Acquirement and characterization of a carotenoid mutant (GM309) of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> 601

A green mutant was obtained among the chemically induced mutants of Rhodobacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular dichroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.     

Activation of K+ channels: an essential pathway in programmed cell death

Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+ efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+ transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+ channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.     

Identification and characterization of a novel cross-link lesion in d(CpC) upon 365-nm irradiation in the presence of 2-methyl-1,4-naphthoquinone

We report the isolation and characterization for the first time of a cross-link lesion between two adjacent cytosines from the 2-methyl-1,4-naphthoquinone (menadione)-sensitized 365-nm irradiation of d(CpC). Electrospray ionization mass spectrometry (ESI-MS), tandem MS and ¹H NMR results indicate that the cross-link occurs between the C5 carbon atom of one cytosine and the N⁴ nitrogen atom of the other cytosine. Furthermore, we synthesized d(CpC) with a ¹⁵N being incorporated on the amino group of either of the two cytosines. We then irradiated the two ¹⁵N-labeled dinucleoside monophosphates, isolated the cross-link products and characterized them by MS and multi-stage tandem MS. The latter results established unambiguously that the N⁴ nitrogen atom of the 3′-nucleobase is involved in the covalent bond formation between the two cytosines. This, in combination with two-dimensional nuclear Overhauser effect spectroscopy (NOESY) results, demonstrates that the cross-link arises from the formation of a covalent bond between the C5 carbon atom of the 5′ cytosine and the N⁴ nitrogen atom of the 3′ cytosine. We also show that the solution pH has a significant effect on the formation of the cross-link lesion, which supports that the deprotonation at the exocyclic amino group of cytosine cation radical is essential for the formation of the cross-link lesion.     

Immunoglobulin treatment suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion

Atherosclerosis is associated with immune activation. Immunoglobulin is used for the treatment of immune-mediated diseases. The mechanisms and importance of the Fc portion of immunoglobulin upon experimental atherosclerosis in apolipoprotein E-deficient mice were examined. Experimental atherosclerosis was induced in mice fed a high-fat diet containing 0.3% cholesterol. Over 8, 12, and 16 wk, on alternate days, mice were treated with an intraperitoneal injection of either 1 g.kg-1.day-1 of human intact immunoglobulin or F(ab')2 fragments of human immunoglobulin. Fatty streak formation and fibrofatty plaques were markedly suppressed in mice that received intact immunoglobulin for 8, 12, and 16 wk. In contrast, atherosclerotic lesions were not ameliorated in mice that received F(ab')2 fragments. Immunohistochemical analysis revealed that macrophage accumulation in the fatty streak lesions was suppressed in mice received intact immunoglobulin but not in those that received F(ab')2 fragments. In addition, the cytotoxic activities of splenocytes from immunoglobulin-treated mice, but not from F(ab')2 fragment-treated mice, were significantly suppressed compared with those from human serum albumin-treated mice. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. Immunoglobulin therapy markedly suppressed atherosclerosis due to Fc receptor-mediated anti-inflammatory and immunomodulating actions. The antiatherosclerotic effects of immunoglobulin may be related to the suppression of cytotoxic activity of atherogenic T cells and the reduction of macrophage accumulation in the lesions.     

Gene expression reveals vulnerability to oxidative stress and interstitial fibrosis of renal outer medulla to nonhypertensive elevations of ANG II

The present study was designed to determine whether nonhypertensive elevations of plasma ANG II would modify the expression of genes involved in renal injury that could influence oxidative stress and extracellular matrix formation in the renal medulla using microarray, Northern, and Western blot techniques. Sprague-Dawley rats were infused intravenously with either ANG II (5 ng. kg(-1). min(-1)) or vehicle for 7 days (n = 6/group). Mean arterial pressure averaged 110 +/- 0.6 mmHg during the control period and 113 +/- 0.4 mmHg after ANG II. The mRNA of 1,751 genes ( approximately 80% of all currently known rat genes) that was differentially expressed (ANG II vs. saline) in renal outer and inner medulla was determined. The results of 12 hybridizations indicated that in response to ANG II, 11 genes were upregulated and 25 were downregulated in the outer medulla, while 11 were upregulated and 13 were downregulated in the inner medulla. These differentially expressed genes, most of which were not known previously to be affected by ANG II in the renal medulla, were found to group into eight physiological pathways known to influence renal injury and kidney function. Particularly, expression of several genes would be expected to increase oxidative stress and interstitial fibrosis in the outer medulla. Western blot analyses confirmed increased expression of transforming growth factor-beta1 and collagen type IV proteins in the outer medulla. Results demonstrate that nonhypertensive elevations of plasma ANG II can significantly alter the expression of a variety of genes in the renal outer medulla and suggested the vulnerability of the renal outer medulla to the injurious effect of ANG II.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

The enigmatic role of angiopoietin-1 in tumor angiogenesis

A tumor vasculature is highly unstable and immature, characterized by a high proliferation rate of endothelial cells, hyper-permeability, and chaotic blood flow. The dysfunctional vasculature gives rise to continual plasma leakage and hypoxia in the tumor, resulting in constant on-sets of inflammation and angiogenesis. Tumors are thus likened to wounds that will not heal. The lack of functional mural cells, including pericytes and vascular smooth muscle cells, in tumor vascular structure contributes significantly to the abnormality of tumor vessels. Angiopoietin-1 (Ang 1) is aphysiological angiogenesis promoter during embryonic development. The function of Angl is essential to endothelial cell survival, vascular branching, and pericyte recruitment. However, an increasing amount of experimental data suggest that Angl-stimulated association of mural cells with endothelial cells lead to stabilization of newly formed blood vessels. This in turn may limit the otherwise continuous angiogenesis in the tumor, and consequently give riseto inhibition of tumor growth. We discuss the enigmatic role of Angl in tumor angiogenesis in this review.     

X-ray crystallographic studies of HemK from Thermotoga maritima, an N5-glutamine methyltransferase

The enzyme HemK (or PrmC) is one of the first identified methyltransferases that modify glutamine. It methylates the highly conserved GGQ motif in class I release factors (RF1 and RF2) in Escherichia coli. HemK from Thermotoga maritima was over-expressed and crystallized in the presence of S-adenosylmethionine at 296 K using ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystal is orthorhombic, belonging to the space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters of a = 104.24, b = 118.73, and c = 146.62 A. Two (or three) monomers of recombinant HemK are likely to be present in the crystallographic asymmetric unit, giving a V(M) of 3.62 A3 Da(-1) (or 2.41 A3 Da(-1)), with a solvent content of 62.7% (or 44.0%).     

Allosteric switching by mutually exclusive folding of protein domains

Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.