Coupled Na/K/Cl efflux. "Reverse" unidirectional fluxes in squid giant axons

Studies of unidirectional Cl-, Na+, and K+ effluxes were performed on isolated, internally dialyzed squid giant axons. The studies were designed to determine whether the coupled Na/K/Cl co-transporter previously identified as mediating influxes (Russell. 1983. Journal of General Physiology. 81:909-925) could also mediate the reverse fluxes (effluxes). We found that 10 microM bumetanide blocked 7-8 pmol/cm2 X s of Cl- efflux from axons containing ATP, Na+, and K+. However, if any one of these solutes was removed from the internal dialysis fluid, Cl- efflux was reduced by 7-8 pmol/cm2 X s and the remainder was insensitive to bumetanide. About 5 pmol/cm2 X s of Na+ efflux was inhibited by 10 microM bumetanide in the continuous presence of 10(-5) M ouabain and 10(-7) M tetrodotoxin if Cl-, K+, and ATP were all present in the internal dialysis fluid. However, the omission of Cl- or K+ or ATP reduced the Na+ efflux, leaving it bumetanide insensitive. K+ efflux had to be studied under voltage-clamp conditions with the membrane potential held at -90 mV because the dominant pathway for K+ efflux (the delayed rectifier) has a high degree of voltage sensitivity. Under this voltage-clamped condition, 1.8 pmol/cm2 X s of K+ efflux could be inhibited by 10 microM bumetanide. All of these results are consistent with a tightly coupled Na/K/Cl co-transporting efflux mechanism. Furthermore, the requirements for cis-side co-ions and intracellular ATP are exactly like those previously described for the coupled Na/K/Cl influx process. We propose that the same transporter mediates both influx and efflux, hence demonstrating "reversibility," a necessary property for an ion-gradient-driven transport process.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Hawksbill turtle terra incognita: conservation genetics of?eastern Pacific rookeries

Prior to 2008 and the discovery of several important hawksbill turtle (Eretmochelys imbricata) nesting colonies in the EP (Eastern Pacific), the species was considered virtually absent from the region. Research since that time has yielded new insights into EP hawksbills, salient among them being the use of mangrove estuaries for nesting. These recent revelations have raised interest in the genetic characterization of hawksbills in the EP, studies of which have remained lacking to date. Between 2008 and 2014, we collected tissue samples from 269 nesting hawksbills at nine rookeries across the EP and used mitochondrial DNA sequences (766 bp) to generate the first genetic characterization of rookeries in the region. Our results inform genetic diversity, population differentiation, and phylogeography of the species. Hawksbills in the EP demonstrate low genetic diversity: We identified a total of only seven haplotypes across the region, including five new and two previously identified nesting haplotypes (pooled frequencies of 58.4% and 41.6%, respectively), the former only evident in Central American rookeries. Despite low genetic diversity, we found strong stock structure between the four principal rookeries, suggesting the existence of multiple populations and warranting their recognition as distinct management units. Furthermore, haplotypes EiIP106 and EiIP108 are unique to hawksbills that nest in mangrove estuaries, a behavior found only in hawksbills along Pacific Central America. The detected genetic differentiation supports the existence of a novel mangrove estuary “reproductive ecotype” that may warrant additional conservation attention. From a phylogeographic perspective, our research indicates hawksbills colonized the EP via the Indo‐Pacific, and do not represent relict populations isolated from the Atlantic by the rising of the Panama Isthmus. Low overall genetic diversity in the EP is likely the combined result of few rookeries, extremely small reproductive populations and evolutionarily recent colonization events. Additional research with larger sample sizes and variable markers will help further genetic understanding of hawksbill turtles in the EP.     

A novel approach to improve specificity of algal biosensors using wild-type and resistant mutants: an application to detect TNT

A new genetic approach was developed for increasing specificity of microalgal biosensors. This method is based on the use of two different genotypes jointly to detect a given pollutant: (i) a sensitive genotype to obtain sensitivity; and (ii) a resistant mutant to obtain specificity. The method was tested by the development of a microalgal biosensor for the detection of the explosive 2,4,6-trinitrotoluene (TNT) using a wild-type strain (DcG1wt) of Dictyosphaerium chlorelloides (Chlorophyceae) as the sensitive organism, and a TNT-resistant mutant, obtained from DcG1wt strain by a modified Luria-Delbrück fluctuation analysis. The inhibition of chlorophyll a fluorescence of PSII by TNT was used as the biological signal. Significant differences in maximal fluorescence of light-adapted algae (F'(m)) between wild-type DcG1wt cells and TNT-resistant mutants, were observed in all the TNT concentrations tested (from 0.5 to 31.3 mg l(-1)) after only 3 min of exposure. Resistant mutants always exhibited significant higher F'(m) values in the presence of TNT than wild-type cells. These results suggest that the use of two different genotypes (sensitive and resistant to a given pollutant) jointly is a useful method to improve microalgal biosensors specificity.     

Presence of Cochlodinium catenatum (Gymnodiniales: Gymnodiniaceae) in red tides of Bahía de Banderas, Mexican Pacific

The evolution of an ichthiotoxic algal bloom caused by the dinoflagellate Cochlodinium catenatum was studied from July to December 2000. The abnormal multiplication of this dinoflagellate occurred in the form of a discoloration spreading between a temperature and salinity interval of 25-32 degrees C and 33-35 ups, respectively. The density of C. catenatum reached 10 841 cells ml(-1). The event was observed in large areas of Banderas Bay affecting 13 fish species, whose massive killing was due to suffocation (gill obstruction and excessive mucus production). The human population around the area did not present respiratory affections or skin irritation. The C. catenatum measurements suggest a hologamic and heterothalic reproduction. Their morphological characteristics suggest that C. polykrikoides, C. heterolobatum and C. catenatum are the same species. It is estimated that the species could be a recent introduction in the Mexican Pacific.     

Stage-dependent sensitivity to ultraviolet radiation in zygotes of the brown alga Fucus serratus

Sensitivity to ultraviolet (UV) radiation (UV-A, lambda = 315-400 nm; plus UV-B, lambda = 280-315 nm) of zygotes of the brown alga Fucus serratus L. (Phaeophyta) has been assessed through effects on growth of developing germlings. Different stages of development were distinguished by considering 5 h periods of time after fertilisation. Both the stage of the zygote and the UV radiation condition significantly affected growth of developing germlings. The negative response of growth rate of early stages of the zygotes to UV radiation seemed to be caused by UV-B rather than UV-A radiation, as the lowest relative growth rates were always estimated for germlings developed from zygotes irradiated with UV-B radiation. As regards the stage of the zygote, those germlings that developed from zygotes irradiated at 5-10 h after fertilisation showed the strongest inhibition of growth compared with the other stages. These results point to polarisation as the most UV-sensitive process during the first 24 h of the development of the zygote. A non-linear relationship between the developmental stage of the zygote and the sensitivity to UV radiation is suggested.