ProDDO: a database of disordered proteins from the Protein Data Bank (PDB)

ProDDO represents a 'pre-screened' database that denotes disorder (or possible disorder) in proteins from the PDB.     

Tyrosine Hydroxylase Dephosphorylation by Protein Phosphatase 2A in Bovine Adrenal Chromaffin Cells

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with ³²P_i and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase 1 inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Beta-carbolines as specific inhibitors of cyclin-dependent kinases

Harmine (3), 7-fluoro-1-methyl beta-carboline (35) and 1-(5-methyl-imidazol-4-yl) beta-carboline (41) were potent and specific inhibitors of cyclin-dependent kinases. The degree of aromaticity of the tricyclic ring and the positioning of substituents are important for inhibitory activity. While most beta-carbolines inhibited CDK2 and CDK5 to the same extent, selective inhibition against CDK2 was observed in 1-(2-chlorophenyl)- (12), 1-(2-fluorophenyl)- (15), and 1-(2-chloro-5-nitrophenyl)- (28) beta-carbolines.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.     

Mannose-binding lectin (MBL) mutants are susceptible to matrix metalloproteinase proteolysis: potential role in human MBL deficiency

Mannose-binding lectin (MBL) plays a critical role in innate immunity. Point mutations in the collagen-like domain (R32C, G34D, or G37E) of MBL cause a serum deficiency, predisposing patients to infections and diseases such as rheumatoid arthritis. We examined whether MBL mutants show enhanced susceptibility to proteolysis by matrix metalloproteinases (MMPs), which are important mediators in inflammatory tissue destruction. Human and rat MBL were resistant to proteolysis in the native state but were cleaved selectively within the collagen-like domain by multiple MMPs after heat denaturation. In contrast, rat MBL with mutations homologous to those of the human variants (R23C, G25D, or G28E) was cleaved efficiently without denaturation in the collagen-like domain by MMP-2 and MMP-9 (gelatinases A and B) and MMP-14 (membrane type-1 MMP), as well as by MMP-1 (collagenase-1), MMP-8 (neutrophil collagenase), MMP-3 (stromelysin-1), neutrophil elastase, and bacterial collagenase. Sites and order of cleavage of the rat MBL mutants for MMP-2 and MMP-9 were: Gly(45)-Lys(46) --> Gly(51)-Ser(52) --> Gly(63)-Gln(64) --> Asn(80)-Met(81) which differed from that of MMP-14, Gly(39)-Leu(40) --> Asn(80)-Met(81), revealing that the MMPs were not functionally interchangeable. These sites were homologous to those cleaved in denatured human MBL. Hence, perturbation of the collagen-like structure of MBL by natural mutations or by denaturation renders MBL susceptible to MMP cleavage. MMPs are likely to contribute to MBL deficiency in individuals with variant alleles and may also be involved in clearance of MBL and modulation of the host response in normal individuals.     

Series of incidents of Listeria monocytogenes non-invasive febrile gastroenteritis involving ready-to-eat meats

AIMS: A series of cases and outbreaks of febrile noninvasive gastrointestinal disease involving 31 identified cases was investigated in terms of the numbers and types of Listeria monocytogenes present in the suspect foods (ready-to-eat meats) and clinical samples from cases. METHODS AND RESULTS: Foods and faecal samples involved in the incidents were tested for the presence and number of L. monocytogenes. Isolates were typed by macrorestriction analysis using pulsed-field gel electrophoresis. The foods contained high levels of L. monocytogenes, in one case 1.8 x 10(7) g-1. Faecal samples contained L. monocytogenes for up to 15 d after the contaminated food was consumed. All isolates from the food and faecal samples were of serotype 1/2 and were indistinguishable from one another by macrorestriction typing. CONCLUSIONS: It is likely that the meats were contaminated either during their manufacture after they had been cooked or by underprocessing. The long shelf lives on these products would have allowed the contaminating L. monocytogenes to grow to the high numbers measured in this study, causing food poisoning as described. SIGNIFICANCE AND IMPACT OF THE STUDY: Outbreaks of febrile noninvasive listeriosis are relatively rare. This report adds ready-to-eat meats to the range of foods that have acted as vehicles for such outbreaks.     

The complex nature of protein phosphatases

Protein phosphatases are integrally associated with the regulation of cellular signaling. The mechanisms underlying the specific regulatory roles are likely to be unique to each cell system. Nevertheless, analysis of phosphatase regulation in a number of systems has identified phosphatase targeting through association with a wide range of binding partners to be a fundamental mechanism of regulation. Using protein phosphatase 2A (PP2A) as an example, this snapshot summarizes these fundamental mechanisms of protein phosphatase regulation.     

Isotonic biaxial loading of fibroblast-populated collagen gels: a versatile, low-cost system for the study of mechanobiology

 We developed a simple, versatile system for applying a range of biaxial loads to cell–matrix constructs for the study of mechanobiology. The system consists of porous polyethylene bars that are polymerized into a square fibroblast-populated gel and loaded by freely hanging weights attached to sutures routed through a custom loading rig. The cost to manufacture each mold/loading rig pair was less than US $250 and the expected life of the components is up to 10 years. Neonatal and adult cardiac fibroblasts contracted gels to a decreasing extent as external load was increased (P=0.003) and achieved contraction forces of up to 1.4 mN per million cells. Strain distributions were reasonably homogeneous in the central region of the gel (25% of gel area), but clearly nonhomogeneous outside that central region. The primary advantages of this system are simplicity, low cost, biaxial loading, and the ability to test for a dose–response effect of mechanical load. The current disadvantages are the inability to apply cycling loading and the inhomogeneities introduced by the use of rigid loading bars. Received: 15 November 2001 / Accepted: 30 January 2002     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001