Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Probing the active site of mitogillin,a fungal ribotoxin

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G₄₃₂₅ and A₄₃₂₆, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.     

Water stress, ammonium, and leaf senescence in detached rice leaves

Ammonium accumulation in relation to water stress-promoted senescence of detached rice leaves was investigated. The effect of water stress on the senescence of detached rice leaves is associated with the accumulation of ammonium. The accumulation of ammonium in detached rice leaves by water stress is attributed to a decrease in glutamine synthetase activity. Ammonium accumulation in detached rice leaves, induced by water stress, was accompanied by an increase in tissue sensitivity to ethylene which, in turn, accelerated leaf senescence.     

Ammonium, calcium, and leaf senescence in rice

The possible involvement of calcium in the regulation of ammonium-promoted senescence of detached rice leaves was investigated. Calcium effectively reduced ammonium-promoted senescence of detached rice leaves. The effect of ammonium on the senescence was also significantly reduced by the calcium ionophore A23187. Ammonium-promoted senescence of detached rice leaves may be mediated through blocking the entrance of calcium ions into the cytosol.     

A Few Bad Apples: A Model of Disease Influenced Agent Behaviour in a Heterogeneous Contact Environment

For diseases that infect humans or livestock, transmission dynamics are at least partially dependent on human activity and therefore human behaviour. However, the impact of human behaviour on disease transmission is relatively understudied, especially in the context of heterogeneous contact structures such as described by a social network. Here, we use a strategic game, coupled with a simple disease model, to investigate how strategic agent choices impact the spread of disease over a contact network. Using beliefs that are based on disease status and that build up over time, agents choose actions that stochastically determine disease spread on the network. An agent’s disease status is therefore a function of both his own and his neighbours actions. The effect of disease on agents is modelled by a heterogeneous payoff structure. We find that the combination of network shape and distribution of payoffs has a non-trivial impact on disease prevalence, even if the mean payoff remains the same. An important scenario occurs when a small percentage (called noncooperators) have little incentive to avoid disease. For diseases that are easily acquired when taking a risk, then even when good behavior can lead to disease eradication, a small increase in the percentage of noncooperators (less than 5%) can yield a large (up to 25%) increase in prevalence.     

Enhanced Bone Tissue Regeneration by Porous Gelatin Composites Loaded with the Chinese Herbal Decoction Danggui Buxue Tang

Danggui Buxue Tang (DBT) is a traditional Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinensis. Pharmacological results indicate that DBT can stimulate bone cell proliferation and differentiation. The aim of the study was to investigate the efficacy of adding DBT to bone substitutes on bone regeneration following bone injury. DBT was incorporated into porous composites (GGT) made from genipin-crosslinked gelatin and β-triclacium phosphates as bone substitutes (GGTDBT). The biological response of mouse calvarial bone to these composites was evaluated by in vivo imaging systems (IVIS), micro-computed tomography (micro-CT), and histology analysis. IVIS images revealed a stronger fluorescent signal in GGTDBT-treated defect than in GGT-treated defect at 8 weeks after implantation. Micro-CT analysis demonstrated that the level of repair from week 4 to 8 increased from 42.1% to 71.2% at the sites treated with GGTDBT, while that increased from 33.2% to 54.1% at GGT-treated sites. These findings suggest that the GGTDBT stimulates the innate regenerative capacity of bone, supporting their use in bone tissue regeneration.     

Clinical Impact of Speed Variability to Identify Ultramarathon Runners at Risk for Acute Kidney Injury

BackgroundUltramarathon is a high endurance exercise associated with a wide range of exercise-related problems, such as acute kidney injury (AKI). Early recognition of individuals at risk of AKI during ultramarathon event is critical for implementing preventative strategies.ObjectivesTo investigate the impact of speed variability to identify the exercise-related acute kidney injury anticipatively in ultramarathon event.MethodsThis is a prospective, observational study using data from a 100 km ultramarathon in Taipei, Taiwan. The distance of entire ultramarathon race was divided into 10 splits. The mean and variability of speed, which was determined by the coefficient of variation (CV) in each 10 km-split (25 laps of 400 m oval track) were calculated for enrolled runners. Baseline characteristics and biochemical data were collected completely 1 week before, immediately post-race, and one day after race. The main outcome was the development of AKI, defined as Stage II or III according to the Acute Kidney Injury Network (AKIN) criteria. Multivariate analysis was performed to determine the independent association between variables and AKI development.Results26 ultramarathon runners were analyzed in the study. The overall incidence of AKI (in all Stages) was 84.6% (22 in 26 runners). Among these 22 runners, 18 runners were determined as Stage I, 4 runners (15.4%) were determined as Stage II, and none was in Stage III. The covariates of BMI (25.22 ± 2.02 vs. 22.55 ± 1.96, p = 0.02), uric acid (6.88 ± 1.47 vs. 5.62 ± 0.86, p = 0.024), and CV of speed in specific 10-km splits (from secondary 10 km-split (10^th – 20^th km-split) to 60^th – 70^th km-split) were significantly different between runners with or without AKI (Stage II) in univariate analysis and showed discrimination ability in ROC curve. In the following multivariate analysis, only CV of speed in 40^th – 50^th km-split continued to show a significant association to the development of AKI (Stage II) (p = 0.032).ConclusionsThe development of exercise-related AKI was not infrequent in the ultramarathon runners. Because not all runners can routinely receive laboratory studies after race, variability of running speed (CV of speed) may offer a timely and efficient tool to identify AKI early during the competition, and used as a surrogate screening tool, at-risk runners can be identified and enrolled into prevention trials, such as adequate fluid management and avoidance of further NSAID use.     

Co-Encapsulating the Fusogenic Peptide INF7 and Molecular Imaging Probes in Liposomes Increases Intracellular Signal and Probe Retention

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging.     

Houttuynia cordata Targets the Beginning Stage of Herpes Simplex Virus Infection

Herpes simplex virus (HSV), a common latent virus in humans, causes certain severe diseases. Extensive use of acyclovir (ACV) results in the development of drug-resistant HSV strains, hence, there is an urgent need to develop new drugs to treat HSV infection. Houttuynia cordata (H. cordata), a natural herbal medicine, has been reported to exhibit anti-HSV effects which is partly NF-κB-dependent. However, the molecular mechanisms by which H. cordata inhibits HSV infection are not elucidated thoroughly. Here, we report that H. cordata water extracts (HCWEs) inhibit the infection of HSV-1, HSV-2, and acyclovir-resistant HSV-1 mainly via blocking viral binding and penetration in the beginning of infection. HCWEs also suppress HSV replication. Furthermore, HCWEs attenuate the first-wave of NF-κB activation, which is essential for viral gene expressions. Further analysis of six compounds in HCWEs revealed that quercetin and isoquercitrin inhibit NF-κB activation and additionally, quercetin also has an inhibitory effect on viral entry. These results indicate that HCWEs can inhibit HSV infection through multiple mechanisms and could be a potential lead for development of new drugs for treating HSV.