Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Chondrodysplasias due to proteoglycan defects

The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.     

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.     

Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris.

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1). Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1). This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.     

Increased acidophilia of eosinophil granules after EDTA treatment

Summary The acidophilic reaction of eosinophil leucocyte granules from human, pig and horse blood smears was investigated by using May—Grünwald—Giemsa staining after previous treatment with EDTA and sodium citrate solutions. The same peak at 530 nm, but absorption values considerably higher than those of controls, were found in eosinophil granules after application of chelating agents, indicating that removal of metal cations could unmask basic groups in these structures.     

Pediatric Drug Safety Surveillance in FDA-AERS: A Description of Adverse Events from GRiP Project

Individual case safety reports (ICSRs) are a cornerstone in drug safety surveillance. The knowledge on using these data specifically for children is limited. We studied characteristics of pediatric ICSRs reported to the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS). Public available ICSRs reported in children (0–18 years) to FAERS were downloaded from the FDA-website for the period Jan 2004-Dec 2011. Characteristics of these ICSRs, including the reported drugs and events, were described and stratified by age-groups. We included 106,122 pediatric ICSRs (55% boys and 58% from United States) with a median of 1 drug [range 1–3] and 1 event [1–2] per ICSR. Mean age was 9.1 years. 90% was submitted through expedited (15-days) (65%) or periodic reporting (25%) and 10% by non-manufacturers. The proportion and type of pediatric ICSRs reported were relatively stable over time. Most commonly reported drug classes by decreasing frequency were ‘nervous system drugs’ (58%), ‘antineoplastics’ (32%) and ‘anti-infectives’ (25%). Most commonly reported system organ classes were ‘general’ (13%), ‘nervous system’ (12%) and ‘psychiatric’ (11%) disorders. Duration of use could be calculated for 19.7% of the reported drugs, of which 14.5% concerned drugs being used long-term (>6 months). Knowledge on the distribution of the drug classes and events within FAERS is a key first step in developing pediatric specific methods for drug safety surveillance. Because of several differences in terms of drugs and events among age-categories, drug safety signal detection analysis in children needs to be stratified by each age group.