国产堇菜属的分子系统学研究

利用基于ITS序列的分子系统学对国产堇菜属(Viola L.)进行了系统发育重建,选择Hybanthus concolor和鳞隔堇(Scyphellandra pierrei)作为外类群,对国产堇菜属植物54种1亚种共66个序列进行分析。分子系统树中获得强烈支持的中国堇菜属属下组级分类群有Sect.Viola、Sect.Diffusae、Sect.Bilobatae、Sect.Vaginatae。Subgen.Melanium与Sect.Viola和Sect.Trigonocarpae聚在一起,说明它们的亲缘关系较近,但Sect.Trigonocarpae支获得的支持率较低。分子系统树证实了Subgen.Dischidium与Subgen.Chamaemelanium的近缘关系,但不支持根据距的长短将Subgen.Dischidium划分为Sect.Longicalcaratae和Sect.Brevicalcaratae的处理。而Pinnatae类群应该作为Sect.Adnatae下的一个亚组处理。根据分子系统发育和形态学特征对V.lucens、V.magnifica、V.dissecta、V.yunnanensis等的分类地位进行了重新评价。     

堇菜属(堇菜科)植物新资料

对堇菜科堇菜属Viola的两个种进行了组合。认为过去在东南亚植物文献上记载的Viola curvistylis实为我国云南、海南等地产的云南堇菜V．yunnanensis,前者为后者的异名;而我国文献上记载的光叶堇菜V．hossei实为东南亚分布的V．sumatrana,前者为后者的异名。文中引用的标本全部存放于邱园(Kew)标本馆。     

河北堇菜属植物的进一步研究

本文着重运用聚类分析的方法,对河北地区堇菜属植物进行研究,确定本属中组和亚组的划分界限,并通过对形态学特征和地理分布式样的分析,确认蒙古堇菜(V. mongolica Franch．)和北京堇菜(V．pekinensis(Regel) W．Beck．)实属同一个种,北京堇菜不应作为独立的种存在。通过将美丽堇菜组(Sect．Melanium Ging．)与其它类群进行比较,发现差异很大,超出了组和组间的相似性范围,因而支持Juzepczuk(1949)将它提升为亚属的分类处理。     

蔓茎堇菜和柔毛堇菜多糖的抑菌抗氧化活性

利用乙醇沉淀法提取蔓茎堇菜Viola diffusa和柔毛堇菜V.principis多糖并分别进行抑菌及抗氧化试验。结果表明,蔓茎堇菜和柔毛堇菜多糖提取率分别为7.0%和8.3%。不同倍数体积无水乙醇沉淀提取的多糖抑菌和抗氧化能力不同。抑菌效果显示,蔓茎堇菜多糖对大肠杆菌和枯草芽孢杆菌的抑菌圈分别可达8.46mm和8.59mm,柔毛堇菜对大肠杆菌和枯草芽孢杆菌的抑菌圈均可达9.13mm,但两种堇菜多糖对黑曲霉和啤酒酵母未呈现抑制活性;抗氧化研究发现,蔓茎堇菜多糖抗氧化活性为243.64U·mL^-1,柔毛堇菜多糖抗氧化活性为411.78U·mL^-1。由此可见,无论是抑菌还是抗氧化活性方面,柔毛堇菜极显著优于蔓茎堇菜(P<0.01)。蔓茎堇菜和柔毛堇菜多糖都具有一定的抑菌抗氧化活性,均可作为食药两用植物资源进行开发利用。     

中国堇菜科小志：（二）堇菜属植物细胞学的初步研究

本文报道了国产堇菜属9种植物的染色体数目,其中5种为染色体计数新纪录。结果表明,该属植物的染色体数目与柱头形态之间具有明显的相关性,它们在属下各类群内是一致的,而在各类群间则存在明显的差异,支持了多数学者根据柱头形态特征所做的属下类群的分类处理。V.kunawarensisRoyle的染色体计数结果2n=20,充分说明根据柱头形态把该种从Sect.Adnatae(W.Beck.)C.J.Wang移到Sect.Trigonocarpae Godr.是正确的。     

中国堇菜（堇菜科）两新异名

将《中国植物志》中未收录的我国堇菜属2种Viola hupeiana W.Beck.和Violapulla W.Beck.分别处理为Violaverecunda A.Gray和Violalucens W.Beck.的异名。     

