Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1

The aim of the present study is to explore whether membrane targeting of K⁺ channel-interacting protein 1 (KChIP1) is associated with its EF-hand motifs and varies with specific phospholipids. Truncated KChIP1, in which the EFhands 3 and 4 were deleted, retained the α-helix structure, indicating that the N-terminal half of KChIP1 could fold appropriately. Compared with wild-type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked loss of truncated KChIP1, suggesting that intact EF-hands 3 and 4 were crucial for the anchorage of KChIP1 on membrane. KChIP1 showed a higher binding capability with phosphatidylserine (PS) than truncated KChIP1. Unlike that of truncated KChIP1, the binding of wild-type KChIP1 with membrane was enhanced by increasing the PS content. Moreover, the binding of KChIP1 with phospholipid vesicles induced a change in the structure of KChIP1 in the presence of PS. Taken together, our data suggest that EF-hands 3 and 4 of KChIP1 are functionally involved in a specific association with PS on the membrane.     

Intervention of cardiomyocyte death based on real-time monitoring of cell adhesion through impedance sensing

Cardiomyocyte death caused by proinflammatory cytokines, such as Tumor necrosis factor α (TNF-α), is one of the hot topics in cardiovascular research. TNF-α can induce multiple cell processes that are dependent on the treatment time although the long-term treatment definitely leads to cell death. The ability to intervene in cell death will be invaluable to reveal the effects of short-term TNF-α treatment to cardiomyocytes. However, a real-time monitoring technique is needed to guide the intervention of cell responses. In this work, we employed the impedance-sensing technique to real-time monitor the equivalent cell–substrate distance of cardiomyocytes via electrochemical impedance spectroscopy (EIS) and electrical cell–substrate impedance sensing (ECIS). In the stabilized cardiomyocyte culture, the sustained TNF-α treatment caused strengthened cell adhesion in the first 2 h which was followed by the transition to cell detachment afterwards. Considering cell detachment was an early morphological evidence of cell death, we removed TNF-α from the cardiomyocyte culture before the transition to achieve the intervention of cell responses. The result of this intervention showed that cell adhesion was continuously strengthened before and after the removal of TNF-α, indicating the short-term treated cardiomyocytes did not undergo death processes. It was also demonstrated in TUNEL and TBE tests that the percentages of apoptosis and cell death were both lowered.     

椒江口滩涂大型底栖动物群落格局与多样性

为了解椒江口滩涂大型底栖动物群落格局与多样性, 揭示其对环境变化的响应规律, 作者于2007年10月、2008年1月、4月和7月在椒江口南岸和北岸潮间带, 沿河流到海洋方向共布设6条采样断面进行大型底栖动物调查。分析了大型底栖动物种类组成、栖息密度和生物量的时空变化特征, 在此基础上运用α, β和γ多样性测度方法对大型底栖动物多样性进行分析, 同时探讨了大型底栖动物群落结构对环境变化的响应方向及程度, 结果显示: (1) 6条断面共记录到大型底栖动物78种, 总种数随季节变化显著, 在空间上沿河流到海洋方向呈升高趋势; (2) 栖息密度的季节变化不显著(P=0.145>0.05), 但空间变化显著(P=0.017<0.05), 生物量的季节变化显著(P=0.012<0.05), 空间变化极显著(P=0.004<0.01); (3) β和γ多样性指数定量显示了椒江河口区域滩涂环境的多变性和大型底栖动物群落的多样性和更替性。     

