Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.     

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

Metabolic and Body Composition Risk Factors Associated with Metabolic Syndrome in a Cohort of Women with a High Prevalence of Cardiometabolic Disease

BackgroundThe aetiology of the metabolic syndrome and the inter-relationship between risk factors for this syndrome are poorly understood. The purpose of this investigation was to determine the risk factors for metabolic syndrome and their interactions in a cohort of women with a high prevalence of metabolic syndrome.ResultsMetabolic syndrome was present in 49.6% of the study cohort. Logistic regression analysis demonstrated that adiponectin (odds ratio [95% CIs]: 0.84 [0.77, 0.92], p<0.0005) and abdominal subcutaneous fat (0.56 [0.39, 0.79], p = 0.001) reduced metabolic syndrome risk whilst insulin resistance (1.31 [1.16, 1.48], p<0.0005) and trunk fat-free soft-tissue mass (1.34 [1.10, 1.61], p = 0.002) increased risk. Within this group of risk factors, the relationship of adiponectin with metabolic syndrome risk, when analysed across adiponectin hexiles, was the least affected by adjustment for the other risk factors.ConclusionsAdiponectin has a significant protective role against metabolic syndrome and is independent of other risk factors. The protective and possible augmentive effects of abdominal subcutaneous fat and lean trunk mass, respectively on metabolic syndrome risk demonstrate the existence of novel interactions between body composition and cardiometabolic disease.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

Identification of a protein that binds to the Ho endonuclease recognition sequence at the yeast mating type locus.

Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a site-specific double-stranded break at MAT, the yeast mating type locus. To identify other proteins involved in this process, we examined whether extracts prepared from ho- mutants contain additional factors that bind near the recognition sequence for Ho. Using an electrophoretic mobility shift assay, we isolated a chromatographic fraction that contains an activity, named YZbp, which binds to two sequences flanking the recognition sequence at MATalpha and to one sequence overlapping it at MATa. MAT plasmids carrying mutations in the YZbp recognition sequence are cleaved by purified Ho at wild-type efficiencies in an in vitro assay. These same plasmids, however, are not cleaved by Ho inside cells, demonstrating that YZbp acts as a positive activator of in vivo cleavage. YZbp is present in all cell types, even those not undergoing mating type switching, suggesting that it has additional cellular functions.     

Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.