Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Alteration of division polarity and preprophase band orientation in stomatogenesis by light

We have developed an experimental system in which the irradiation with a red light pulse induces stomatal disorientation in the hypocotyl epidermis ofCucumis sativus L. In this system, the orientation of the division plane in guard mother cells was not defined correctly. Preprophase bands formed in these cells but their orientation was abnormal.     

The structure of 180° supernumerary limbs and a hypothesis of their formation

The results of a detailed analysis of 100 supernumerary limbs generated by 180° ipsilateral rotation (on the same limb stump) of regeneration blastemas is presented. The limbs were analyzed in terms of their position of origin, frequency, cartilage structure by Victoria blue staining, and muscle structure by serial sections. Single, double, or triple supernumeraries can be produced at no unique position of origin, although the posterodorsal quadrant was preferred. Four classes of supernumerary limbs were generated by such operations—normal; double dorsal or double ventral; part normal/part mirror imaged; part normal/part inverted in approximately equal frequencies. After amputation of these supernumeraries the same muscle patterns are faithfully regenerated. A hypothesis to explain the production of these abnormal limbs is proposed based on the observed phenomenon of fusion of supernumerary blastemata, but their regenerative behaviour presents problems for current models of pattern formation. Similar results have been obtained with developing limb buds and the relation between development and regeneration is discussed.     

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.     

Creatine kinase of heart mitochondria: Changes in its kinetic properties induced by coupling to oxidative phosphorylation

A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: K_ib = 29 mm; K_b = 5.3 mM, and the maximum velocity of the reaction was unchanged: V₁ = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant (K_ia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 mm in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant (K_a) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 mm with exogenous ATP and 0.014 mm with oxidative phosphorylation. Since K_ia × K_b does not equal K_a × K_ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.     

Temperature effects of intraventricular serotonin,norepinephrine and pilocarpine in the morphine-tolerant rat

Studies were undertaken to examine the possibility that changes occur in the responsiveness of thermoregulatory neurons in the anterior hypothalamus to endogenously released neurotransmitters upon the development of tolerance to morphine. In experiments conducted at an environmental temperature of 20–21°C, tolerance produced by the subcutaneous implantation of morphine alkaloid pellets failed to alter the temperature response of rats to intraventricular injections of 5-HT (10 μg), NE (20 μg) or pilocarpine (100 μg). It is concluded that tolerance development to morphine-induced hypothermia is not a result of changes in the postsynaptic sensitivity to the putative neurotransmitters in the thermoregulatory center.     

Begg矫治器和直丝弓矫治器联合治疗安氏Ⅱ类1 分类错颌畸形的临床 疗效分析

目的:探讨Begg矫治器和直丝弓矫治器联合治疗安氏Ⅱ类1分类错颌畸形的临床疗效。方法:采取回顾性分析方法,调阅2010-2013年徐州矿务集团总医院安氏Ⅱ类1分类错颌畸形患者病历资料,选择病历资料完整且符合本研究要求的正畸病人病历44例进行分析,实验组22例为Begg矫治器和直丝弓矫治器联合治疗组,对照组22例为单纯直丝弓治疗组。对治疗前后的头影测量片进行扫描分析,对治疗时间及辅助支抗应用情况进行统计分析。结果:治疗完成时,Begg矫治器和直丝弓矫治器联合治疗组和单纯直丝弓治疗组的前牙覆合覆盖均减少,上切牙切缘均向远中移动,上下磨牙均伸长,前下面高度均增加,两组治疗结束后硬组织的变化无统计学差异(P0.05)。Begg矫治器和直丝弓矫治器联合治疗组与单纯直丝弓治疗组相比,治疗时间短,使用辅助支抗少,差异具有统计学意义(P0.05)。结论:Begg矫治器和直丝弓矫治器联合矫治安氏Ⅱ类1分类错颌畸形是临床上一种速度快,费用低,效果优的治疗方法。     

The induction of cell-mediated immunity and tolerance to insulin with insulin coupled to syngeneic lymphoid cells

The induction of delayed type hypersensitivity (DTH) and tolerance to DTH against bovine insulin in mice were explored. DTH was induced with insulin in complete Freund's adjuvant (CFA) and was assessed by ear swelling in vivo and by antigen-driven cell proliferation in vitro. Using the concept that thymus cell unresponsiveness is most easily accomplished via antigen on syngeneic membranes, tolerance was induced by iv injection of syngeneic lymphoid cells which had been coupled to insulin with carbodiimide. Mice tolerized with insulin-coupled cells and then sensitized with insulin-CFA had diminished ear swelling in vivo and decreased insulin-driven cell proliferation in vitro. This unresponsiveness was antigen specific but was also inconstant in degree with regard to suppression of ear swelling, most likely because of variability in coupling of insulin to cells. Proliferative responses were more uniformly suppressed, suggesting the possibility that two target cells were being tolerized. Thus, as with other proteins, the biologically active insulin can be used to induce tolerance.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

MinC Protein Shortens FtsZ Protofilaments by Preferentially Interacting with GDP-bound Subunits

The interaction of MinC with FtsZ and its effects on FtsZ polymerization were studied under close to physiological conditions by a combination of biophysical methods. The Min system is a widely conserved mechanism in bacteria that ensures the correct placement of the division machinery at midcell. MinC is the component of this system that effectively interacts with FtsZ and inhibits the formation of the Z-ring. Here we report that MinC produces a concentration-dependent reduction in the size of GTP-induced FtsZ protofilaments (FtsZ-GTP) as demonstrated by analytical ultracentrifugation, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. Our experiments show that, despite being shorter, FtsZ protofilaments maintain their narrow distribution in size in the presence of MinC. The protein had the same effect regardless of its addition prior to or after FtsZ polymerization. Fluorescence anisotropy measurements indicated that MinC bound to FtsZ-GDP with a moderate affinity (apparent K_D ∼10 μm at 100 mm KCl and pH 7.5) very close to the MinC concentration corresponding to the midpoint of the inhibition of FtsZ assembly. Only marginal binding of MinC to FtsZ-GTP protofilaments was observed by analytical ultracentrifugation and fluorescence correlation spectroscopy. Remarkably, MinC effects on FtsZ-GTP protofilaments and binding affinity to FtsZ-GDP were strongly dependent on ionic strength, being severely reduced at 500 mm KCl compared with 100 mm KCl. Our results support a mechanism in which MinC interacts with FtsZ-GDP, resulting in smaller protofilaments of defined size and having the same effect on both preassembled and growing FtsZ protofilaments.