Critical Factors Determining Dimerization of Human Antizyme Inhibitor

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K_d, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K_d value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K_d value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1–3). Ornithine decarboxylase (ODC,³ EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8–15). They also have been associated with human diseases and a variety of cancers (16–26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6, 11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity requires the formation of a dimer (29–31). X-ray structures of the ODC enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in PLP interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate (27, 32–41).ODC undergoes a unique ubiquitin-independent proteasomal degradation via a direct interaction with the regulatory protein antizyme (AZ). Binding of AZ promotes the dissociation of the ODC homodimers and targets ODC for degradation by the 26 S proteasome (42–46). Current models of antizyme function indicate that increased polyamine levels promote the fidelity of the AZ mRNA translational frameshift, leading to increased concentrations of AZ (47). The AZ monomer selectively binds to dimeric ODC, thereby inactivating ODC by forming inactive AZ-ODC heterodimers (44, 48–50). AZ acts as a regulator of polyamine metabolism that inhibits ODC activity and polyamine transport, thus restricting polyamine levels (4, 5, 51, 52). When antizymes are overexpressed, they inhibit ODC and promote ubiquitin-independent proteolytic degradation of ODC. Because elevated ODC activity is associated with most forms of human malignancies (1), it has been suggested that antizymes may function as tumor suppressors.In contrast to the extensive studies on the oncogene ODC, the endogenous antizyme inhibitor (AZI) is less well understood. AZI is homologous to the enzyme ODC. It is a 448-amino acid protein with a molecular mass of 50 kDa. However, despite the homology between these proteins, AZI does not possess any decarboxylase activity. It binds to antizyme more tightly than does ODC and releases ODC from the ODC-antizyme complex (53, 54). Both the AZI and AZ proteins display rapid ubiquitin-dependent turnover within a few minutes to 1 h in vivo (5). However, AZ binding actually stabilizes AZI by inhibiting its ubiquitination (55).AZI, which inactivates all members of the AZ family (53, 56), restores ODC activity (54), and prevents the proteolytic degradation of ODC, may play a role in tumor progression. It has been reported that down-regulation of AZI is associated with the inhibition of cell proliferation and reduced ODC activity, presumably through the modulation of AZ function (57). Moreover, overexpression of AZI has been shown to increase cell proliferation and promote cell transformation (58–60). Furthermore, AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function (60, 61). These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis.It is now known that ODC exists as a dimer and that AZI may exist as a monomer physiologically (62). Fig. 1 shows the dimeric structures of ODC (Fig. 1A) and AZI (Fig. 1B). Although structural studies indicate that both ODC and AZI crystallize as dimers, the dimeric AZI structure has fewer interactions at the dimer interface, a smaller buried surface area, and a lack of symmetry of the interactions between residues from the two monomers, suggesting that the AZI dimer may be nonphysiological (62). In this study, we identify the critical amino acid residues governing the difference in dimer formation between ODC and AZI. Our preliminary studies using analytical ultracentrifugation indicated that ODC exists as a dimer, whereas AZI exists in a concentration-dependent monomer-dimer equilibrium. Multiple sequence alignments of ODC and AZI from various species have shown that residues 277, 331, 332, and 389 are not conserved between ODC and AZI (Open in a separate windowFIGURE 1.Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).

TABLE 1

Amino acid residues at the dimer interface of human ODC and AZI

An efficient regeneration system of barley cultivars from leaf base segments

A simple and reliable regeneration system from leaf bases of barley (Hordeum vulgare L.) has been developed. The in vitro regeneration frequencies of seven commercial barley genotypes were compared using segments from the first leaves of 5-d-old seedlings. The regeneration frequency ranged from 31.56 to 72.22 % among the barley genotypes. Murashige and Skoog medium supplemented with 6-benzyladenine (0.5 mg dm⁻³) and kinetin (0.5 mg dm⁻³) was optimum for the regeneration. Longitudinal cut of the segments or the removal of coleoptiles further increased plantlet regeneration frequency.     

Tissue softening of guinea pig oesophagus tested by the tri-axial test machine

Tissue softening is commonly reported during mechanical testing of biological tissues in vitro. The loss of stiffness may be due to viscoelasticity-induced softening (the time-history of load-caused softening) and strain-induced stress softening (the maximum previous load-caused softening). However, the knowledge about tissue softening behaviour is presently poor. The aims of this study were to distinguish whether the loss of the stiffness during preconditioning was due to strain softening or viscoelasticity and to test the tissue softening in circumferential and longitudinal direction in the guinea pig oesophagus. Eight repeated pressure controlled ramp distensions and eight uniaxial tensile-release ramp stretches in three series were done on eight guinea pig oesophagi. The stress–strain curves were used to display the time-dependency (viscoelasticity) and the maximum previous load-caused softening (strain softening) in circumferential and longitudinal directions. For both the longitudinal and the circumferential softening, the peak stress and stiffness produced during the first loading were bigger than those produced in the remaining loadings. The stress loss due to strain softening was about three times more than that due to viscoelasticity in the longitudinal direction. The strain increased more than two times between the strain softening and viscoelastic softening in the circumferential direction. With a stress level of 20 kPa, the stiffness in the circumferential direction lost more than that in the longitudinal direction (P<0.05), indicating the anisotropic softening properties in the oesophagus. In conclusion, the stiffness loss during preconditioning is mainly attributed to strain softening, appears irreversible and is anisotropic.     

RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.