Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus)

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod^#5 and ^#7). T. gondii DNA was obtained from the heart and lungs of Macropod^#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype^#3, and TgRooCHn3 was ToxoDB genotyp^#2. Both 10⁴ TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB^#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.     

Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover

During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121-131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC₇PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (E_i^#) of SMase with DC₇PC. Biophysical studies indicate that in E_i^# monodisperse DC₇PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC₇PC (E_i^# with i = 1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction (X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC₇PC will be monodisperse, E_i^# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC₇PC in solution is in a rapid exchange with that bound to E_i^# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the E_i^# complexes for the interpretation of the stationary phase reaction progress is discussed.     

Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH^#), and activation entropy (ΔS^#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH^# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro¹¹⁴ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp¹¹⁵. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.     

Optimising a coal rail network under capacity constraints

This research deals with an innovative methodology for optimising the coal train scheduling problem. Based on our previously published work, generic solution techniques are developed by utilising a ??toolbox?? of standard well-solved standard scheduling problems. According to our analysis, the coal train scheduling problem can be basically modelled a Blocking Parallel-Machine Job-Shop Scheduling (BPMJSS) problem with some minor constraints. To construct the feasible train schedules, an innovative constructive algorithm called the SLEK algorithm is proposed. To optimise the train schedule, a three-stage hybrid algorithm called the SLEK-BIH-TS algorithm is developed based on the definition of a sophisticated neighbourhood structure under the mechanism of the Best-Insertion-Heuristic (BIH) algorithm and Tabu Search (TS) metaheuristic algorithm. A case study is performed for optimising a complex real-world coal rail system in Australia. A method to calculate the lower bound of the makespan is proposed to evaluate results. The results indicate that the proposed methodology is promising to find the optimal or near-optimal feasible train timetables of a coal rail system under network and terminal capacity constraints.     

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (E_a, ΔG^#, ΔS^#, ΔH^#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.     

拉萨拉鲁湿地夏季土壤动物的群落特征

2010年6～7月,对拉鲁湿地5个生境的土壤动物进行了调查,共捕获大型和中小型干生土壤动物41类,18 446只,隶属于3门7纲14目32科17属。土壤动物的优势类群为线蚓科,占总捕获量的10.95%。常见类群有27类,占总捕获量的84.63%,其中螨类占总捕获量的32.5%,弹尾类占总捕获量的31.43%。各生境的土壤动物数量和种类在土壤各层的垂直分布具有明显的表聚性。5个生境分布的土壤动物Shannon-Weiner多样性指数(H’)从大到小依次为3#>5#>4#>1#>2#。5个生境间土壤动物的相似程度由极不相似到中等不相似(S值为0.105～0.400)。     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Kinetics and mechanism of the oxidation of L-ascorbic acid by trisoxalatocobaltate(III) in basic aqueous solution

The kinetics and mechanism of the oxidation of L- ascorbic acid by trisoxalatocobaltate(III) were studied as a function of pH, ascorbate concentration, ionic strength and temperature in a weakly basic aqueous solution. The pH dependence of the process can be ascribed to the oxidation of the doubly deprotonated ascorbate ion for which k = 20 M⁻¹ s⁻¹ at 25 °C, ΔH^# = 34 ± 2 kJ mol⁻¹ and ΔS^# = −108 ± 7 J K⁻¹ mol⁻¹. The results are discussed in reference to literature data for this reaction in weakly acidic medium and for the oxidation by a series of other oxidants.     

Kinetics of the decomposition of hydrogen peroxide in the presence of ethylenediaminetetraacetatocerium(IV) complex

The rate of reaction of [Ce(EDTA)(OH)_nⁿ⁻] with H₂O₂ in 0.10 M KNO₃ solution was investigated at various temperatures. The presence of a peroxy intermediate is inferred from spectrophotometric measurements. The general rate equation,

is valid for pH 7-9 with n= 1 and 2 complexes involved. The rate constants k_l and k₂ were determined at 25 °C to be 0.054 and 0.171 M⁻¹ s⁻¹ respectively. The corresponding activation enthalpies, as calculated from Arrhenius plots, were δH₁^#= 51.3 ± 14.8 and δH₂^#= 41.8 ± 5.3 kJ m⁻¹ and the activation entropies were δS₁^#=-97 ± 47 and ΔS₂^#=−119±17 J K⁻¹ m⁻¹.     

The kinetics and mechanism of the reaction of 2′-deoxy-5′-guanosinemonophosphoric acid and the diaqua form of cis-platin

2′-Deoxy-5′-guanosinemonosphoric acid (B) reacts with cis-[Pt(NH₃)₂(OH₂)₂]²⁺ in two steps to form the cis-[Pt(NH₃)₂B₂]^y+ ion. In the first step 2′-d-5′- GMPH₂ reacts some ten times faster than 5′-GMPH₂ does. Rate constants, ΔH^#, ΔS^# and ΔV^# are very similar for the two bases in the second reaction. It is proposed that the product in the first step contains no water and is cis-[Pt(NH₃)₂B]^x+ in which the nucleobase is bidentate bonding through both N(7) of guanine and an oxygen atom of the phosphate group.     

