A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Macromolecular organization of the Yersinia pestis capsular F1 antigen: insights from time-of-flight mass spectrometry

Mass spectrometry has been used to examine the subunit interactions in the capsular F1 antigen from Yersinia pestis, the causative agent of the plague. Introducing the sample using nanoflow electrospray from solution conditions in which the protein remains in its native state and applying collisional cooling to minimize the internal energy of the ions, multiple subunit interactions have been maintained. This methodology revealed assemblies of the F1 antigen that correspond in mass to both 7-mers and 14-mers, consistent with interaction of two seven-membered units. The difference between the calculated masses and those measured experimentally for these higher-order oligomers was found to increase proportionately with the size of the complex. This is consistent with a solvent-filled central cavity maintained on association of the 7-mer to the 14-mer. The charge states of the ions show that an average of one and four surface accessible basic side-chains are involved in maintaining the interactions between the 7-mer units and neighboring subunits, respectively. Taken together, these findings provide new information about the stoichiometry and packing of the subunits involved in the assembly of the capsular antigen structure. More generally, the data show that the symmetry and packing of macromolecular complexes can be determined solely from mass spectrometry, without any prior knowledge of higher order structure     

Tolrestat对高糖所致肾小球系膜细胞肌动蛋白去组装的影响

研究了醛糖还原酶抑制剂Tolrestat对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白(actin)组装的影响。结果证明,与正常浓度葡萄糖(NG)相比,在HG培养的MC,F-actin失去束状外观呈不规则网状,显示F-actin部分去组装;F-actin荧光强度降低,G-actin荧光强度升高和F-/G-actin荧光强度比值下降。Tolrestat加入培养后,明显防止HG引起的F-actin去组装及F-和G-actin荧光强度的变化。提示多元醇通路激活在HG引起的MCactin去组装改变中起一定作用。     

Addition of HOXt (X = A or B) improves the efficiency of phenol-based coupling reagents during peptide synthesis

Summary In the course of comparing the effectiveness of HATU, HBTU, and phenol-based coupling reagents, such as the pentafluorophenyl, 2-nitrophenyl, and 2,4,5-trichlorophenyl uronium salts by (a) formation of Fmoc-Ala-Val-OtBu, (b) (2+1) segment coupling and (c) stepwise solid phase peptide assembly of typical model peptides such as the pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH₂ and ACP decapeptide (65–74), we found a striking improvement of the less effective phenol-based coupling reagents (HPyOPfp, HPyONp, and HPyOTcp), both with regard to reaction rate and extent of epimerization, when HOAt was added and a clear superiority of HAPyU (in the presence and absence of HOAt) relative to the compounds derived from HOBt, HOPfp, HONp, and HOTcp. Abbreviations: Aib, α-aminoisobutyric acid; DIEA, diisopropylethylamine; TMP, collidine, 2,4,6,-tri-methylpyridine; DMF, N, N-dimethylformamide; HOBt, 1-hydroxybenzotriazole; HOAt, 7-aza-1-hydroxybenzotriazole; HAPyU, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium hexafluorophosphate N-oxide; HOPfp, pentafluorophenol; HONp, 2-nitrophenol; HOTcp, 2,4,5-trichlorophenol; HPyOPfp,bis(tetramethylene)pentafluorophenoxyformamidinium hexafluorophosphate; HpyONp,bis(tetramethylene)-2-nitrophenoxyformamidinium hexafluorophosphate; HPyOTcp,bis(tetramethylene)-2,4,5-trichlorophenoxyformamidinium hexafluorophosphate; BTCFHbis(tetramethylene)chloroformamidinium hexafluororophosphate. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977]     

Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.     

The effect of ruthenium red on the assembly and disassembly of microtubules and on rapid axonal transport

The assembly of microtubules was found to decrease in proportion to the amount of added ruthenium red, indicating a high affinity of ruthenium red for the microtubule system. An equimolar amount of ruthenium red per tubulin dimer inhibited the microtubule assembly completely and disassembled existing microtubules. Binding of ruthenium red to tubulin is accompanied by a shift in the absorption maximum from 535 to 538 nm. The binding is very strong, as shown by the finding that ruthenium red could not be displaced from tubulin by gel chromatography on Sephadex, or by the addition of Ca²⁺ or Mg²⁺. The binding of ruthenium red to tubulin did not affect the single colchicine site, nor the Mg²⁺ site(s), as shown by use of Mn²⁺ as an EPR probe. Ruthenium red also interfered with microtubules in an intact cell system, as it inhibited rapid axonal transport in the frog sciatic nerve, measured by the accumulation of [³H]leucine-labelled proteins in front of a ligature.     

Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation

Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24 h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic dsDNA helix could be advantageous for cell replication such as bacterial binary fission and cell mitosis without the need for topoisomerase or helicase to consume additional energy.