Physical bioenergetics: Energy fluxes, budgets, and constraints in cells

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics

ショウレイ ホウコク 100サイ ソウセイジ ト チョウジュ

日本では,各自治体の長が敬老の日に通常100歳以上の老人を訪間して,その長寿を祝う習わしがある。1992年には女性の100歳以上長寿者が3330人あり,この中に1組の100歳双生児姉妹がいた。この双生児は多くの遺伝マーカーによって1卵生双生児と診断された。健康診査により同年齢の者に比べて,極めて良好な健康状態であることが明らかになった。この理由としてバランスのとれた食事,規則正しい3回の食事摂取,十分な睡眠,毎日30分の運動等にみられる良好な生活習慣,ふたりに共通にみられる強い性格,ふたりのよい意味でのライバル意識などによると考えられた。journal articl

Observation of B0→D*-(5π)+, B+→D*-(4π)++ and B+→D̅*0 (5π)+

journal articl

Data_Sheet_1_Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain.docx

Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′ (R, A, or G) or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn2+ or Ni2+, and it has low activity in Mg2+ buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications.</p

＜研究ノート＞米国内国歳入法83 条の意義と機能 : リストリクテッド・ストックを中心に

departmental bulletin pape

ゲンゴ キノウ カイメイ ニ ムケタ トリクミ ニ カンスル チョウサ

奈良先端科学技術大学院大学修士(工学)master thesi

Data_Sheet_1_Expression and Purification of BsaXI Restriction Endonuclease and Engineering New Specificity From BsaXI Specificity Subunit.PDF

It is stated that BsaXI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5′↓N9 AC N5 CTCC N10↓ 3′ (complement strand 5′ ↓N7 GGAG N5 GT N12↓ 3′), creating 3-base 3′ overhangs. Here we report the cloning and expression of bsaXIS and bsaXIRM genes in Escherichia coli. The BsaXI activity was successfully reconstituted by mixing the BsaXI RM fusion subunit with the BsaXI S subunit and the enzyme complex further purified by chromatography over 6 columns. As expected, the S subunit consisted of two subdomains encoding TRD1-CR1 [target recognition domain (TRD), conserved region (CR)] for 5′ AC 3′, and TRD2-CR2 presumably specifying 5′ CTCC 3′. TRD1-CR1 (TRD2-CR2 deletion) or duplication of TRD1 (TRD1-CR1-TRD1-CR2) both generated a new specificity 5′ AC N5 GT 3′ when the S variants were complexed with the RM subunits. The circular permutation of TRD1 and TRD2, i.e., the relocation of TRD2-CR2 to the N-terminus and TRD1-CR1 to the C-terminus generated the same specificity with the RM subunits, although some wobble cleavage was detected. The TRD2 domain in the BsaXI S subunit can be substituted by a close homolog (∼59% sequence identity) and generated the same specificity. However, TRD2-CR2 domain alone failed to express in E. coli, but CR1-TRD2-CR2 protein could be expressed and purified which showed partial nicking activity with the RM subunits. This work demonstrated that like Type I restriction systems, the S subunit of a Type IIB system could also be manipulated to create new specificities. The genome mining of BsaXI TRD2 homologs in GenBank found more than 36 orphan TRD2 homologs, implying that quite a few orphan TRD2s are present in microbial genomes that may be potentially paired with other TRDs to create new restriction specificities.</p

Double digestion of the PEAR product by PspGI and Hpy99I.

<p>The sense and the antisense strand are indicated respectively by (+) and (<b>−</b>). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“<b> ̂</b>”). The antisense strands (<i>A</i>) are black boxed, the sense strands (<i>B</i> and <i>C</i>) are boxed, and the by-products (<i>D</i> and <i>E</i>) are grey boxed. The expected length for each strand is indicated in parenthesis.</p

Average recoverable concentration, purity and yield of the purified antisense oligonucleotide.

a<p>The product of round 1 was used as seeds for round 2, and that of round 2 for round 3.</p>b<p>Each run consisted of 95×100 µl reactions. Target and probe concentration are at 1 nM and 100 nM respectively.</p>c<p>Round 1 had two duplicate runs. As the first run had been used as seeds, the second run was used for purification and analysis.</p>d<p>Purity was calculated as peak area % at UV 260 nm.</p

Analytical HPLC analysis of purified antisense oligonucleotide.

<p>Sample preparation: 25 µL purified antisense oligonucleotide; Column: DNAPac PA-100 (4/250); Flow rate: 1 ml/min; Buffer A: 10 mM NaClO₄+1 mM Tris; Buffer B: 300 mM NaClO₄+1 mM Tris; Gradient: 10–70% B, 7.6 CV.</p