杨树无性系光合特征的研究

对新培育的金科系列的4个杨树无性系（3^#、6^#、8^#、9^#）的光合特征及净光合速率、水分利用效率与主要影响因子的关系进行了研究。结果表明：不同月份4个无性系的净光合速率的日进程一般在10:00左右达到峰值,然后逐渐降低;蒸腾速率日进程不尽一致;气孔导度在7:00～9:00出现峰值后,缓慢下降。在6～8月,4个无性系的净光合速率的总平均值(μmol CO₂·m^-2·s^-1)排序为：无性系9^#（8.53）>6^#>（7.21）3^#（6.47）>8^#（4.98）;蒸腾速率的总平均值(mmol H₂O·m^-2·s^-1)排序为：无性系9^#（3.74）>3^#（2.76）>6^#（1.76）>8^#（1.47）;水分利用效率的总平均值(mmol CO₂·mol^-1 H₂O)排序为：无性系8^#（4.77）>6^#（4.35）>3^#（2.99）>9^#（2.40）。4个无性系的净光合速率与水分利用效率的排序并不一致。9^#属于高光合、高蒸腾、低水分利用效率类型,8^#属低光合、低蒸腾、高水分利用效率类型。在6月份6^#无性系的净光合速率和水分利用效率与温度、湿度显著相关,3^#、8^#、9^#无性系与光合有效辐射和气孔导度密切相关。     

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (x _β), lifetimes in the absence of external perturbation (τ ⁰) and free energies (ΔG ^#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG ^# for these complexes. The values determined were up to 8 k_BT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.     

Reactivity of metal chelates of sulphur containing ligands towards Lewis bases. Part III. Reaction of bis(1,2-dimethyl-and 1-phenyl-1,2-ethanediylidene)bis(S-methylhydrazinecarbodithioate)NN′SS′(−2)dizinc(II) with pyridines

The reaction of the dimeric zinc(II) chelates of the type I (R₁ = R₂ = CH₃, R₁ = H, R₂ = Ph) with pyridine, 2-methylpyridine, 3-methylpyridine and 4-methylpyridine afforded the monomeric monobase adducts. The isolated adducts were characterized by their electronic and ¹H NMR spectra, and a five coordinate square pyramidal structure was tentatively assigned for these adducts.The adduct formation reaction was followed spectrophotometrically and the reaction kinetics were studied using a stopped flow technique. From the available kinetic data, as well as the measured activated parameters (ΔH^#, ΔS^#), a mechanism for the adduct formation reaction is proposed.     

Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s

Geometrical configuration of the polyene chain of approximately 40 mono- and di-cis carotenoids was determined from 1970 through 1990. Subsequently, the kinetic, equilibrium and thermodynamic parameters (k, K, A, E_A, ΔH^#, ΔG^#, ΔS^#) of the reversible thermal isomerization of several symmetrical and unsymmetrical carotenoids were calculated. The rate of the iodine-catalyzed photoisomerization of (all-E)-, (9Z)- and (13Z)-zeaxanthin was compared and the ‘specific rate’ (per unit light energy at given wavelengths) of the iodine-catalyzed photoisomerization for several (13Z)-carotenoids was investigated. As the missing links of the biosynthetic pathway of paprika-carotenoids, carotenoids containing new end groups were isolated; their sterically unhindered mono-cis isomers were also prepared and their geometrical configuration was determined. The investigation concentrated on the substrate specificity of the enzyme violaxanthin-deepoxidase, the light-induced formation of (13Z)-violaxanthin in green leaves, the binding of xanthophylls to the bulk light-harvesting complex (LHC) of photosystem II in higher plants, the biochemical basis of color as an aesthetic quality in Citrus-fruits and the (9Z)-epoxycarotenoid cleavage enzyme activity for ABA biosynthesis. Recently (9Z)-capsanthin-5,6-epoxide and capsoneoxanthin, two novel carotenoids have been isolated from natural sources.     

Thermodynamic compensation in microbial thermal death

Sixty eight Arrhenius plots of thermal death in six mesophilic yeast species, tested at various concentrations of NaCl, lacked an isokinetic temperature. Nevertheless the H ^#/S ^# plot was apparently linear with a slope corresponding to 314° K. It was concluded that linear thermodynamic compensation of thermal death is non-existent in heterogeneous groups of yeasts and is unlikely to occur in heterogeneous groups of other organisms and that H ^#/S ^# plots lack sensitivity for the detection of non-linearity over narrow temperature ranges.However, the H ^# and S ^# parameters of thermal death displayed non-linear compensation in such a way that the extrapolated Arrhenius plots of death attained nearly identical values near the respective maximum temperatures for growth.Linear thermodynamic compensation occurred in each of the six strains, when stationary populations of the same strain were tested at various NaCl concentrations. On the other hand, exponential populations of each of the strains, tested in the same way, lacked an isokinetic temperature of thermal death.The significance of linear and non-linear thermodynamic compensation in biological rate processes is discussed.     

