19975781
Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.
The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.
24638854
Structure determination of an FMN reductase from Pseudomonas aeruginosa PA01 using sulfur anomalous signal.
The availability of high-intensity synchrotron facilities, technological advances in data-collection techniques and improved data-reduction and crystallographic software have ushered in a new era in high-throughput macromolecular crystallography. Here, the de novo automated crystal structure determination at 1.28 A resolution of an NAD(P)H-dependent FMN reductase flavoprotein from Pseudomonas aeruginosa PA01-derived protein Q9I4D4 using the anomalous signal from an unusually small number of S atoms is reported. Although this protein lacks the flavodoxin key fingerprint motif [(T/S)XTGXT], it has been confirmed to bind flavin mononucleotide and the binding site was identified via X-ray crystallography. This protein contains a novel flavin mononucleotide-binding site GSLRSGSYN, which has not been previously reported. Detailed statistics pertaining to sulfur phasing and other factors contributing to structure determination are discussed. Structural comparisons of the apoenzyme and the protein complexed with flavin mononucleotide show conformational changes on cofactor binding. NADPH-dependent activity has been confirmed with biochemical assays.
205373753
SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.
Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.
12841382
Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.
124927
Oxidative stress in hypertension and chronic kidney disease: role of angiotensin II.
Angiotensin II, via the type 1 (AT1) receptor, stimulates oxidative stress. The vasculature, interstitium, juxtaglomerular apparatus, and the distal nephron in the kidney express nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that generates superoxide anion, which is an important component of angiotensin II-induced oxidative stress. The angiotensinogen gene is stimulated by NF-kappaB activation, which is sensitive to the redox ratio, providing a positive feedback loop that can upregulate angiotensin II production. Oxidative stress can accompany hypertension in many models, including the spontaneously hypertensive rat (SHR), angiotensin II-infused rats, renovascular hypertension, and the deoxycorticosterone acetate (DOCA) salt model of hypertension. AT1 receptor antagonists can abrogate the effects of angiotensin II on oxidative stress, thus providing an important mechanistic insight onto the renal protective effects of these agents in conditions associated with angiotensin II excess.
37510627
Evidence for the involvement of singlet oxygen in the photodestruction by chloroaluminum phthalocyanine tetrasulfonate.
In recent years, choloroaluminum phthalocyanine tetrasulfonate (A1PCTS) has been shown to be a promising photosensitizer for the photodynamic therapy (PDT) of cancer. Although its mechanism of photodynamic action is not well defined, A1PCTS is going to be under clinical trials of PDT. In this study, in vitro addition of A1PCTS to a suspension of rat epidermal microsomes followed by irradiation with red light (approximately 675 nm) resulted in significant destruction of cytochrome P-450 and associated monooxygenase activities. The photodestructive effect was dependent on both the dose of A1PCTS and the duration of light exposure. Studies using various quenchers of reactive oxygen species showed that only scavengers of singlet oxygen such as histidine, 2,5-dimethylfuran, beta-carotene and sodium azide afforded substantial protection against photodestruction. Our data indicate the direct involvement of singlet oxygen in the A1PCTS-mediated photodestructive process.
30954752
Salt, salt sensitivity, and the endothelium: a pathway to discovery of molecular mechanisms.
See related article, pp 951–956

This issue of Hypertension contains a report of molecular mechanisms transducing dietary salt intake1 that may have broad clinical relevance. These molecular mechanisms are complex, but to begin to understand this study we have to understand the importance of dietary salt intake and salt sensitivity. This background will be discussed in some detail before discussing the molecular mechanisms.

Although cohort studies have called into question the role of dietary salt intake in causing hypertension,2 cause and effect relationships are most convincing when demonstrated using randomized clinical trials. It would be unethical to perform a randomized clinical trial in which the dietary intake of salt (sodium chloride) is increased for years in humans to observe an increase in blood pressure. However, such an experiment in primates has been performed by Denton et al,3 who studied in Gabon, Africa, 26 chimpanzees. Chimpanzees have a native diet that is vegetarian and is very high in potassium content. The baseline sodium intake in these primates was only 6 mEq/d; in contrast, that of K was 235 mEq/d. Blood pressure was measured in the 26 animals for 1 year. Subsequently, among half the animals, increasing amounts of salt (in increments of 5 g/d sodium) were added to a diet to a steady-state level of 15 g/d. This dietary intake was maintained for 16 months. After this period of dietary supplementation, the diet was switched to baseline and observations made for another 6 months. A dose–response relationship between salt supplementation and blood pressure increment was seen. With 15 g/d sodium supplementation for 67 weeks, the change from baseline in blood pressure was 33/10 mm Hg. In comparison, 5 g/d sodium supplementation for 19 weeks provoked blood pressure change of 12/0 mm Hg. Notably, plasma renin activity was reduced with salt …
21512706
Experience with ARDS caused by tuberculosis in a respiratory intensive care unit
ObjectiveAcute respiratory distress syndrome (ARDS) is an important cause of morbidity and mortality in intensive care units. Tuberculosis (TB) commonly causes respiratory failure in patients with extensive pulmonary parenchymal involvement, but it is a rare cause of ARDS. We report our experience of TB presenting with ARDS.MethodsRetrospective analysis of 187 patients admitted with a diagnosis of ARDS over the previous 7 years. Data are presented in a descriptive fashion using mean±SD or median (range).ResultsNine (4.9%) of 187 patients had ARDS secondary to tuberculosis. All patients were mechanically ventilated. The diagnosis was made on clinico-radiological grounds and confirmed later using fiberoptic bronchoscopy and transbronchial biopsy in seven patients, and lymph node biopsy and examination of the joint aspirate in the remaining two. All patients were empirically started on anti-tubercular therapy with a median time to initiation of therapy being 3 days (range 2–8 days). Three patients had multi-organ dysfunction syndrome (MODS) without any evidence of bacterial infection. Seven of nine (77.8%) patients survived; two died because of severe ARDS, MODS, and respiratory failure.ConclusionsTuberculosis is an uncommon but definite cause of ARDS, and in patients with ARDS of obscure aetiology where the clinical features suggest tuberculosis as the inciting cause, antitubercular therapy should be started empirically and the diagnosis actively pursued later.
7762587
The role of statins in chronic kidney disease.
Chronic kidney disease is associated with cardiovascular event rates that are at least as high as in patients with established atherosclerotic cardiovascular disease or in those with diabetes mellitus. Chronic kidney disease is therefore considered a cardiovascular disease risk equivalent. Treatment of dyslipidemia, which is very common in this population and reflects the pattern seen in the metabolic syndrome, reduces cardiovascular events in patients with chronic kidney disease. Thus, patients with chronic kidney disease should be evaluated and treated for dyslipidemia. Dyslipidemia is a risk factor for the development of impaired kidney function. Dyslipidemia is also associated with progressive renal disease in subjects with no overt renal disease, as well as those with diabetic and nondiabetic kidney disease. Although definitive randomized controlled trials are lacking, the collective evidence suggests that treatment of dyslipidemia is associated with less decline in renal function. The use of potent statins in high doses can lead to transient proteinuria via impairment of proximal tubular receptor--mediated endocytosis, in a dose-dependent manner. Over the long term, however, the use of statins results in a reduction in proteinuria and in the rate of decline of renal function. Several large definitive trials that are currently underway to examine the safety and efficacy of statins in cardiovascular and renal protection should provide more definitive answers on the role of these drugs in this very high risk population.