Jesper Larsen

A Proteome-wide Screen for Mammalian SxIP Motif-Containing Microtubule Plus-End Tracking Proteins

Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and control microtubule dynamics [1, 2]. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) [3]. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.

Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response.

n Abstractn n Trans-tracheal aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and shown to be specific for the two species M. haemolytica and Mannheimia glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.n n

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms.

The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1–EB3 dimer species have distinct functions within cells.

Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark.

During 2001–2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans.

Reaction between drug substances and pharmaceutical excipients: Formation of citric acid esters and amides of carvedilol in the solid state

The reactivity of citric acid towards drug substances in the solid state was examined using the beta-blocker carvedilol as a model compound. The reaction mixtures were analysed by LC-MS, the reaction products were isolated by preparative HPLC, and the structures were elucidated by microprobe NMR spectroscopy. Heating a mixture of solid carvedilol and solid citric acid monohydrate for 96 h at 50 degrees C resulted in the formation of about 3% of a symmetrical ester as well as of a number of other reaction products in smaller amounts. Formation of the symmetrical ester was also observed at room temperature. At 70 degrees C, the amounts of three isomeric esters formed reached 6-8%. The minor reaction products were citric acid amides, O-acetylcarvedilol, and esters of itaconic acid.

Identification of reaction products between drug substances and excipients by HPLC-SPE-NMR: ester and amide formation between citric acid and 5-aminosalicylic acid.

The reaction between the high-dose drug substance 5-aminosalicylic acid (5-ASA) and the excipient citric acid during storage of an experimental enema preparation has been studied and three isobaric reaction products, i.e., an ester and an amide with non-symmetrically substituted citric acid moieties and a symmetrical amide, were identified by combined use of HPLC-SPE-NMR and HPLC-MS. After storage for 1 week at 70 degrees C, approximately 5% of the 5-ASA present in the formulation was transformed into these impurities. Storage of the enema for 32 months at room temperature led to loss of approximately 10% of the original amount of 5-ASA, with the ester as the main reaction product.

Reaction between drug substances and pharmaceutical excipients: Formation of esters between cetirizine and polyols

Reactions between active drug substances and excipients are of interest in the drug formulation process and should also be considered in the following storage of final preparations. Some excipients react more readily with certain chemical groups in drug substances and in the present paper the ester formation between a drug substance having a carboxylic acid moiety and some polyols are described. The drug substance cetirizine was chosen as the model substance as it is already marketed and used as a common drug for treatment of allergic reactions. Among the marketed products are oral solutions and oral drops containing excipients like sorbitol and glycerol. It was found that the carboxylic acid cetirizine readily reacts with sorbitol and glycerol to form monoesters. At a temperature as low as 40 degrees C, more than 1% of the cetirizine content was transformed into a monoester within 1 week using concentrations similar to those used in marketed preparations. The kinetic studies of the reaction performed at 40, 60 and 80 degrees C also revealed that the esters were unstable and they degraded especially at higher temperatures. Analysis of two marketed preparations having expiry dates in 2011 showed content of the cetirizine esters corresponding to a range from 0.1 to 0.3% of the declared cetirizine content.

Escherichia coli Producing CTX-M-1, -2, and -9 Group β-Lactamases in Organic Chicken Egg Production

