Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.Australian Peptide Conference Issue.     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.     

The message of nitric oxide in cadmium challenged plants

During the last decade it has been found that cadmium (Cd), one of the most toxic elements occurring in polluted environments, interferes with nitric oxide (NO), a multifunctional signaling molecule in living organisms. The formation of NO has been demonstrated in vivo in various plant tissues exposed to Cd stress, but unfortunately, the time and intensity of NO generation, relatively frequently shows conflicting data. What is more, there is still limited information regarding the functional role of endogenously produced NO in plants challenged with heavy metals. The first pharmacological approaches revealed that exogenously applied NO can alleviate cadmium toxicity in plants, promoting the direct scavenging of reactive oxygen species (ROS) or activating antioxidant enzymes. However, recent reports have indicated that NO even contributes to Cd toxicity by promoting Cd uptake and participates in metal-induced reduction of root growth. In view of this heterogeneous knowledge, much more puzzling if we consider results first obtained using exogenous NO sources, this review is focused mainly on the implication of endogenous NO in plant response to Cd exposure. Furthermore, a basic draft for NO mode of action during cadmium stress is proposed.     

PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies

Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10 s for all actinic flash energies (the maximum energy of 7.5 × 10¹⁶ photons/(cm² flash) is set to 100%, the relative energies of weaker actinic flashes were of ∼8%, 4%, ∼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4 × 10¹⁴ photons/(cm² flash) to 7.5 × 10¹⁶ photons/(cm² flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe⁻) and P680⁺ by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant.The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.     

Red/ET重组及其在生物医学中的应用

通过应用Rac噬菌体的RecE RecT和λ噬菌体的RedαRedβ系统而建立的DNA工程平台———Red ET重组,是一种不依赖于限制性内切酶的分子克隆新技术。运用该技术能够介导PCR产物或寡核苷酸对目标基因进行剪切、插入、融合及突变等多种操作,在生物医学领域里具有广阔的应用前景,尤其在基因组功能研究中对BACs、PACs和细菌染色体的打靶修饰以及基因敲除动物DNA靶分子的快速构建等方面最有效。随着Red ET重组的推广与应用,该技术本身也在不断被改进,在工作效率得到显著提高的同时,其操作也变得更加简单、省时、省力。结合自身的一些研究结果,对Red ET重组的技术特点、发展现状和在生物医学中的应用进行了详细阐述。     

Chemical rescue of H+ delivery in proton transfer mutants of reaction center of photosynthetic bacteria

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of Q_B^??→?Q_BH₂. In the presence of divalent metal ions (e.g. Cd²⁺) or in some (“proton transfer”) mutants (L210DN/M17DN or L213DN), the proton delivery to Q_B^? is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1?M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H?+?Cd²⁺), beneath the surface (M17DN?+?Cd²⁺, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including Q_B^? itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100–200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.     

体内直接克隆大片段DNA的研究进展

基因组序列的功能分析以及代谢途径的构建改造等都需要克隆目的DNA。获得大片段DNA序列的方法有构建和筛选基因文库,PCR扩增,体外大片段DNA合成和组装等,但体内重组直接克隆的方法在操作、克隆长片段和应用等方面更具优势。介绍了Red/ET重组介导的大片段DNA体内直接克隆的主要方法及其应用。     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.     

Two Y chromosomes with duplication of the distal long arm including the entire AZFc region

Chromosome anomalies/rearrangements of the Y chromosome seldom threaten life and are quite common. The 4.5 Mb AZFc region on Yq11.2 is one of the most polymorphic regions in the human genome. AZFc partial deletion is almost inevitably associated with impaired fertility while the functional significance of AZFc partial duplications remains controversial. In this report, a large Y chromosome with one centromere and two heterochromatic blocks was identified incidentally in two men. The first case was ascertained through surveillance for recurrent miscarriage. The second case was ascertained through amniocytes of a fetus and his father. FISH and array-based comparative genomic hybridization showed duplications of the entire AZFc region as well as Yq euchromatic region. The duplications in Case 1 and Case 2 spanned 4.8 Mb and 6.2 Mb, respectively, of the Yq euchromatic region. These two cases suggest that complete AZFc duplication could be completely benign. It awaits further investigation whether this is a bona fide chromosomal polymorphism.     

An Evaluation of Reproductive and Developmental Toxicity of Sitaxentan (Thelin) in Rats

Sitaxentan sodium (Thelin) is a once daily, orally bioavailable, highly selective endothelin A receptor antagonist. Initially approved for the treatment of pulmonary arterial hypertension, sitaxentan was withdrawn in 2010 following the recognition of a pattern of idiosyncratic liver injury. During development of this drug, a series of nonclinical studies investigated the effects of orally administered sitaxentan on fertility, embryofetal development, and pre‐ and postnatal development in the rat; results of these studies are reported here. In the fertility study, sitaxentan did not affect mating behavior, fertility, sperm morphology, or estrous cycle. Sitaxentan was teratogenic in the embyrofetal development study, which was expected based on its pharmacologic mechanism of action. Teratogenic effects included malformations of the head, mouth, face, and large blood vessels. In the pre‐ and postnatal study, sitaxentan administration was associated with reduced pup survival, large or abnormally shaped livers, and delays in markers of auditory and sexual development. Sitaxentan was detected in plasma of suckling pups receiving milk from females dosed with sitaxentan. These nonclinical study findings were reflected in the sitaxentan product label warnings. Birth Defects Res (Part B) 00:1‐10, 2012. © 2012 Wiley Periodicals, Inc.