Effect of indomethacin on prostaglandin content of several rabbit tissues

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle . PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.     

Heparan sulfate 2-O-sulfotransferase (Hs2st) and mouse development

Heparan sulphate 2-O-sulphotransferase (Hs2st) acts at an intermediate stage in the pathway of biosynthesis of heparan sulphate (HS), catalysing the transfer of sulphate from 3-phosphoadenosine-5-phosphosulfate (PAPS) to the C2-position of selected hexuronic acid residues within the maturing HS chain. It is well established that 2-O-sulphation within HS, particularly of iduronate residues, is essential for HS to participate in a variety of high-affinity ligand-binding interactions. HS plays a central role in embryonic development and cellular function, modulating the activities of an extensive range of growth factors. Interestingly, in contrast to the early failure of embryos entirely lacking HS, Hs2st ^–/– mice survive until birth, but die perinatally due to a complete failure of kidney formation. The phenotype of Hs2st ^–/– mutant kidneys suggests that signalling between two tissues, ureteric bud and metanephric mesenchyme, is disrupted. We discuss candidate signalling molecules that may mediate this interaction. The HS generated by these mice lacks 2-O-sulphate groups but is extensively modified above wild type levels by O-sulphation at C-6 of glucosamine-N-sulfate (GlcNS) residues. We will discuss the potentially altered role of this atypical HS in growth factor signalling. Published in 2003.     

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.     

Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake

Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor tyrosine kinase B (TrkB) are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF in the hypothalamic paraventricular nucleus (PVN) on feeding. BDNF injected unilaterally or bilaterally into the PVN of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and 24- to 48-h postinjection intervals. Effective doses producing inhibition of feeding behavior did not establish a conditioned taste aversion. PVN BDNF significantly decreased PVN neuropeptide Y (NPY)-induced feeding at 1, 2, and 4 h following injection. BDNF administration in the PVN abolished food-restriction-induced NPY gene expression in the hypothalamic arcuate nucleus. In conclusion, BDNF in the PVN significantly decreases food intake and body weight gain, suggesting that the PVN is an important site of action for BDNF in its effects on energy metabolism. Furthermore, BDNF appears to interact with NPY in its anorectic actions, although a direct effect on NPY remains to be established.     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.