The Eag Domain Regulates the Voltage-Dependent Inactivation of Rat Eag1 K+ Channels

Eag (Kv10) and Erg (Kv11) belong to two distinct subfamilies of the ether-à-go-go K⁺ channel family (KCNH). While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N)-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1) and human Erg (hERG1) channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4–S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K⁺ channels.     

Modular synthesis of Petri nets for modeling flexible manufacturing systems

This paper proposes a modular Petri net synthesis method for modeling flexible manufacturing systems based on synchronization among control processes of the manufacturing resources (such as robots and machines). In the method, the target system is modeled in a bottom-up and uniform manner by first describing the system's control processes using strongly connected state machines (SCSMs) as the basic modules. Each SCSM may contain multiple tokens to represent resources from the same type such as spaces in a buffer. Next, the common transitions and common transition subnets of the modules are merged to represent their synchronization. The system model constructed is proven to be conservative and thus bounded. Moreover, a restricted class of merged nets is proven to be live and reversible. For general classes of merged nets, this paper shows theorems that easily calculateP-invariants of the final net without solving the linear system equations. TheseP-invariants can be used to help in verifying the model's qualitative properties such as liveness.     

Visual orientation of the symbiotic snapping shrimp Synalpheus demani

Visual cues play an important role in crustacean shelter-seeking behavior. We hypothesize that Synalpheus demani, an obligate crinoid-dwelling snapping shrimp, uses visual cues in host location. We tested shrimp response to rectangular visual targets that subtended 10°, 30°, 90°, 180°, and 270° in a circular arena in background seawater and in seawater containing host odor. In background seawater, S. demani oriented to solid visual targets of 90° and larger, and avoided the 10° and 30° target. Orientation to targets is interpreted as refuge-seeking behavior. Avoidance is speculated as predator-avoidance behavior. Shrimp oriented to large visual targets in sea water and host odor. The presence of host odor altered avoidance responses of a significant number, but not all, shrimp to small targets. Host odor also increased shrimp activities in the arena. Synalpheus demani oriented to 90° patterned (vertical and horizontal stripes, and checker board) targets, but they did not orient to 270° patterned targets. This was interpreted as positive scototaxis, which is common in many crustaceans. Thus we conclude that visual orientation of S. demani is similar to that of free-living snapping shrimp.     

A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication.

The ribozymes of hepatitis delta virus (HDV) have so far been studied primarily in vitro. Several structural models for HDV ribozymes based on truncated HDV RNA fragments, which are different from the hammerhead or the hairpin/paperclip ribozyme model proposed for plant viroid or virusoid RNAs, have been proposed. Whether these structures actually exist in vivo and whether ribozymes actually function in the HDV replication cycle have not been demonstrated. We have now developed an in vivo ribozyme self-cleavage assay capable of detecting self-cleavage of dimer or trimer HDV RNA in vivo. By site-directed mutagenesis and compensatory mutations to disrupt and restore potential base pairing in the ribozyme domain of the full-length HDV RNA according to the various structural models, a close correlation between the detected in vivo and the predicted in vitro ribozyme activities of various mutant RNAs was demonstrated. These results suggest that the proposed in vitro ribozyme structure likely exists and functions during the HDV replication cycle in vivo. Furthermore, the pseudoknot model most likely represents the structure responsible for the ribozyme activity in vivo. All of the mutants that had lost the ribozyme activity could not replicate, indicating that the ribozyme activities are indeed required for HDV RNA replication. However, some of the compensatory mutants which have restored both the cleavage and ligation activities could not replicate, suggesting that the ribozyme domains are also involved in other unidentified functions or in the formation of an alternative structure that is required for HDV RNA replication. This study thus established that the ribozyme has important biological functions in the HDV life cycle.     

Evidence for CD8+ antiviral activity in cats infected with feline immunodeficiency virus.

Human immunodeficiency virus (HIV) causes a long, asymptomatic infection characterized by normal to elevated numbers of circulating CD8+ cells and a progressive decline in CD4+ cells. It has been speculated that HIV-specific antiviral activity driven by CD8+ T cells may control viral replication during this period and maintain the clinically asymptomatic stage of disease. The disease induced in cats by feline immunodeficiency virus (FIV) is similar to HIV in that it is characterized by a long asymptomatic stage with a progressive decline in CD4+ cells, culminating in AIDS. In the present study, we demonstrate that FIV is more readily isolated from CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMC) of FIV-infected cats than from unfractionated PBMC cultures. In addition, CD8+ T cells isolated from FIV-positive cats demonstrating anti-FIV activity in PBMC cultures inhibit FIV infection of FCD4E cells in vitro. Anti-FIV activity is not found in FIV- negative cats and is not characteristic of cats acutely infected with FIV but is present in the majority of chronically infected, clinically asymptomatic and symptomatic cats. Decreases in plasma and cell-associated viremia during the acute-stage FIV infection appears to precede the appearance of CD8+ anti-FIV cells in the circulation. In summary, this study demonstrates a population(s) of CD8+ T cells in chronically FIV-infected cats capable of suppressing FIV replication in cultured PBMC. The significance of anti-FIV CD8+ cells in the immunopathogenesis of the infection and disease progression has yet to be determined.     

