Functional Roles of Charged Amino Acid Residues on the Wall of the Cytoplasmic Pore of Kir2.1

It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance

Perancangan Perangkat Lunak Diagnostik untuk Analisis Ligamen Bahu dengan Pencitraan Ultrasonik

Makalah ini menjelaskan implementasi perangkat lunak dengan antar muka pengguna grafis (GUI) untuk menganalisis citra bergerak ultrasonik dari ligament bahu. Perangkat lunak yang dirancang dapat bermanfaat di bidang medis khususnya sebagai alat bantu identifikasi penyakit yang berhubungan dengan kelainan gerak dan nyeri pada bahu. Pada prinsipnya, perangkat lunak yang dirancang bekerja dengan cara memvisualisasikan lapisan jaringan di bahu berdasarkan kecepatannya selama gerakan melalui metode speckle tracking citra B-mode ultrasonik. Ligamen dalam bahu yang mengalami pelekatan dapat diamati dan diukur secara kuantitatif dalam besaran indeks dengan cara tersebut. Indeks tersebut dihitung dari perbedaan kecepatan antara otot subscapularis dan otot deltoid bahu. Hasil pengamatan menunjukkan bahwa pasien dengan restriksi gerakan pada bahu mengalami pelekatan subscapularis yang ditunjukkan oleh nilai Indeks Adhesi (IA) yang tinggi. Hal ini menunjukkan bahwa keterbatasan gerak bahu dapat dikuantisasi dengan besaran indeks. Kelebihan dari penggunaan perangkat lunak analisis citra ultrasonik sebagai alat bantu diagnosis adalah sifatnya yang non-invasif dan semua data pengukuran dapat diambil dengan menggunakan mesin ultrasonografi konvensional

Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X2 channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X2 is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X2 even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X2-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP]

C-Arm-Free Minimally Invasive Cervical Pedicle Screw Fixation (MICEPS): A Technical Note

A minimally invasive posterolateral approach designed to avoid the lateral misplacement of midcervical pedicle screws was reported, but there is no technical report that describes this technique without C-arm fluoroscopy. We report the results of a 2.5 years follow-up of a 62-year-old female patient with C4 metastatic breast cancer. The patient suffered from severe neck pain and impending quadriplegia for 2 months after radiation therapy. We performed C-arm-free minimally invasive cervical pedicle screw fixation (MICEPS). The patient was suc-cessfully treated with surgery, and her neck pain was well controlled. She had neither neurological deficits nor neck pain at the final (2.5-year) follow-up. C-arm-free MICEPS is a useful technique; in addition, the sur-geons and staff have no risk of radiation exposure, there is a reduced need for postoperative imaging, and a decreased revision rate can be expected with C-arm-free MICEPS

海底に沈下した鯨骨下に棲むゲイコツケシハマグリ（新種）

2002年に薩摩半島野間岬沖に沈設した鯨の遺骸下の硫化水素を豊富に含んだ海底沈積物中から発見された，微小なケシハマグリ科の1新種を記載する。 “Kelliella” ossisocian. sp.　ゲイコツケシハマグリ（和名新称） 殻長7 mmの微小種で，殻はハマグリ型で，同属の他種に比しやや前後に長く，且つ殻頂も秀いでない。殻表は白色で成長線は時に弱い褶状になり，極めて薄い殻皮を被る。小月面は浅い溝で区切られ，楯面は不分明。前後の閉殻筋痕は同型同大。外套湾入は無い。右殻の主歯は半月形で，弧状の殻頂下歯との間に深い歯槽が出来る。左殻の殻頂下歯は前肢は溝を伴った半月形であるが，後肢は逆V字型。後主歯は太く斜位。本種は野間崎沖の水深226 mから得られたホロタイプのみ知られる。鯨骨下の還元層に棲むと思われるが，本属の他種は漸深海底帯から超深海底帯から知られ，このように浅海からの出現は極めて異例である。 本種は整ったハマグリ型であるが，既知種の多くは丸みが強く，殻頂部は著しく聳えて強く前傾する。Kelliella属 は多系統と思われるので，本種の属位は決定的ではない。チトクロームオキシダーゼ サブユニット1 （COI） 遺伝子の部分長塩基配列に基づく予察的な分子系統解析（藤原・他，未発表）によれば，本種はオトヒメハマグリ類のクレードの外群となることが示されている。http://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt07-09/