A zoonotic contribution to the spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli at the community level has been proposed repeatedly (3), but information on the origin and frequency of ESBLs within different animal reservoirs is still limited. To improve the current understanding of the evolution and epidemiology of ESBLs in poultry farming, we investigated the occurrence of ESBL-producing E. coli in flocks of chicken egg layers reared in Danish organic systems, where low flock density, restricted antimicrobial use, and access to outdoor areas are regulated at the European level (4). From June to August 2009, we visited four randomly selected healthy layer flocks (flocks A, B, C, and D), reared at independent farms with no history of antimicrobial and biocide use. E. coli bacteria with reduced susceptibility to cefotaxime were recovered from flocks B and C using sock samples enriched in MacConkey broth supplemented with cefotaxime (2 μg/ml) (1). PCR analysis (6, 7) followed by partial sequencing revealed the presence of blaTEM-1 (n = 7), blaCTX-M-1 group (compatible with blaCTX-M-1/61) (n = 9), blaCTX-M-2 group (compatible with blaCTX-M-2/20/44) (n = 3), and blaCTX-M-9 group (compatible with blaCTX-M-14/17) (n = 4) genes (http://www.ncbi.nlm.nih.gov/ and http://www.lahey.org/Studies/) in randomly selected colonies (Table u200b(Table1).1). We revisited farms B and C after 10 and 11 weeks, respectively, aiming to quantify the prevalence of CTX-M-positive chickens and the concentration of CTX-M-producing E. coli in feces. The study population remained unchanged, since no chickens had entered the farms in the interval between the two sampling points. At both farms, 2 (3%) out of 60 cloacal swabs collected from individual animals and processed as described previously (1) were positive for CTX-M-producing E. coli. Of 10 fresh fecal droppings collected from the floor on each farm, 7 (farm B) and 3 (farm C) samples yielded CTX-M-producing E. coli at concentrations ranging between 102 and 103 CFU per gram of feces, which accounted for ≤0.03% of total E. coli. By XbaI pulsed-field gel electrophoresis (PFGE) analysis, 30 CTX-M producers obtained from different sample types (sock samples, swabs, and fecal droppings) displayed 16 epidemiologically unrelated band patterns according to Tenover et al. (8). Two isolates were untypeable. Twenty strains representing distinct PFGE profiles and sample types carried blaCTX-M on transferable IncI1 (n = 7), IncN (n = 3), and untypeable (n = 10) plasmids, often containing additional determinants conferring resistance to tetracycline, sulfonamides, and trimethoprim, as demonstrated by conjugation and PCR-based replicon typing (PBRT) (2, 5) (Table u200b(Table1).1). Restriction fragment length polymorphism (RFLP) analysis using ClaI and BglII showed that farm-specific IncI1 and IncN plasmids were widespread among different E. coli lineages in association with specific blaCTX-M genes (Table u200b(Table1).1). All PBRT-untypeable plasmids, which demonstrated poor quality images by RFLP analysis, were approximately 149 kb in size. n n n nTABLE 1. n nGenetic and phenotypic traits of CTX-M-producing Escherichia coli from organic layers n n n nTo our knowledge, this is the first description of CTX-M-producing E. coli in the Danish chicken production. The factors leading to the origin and persistence of these resistance genes of high clinical relevance in organic poultry not exposed to antimicrobial agents remain unknown. Further research based on plasmid sequencing and gene characterization is needed to elucidate the nature of the nonantimicrobial resistance genes located on blaCTX-M gene-carrying plasmids and their possible role in favoring the spread and maintenance of such plasmids in the absence of antimicrobial selective pressure.

IDENTIFICATION OF A NOVEL MANNHEIMIA GRANULOMATIS LINEAGE FROM LESIONS IN ROE DEER (CAPREOLUS CAPREOLUS)

Eight atypical Mannheimia isolates were isolated from lesions in roe deer (Capreolus capreolus). Traditional classification based on morphologic and physiologic traits showed that they belong to a distinct biogroup (taxon) within genus Mannheimia. Extensive phenotypic characterization suggested that the isolates should be classified as M. granulomatis, although the presence of distinct traits justified their classification into a separate biogroup within this species. Phylogenetic analyses based on 16S rRNA sequences from two roe deer isolates and 41 other Mannheimia strains supported that the roe deer isolates form a monophyletic group within M. granulomatis. The lktA genotype was present in all roe deer isolates based on Southern blot analysis, whereas the corresponding β-hemolytic phenotype was absent in one of these isolates.

Evidence for Vertical Inheritance and Loss of the Leukotoxin Operon in Genus Mannheimia

The Mannheimia subclades belong to the same bacterial genus but have taken divergent paths toward their distinct lifestyles. M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that horizontal gene transfer of the leukotoxin operon has catalyzed pathogenic adaptation and speciation of M. haemolytica + M. glucosida, or other major subclades, by using a strategy that combines compositional and phylogenetic methods. We show that it has been vertically inherited from the last common ancestor of the diverging Mannheimia subclades, although several strains belonging to M. ruminalis have lost the operon. Our analyses support that divergence within M. ruminalis following colonization of the ovine rumen was very rapid and that functional decay of most of the leukotoxin operons occurred early when the adaptation to the rumen was fastest, suggesting that antagonistic pleiotropy was the main contributor to losses in the radiating lineages of M. ruminalis. To sum up, the scenario derived from these analyses reflects two aspects. On one hand, it opposes the hypothesis of horizontal gene transfer as a catalyst of pathogenic adaptation and speciation. On the other hand, it indicates that losses of the leukotoxin operons in the radiating lineages of M. ruminalis have catalyzed their adaptation to a commensal environment and reproductive isolation (speciation).