Exploring the activity of tobacco etch virus protease in detergent solutions

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.     

The genetics of rheumatoid arthritis and the need for animal models to find and understand the underlying genes

The causes of rheumatoid arthritis (RA) are largely unknown. However, RA is most probably a multifactorial disease with contributions from genetic and environmental factors. Searches for genes that influence RA have been conducted in both human and experimental model materials. Both types of study have confirmed the polygenic inheritance of the disease. It has become clear that the features of RA complicate the human genetic studies. Animal models are therefore valuable tools for identifying genes and determining their pathogenic role in the disease. This is probably the fastest route towards unravelling the pathogenesisis of RA and developing new therapies.     

Network organization of interstitial connective tissue cells in the human endolymphatic duct.

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Chromosomal localization of three human genes encoding bone morphogenetic protein receptors

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily that play a pivotal role in bone formation during embryogenesis and fracture repair. BMP signaling occurs via hetero-oligomeric serine/threonine kinase complexes of BMP type I (BMPR-IA or BMPR-IB) and type II receptors (BMPR-II). BMPR-IA and IB are closely related receptors, with sequence differences conserved between different species, suggesting that they serve distinct functions. Here we report the cDNA cloning of human BMPR1B and the chromosomal localization of all three BMPR genes. Using somatic cell hybrid and FISH analyses, the BMPR1A, BMPR1B, and BMPR2 genes were assigned to 10q23, 4q22-24, and 2q33-34, respectively. A processed BMPR1A pseudogene was mapped to 6q23. Received: 17 February 1997 / Accepted: 15 October 1998     

Expression and purification of the recombinant membrane protein YidC: a case study for increased stability and solubility

YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 microg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies.     

Host resistance elicited by methyl jasmonate reduces emission of aggregation pheromones by the spruce bark beetle, Ips typographus

We treated Norway spruce (Picea abies) stems with methyl jasmonate (MeJA) to determine possible quantitative and qualitative effects of induced tree defenses on pheromone emission by the spruce bark beetle Ips typographus. We measured the amounts of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two main components of the beetle's aggregation pheromone, released from beetle entrance holes, along with phloem terpene content and beetle performance in MeJA-treated and untreated Norway spruce logs. As expected, phloem terpene levels were higher and beetle tunnel length was shorter (an indication of poor performance) in MeJA-treated logs relative to untreated logs. Parallel to the higher phloem terpene content and poorer beetle performance, beetles in MeJA-treated logs released significantly less 2-methyl-3-buten-2-ol and (S)-cis-verbenol, and the ratio between the two pheromone components was significantly altered. These results suggest that host resistance elicited by MeJA application reduces pheromone emission by I. typographus and alters the critical ratio between the two main pheromone components needed to elicit aggregation. The results also provide a mechanistic explanation for the reduced performance and attractivity observed in earlier studies when bark beetles colonize trees with elicited host defenses, and extend our understanding of the ecological functions of conifer resistance against bark beetles.     

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17β-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.     

Characterization of the rates of testosterone metabolism to various products and of glutathione transferase and sulfotransferase activities in rat intestine and comparison to the corresponding hepatic and renal drug-metabolizing enzymes

Metabolism of testosterone to various products (catalyzed by several different CYP isozymes) and the activities of phenol sulfotransferase (pST) and glutathione transferase (GST) in S9 fractions prepared from the mucosa of the duodenum, jejunum, ileum, caecum and upper and lower colon of male Sprague-Dawley rats were determined and compared to the corresponding hepatic and renal activities. Incubation of the S9 fraction prepared from the jejunum with testosterone and NADPH resulted in the formation of 2alpha-, 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione at rates that were 1.6, 24, 1.3, 0.6 and 1.3%, respectively, of the corresponding hepatic values. The production of 2alpha-hydroxytestosterone was catalyzed only by the preparations from the duodenum and jejunum; whereas 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione were produced in all regions of the intestine. In the case of the rat kidney, the rates of formation of the different testosterone metabolites were between 0.6 and 35% of the corresponding liver activity. The activity of glutathione transferase was approximately 12-26% of the corresponding hepatic activity throughout the intestine. The highest activity of phenol sulfotransferase was observed in the lower colon (almost 6% of the liver activity) and the lowest activity in the duodenum (1%). The renal activities of GST and pST were 70 and 1%, respectively, of the corresponding liver values. In summary, the metabolism of testosterone and the activities of GST and pST in rat intestine are generally low to very low in comparison to the corresponding activities in rat liver. In most cases, these activities are present throughout the entire intestine and not restricted to a particular portion(s) of this organ.     

The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.