Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Immunomodulatory effect of selenosemicarbazides and selenium inorganic compounds, distribution in organs after selenium supplementation

Antioxidant properties of selenium producing a protective barrier against free radicals play an important role in numerous metabolic and immunologic processes associated with oxidation- reduction reactions which take place during intracellular digestion of phagocyted bacteria. The aim of our study was to examine the properties of an organic compound of selenium, 4-(o-tolilo)- selenosemicarbazide of p-chlorobenzoic acid in terms of its retention in organs, effect on erythropoesis and phagocytic abilities of neutrophiles as well as antioxidant properties in neutrophiles tested with NBT test. This compound as well as inorganic sodium selenate was given to Swiss mice at the dose of 10^–3 g Se/kg for the period of 10 days. The concentrations of selenium in livers of mice treated with sodium selenate and selenosemicarbazide were found to be higher than in controls (18,7 g lg^–1 and 23.2 g lg^–1 vs. 12 g lg^–1, respectively). Analysis of blood cells count has shown a significant decrease in neutrophile levels in both groups treated with selenium. The influence of selenium compounds on phagocytosis and especially NBT test has been determined (3.8% of positive cells in the controls vs. 2.2% and 0.9% in the groups treated with sodium selenate and selenosemicarbazide, respectively). Our preliminary investigations suggest that selenosemicarbazides are biologically active compounds and can modify neutrophile functions.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Factors affecting abundance of Triaenophorus infection in Cyclops strenuus, and parasite-induced changes in host fitness

Factors affecting the abundance of Triaenophorus crassus and Triaenophorus nodulosus procercoids in their copepod first intermediate host, Cyclops strenuus, and effects of infection on feeding behaviour, reproduction and survival of the host were studied experimentally. When exposed to the same number of coracidia, copepods harboured considerably less procercoids in the trials where ciliates or Artemia salina nauplii were given as alternative food items. The prevalence of infection was higher in adult copepods as compared with copepodite stage IV and stage V, and higher in stage V than in stage IV. The prevalences in adult females and males did not differ significantly from each other. The frequency of females carrying egg sacs was lower among infected than among exposed uninfected and unexposed copepods. The rate of feeding on Artemia nauplii remained at the same level in uninfected copepods, but decreased strongly in infected copepods during 7 days p.i. The survival of unexposed, exposed uninfected and infected copepods did not differ significantly from each other for the first 11 days post-exposure, but the mortality of infected copepods increased significantly after 3 weeks post-exposure. However, the rate of development and mortality of copepods might have been affected by the apparently arrested development of stage IV copepodites found in the experiment. Some of the contradictions between these results and earlier observations are suggested to be caused by the differences in the duration of exposure, intensity of infection and duration of observation post-exposure in the present study as compared with other experiments.     

Thioredoxin can influence gene expression by affecting gyrase activity

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.     

Identification and characterization of two new human mu opioid receptor splice variants,hMOR-1O and hMOR-1X

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.     

Proton and metal ion-dependent assembly of a model diiron protein

DF1 is a small, idealized model for carboxylate-bridged diiron proteins. This protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structure has confirmed that it adopts the intended conformation. However, the protein showed limited solubility in aqueous buffer, and access to its active site was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ions in a rapid and reversible manner. Furthermore, the DF2 protein shows significant ferroxidase activity, suggesting that its dimetal center is accessible to oxygen. The affinity of DF2 for various first-row divalent cations deviates from the Irving-Willliams series, suggesting that its structure imparts significant geometric preferences on the metal ion-binding site. Furthermore, in the absence of metal ions, the protein folds into a dimer with concomitant binding of two protons. The uptake of two protons is expected if the structure of the apo-protein is similar to that of the crystal structure of dizinc DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions.     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.