The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears >90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an =9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hHl exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hHl to sodium ions is about twice that of the homologous rat channel and hHl is more resistant to block by tetrodotoxin (ICso = 5.7 pM). hHl is also resistant to Iu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.

It has been postulated that the K+ channel isoform Kv1.3 plays a role in regulatory volume decrease (RVD) in response to hypotonic shock. We show that a mouse cytotoxic T-lymphocyte line, CTLL-2, is devoid of voltagedependent K+ channels and is unable to volume regulate. Transient transfection of these cells with Kv1.3 reconstitutes their ability to volume regulate. As predicted by our model, this ability depends critically on volume-induced changes in membrane potential and the isoform of the K+ channel used. When the cells were transfected with Kv3.1, an isoform believed to be expressed in a specific subclass of mouse thymocytes, the CTLL-2 cells did not show RVD. The difference in the ability of the two isoforms to confer the capacity for RVD is expected from differences in the voltage dependence of activation of the channels, according to our proposed model for RVD. The experimental approach that we use, transient transfection and panning to select positive transfectants, is highly effective; it has a >95% efficiency. This method, and this cell line, may be important tools in studying lymphocyte K+ channels and their function in situ. MATERIALS AND METHODSCytotoxic murine T cells (CTLL-2) were transiently cotransfected with a plasmid using a cytomegalovirus (CMV) promoter (pRc/CMV; 5.4 kb) and a Kv1.3 insert (1.8 kb) or a Kv3.1 insert (1.8 kb; gift from Teresa Pemey, Yale University, New Haven, CT) along with a Ccd4neo plasmid (5.0 kb; pUC-based plasmid with a CMV promoter driving CD4; gift from Ray Sweet, SmithKline Beecham) containing human surface membrane CD4 (1.7 kb). Transfection. Using sterile techniques, cells were collected and suspended in Opti-MEM plus recombinant interleukin 2 (rIL-2) (8.3 ng/ml; Cetus) at 2 x 107 cells per ml with Ccd4neo (10 ,ug/ml) and pCMV/Kvl.3 or pCMV/Kv3.1 (80 ug/ml). Control cells were transfected only with Ccd4neo at 10 ng/ml. These suspensions were incubated on ice for 10 min in 0.4-cm electrode gap cuvettes (400 ,ul) and then electroporated using a BTX electroporator (San Diego) with settings determined previously to give :50% viability (voltage, 220-320 V; capacitance, 800 ,LF; resistance, 72 fQ). The resultant time constants were 24-30 ms. Cells were incubated for 10 min more on ice and then transferred back to culture medium (14) in the incubator and grown for 48 hr.Harvesting of Cells. Cells were collected from culture and incubated with monoclonal anti-human CD4 antibody (AMAC, Westbrook, ME; 0.5 tLg/106 cells) and adhered to Petri dishes (35 mm) coated with goat anti-mouse IgG as described in ref. 15. Dishes were washed gently five times with 1 ml of phosphate-buffered saline (PBS) containing 5% fetal bovine serum and rIL-2 (8.3 ng/ml) for the RVD experiments or with 1 ml of normal extracellular medium plus rIL-2 (8.3 ng/ml) for the patch-clamp experiments. All experiments were carried out at room temperature.Electrophysiology. Standard patch-clamp techniques were used, as described in ref. 2. Pipettes were made from 0010 glass, coated with Sylgard, and ...

[1] A robust retrieval algorithm to estimate concentrations of total suspended sediments (TSS) in Poyang Lake (the largest freshwater lake in China) was developed using Moderate Resolution Imaging Spectroradiometer (MODIS) medium-resolution (250 m) data from 2000 to 2010 and in situ data collected during two cruise surveys. The algorithm was based on atmospherically corrected surface reflectance at 645 nm, with 1240 nm data serving as a reference for aerosols and a nearest-neighbor method was used to avoid land adjacency effect. The algorithm showed an uncertainty of 30-40% for TSS ranging between 3 and 200 mg L À1. Long-term TSS distribution maps derived from MODIS data and the customized TSS algorithm showed significant variations in both space and time, with low TSS (<10 mg L À1) in wet seasons and much higher TSS (>15-20 mg L À1 ) in dry seasons for the south lake, and generally higher TSS in the north lake. The TSS difference between the north and the south increased significantly after 2002, with mean TSS often reaching >40 mg L À1 in the north. While the TSS seasonality was attributed to the seasonal changes of the lake's circulation, the inter-annual variations were primarily driven by sand dredging activities, regulated by management policies. The case study here provides baseline water quality information for future restoration efforts in Poyang Lake, and more generally, an approach to assess water quality changes in similar water bodies, which have resulted from either climate variability or human activities.

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (y1494ya495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/ QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 inV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 inV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

The mechanism of inhibition of Na+ channels by lidocaine has been suggested to involve low-affinity binding to rested states and high-affinity binding to the inactivated state of the channel, implying either multiple receptor sites or allosteric modulation of receptor affinity. Alternatively, the lidocaine receptor may be guarded by the channel gates. To test these distinct hypotheses, inhibition of Na+ channels by lidocaine was studied by voltage-clamp methods in both native and heterologous expression systems. Native Na+ channels were studied in guinea pig ventricular myocytes, and recombinant human heart Na+ channels were expressed in Xenopus laevis oocytes. Fast inactivation was eliminated by mutating three amino acids (isoleucine, phenylalanine, and methionine) in the III-IV interdomain to glutamines or by enzymatic digestion with alpha-chymotrypsin. In channels with intact fast inactivation, lidocaine block developed with a time constant of 589 +/- 42 ms (n = 7) at membrane potentials between -50 and +20 mV, as measured by use of twin pulse protocols. The IC50 was 36 +/- 1.8 mumol/L. Control channels inactivated within 20 ms, and slow inactivation developed much later (time constant of slow inactivation, 6.2 +/- 0.36 s). The major component of block developed long after activated and open channels were no longer available for drug binding. Control channels recovered fully from inactivation in < 50 ms at -120 mV (time constant, 11 +/- 0.5 ms; n = 50).(ABSTRACT TRUNCATED AT 250 WORDS)

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

No abstract

In order to improve software reliability, software defect prediction is applied to the process of software maintenance to identify potential bugs. Traditional methods of software defect prediction mainly focus on designing static code metrics, which are input into machine learning classifiers to predict defect probabilities of the code. However, the characteristics of these artificial metrics do not contain the syntactic structures and semantic information of programs. Such information is more significant than manual metrics and can provide a more accurate predictive model. In this paper, we propose a framework called defect prediction via attention-based recurrent neural network (DP-ARNN). More specifically, DP-ARNN first parses abstract syntax trees (ASTs) of programs and extracts them as vectors. Then it encodes vectors which are used as inputs of DP-ARNN by dictionary mapping and word embedding. After that, it can automatically learn syntactic and semantic features. Furthermore, it employs the attention mechanism to further generate significant features for accurate defect prediction. To validate our method, we choose seven open-source Java projects in Apache, using F1-measure and area under the curve (AUC) as evaluation criteria. The experimental results show that, in average, DP-ARNN improves the F1-measure by 14% and AUC by 7% compared with the state-of-the-art methods, respectively.