di-O-Methylglucosyl Disaccharides with Methyl 3-(p-Hydroxyphenyl)propionate as a Linker Arm and Their Use in the Serodiagnosis of Leprosy

The disaccharide, 2,3-di-O-methyl-4-O-(3,6-di-O-methyl-β-d-glucopyranosyl)-l-rhamno-pyranose, the distal segment of phenolic glycolipid I, that is a specific antigen from Mycobacterium leprae, and some related disaccharides were synthesised as the glycosides of methyl 3-(p-hydroxyphenyl)propionate. The methyl 3-(p-hydroxyphenyl)propionate was coupled with 2,3,4-tri-O-acetyl-l-rhamnosyl bromide, deacetylated, acetonated, coupled with 2,4,6-tri-O-acetyl-3-O-methyl-d-glucosyl bromide, and converted into a variety of p-(2-methoxycarbonylethyl)phenyl 4-O-(3,6-di-O-methyl-d-glucopyranosyl)-containing disaccharides that are amenable to ready conjugation with protein carriers, thereby providing neo-glycoconjugates for the specific serodiagnosis of leprosy.     

The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon

The arcA (dye) and arcB genes of Escherichia coli are responsible for anaerobic repression of target operons and regulons of aerobic function (the arc modulon). The amino acid sequence of ArcA (Dye) indicated that it is the regulator protein of a two-component control system. Here we show that ArcB is a membrane sensor protein on the basis of its deduced amino acid sequence (778 residues), hydropathicity profile, and cellular distribution. On the carboxyl end of the ArcB sequence there is an additional domain showing homology with conserved regions of regulator proteins. Deletion into this domain destroyed ArcB function. ArcB conserved a histidine residue for autophosphorylation of the sensor proteins, and aspartic residues important for the regulator proteins.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Shear Stress and Atherosclerosis

Hemodynamic shear stress, the frictional force acting on vascular endothelial cells, is crucial for endothelial homeostasis under normal physiological conditions. When discussing blood flow effects on various forms of endothelial (dys)function, one considers two flow patterns: steady laminar flow and disturbed flow because endothelial cells respond differently to these flow types both in vivo and in vitro. Laminar flow which exerts steady laminar shear stress is atheroprotective while disturbed flow creates an atheroprone environment. Emerging evidence has provided new insights into the cellular mechanisms of flow-dependent regulation of vascular function that leads to cardiovascular events such as atherosclerosis, atherothrombosis, and myocardial infarction. In order to study effects of shear stress and different types of flow, various models have been used. In this review, we will summarize our current views on how disturbed flow-mediated signaling pathways are involved in the development of atherosclerosis.     

Risk Factors for Cisplatin-Induced Nephrotoxicity and Potential of Magnesium Supplementation for Renal Protection

Background

Nephrotoxicity remains a problem for patients who receive cisplatin chemotherapy. We retrospectively evaluated potential risk factors for cisplatin-induced nephrotoxicity as well as the potential impact of intravenous magnesium supplementation on such toxicity.

Patients and Methods

We reviewed clinical data for 401 patients who underwent chemotherapy including a high dose (≥60 mg/m²) of cisplatin in the first-line setting. Nephrotoxicity was defined as an increase in the serum creatinine concentration of at least grade 2 during the first course of cisplatin chemotherapy, as assessed on the basis of National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. The severity of nephrotoxicity was evaluated on the basis of the mean change in the serum creatinine level. Magnesium was administered intravenously to 67 patients (17%).

Results

Cisplatin-induced nephrotoxicity was observed in 127 patients (32%). Multivariable analysis revealed that an Eastern Cooperative Oncology Group performance status of 2 (risk ratio, 1.876; P = 0.004) and the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) (risk ratio, 1.357; P = 0.047) were significantly associated with an increased risk for cisplatin nephrotoxicity, whereas intravenous magnesium supplementation was associated with a significantly reduced risk for such toxicity (risk ratio, 0.175; P = 0.0004). The development of hypomagnesemia during cisplatin treatment was significantly associated with a greater increase in serum creatinine level (P = 0.0025). Magnesium supplementation therapy was also associated with a significantly reduced severity of renal toxicity (P = 0.012).

Conclusions

A relatively poor performance status and the regular use of NSAIDs were significantly associated with cisplatin-induced nephrotoxicity, although the latter association was marginal. Our findings also suggest that the ability of magnesium supplementation to protect against the renal toxicity of cisplatin warrants further investigation in a prospective trial.     

Developmental profiles of wing imaginal discs of flügellos(fl), a wingless mutant of the silkworm, Bombyx mori

 Mutations at the flügellos (fl) locus in Bombyx mori give rise to wingless pupae and moths. To understand the developmental steps responsible for the fl wing defect, we compared the morphological changes and protein synthesis profiles between fl and wild-type (WT) wing discs during larval development. Morphologically, the four wing discs in the fl homozygote larva developed normally at least until the fourth instar, but they were slightly smaller than those of the WT. After the last larval ecdysis, wing epithelial invagination and tracheal migration into the lacunar spaces evidently occurred in the WT wing discs. However, there was no apparent morphological change in fl discs through the fifth instar. The fl wing discs cultured in medium containing 20-hydroxyecdysone (20E) did not grow and develop, although the WT wing discs extended and differentiated under the same conditions. A comparison of protein synthesis in the wing discs revealed that several bands were differentially expressed between the fl and WT. A 41-kDa band expressed abundantly from larval to pharate pupal stages in the WT wing discs was rarely observed in fl discs. Furthermore, in vitro culture studies showed that the 41-kDa protein was induced by 20E and specifically synthesized in WT wing discs after the wandering stage, but not in fl discs. The wing-specific protein synthesis and morphogenesis in fl wing discs may be blocked due to aberrant expression of the fl gene. Received: 6 November 1996 / Accepted: 5 February 1997     

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact form Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and W. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8-to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [¹⁴C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydroplulic agent NDE.