Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Kinetics of veratridine action on Na channels of skeletal muscle

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes

Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82–89% similarity at the amino acid level to other F3H proteins. This is the first report on the gennomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.     

Freeze-Thaw Stress: Effects of Temperature on Hydraulic Conductivity and Ultrasonic Activity in Ten Woody Angiosperms

Freeze-thaw events can affect plant hydraulics by inducing embolism. This study analyzed the effect of temperature during the freezing process on hydraulic conductivity and ultrasonic emissions (UE). Stems of 10 angiosperms were dehydrated to a water potential at 12% percentage loss of hydraulic conductivity (PLC) and exposed to freeze-thaw cycles. The minimal temperature of the frost cycle correlated positively with induced PLC, whereby species with wider conduits (hydraulic diameter) showed higher freeze-thaw-induced PLC. Ultrasonic activity started with the onset of freezing and increased with decreasing subzero temperatures, whereas no UE were recorded during thawing. The temperature at which 50% of UE were reached varied between −9.1°C and −31.0°C across species. These findings indicate that temperatures during freezing are of relevance for bubble formation and air seeding. We suggest that species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of water potential in the ice, while bubble expansion and the resulting PLC occur during thawing. UE analysis can be used to monitor the cavitation process and estimate freeze-thaw-induced PLC.Xylem embolism is a limiting factor for plant survival and distribution (Choat et al., 2012; Charrier et al., 2013). Two major factors can induce embolism in the xylem of plants: drought and freeze stress. Freeze-thaw-induced embolism is caused by bubbles formed during freezing that then expand on thawing (Lemoine et al., 1999; Hacke and Sperry, 2001; Cruiziat et al., 2002; Tyree and Zimmermann, 2002). As wider conduits contain more gas and form larger bubbles, which expand at less negative tension, conduit diameter and xylem sap tension are critical for the formation of freeze-thaw-induced embolism (Davis et al., 1999; Pittermann and Sperry, 2003). Accordingly, Mayr and Sperry (2010) observed a loss of conductivity only when samples were under critical tension during thawing. Under drought stress, tension in the xylem sap increases the sensitivity to embolism generated by successive freeze-thaw cycles (Mayr et al., 2003, 2007).Ultrasonic emissions (UE) analysis can be used to detect cavitation events in wood. It is unclear how well related UE are to cavitation events, as they are extracted from continuous acoustic emissions and depend on set definitions. However, UE analysis has been proven effective for monitoring drought-induced embolism in the laboratory (Pena and Grace, 1986; Salleo and Lo Gullo, 1986; Borghetti et al., 1993; Salleo et al., 2000) as well as in field experiments (Ikeda and Ohtsu, 1992; Jackson et al., 1995; Jackson and Grace, 1996; Hölttä et al., 2005; Ogaya and Penuelas, 2007). In a cavitating conduit, signals are probably produced by the disruption of the water column and subsequent tension relaxation of cell walls.UE have also been detected during freezing events, but the origin of these signals was less clear. In some cases, UE were observed during thawing, which are thus probably related to embolism formation according to the classic thaw-expansion hypothesis (Mayr and Sperry, 2010); however, all species studied have produced UE on freezing, which cannot yet be explained (Raschi et al., 1989; Kikuta and Richter, 2003; Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010). The low solubility of gases in ice prompted the idea that air bubbles expulsed from the ice structure produce UE near the ice-liquid interface (Sevanto et al., 2012). As the water potential of ice is strongly temperature dependent, the minimum temperature during freezing might be a relevant factor. Numerous studies have analyzed UE patterns during freeze-thaw cycles in conifers (Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010) or angiosperms (Weiser and Wallner, 1988; Kikuta and Richter, 2003), but few of them measured embolism concomitantly. Percentage loss of hydraulic conductivity (PLC) was only measured in a few studies and only in conifers (Mayr et al., 2007; Mayr and Sperry, 2010).In this study, we analyzed the effect of freeze-thaw cycles on the hydraulic conductivity and ultrasonic activity in 10 angiosperm species. We hypothesized that (1) the extent of freeze-thaw-induced embolism depends on xylem anatomy (related to conduit diameter) and minimal temperature (related to the water potential of ice); (2) ultrasonic activity is also influenced by anatomy and temperature; and (3) PLC and UE are positively correlated. PLC was measured in 10 angiosperm species after freeze-thaw cycles at different minimal temperatures (−10 to −40°C). Furthermore, UE were recorded during a freeze-thaw cycle down to −40°C.