A new approach for detecting riboswitches in DNA sequences.

MOTIVATION: Riboswitches are short sequences of messenger RNA that can change their structural conformation to regulate the expression of adjacent genes. Computational prediction of putative riboswitches can provide direction to molecular biologists studying riboswitch-mediated gene expression. RESULTS: The Denison Riboswitch Detector (DRD) is a new computational tool with a Web interface that can quickly identify putative riboswitches in DNA sequences on the scale of bacterial genomes. Riboswitch descriptions are easily modifiable and new ones are easily created. The underlying algorithm converts the problem to a 'heaviest path' problem on a multipartite graph, which is then solved using efficient dynamic programming. We show that DRD can achieve ∼ 88-99% sensitivity and >99.99% specificity on 13 riboswitch families. AVAILABILITY AND IMPLEMENTATION: DRD is available at http://drd.denison.edu.

TSG-6 and calcium ions are essential for the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human synovial fluid.

OBJECTIVE: To investigate the role of tumor necrosis factor stimulated gene 6 (TSG-6) and metal ions in the coupling of inter-alpha-trypsin inhibitor (ITI) to hyaluronan in human synovial fluid. DESIGN: The concentration of ITI heavy chains bound to hyaluronan was determined by a two-step electrophoretic technique. Synovial fluid, TSG-6 depleted synovial fluid and metal chelated synovial fluid were tested for their ability to support the coupling of ITI heavy chains to hyaluronan. RESULTS: When synovial fluid was mixed with an ITI-source (serum or purified ITI), coupling of ITI heavy chains to hyaluronan took place. TSG-6 immunodepleated synovial fluid lost the coupling activity, but addition of recombinant TSG-6 restored the activity. EDTA inhibited the coupling activity, but combinations of the metal-ion chelators Mg-EGTA and Ca-EGTA demonstrated, that Ca++ is essential for the coupling of ITI heavy chains to hyaluronan. CONCLUSIONS: Tumor necrosis factor stimulated gene 6 (TSG-6) and calcium ions are both essential for the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human synovial fluid.

Role of tumour necrosis factor stimulated gene 6 (TSG-6) in the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human follicular fluid.

Binding of the plasma proteinase inhibitor inter-alpha-trypsin inhibitor (ITI) to hyaluronan is necessary for normal expansion of the cumulus-oocyte complex. Lack of ITI causes severe infertility. Binding of ITI to hyaluronan depends on calcium ions and coupling activity present in follicular fluid (Ødum et al., 2002). The complexes formed by this process contain ITI heavy chains bound to hyaluronan, and bikunin is detached from ITI during the coupling reaction. In the present study, an electrophoretic technique by which hyaluronan-bound ITI is immobilized was used to demonstrate that tumour necrosis factor stimulated gene 6 (TSG-6) is necessary for the coupling reaction. Thus, immunoprecipitation of TSG-6 in human follicular fluid eliminates the coupling reaction and re-addition restores the activity. However, it appears that components other than hyaluronan, ITI, calcium ions and TSG-6 are involved in the coupling reaction, as in vitro incubation of these components does not generate stable complexes between ITI heavy chains and hyaluronan unless some follicular fluid is added. In conclusion, TSG-6 is necessary for the coupling of ITI to hyaluronan, but at least one additional component in follicular fluid is essential.

Characterization of the coupling activity for the binding of inter-alpha-trypsin inhibitor to hyaluronan in human and bovine follicular fluid.