中国云南8种堇菜属植物的叶形态解剖特征及分类学意义

堇菜属（Viola）植物种类多、分布广、形态变异大,物种鉴定和分类系统争议较大。因此,为解决该属物种的分类学问题,以中国云南产的8种堇菜属植物为研究对象,利用显微镜和石蜡切片技术观察叶形态解剖特征,结果如下（：1）叶型可分为4种：肾形、戟形、卵形和三深裂（;2）叶片边缘和叶脉处稀疏分布着单细胞单列毛状体,可分为3类：短柱状、中柱状和长柱状（;3）叶表皮细胞垂周壁式样有平直-弓形、浅波状和深波状,气孔器类型有平列型、横列型和无规则型;气孔多为椭圆形,少数为近圆形;（4）叶中脉处上下表皮突起有均等型和不等型（;5）叶柄横切面轮廓和主维管束形态相关,前者为椭圆形、半圆形和近圆形,后者为浅U型、深U型和圆形。分析结果表明,堇菜属的叶型、表皮毛被、叶表皮细胞形态、气孔类型、叶中脉处的上下表皮形态、叶柄横切面及主维管束轮廓等,在种间差异明显,可以用作属内物种划分和近似种间的区别,支持长萼堇菜（V. inconspicua）、早开堇菜（V. prionantha）和紫花地丁（V. philippica）是一组有近缘关系的独立种。     

球果堇菜一新变种

本文发表了堇菜属球果堇菜一新变种,即光果球果堇菜Viola collina Bess.var.glabricarpa K.Sun.     

广东堇菜属植物新记录

报道了广东省3种新分布堇菜属植物,分别为犁头叶堇菜Viola magnifica Ching J.Wanget X.D.Wang、白花戟叶堇菜Viola betonicifolia Sm.var.albescens(Nakai)F.Maek.et T.Hashim.和假如意草Viola pseudo-arcuata C.C.Chang。     

中国堇菜属美丽堇菜亚属(堇菜科)植物分类学修订

对中国堇菜属美丽堇菜亚属植物进行了系统学修订,确认中国堇菜属美丽堇菜亚属植物共有6种,列出分种检索表,对《中国植物志》51卷中未记载的3种进行了详细的描述,并对塔城堇菜、深紫堇菜及隐距堇菜进行了绘图。     

Mast cell-independent impairment of host defense and muscle contraction in T. spiralis-infected W/W(V) mice

In response to nematode infection, the host presumably attempts to create an unfavorable environment to prevent larval penetration of the host and to expedite parasite expulsion from the gut. In this study, we have used W/W(V) mice with or without mast cells after bone marrow reconstitution (BMR-W/W(V)) to examine the role of mast cells in the host response. W/W(V), BMR-W/W(V), and wild-type (+/+) mice were infected with Trichinella spiralis. Infected W/W(V) mice exhibited less tissue damage and experienced a delay in worm expulsion and a greater degree of larval penetration of the gut leading to encystment in skeletal muscle. Tissue injury was greater and worm expulsion was normalized in BMR-W/W(V) mice, but larval penetration remained unchanged. Spontaneous contractile activity of jejunal muscle was disrupted in W/W(V) mice, as was the contractile response to carbachol. These abnormalities were also present in BMR-W/W(V) mice. These results indicate that mast cells mediate tissue damage and contribute to the timely expulsion of nematodes from the gut during primary infection.     

Genetically Altering the Thermodynamics and Kinetics of Hepatitis B Virus Capsid Assembly Has Profound Effects on Virus Replication in Cell Culture

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.     

Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.     

Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-beta) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-alpha 3 and karyopherin-alpha 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.     

