Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel.We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from ∼4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm³, i.e. about 40% of the channel's volume in the high conductance state.     

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 µm²). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta_1-40 (Aβ_1-40) with or without a therapeutic intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.     

液体复苏对于严重脓毒症患者生存率的预后分析

目的:严重脓毒症中液体复苏的生理病理学理论支持及动物实验证据不足,有必要进行相关临床试验来确定液体复苏对于严重脓毒症患者预后的影响。方法:将26名严重脓毒症和脓毒症休克患者分为液体复苏组和非液体复苏组。比较两组的28天生存率,ICU住院天数的差别,对两组在院生存天数进行生存函数分析。结果:液体复苏组的28天生存率为41.7%,未液体复苏组的28天生存率为50.0%,之间没有统计学差异(P=0.713)。液体复苏组的平均住院天数为24.7±6.0天,未液体复苏组的平均住院天数为17.7±3.4天,两组之间没有统计学差异(P=0.308)。液体复苏组的中位在院生存天数为38.0[28.0,48.0],液体复苏组的中位在院生存天数为25.0[22.8,27.2]。两组之间有显著统计学差异,(Log Rank P=0.044,Breslow P=0.025)。结论:在严重脓毒症及脓毒症休克患者中,采取液体复苏能够延长患者的中位在院生存天数,可见液体复苏在严重脓毒症的治疗中有重要意义。尚未发现液体复苏能提高严重脓毒症患者28天生存率的相关证据。采取液体复苏究竟能否改善严重脓毒症患者预后,还需要大样本随机临床对照试验来证明。     

Search and Analysis of Identical Reverse Octapeptides in Unrelated Proteins

For the past few decades, intensive studies have been carried out in an attempt to understand how the amino acid sequences of proteins encode their three dimensional structures to perform their specific functions. In order to understand the sequence-structure relationship of proteins, several sub-sequence search studies in non-redundant sequence-structure databases have been undertaken which have given some fruitful clues. In our earlier work, we analyzed a set of 3124 non-redundant protein sequences from the Protein Data Bank (PDB) and retrieved 30 identical octapeptides having different secondary structures. These octapeptides were characterized by using different computational procedures. This prompted us to explore the presence of octapeptides with reverse sequences and to analyze whether these octapeptides would adopt similar structures as that of their parent octapeptides. Our identical reverse octapeptide search resulted in the finding of eight octapeptide pairs (octapeptide and reverse octapeptide) with similar secondary structure and 23 octapeptide pairs with different secondary structures. In the present work, the geometrical and biophysical characteristics of identical reverse octapeptides were explored and compared with unrelated octapeptide pairs by using various computational tools. We thus conclude that proteins containing identical reverse octapeptides are not very abundant and residues in the octapeptide pairs do not contribute to the stability of the protein. Furthermore, compared to unrelated octapeptides, identical reverse octapeptides do not show certain biophysical and geometrical properties.     

Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers,Apis mellifera

Pollinators, honey bees in particular, are continuously exposed to various mixtures of pesticides, which contribute to their population decline. Both amitraz and thiacloprid have been proven less toxic to honey bees and are frequently applied in- and out-hive, respectively. We examined the sub-lethal effects of amitraz, thiacloprid and their sequential exposure on learning, memory and sugar responsiveness in Apis mellifera using the Proboscis extension response (PER). Sub-lethal doses of amitraz (0.1, 0.2 and 0.4 µg/bee) and thiacloprid (0.05, 0.1 and 0.2 µg/bee) were tested. Sub-lethal effects were observed only at the highest doses of each pesticide treatment; amitraz (0.4 µg/bee) and thiacloprid (0.2 µg/bee) but not in lower doses. In sequential treatment of amitraz and thiacloprid, reduced acquisition and memory retention were significant across all tested doses. The same profile was also obtained on sugar responsiveness of foragers. Our results suggest that the sequential exposure would pose higher risk to honey bee compared to single pesticide exposure by reducing the bees’ appetitive olfactory learning, memory and sugar acuity more than individual pesticide exposures.     

EXPERIMENTAL EVOLUTION OF HETEROTROPHY IN A GREEN ALGA

Laboratory populations of a green alga cultured in the dark with an organic substrate evolved into efficient heterotrophs with faster growth, higher fitness, and increased responsiveness to substrate concentration. Their phenotypes were almost entirely attributable to selection, rather than to history or ancestry. The fitness of evolved lines in the light was uniformly depressed, presumably through the accumulation of conditionally deleterious genes governing photosynthesis. Some evolved lines were no longer able to grow in the light and are thereby permanently isolated from their ancestors. Specialized autotrophs and heterotrophs may often evolve in algae through long‐term shifts in the conditions of growth.     

IKCa agonist (NS309)-elicited all-or-none dehydration response of human red blood cells is cell-age dependent

Elevated [Ca²⁺]_i in human red blood cells (RBCs) activates IK1 K⁺ channels leading to cell dehydration. NS309, a powerful IK1 agonist, has been shown to activate IK1 channels even at sub-physiological [Ca²⁺]_i levels. An intriguing feature of this response is its all-or-none nature, with responsive cells dehydrating fully and refractory cells retaining normal volume. We investigated the mechanism of this response suspecting cell-age involvement. We expected the younger cells, with the more vigorous plasma membrane Ca²⁺ pumps (PMCA), to be the refractory cells because of their lower [Ca²⁺]_i. Osmotic fragility measurements and density separation through phthalate oil were used to monitor red cell dehydration. The fraction of glycosilated haemoglobin (Hb A1c) was used to estimate the mean age of density fractionated cells. The results showed that inhibition of the PMCA by vanadate abolished the all-or-none response, that the mean age of the responsive cells was young, contrary to expectations, and that pump inhibition subsequent to an all-or-none response caused the refractory cells to dehydrate fully, suggesting that the all-or-none response resulted from a reduced efficiency of NS309 to increase the Ca²⁺ sensitivity of IK1 channels in aged RBCs.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

The Lens Circulation

The lens is the largest organ in the body that lacks a vasculature. The reason is simple: blood vessels scatter and absorb light while the physiological role of the lens is to be transparent so it can assist the cornea in focusing light on the retina. We hypothesize this lack of blood supply has led the lens to evolve an internal circulation of ions that is coupled to fluid movement, thus creating an internal micro-circulatory system, which makes up for the lack of vasculature. This review covers the membrane transport systems that are believed to generate and direct this internal circulatory system.