The plasma proteinase inter-alpha-trypsin inhibitor is necessary for normal expansion of the cumulus-oocyte complex (COC) and lack of inter-alpha-trypsin inhibitor results in severe infertility. After diffusion from the circulation into the follicles, inter-alpha-trypsin inhibitor is incorporated into the extracellular hyaluronan network of the expanding COC. However, mixing isolated inter-alpha-trypsin inhibitor with hyaluronan in vitro does not result in coupling to hyaluronan. Other components must be present. A recently developed electrophoretic technique by which hyaluronan-bound inter-alpha-trypsin inhibitor is immobilized was used to demonstrate coupling activity in human and bovine follicular fluid that is necessary for the formation of a firm binding between inter-alpha-trypsin inhibitor heavy chains and hyaluronan, as observed in vivo. No coupling activity could be detected in human serum. Coupling occurred only in the presence of follicular fluid. The coupling activity of follicular fluid was irreversibly destroyed by heat treatment, lowering of pH or tryptic digestion, indicating that the coupling activity is associated with a protein. Calcium ions are essential for the coupling reaction. The binding reaction in vitro using intact inter-alpha-trypsin inhibitor is slow and occurs over 24 h. The early-formed complexes between inter-alpha-trypsin inhibitor and hyaluronan contain small amounts of bikunin, whereas the end product contains heavy chains and essentially no bikunin. The heavy chains released from inter-alpha-trypsin inhibitor by NaOH treatment bound immediately to hyaluronan, indicating that the dissociation of heavy chains from inter-alpha-trypsin inhibitor is the rate-limiting step. In conclusion, at least four components are essential for the covalent binding of heavy chains to hyaluronan: inter-alpha-trypsin inhibitor and calcium from plasma, hyaluronan and one or more proteins found in follicular fluid.

Concurrent expression of recombination activating genes 1 and 2 in zebrafish olfactory sensory neurons.

Each olfactory sensory neuron (OSN) expresses a single odorant receptor (OR) from a large repertoire of clustered OR genes. It has been hypothesized that OR gene regulation may involve stochastic DNA rearrangement, which in lymphocytes requires the recombination activating genes, rag1 and rag2. We have recently demonstrated that rag1 is expressed in zebrafish OSNs. Here we report that rag2, the obligate partner for rag1 function, is also expressed in OSNs and that its expression pattern mimics that of rag1. The onset of rag1 and rag2 expression preceded that of known zebrafish ORs and the number of rag1-positive OSNs corresponded with the number expressing the olfactory cyclic nucleotide-gated cation channel, an OSN marker. Zebrafish OSNs are the first example of concurrent rag expression in a nonlymphoid tissue. The expression of rag1 and rag2 in OSNs adds to the list of similarities between the olfactory and immune systems that includes monoallelic and mutually exclusive gene expression.

[Risk factors for low back pain among 1.389 pupils in the 8th and 9th grade. An epidemiologic study]. / Risikofaktorer for laendesmerter hos 1.389 skoleelever i 8.- og 9.-klasse. Et epidemiologisk studie.

INTRODUCTION: The aim of this study was to investigate the frequency and severity of low back pain (LBP) in 13-16-year-old pupils in relation to selected factors, such as anthropometry, physical activity, smoking, hypermobility, and tightness of hamstring muscles. METHODS: The study was designed as a cross-sectional questionnaire-based survey and all the pupils were examined by the school doctor for height, weight, mobility of joints, and general health status. RESULTS: The lifetime prevalence of non-specific LBP was 58.9% and the one-year prevalence was 50.8% with no differences between the sexes. Recurrent/continuous LBP in a moderate to severe degree was recorded in 19.4% of the school children (182 F, 88 M). This was positively correlated to a female gender, a BMI of more than 25 kg/square meter, competitive sports for boys, poor physical fitness, daily smoking, heavy jobs in leisure time, increased use of the health system, and reduced life quality. DISCUSSION: Stepwise logistic regression analysis indicates that a female gender, daily smoking, and heavy jobs are important associated factors for severe LBP in adolescents, with an observed probability of 46% if all factors are present. We do not know yet whether these factors have any causal importance for the development of severe LBP.

Glycosaminoglycan-bound and free inter-alpha-trypsin inhibitor components of follicular fluid.

The proteinase inhibitor inter-alpha trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.

Introduction of plasmid DNA into cells.