The complete mitochondrial genome sequence of the hydrothermal vent galatheid crab Shinkaia crosnieri (Crustacea: Decapoda: Anomura): A novel arrangement and incomplete tRNA suite

<p>Abstract</p> <p>Background</p> <p>Metazoan mitochondrial genomes usually consist of the same 37 genes. Such genes contain useful information for phylogenetic analyses and evolution modelling. Although complete mitochondrial genomes have been determined for over 1,000 animals to date, hydrothermal vent species have, thus far, remained excluded due to the scarcity of collected specimens.</p> <p>Results</p> <p>The mitochondrial genome of the hydrothermal vent galatheid crab <it>Shinkaia crosnieri </it>is 15,182 bp in length, and is composed of 13 protein-coding genes, two ribosomal RNA genes and only 18 transfer RNA genes. The total AT content of the genome, as is typical for decapods, is 72.9%. We identified a non-coding control region of 327 bp according to its location and AT-richness. This is the smallest control region discovered in crustaceans so far. A mechanism of cytoplasmic tRNA import was addressed to compensate for the four missing tRNAs. The <it>S. crosnieri </it>mitogenome exhibits a novel arrangement of mitochondrial genes. We investigated the mitochondrial gene orders and found that at least six rearrangements from the ancestral pancrustacean (crustacean + hexapod) pattern have happened successively. The codon usage, nucleotide composition and bias show no substantial difference with other decapods. Phylogenetic analyses using the concatenated nucleotide and amino acid sequences of the 13 protein-coding genes prove consistent with the previous classification based upon their morphology.</p> <p>Conclusion</p> <p>The present study will supply considerable data of use for both genomic and evolutionary research on hydrothermal vent ecosystems. The mitochondrial genetic characteristics of decapods are sustained in this case of <it>S. crosnieri </it>despite the absence of several tRNAs and a number of dramatic rearrangements. Our results may provide evidence for the immigrating hypothesis about how vent species originate.</p

Phylogenetic position of a whale-fall lancelet (Cephalochordata) inferred from whole mitochondrial genome sequences

<p>Abstract</p> <p>Background</p> <p>The lancelet <it>Asymmetron inferum </it>(subphylum Cephalochordata) was recently discovered on the ocean floor off the southwest coast of Japan at a depth of 229 m, in an anaerobic and sulfide-rich environment caused by decomposing bodies of the sperm whale <it>Physeter macrocephalus</it>. This deep sulfide-rich habitat of <it>A. inferum </it>is unique among the lancelets. The distinguishing adaptation of this species to such an extraordinary habitat can be considered in a phylogenetic framework. As the first step of reconstruction of the evolutionary processes in this species, we investigated its phylogenetic position based on 11 whole mitochondrial genome sequences including the newly determined ones of the whale-fall lancelet <it>A. inferum </it>and two coral-reef congeners.</p> <p>Results</p> <p>Our phylogenetic analyses showed that extant lancelets are clustered into two major clades, the <it>Asymmetron </it>clade and the <it>Epigonichthys </it>+ <it>Branchiostoma </it>clade. <it>A. inferum </it>was in the former and placed in the sister group to <it>A. lucayanum </it>complex. The divergence time between <it>A. inferum </it>and <it>A. lucayanum </it>complex was estimated to be 115 Mya using the penalized likelihood (PL) method or 97 Mya using the nonparametric rate smoothing (NPRS) method (the middle Cretaceous). These are far older than the first appearance of large whales (the middle Eocene, 40 Mya). We also discovered that <it>A. inferum </it>mitogenome (mitochondrial genome) has been subjected to large-scale gene rearrangements, one feature of rearrangements being unique among the lancelets and two features shared with <it>A. lucayanum </it>complex.</p> <p>Conclusion</p> <p>Our study supports the monophyly of genus <it>Asymmetron </it>assumed on the basis of the morphological characters. Furthermore, the features of the <it>A. inferum </it>mitogenome expand our knowledge of variation within cephalochordate mitogenomes, adding a new case of transposition and inversion of the <it>trnQ </it>gene. Our divergence time estimation suggests that <it>A. inferum </it>remained a member of the Mesozoic and the early Cenozoic large vertebrate-fall communities before shifting to become a whale-fall specialist.</p