SYNTHETIC OLIGODEOXYNUCLEOTIDE PURIFICATION VIA CATCHING BY POLYMERIZATION

Large quantities of pure synthetic oligodeoxynucleotides (ODNs) are important for preclinical research, drug development, and biological studies. These ODNs are synthesized on an automated synthesizer. It is inevitable that the crude ODN product contains failure sequences which are not easily removed because they have the same properties as the full length ODNs. Current ODN purification methods such as polyacrylamide gel electrophoresis (PAGE), reversed-phase high performance liquid chromatography (RP HPLC), anion exchange HPLC, and affinity purification can remove those impurities. However, they are not suitable for large scale purification due to the expensive aspects associated with instrumentation, solvent demand, and high labor costs. To solve these problems, two non-chromatographic ODN purification methods have been developed. In the first method, the full-length ODN was tagged with the phosphoramidite containing a methacrylamide group and a cleavable linker while the failure sequences were not. The full-length ODN was incorporated into a polymer through radical acrylamide polymerization whereas failure sequences and other impurities were removed by washing. Pure full-length ODN was obtained by cleaving it from the polymer. In the second method, the failure sequences were capped by a methacrylated phosphoramidite in each synthetic cycle. During purification, the failure sequences were separated from the full-length ODN by radical acrylamide polymerization. The full-length ODN was obtained via water extraction. For both methods, excellent purification yields were achieved and the purity of ODNs was very satisfactory. Thus, this new technology is expected to be beneficial for large scale ODN purification.

Proteomic analysis in aortic media of patients with Marfan syndrome reveals increased activity of calpain 2 in aortic aneurysms

BACKGROUND: Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. METHODS AND RESULTS: Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. CONCLUSIONS: Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

Projection structure of DtpD (YbgH), a prokaryotic member of the peptide transporter family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as beta-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 A resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Stenotrophomonas maltophilia isolated from the airways of animals with chronic respiratory disease

Stenotrophomonas maltophilia (S. maltophilia) is a nonfermentative bacterium, which is naturally resistant against a panel of commonly-used antibiotics. It is frequently isolated from humans with chronic respiratory disease, e.g. cystic fibrosis or chronic obstructive pulmonary disease. In veterinary medicine S. maltophilia is perceived to be a mere coloniser. We herewith report 7 strains of S. maltophilia isolated from animals, of which 5 strains were harvested from 3 horses, a dog and a cat with chronic respiratory disease. The dog isolate showed resistance to trimethoprim / sulphamethoxazole, which was confirmed by detection of the sul 1 gene. Analysis with pulsed field gel electrophoresis revealed that 2 horses, which were boarded in the same clinic but two years apart, harboured the same strain of S. maltophilia. This is indicative of a hospital acquired colonisation / infection, which contradicts involvement in the pre-existing chronic disease.

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock-down results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.

Laribacter hongkongensis: a potential cause of infectious diarrhea

In this study, we describe the isolation of Laribacter hongkongensis, a recently described genus and species of bacterium, in pure culture on charcoal cefoperazone deoxycholate agar from the stool of six patients with diarrhea. Three patients were residents of Hong Kong, and three of Switzerland. In none of the stool samples obtained from these six patients was Salmonella, Shigella, enterohemorrhagic Escherichia coli, Vibrio, Aeromonas, Plesiomonas, or Campylobacter recovered. Rotavirus antigen detection, electron microscopic examination for viruses, and microscopic examinations for ova and cysts were all negative for the stool samples obtained from the three patients in Hong Kong. Enterotoxigenic E. coli was recovered from one of the patients in Hong Kong. Unlike L. hongkongensis type strain HKU1, all the six strains were motile with bipolar flagellae. Sequencing of the 16S ribosomal RNA genes of the six strains showed that they all had sequences with only 0-2 base differences to that of the type strain. Pulsed field gel electrophoresis of the SpeI digested genomic DNA of the six isolates and that of the type strain revealed that the seven isolates were genotypically unrelated strains. More extensive epidemiologic studies should be carried out to ascertain the causative association between L. hongkongensis and diarrhea and to define the reservoir and modes of transmission of L. hongkongensis.

Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency

BACKGROUND Defects of the mitochondrial respiratory chain complex II (succinate dehydrogenase (SDH) complex) are extremely rare. Of the four nuclear encoded proteins composing complex II, only mutations in the 70 kDa flavoprotein (SDHA) and the recently identified complex II assembly factor (SDHAF1) have been found to be causative for mitochondrial respiratory chain diseases. Mutations in the other three subunits (SDHB, SDHC, SDHD) and the second assembly factor (SDHAF2) have so far only been associated with hereditary paragangliomas and phaeochromocytomas. Recessive germline mutations in SDHB have recently been associated with complex II deficiency and leukodystrophy in one patient. METHODS AND RESULTS We present the clinical and molecular investigations of the first patient with biochemical evidence of a severe isolated complex II deficiency due to compound heterozygous SDHD gene mutations. The patient presented with early progressive encephalomyopathy due to compound heterozygous p.E69 K and p.*164Lext*3 SDHD mutations. Native polyacrylamide gel electrophoresis and western blotting demonstrated an impaired complex II assembly. Complementation of a patient cell line additionally supported the pathogenicity of the novel identified mutations in SDHD. CONCLUSIONS This report describes the first case of isolated complex II deficiency due to recessive SDHD germline mutations. We therefore recommend screening for all SDH genes in isolated complex II deficiencies. It further emphasises the importance of appropriate genetic counselling to the family with regard to SDHD mutations and their role in tumorigenesis.

Unilateral epididymitis associated with salmonella bacteremia in a dog

This report describes the clinical features, diagnosis and treatment of a dog with unilateral epididymitis associated with Salmonella spp. bacteremia. Fever and an enlarged and painful testicle were the main clinical signs that resulted in referral for diagnostic evaluation. Unilateral septic epididymitis was diagnosed via ultrasonography of the genitourinary tract and aerobic culture of scrotal fluid, urine and blood, which yielded heavy growth of Salmonella spp. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of Salmonella javiana. Following antibiotic therapy there was total resolution of clinical signs, and no Salmonella was isolated from a post-treatment urine culture. The source of infection was unknown, however an environmental exposure was suspected. Although infrequent, infection with Salmonella spp. should be included in the differential diagnosis of canine epididymitis. Given the major zoonotic importance of salmonellosis, and to prevent re-infection after treatment, the source of the infection should be investigated and eliminated, if possible.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America.

BACKGROUND: Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. METHODS: S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsed-field gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CA-MRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. RESULTS: A total of 1570 S. aureus were included; 651 were MRSA (41%)--with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)--and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a < or =6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). CONCLUSION: A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals.

Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years.

Elevated albumin in retinas of monkeys with experimental glaucoma.

PURPOSE: To establish the identity of a prominent protein, approximately 70 kDa, that is markedly increased in the retina of monkeys with experimental glaucoma compared with the fellow control retina, the relationship to glaucoma severity, and its localization in the retina. METHODS: Retinal extracts were subjected to 2-D gel electrophoresis to identify differentially expressed proteins. Purified peptides from the abundant 70 kDa protein were analyzed and identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) separation, and collision-induced dissociation sequencing. Protein identity was performed on MASCOT (Matrix Science, Boston, MA) and confirmed by Western blot. The relationship between the increase in this protein and glaucoma severity was investigated by regression analyses. Protein localization in retina was evaluated by immunohistochemistry with confocal imaging. RESULTS: The abundant protein was identified as Macaca mulatta serum albumin precursor (67 kDa) from eight non-overlapping proteolytic fragments, and the identity was confirmed by Western blot. The average increase in retinal albumin content was 2.3 fold (P = 0.015). In glaucoma eyes, albumin was localized to some neurons of the inner nuclear layer, in the inner plexiform layer, and along the vitreal surface, but it was only found in blood vessels in control retinas. CONCLUSIONS: Albumin is the abundant protein found in the glaucomatous monkey retinas. The increased albumin is primarily localized to the inner retina where oxidative damage associated with experimental glaucoma is known to be prominent. Since albumin is a major antioxidant, the increase of albumin in the retinas of eyes with experimental glaucoma may serve to protect the retina against oxidative damage.