Kinetic analysis of the effects of mutagenesis of W501 and V432 of the hepatitis C virus NS3 helicase domain on ATPase and strand-separating activity

Two hydrophobic residues, W501 and V432, in the nucleic acid (NA) binding pocket of the HCV helicase domain (E) were mutagenized in an effort to investigate contributions of these residues to substrate affinities and to enzymatic activities. The affinities of wild-type [hE(wt)] and mutant enzymes [hE(W501F), hE(W501A), and hE(V432A)] for NA and ATP were determined by monitoring changes in the intrinsic protein fluorescence, in the fluorescence of fluorescently tagged nucleic acid, and in the enzymatic activity. The steady-state kinetic parameters of the mutant enzymes for ATP hydrolysis (at saturating concentrations of NA) were similar to those of hE(wt). hE(W501F), hE(W501A), and hE(V432A) had strand-separating activities that were 136%, 3.8%, and 3.1% of that of hE(wt). The processivities of hE(W501F), hE(W501A), and hE(V432A) were reduced relative to that of hE(wt). The reduced processivities of hE(W501F) and hE(W501A) were primarily due to an increase in the rate of dissociation of E. ATP from E.ATP.NA. The reduced processivity of hE(V432A) was primarily due to a reduction in the intrinsic forward rate constant for strand separation. This result suggested that V432 may constitute part of the forward "stepping" motor of E. hE(W501A) and hE(V432A) did not display a dominant negative phenotype in a steady-state helicase assay with hE(wt). hE(wt) stored in the presence of beta-mercaptoethanol was covalently modified at three cysteinyl residues. The biological significance of the potential reactivity of these cysteinyl residues on hE(wt) is unknown.     

Dietary unripe apple polyphenol inhibits the development of food allergies in murine models

The incidence of type I allergic disorders has been increasing worldwide, particularly, the hypersensitivity to food. We first showed that apple condensed tannin (ACT) intake would inhibit the development of the oral sensitization and that the inhibition could correlate with the rise in the population of TCR(gamma)delta-T cells in the intestinal intraepithelial lymphocytes (IEL) using W/W(V) mice and B10A mice which were ovalbumin (OVA)-orally sensitized. Serum OVA-specific immunoglobulin E and immunoglobulin G1 titers in the OVA-orally sensitized W/W(V) and B10A mice ad libitium fed ACT were extremely inhibited compared to those of the control. The ACT intakes of OVA-sensitized W/W(V) and B10A mice inhibited the immediate reduction of the body temperature or the rise in serum histamine induced by active systemic anaphylaxis. The proportions of the TCR(gamma)delta-T cells in the IEL of the OVA-orally sensitized W/W(V) and B10A mice ad libitium fed ACT were significantly greater than that in the controls. Furthermore, ACT feeding by itself could induce the rise in the percentage of the TCR(gamma)delta-T cells among the IEL of the W/W(V) and B10A mice. This suggests that the ACT intake may prevent the development of food allergies and this effect could be correlated with the rise in the percentage of TCR(gamma)delta-T cells among the IEL.     

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28-41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its K(m) upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.     

Stabilizing the closed S6 gate in the Shaker Kv channel through modification of a hydrophobic seal

The primary activation gate in K+ channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker (Hackos et al., 2002), we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K+ channels.     

Non-linear relationship between oxygen uptake and power output in the Astrand nomogram-old data revisited.

For the last decade there have been considerable discussion concerning the linearity / non-linearity of the oxygen uptake (V(O2)) - power output (W) relationship with strong experimental evidence of non-linearity provided mainly by breath-by-breath measurements. In this study, we attempted to answer the question whether the V(O2) - W relationship in the Astrand nomogram, as presented in the Textbook of Work Physiology, P.-O. Astrand et al. (2003), page 281, based on the Douglas bag method, is indeed linear, as stated by the authors before, or if a change point in V(O2), described by Zoladz et al. (1998) Eur J Appl Physiol 78: 369-377, can possibly be detected in those data. The V(O2) - W data were taken from the Astrand nomogram referenced above and from the Table 9.5 on page 282 in the same reference and tested for the presence of the change point in V(O2), using our two-phase model (see the reference above). In the first phase, a linear V(O2) - W relationship was assumed, whereas in the second one (above the so-called change point) an additional increase in V(O2) above the values expected from the linear model was allowed. It was found that in the data taken from the Astrand nomogram (data for men), as well as in the data taken from the Table 9.5, statistically significant change points in V(O2) were present at the power output of 150 W. The documentation of the presence of a change point in the V(O2) - W relationship in the Astrand data provides further evidence for the existence of a non-linearity in the V(O2) - W relationship in incremental exercise tests of humans, also in V(O2) data based upon the Douglas bag method.