Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, beta-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Gas-phase Dissociation of homo-DNA Oligonucleotides

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS(3) of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Clinical response to chemotherapy in oesophageal adenocarcinoma patients is linked to defects in mitochondria

Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

The Family Pasteurellaceae

This chapter describes the systematics and evolution of Pasteurellaceae with emphasis on new information generated since the 3rd edition of The Prokaryotes which only included chapters dealing with Haemophilus, Actinobacillus, and Pasteurella. A major source of new information for the current chapter has been provided by whole genome sequences now available for many taxa of the family. Some 100 species and species-like taxa have been documented and 18 genera of Pasteurellaceae reported so far. Members of the family include specialized commensals, potential pathogens, or pathogens of vertebrates and mainly survive poorly in other habitats including the external environment. The pathogenic members are of major importance to animal production and human health. Members of Pasteurellaceae have relatively small genomes, probably as a result of adaption to a special habitat. The most important species in veterinary microbiology include Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Bibersteinia trehalosi, and Avibacterium paragallinarum, while Haemophilus influenzae and Aggregatibacter actinomycetemcomitans represent the most important species as to human disease. Traditional isolation techniques are still used in both human and veterinary clinical diagnostic laboratories although genetically based diagnostic methods have replaced traditional biochemical/physiological methods for characterization and identification. For all species, MALDI-TOF can now be used as a diagnostic tool. As control and if MALDI-TOF equipment is not at hand, PCR-based specific detection is possible for Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Avibacterium paragallinarum, Gallibacterium anatis, Haemophilus influenzae, and Aggregatibacter actinomycetemcomitans. A lot of work has been directed towards identification of virulence factors and understanding host microbe interactions involved in disease.

Outbreak investigation identifies a single Listeria monocytogenes strain in sheep with different clinical manifestations, soil and water.

Listeria (L.) monocytogenes causes orally acquired infections and is of major importance in ruminants. Little is known about L. monocytogenes transmission between farm environment and ruminants. In order to determine potential sources of infection, we investigated the distribution of L. monocytogenes genetic subtypes in a sheep farm during a listeriosis outbreak by applying four subtyping methods (MALDI-TOF-MS, MLST, MLVA and PFGE). L. monocytogenes was isolated from a lamb with septicemia and from the brainstem of three sheep with encephalitis. Samples from the farm environment were screened for the presence of L. monocytogenes during the listeriosis outbreak, four weeks and eight months after. L. monocytogenes was found only in soil and water tank swabs during the outbreak. Four weeks later, following thorough cleaning of the barn, as well as eight months later, L. monocytogenes was absent in environmental samples. All environmental and clinical L. monocytogenes isolates were found to be the same strain. Our results show that the outbreak involving two different clinical syndromes was caused by a single L. monocytogenes strain and that soil and water tanks were potential infection sources during this outbreak. However, silage cannot be completely ruled out as the bales fed prior to the outbreak were not available for analysis. Faeces samples were negative, suggesting that sheep did not act as amplification hosts contributing to environmental contamination. In conclusion, farm management appears to be a crucial factor for the limitation of a listeriosis outbreak.

Distamycin-NA: A DNA analog with an aromatic heterocyclic polyamide backbone. Part 2. Solid-phase synthesis of distamycin-NAs containing the nucleobase uracil: Unexpected solvent participation in the coupling step

10.1002/hlca.19980810512.abs The synthesis of the Fmoc-protected amino acid 2 is presented. First attempts of amide-bond formation to the homodimer 4 in solution showed only poor coupling yields indicative for the low reactivity of the amino and carboxy groups in the building blocks 1 and 2, respectively (Scheme 1). Best coupling yields were found using dicyclohexylcarbodiimide (DCC) without any additive. The oligomerization of building block 2 adopting the Fmoc ((9H-fluoren-9-ylmethoxy)carbonyl) solid-phase synthesis yielded a mixture of N-terminal-modified distamycin-NA derivatives. By combined HPLC and MALDI-TOF-MS analysis, the N-terminal functional groups could be identified as acetamide and N,N-dimethylformamidine functions, arising from coupling of the N-terminus of the growing chain with residual AcOH or DCC-activated solvent DMF. An improved preparation of building block 2 and coupling protocol led to the prevention of the N-terminal acetylation. However, ‘amidination’ could not be circumvented. A thus isolated tetramer of 2, containing a lysine unit at the C-terminus and a N,N-dimethylformamidine-modified N-terminus, not unexpectedly, showed no complementary base pairing to DNA and RNA, as determined by standard UV-melting-curve analysis.