Thoughts on thiolate tethering. Tribute and thanks to a teacher

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.     

Fourier transform IR spectroscopy study for new insights into molecular properties and activation mechanisms of visual pigment rhodopsin

Fourier transform IR (FTIR) spectroscopy has been successfully applied in recent years to examine the functional and structural properties of the membrane protein rhodopsin, a prototype G protein coupled receptor. Unlike UV-visible spectroscopy, FTIR spectroscopy is structurally sensitive. It may give us both global information about the conformation of the protein and very detailed information about the retinal chromophore and all other functional groups, even when these are not directly related to the chromophore. Furthermore, it can be successfully applied to the photointermediates of rhodopsin, including the active receptor species, metarhodopsin II, and its decay products, which is not expected presently or even in the near future from crystallographic approaches. In this review we show how FTIR spectroscopy has significantly contributed to the understanding of very different aspects of rhodopsin, comprising both structural properties and the mechanisms leading to receptor activation and deactivation.     

Molecular mechanisms of gene expression regulation by the apoptosis-promoting protein TIA-1

TIA-1 and the related protein TIAR promote DNA fragmentation in digitonin-permeabilized thymocytes. These proteins contain RNA Recognition Motifs (RRMs) and bind uridine-rich sequences. These observations suggested that TIA-1/TIAR are pro-apoptotic factors that influence some aspect of RNA metabolism. Here we review recent data implicating TIA-1 as a regulator of translation of Tumor Necrosis Factor- mRNA and as regulator of alternative splicing of a variety of pre-mRNAs, including those of the Fibroblast Growth Factor Receptor 2 and the Fas receptor. We also discuss how some of these activities could be integrated in the control of programmed of cell death.     

电刺激大鼠脊神经皮支对远距离机械感受单位电活动的影响

实验采用分离神经细束的方法,观察逆行电刺激大鼠脊神经背侧皮支后,在相距较远的神经细束上记录到的Aδ和C类机械感受单位电活动的变化。刺激T9脊神经背侧皮支,在T12神经细束上记录到59.3%（16/27）的Aδ和71.2%(37/52）的C类单位在刺激后90～120s放电显著增加。刺激T8脊神经背侧皮支,在T12神经细束上记录到47.8%(11/23）的Aδ单位和36.6%(15/41）的C类单位在刺激后120～150s放电显著增加。大多数单位（18/23）的机械感受阈值在电刺激远距离脊神经背侧皮支后降低。结果表明,逆行电刺激外周感觉神经,可以使相距较远的Aδ和C类机械感受单位致敏,其传入放电增加。     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.     

The development and use of vaccine adjuvants

Interest in vaccine adjuvants is intense and growing, because many of the new subunit vaccine candidates lack sufficient immunogenicity to be clinically useful. In this review, I have emphasized modern vaccine adjuvants injected parenterally, or administered orally, intranasally, or transcutaneously with licensed or experimental vaccines in humans. Every adjuvant has a complex and often multi-factorial immunological mechanism, usually poorly understood in vivo. Many determinants of adjuvanticity exist, and each adjuvanted vaccine is unique. Adjuvant safety is critical and can enhance, retard, or stop development of an adjuvanted vaccine. The choice of an adjuvant often depends upon expensive experimental trial and error, upon cost, and upon commercial availability. Extensive regulatory and administrative support is required to conduct clinical trials of adjuvanted vaccines. Finally, comparative adjuvant trials where one antigen is formulated with different adjuvants and administered by a common protocol to animals and humans can accelerate vaccine development.     

The pathogenicity of strains of Salmonella paratyphi B and Salmonella java

AIMS: To relate the diseases caused by strains of Salmonella paratyphi B and S. java to pathogenic mechanisms expressed by these bacteria for the purpose of organism discrimination. METHODS AND RESULTS: Epidemiological data relating to cases of disease caused by strains of S. paratyphi B and S. java, isolated over a 10-year period, were analysed with respect to patients' symptoms, particularly those involving enteric fever. Strains of S. paratyphi B and S. java were also examined for a range of known pathogenic mechanisms. Infection with S. paratyphi B involved pyrexia in 12.5% of patients compared with 2.2% of patients infected with S. java. These organisms could not be differentiated based on the pathogenic properties examined. CONCLUSIONS: Strains of S. paratyphi B appear not to be a major cause of enteric fever but primarily a cause of gastroenteritis, in common with S. java. Both organisms express similar pathogenic mechanisms, and strains of S. java are probably d-tartrate utilizing variants of S. paratyphi B. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of S. paratyphi B are very closely related organisms, primarily causing gastroenteritis. From this study it would appear that strains of S. paratyphi B are not a major cause of enteric fever.     

神经元烟碱受体在全身麻醉机制中的作用

作为配体－门控离子通道超家族成员的神经元烟碱受体分布于中枢和外周神经系统,包括多种亚型,具有广泛的生理作用,可以成为多种疾病的药物治疗靶点。它在全身麻醉原理中的作用也被日益重视。部分全身麻醉药物（挥发性吸入、气体吸入麻醉药、硫喷妥钠、氯胺酮等）在低于临床麻醉剂量时能够明显抑制该受体功能,神经元烟碱受体可能参与了这些药物的临床作用机制。     

Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera

Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95-117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733-779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.     

Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts

Growth factors and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac fibroblasts, we tested the involvement of growth factors and hormones in this process.We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 ± 3%. Moreover, CM derived from 2 h stretched fibroblasts increased the rate of protein synthesis in static fibroblasts as well as in static cardiomyocytes by 8 ± 2 and 6 ± 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and fibroblasts released at least three factors with MW 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGF₁) being increased by 45 ± 17 and 21 ± 4% upon 4 h of stretch, respectively. Fibroblasts released TGF₁ and very low quantity of endothelin-1 (ET-1). The release of TGF₁ was significantly increased by 18 ± 4% after 24 h of stretch in fibroblasts. Both cell types released no detectable amount of angiotensin II (Ang II).In conclusion, upon cyclic stretch cardiomyocytes and fibroblasts secrete growth factors and hormones which induce growth responses in cardiomyocytes and fibroblasts in an autocrine/paracrine way. TGF secreted by cardiomyocytes and fibroblasts, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.