Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase

AbstractPlant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R→A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R→A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F− inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+–PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment

Difference in imipenem, meropenem, sulbactam, and colistin nonsusceptibility trends among three phenotypically undifferentiated Acinetobacter baumannii complex in a medical center in Taiwan, 1997–2007

BackgroundTo determine whether the susceptibilities and the trends of nonsusceptibility of imipenem, meropenem, sulbactam, and colistin differed among Acinetobacter baumannii, Acinetobacter genomic species 3 (AGS 3), and Acinetobacter genomic species 13TU (AGS 13TU) over 11 years.MethodsA total of 1,039 nonduplicate blood isolates of A baumannii complex from bacteremic patients between 1997 and 2007 were collected at Taipei Veterans General Hospital and were identified to the species level using a multiplex polymerase chain reaction method and sequence analysis of 16S–23S intergenic spacer. The minimal inhibitory concentrations of antibiotics were determined by the agar dilution method.ResultsThe nonsusceptibility rates of carbepenems and sulbactam were highest in A baumannii, which also showed a trend toward increasing rate of carbapenems nonsusceptibility over the 11-year period of the study. AGS 13TU had the highest nonsusceptible rate to colistin, comparably increasing trend of carbapenem nonsusceptiblity as that of A baumannii, and is the only species with increasing sulbactam nonsusceptibility. AGS 3 had the lowest rate of nonsusceptibility to all four antimicrobial agents.ConclusionAlthough A baumannii had the highest nonsusceptibility rate to imipenem, meropenem, and sulbactam over the years, the higher rate of colistin nonsusceptibility and the emergence of nonsusceptibility of carbapenems and sulbactam in AGS 13TU suggested that this species might cause a great problem in the near future

Urinary angiotensin-converting enzyme 2 and its activity in cats with chronic kidney disease

IntroductionAngiotensin-converting enzyme 2 (ACE2) played an important role in the renin-angiotensin-aldosterone system (RAAS) and it was proved to be renoprotective in renal disease. Urinary angiotensin-converting enzyme 2 (uACE2) has been shown to reflect renal injury in human and experimental studies, but its role in feline kidney disease remains unknown.AimsOur objectives involve comparing uACE2 concentrations and activities in cats across CKD stages with healthy controls, investigating the relationship between uACE2 concentrations, activities, and clinicopathological data in feline CKD patients, and assessing the predictive abilities of both for CKD progression.MethodsA retrospective, case–control study. The concentration and activity of uACE2 were measured by commercial ELISA and fluorometric assay kits, respectively. The concentration was adjusted to give uACE2 concentration-to-creatinine ratios (UACCRs).ResultsIn total, 67 cats consisting of 24 control and 43 chronic kidney disease (CKD), including 24 early-stage CKD and 19 late-stage CKD, were enrolled in this study. UACCR values were significantly higher in both early-stage (2.100 [1.142–4.242] x 10−6) and late-stage feline CKD (4.343 [2.992–5.0.71] x 10−6) compared to healthy controls (0.894 [0.610–1.076] x 10−6; p < 0.001), and there was also significant difference between-early stage group and late-stage group (p = 0.026). Urinary ACE2 activity (UAA) was significantly lower in CKD cats (1.338 [0.644–2.755] x pmol/min/ml) compared to the healthy cats (7.989 [3.711–15.903] x pmol/min/ml; p < 0.001). UACCR demonstrated an independent, positive correlation with BUN (p < 0.001), and UAA exhibited an independent, negative correlation with plasma creatinine (p < 0.001). Both UACCR and UAA did not yield significant results in predicting CKD progression based on the ROC curve analysis.Conclusion and clinical importanceuACE2 concentration and activity exhibit varying changes as renal function declines, particularly in advanced CKD cats

Fak56 functions downstream of integrin alphaPS3betanu and suppresses MAPK activation in neuromuscular junction growth

Background: Focal adhesion kinase (FAK) functions in cell migration and signaling through activation of the mitogen-activated protein kinase (MAPK) signaling cascade. Neuronal function of FAK has been suggested to control axonal branching; however, the underlying mechanism in this process is not clear. Results: We have generated mutants for the Drosophila FAK gene, Fak56. Null Fak56 mutants display overgrowth of larval neuromuscular junctions (NMJs). Localization of phospho-FAK and rescue experiments suggest that Fak56 is required in presynapses to restrict NMJ growth. Genetic analyses imply that FAK mediates the signaling pathway of the integrin αPS3βν heterodimer and functions redundantly with Src. At NMJs, Fak56 downregulates ERK activity, as shown by diphospho-ERK accumulation in Fak56 mutants, and suppression of Fak56 mutant NMJ phenotypes by reducing ERK activity. Conclusion: We conclude that Fak56 is required to restrict NMJ growth during NMJ development. Fak56 mediates an extracellular signal through the integrin receptor. Unlike its conventional role in activating MAPK/ERK, Fak56 suppresses ERK activation in this process. These results suggest that Fak56 mediates a specific neuronal signaling pathway distinct from that in other cellular processes

Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons

We herein describe an integrated system for the high-throughput analysis of splicing events and the identification of transcript variants. The system resolves individual splicing events and elucidates transcript variants via a pipeline that combines aspects such as bioinformatic analysis, high-throughput transcript variant amplification, and high-resolution capillary electrophoresis. For the 14 369 human genes known to have transcript variants, minimal primer sets were designed to amplify all transcript variants and examine all splicing events; these have been archived in the ASprimerDB database, which is newly described herein. A high-throughput thermocycler, dubbed GenTank, was developed to simultaneously perform thousands of PCR amplifications. Following the resolution of the various amplicons by capillary gel electrophoresis, two new computer programs, AmpliconViewer and VariantAssembler, may be used to analyze the splicing events, assemble the consecutive exons embodied by the PCR amplicons, and distinguish expressed versus putative transcript variants. This novel system not only facilitates the validation of putative transcript variants and the detection of novel transcript variants, it also semi-quantitatively measures the transcript variant expression levels of each gene. To demonstrate the system’s capability, we used it to resolve transcript variants yielded by single and multiple splicing events, and to decipher the exon connectivity of long transcripts

Antitumor Agents. 280. Multidrug Resistance-Selective Desmosdumotin B Analogues

6,6,8-Triethyldesmosdumotin B (2) was discovered as a MDR–selective flavonoid with significant in vitro anticancer activity against a multi-drug resistant (MDR) cell line (KB-VIN) but without activity against the parent cells (KB). Additional 2-analogues were synthesized and evaluated to determine the effect of B-ring modifications on MDR-selectivity. Analogues with a B-ring Me (3) or Et (4) group had substantially increased MDR–selectivity. Three new disubstituted analogues, 35, 37 and 49, also had high collateral sensitivity (CS) indices of 273, 250 and 100, respectively. Furthermore, 2–4 also displayed MDR-selectivity in an MDR hepatoma-cell system. While 2–4 showed either no or very weak inhibition of cellular P-glycoprotein (P-gp) activity, they either activated or inhibited the actions of the first generation P-gp inhibitors verapamil or cyclosporin, respectively