Cadmium status of soils and plants from a long-term fertility experiment in southeast Norway

To determine whether the use of phosphate fertilizer resulted in measurable cadmium accumulation in soils and crops harvested, soil and plant samples were collected from some selected treatments of a seventy year-old fertilizer experiment at Møystad, southeast Norway. Soil samples after extraction with Aqua Regia or 1M NH₄NO₃ and plant samples after digestion were analyzed for Cd. Cadmium balance based on fertilizer and atmospheric inputs and crop removal and leaching losses was worked out. Neither the total nor the available (NH₄NO₃-extractable) Cd in the soil was significantly affected by Cd added through fertilizer, though a tendency of higher Cd in soils from the plots receiving higher amounts of fertilizer was seen. The same trend was also observed for the Cd concentration in plants. Annual Cd input rates (fertilizer and atmosphere) varied from 1.20 to 2.57 g Cd ha^-1 y^-1 and the Cd removal (crops and leaching) rates varied from 1.16 to 1.79 g Cd ha^-1 y^-1. The balance calculations based on the seventy years data indicated Cd accumulation in the soil was <1 g Cd ha^-1 y^-1, but that increasing the doses of either commercial fertilizer or farm yard manure would likely result in increased accumulation of the element. This may have a negative impact because the available soil Cd content would be increased at a faster rate, resulting in increased plant uptake. Although Cd tended to accumulate as a result of P fertilization, the rate of increase was slow. The annual increase in the total Cd content of fertilized plots varied from 0.04 to 0.12% indicating that it may take from 800 to 2000 years, depending upon the fertilizer input, to accumulate Cd equivalent to that currently present in the soil.     

Comparison of calcium-current in isolated atrial myocytes from failing and nonfailing human hearts

To identify possible alterations of the L-type calcium currents (I_Ca,L) in cardiomyopathy, I_Ca,L were recorded in atrial myocytes dissociated from the nonfailing heart (NF) of patients undergoing corrective open-heart surgery and explanted failing heart (FH) of patients with dilated cardiomyopathy undergoing heart transplantation. The patch-clamp technique was applied in the single-electrode whole-cell mode. The electrophysiological properties of I_Ca,L, including cell capacitance and current density, were similar in atrial myocytes from both groups of patients. Further to identify possible alterations of the myocardial beta-adrenergic pathway in cardiomyopathy, we examined the effects of isoproterenol, forskolin, 8-Br-cAMP and IBMX on I_Ca,L in both groups of atrial myocytes. Perfusion of isoproterenol (1 M) significantly increased the peak I_Ca,L by 515 ± 44% in 6 atrial myocytes from NF but increased only by 135 ± 25% in 27 atrial myocytes from FH. However, forskolin (1 M) or 8-Br-cAMP (0.1 mM) increased the peak I_Ca,L to a similar extent in atrial myocytes from NF and FH. IBMX (20 M) also induced a comparable increase in the peak I_Ca,L by 213 ± 31% (n=5) and 207 ± 59% (n=4) in atrial myocytes from NF and FH, respectively. The above findings suggest that in atrial myocytes obtained from FH the beta-adrenoceptor numbers might be decreased but no impairment of the signal transduction cascade occurred beyond the GTP binding proteins level.     

Signal transduction and regulation of IbpreproHypSys in sweet potato

Hydroxyproline‐rich glycopeptides (HypSys) are small signalling peptides containing 18–20 amino acids. The expression of IbpreproHypSys, encoding the precursor of IbHypSys, was induced in sweet potato (Ipomoea batatas cv. Tainung 57) through wounding and IbHypSys treatments by using jasmonate and H₂O₂. Transgenic sweet potatoes overexpressing (OE) and silencing [RNA interference (RNAi)] IbpreproHypSys were created. The expression of the wound‐inducible gene for ipomoelin (IPO) in the local and systemic leaves of OE plants was stronger than the expression in wild‐type (WT) and RNAi plants after wounding. Furthermore, grafting experiments indicated that IPO expression was considerably higher in WT stocks receiving wounding signals from OE than from RNAi scions. However, wounding WT scions highly induced IPO expression in OE stocks. These results indicated that IbpreproHypSys expression contributed towards sending and receiving the systemic signals that induced IPO expression. Analysing the genes involved in the phenylpropanoid pathway demonstrated that lignin biosynthesis was activated after synthetic IbHypSys treatment. IbpreproHypSys expression in sweet potato suppressed Spodoptera litura growth. In conclusion, wounding induced the expression of IbpreproHypSys, whose protein product was processed into IbHypSys. IbHypSys stimulated IbpreproHypSys and IPO expression and enhanced lignin biosynthesis, thus protecting plants from insects.