Detection of Patient Subgroups with Differential Expression in Omics Data: A Comprehensive Comparison of Univariate Measures

Detection of yet unknown subgroups showing differential gene or protein expression is a frequent goal in the analysis of modern molecular data. Applications range from cancer biology over developmental biology to toxicology. Often a control and an experimental group are compared, and subgroups can be characterized by differential expression for only a subgroup-specific set of genes or proteins. Finding such genes and corresponding patient subgroups can help in understanding pathological pathways, diagnosis and defining drug targets. The size of the subgroup and the type of differential expression determine the optimal strategy for subgroup identification. To date, commonly used software packages hardly provide statistical tests and methods for the detection of such subgroups. Different univariate methods for subgroup detection are characterized and compared, both on simulated and on real data. We present an advanced design for simulation studies: Data is simulated under different distributional assumptions for the expression of the subgroup, and performance results are compared against theoretical upper bounds. For each distribution, different degrees of deviation from the majority of observations are considered for the subgroup. We evaluate classical approaches as well as various new suggestions in the context of omics data, including outlier sum, PADGE, and kurtosis. We also propose the new FisherSum score. ROC curve analysis and AUC values are used to quantify the ability of the methods to distinguish between genes or proteins with and without certain subgroup patterns. In general, FisherSum for small subgroups and -test for large subgroups achieve best results. We apply each method to a case-control study on Parkinson''s disease and underline the biological benefit of the new method.     

我国东部温带植物群落的季相及其时空变化特征

 植物群落季相阶段的划分,对于诊断地方、区域和全球尺度上生态系统对气候变化的快速响应和进行遥感植被生长季节的地面检验,具有重要 的科学意义。该文利用物候累积频率拟合法对我国东部温带地区7个站点1982～1996年的植物群落季相阶段进行划分,并分析了植物群落季相的 空间差异和年际变化及其与气候因子的关系。结果表明：1）各站点多年平均变绿期和旺盛光合期初日随纬度的升高而推迟,凋落期和休眠期初 日随纬度的升高而提前;多年平均变绿期、旺盛光合期和凋落期长度随纬度的变化不甚明显,而休眠期则随纬度的升高明显延长;2）在研究期 间内,站点平均变绿期初日以0.6 d&;#8226;a^－1的平均速率显著提前,且长度以0.7 d&;#8226;a^－1的平均速率显著延长;旺盛光合期初日呈不显著推迟,长 度呈不显著缩短;凋落期初日呈微弱提前,长度呈微弱延长;休眠期初日呈微弱提前,但长度却以0.9 d&;#8226;a^－1的平均速率显著缩短;3）站点平 均变绿期初日与当月平均气温的负相关显著,平均气温每升高1 ℃,初日提前约4.3 d;站点平均旺盛光合期初日与初日前第二个月到初日当月 平均气温的负相关显著,平均气温每升高1 ℃,初日提前约4.4 d;站点平均凋落期和休眠期初日与气温的相关均不显著。     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

氮、硅限制对赤潮藻扁面角毛藻休眠孢子形成的影响

休眠孢子的形成对于赤潮藻种群的保存、延续以及分布扩散等均具有重要的意义。通过单因子营养限制研究氮、硅对赤潮藻扁面角毛藻 (Chaetoceros compressus )休眠孢子形成的影响, 结果表明: 培养基中氮的初始浓度对休眠孢子的出现时间有一定影响。氮的初始浓度越低, 休眠孢子出现的时间越早; 反之, 氮的初始浓度越高, 休眠孢子出现的时间越晚。氮缺乏是硅藻形成休眠孢子的必需条件之一, 当培养基中氮含量低于10 mmol.L^-1时, 扁面角毛藻可以形成休眠孢子。氮缺乏诱发的休眠孢子的形成需要大量的硅, 当培养基中硅含量低于23 mmol.L^-1时, 即使氮缺乏, 扁面角毛藻也几乎不再继续形成休眠孢子。这说明硅藻休眠孢子的形成不仅受氮浓度的影响, 还与硅浓度有关。     

Phylogenetic patterns and genetic diversity of Indian Tinospora species based on chloroplast sequence data and cytochrome P450 polymorphisms

Based on morphology, the species status and taxonomic affinities of three species of Tinospora (T. cordifolia, T. sinensis, and T. crispa) with ranges in India, have been questioned. To evaluate species delimitation and population structure among 40 accessions of the three species, a relatively new marker, cytochrome P450, was used. Five out of nine primers generated polymorphisms with 39 out of 47 bands polymorphic. The P450 binary data, when analyzed using distance methods, strongly supported the monophyly of each Indian species and were congruent with previous RAPD work. To further investigate the status of these species, we combined P450 and RAPD data. The resulting unrooted phylogram highly supports the monophyly of each species but with little population structure within each species. To understand the phylogenetic placement of the three Indian Tinospora species within Menispermaceae, chloroplast atpB and rbcL sequence data for a large sampling of the family were analyzed using likelihood and parsimony methods. The resulting phylogenies highly support the Indian Tinospora species as part of a clade (expanded Tinosporeae), consisting of diverse Menispermaceae from around the world. The three Indian species are monophyletic and are most closely related to Tinospora species from Australia (T. esiangkara and T. smilacina).