Transformation of E. coli can be achieved using any of the four protocols in this unit. The first method using calcium chloride gives good transformation efficiencies, is simple to complete, requires no special equipment, and allows storage of competent cells. The alternate one-step method is considerably faster and also gives good transformation efficiencies (although they are somewhat lower). If considerably higher transformation efficiencies are needed, the third method using electroporation is simple, fast, and reliable. As in the calcium chloride protocol, prepared cells can be stored. The final method described is an adaptation of the electroporation protocol that allows direct transfer of vector DNA from yeast into E. coli.

Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR.

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose. The fractional enrichment pattern of teicoplanin produced in the medium containing [1-(13)C]glucose was obtained from a one-dimensional (13)C spectrum. The enrichment pattern showed characteristic peaks indicating that amino acids 3 and 7 are derived from acetate, whereas amino acids 1, 2, 4, 5, and 6 are derived from tyrosine. Multiplet structures in heteronuclear single quantum coherence spectra of teicoplanin produced in the medium containing [U-(13)C]glucose showed characteristic coupling patterns supporting these results. Fractional enrichment patterns and multiplet structures of the three sugars in teicoplanin showed that about 50% of the sugars have the same labeling pattern as the glucose substrate whereas the rest have a labeling pattern showing that they are reassembled, probably from precursors in the primary metabolism.

High-throughput screening brings in the crops.

Screening is the acknowledgement of ignorance. Such a provocative statement might not amuse our community and, in fact, an incredible quantity of knowledge does go into each screen. However, from a structural point of view, screening is where serendipity meets prediction.

Growth and production kinetics of a teicoplanin producing strain of Actinoplanes teichomyceticus.

The growth and production kinetics of a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) was investigated during batch cultivations on defined media. The growth was characterised by two exponential growth phases (EGPs), with a higher specific growth rate in the first than in the second phase. Also the specific rate of formation of teicoplanin was significantly lower in the second phase than in the first phase. This two-phased growth pattern was suggested to be caused by inhibition of growth by teicoplanin accumulated. Furthermore high concentrations of ammonia or phosphate reduced both the specific growth rate in the first EGP and the total production of teicoplanin.

Risk factors for low back pain in a cohort of 1389 Danish school children: an epidemiologic study.

This study was designed as a cross-sectional questionnaire-based survey of low back pain (LBP) in 13- to 16-year-old Danish school children. The cohort consisted of 671 boys and 718 girls in eighth and ninth grade in 46 municipal schools in three counties of Sealand. All the pupils filled in a questionnaire with LBP as the main topic and were at the same time examined by the school doctors. The first part of the questionnaire contained questions about leisure time sports activity, TV watching, PC use, job in leisure time and smoking. The second part dealt with LBP in relation to frequency and severity, influence on daily living and use of the health system. The school doctor measured body height and weight, (BMI), degree of hypermobility and the tightness of the hamstring muscles. The results showed a cumulative life-time prevalence of LBP of 58.9%, a 1-year prevalence of 50.8% and an increase in LBP prevalence of 6.4% from 14 to 15 years of age, independent of gender. Fourteen percent (141 F, 54 M) fulfilled the criteria for general hypermobility and 12.2% (45 F, 124 M) had tightness of hamstring muscles of more than 40 degrees. Recurrent/continuous LBP in a moderate to severe degree was recorded in 19.4% of children (182 F, 88 M). This was positively correlated to female gender, BMI more than 25 kg/m(2), competitive sport for boys, poor physical fitness, daily smoking, heavy jobs in leisure time, increased use of the health system and reduced life quality. Stepwise logistic regression analysis indicates that female gender, daily smoking and heavy jobs are important associated factors for severe LBP in adolescents, with an observed probability of 46% if all factors are present. We don't know yet whether these factors are of any causal importance in the development of severe LBP.

Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene.

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.

Long-term ACE inhibition doubles lifespan of hypertensive rats.

BACKGROUND: We compared the outcome of lifelong treatment with the ACE inhibitor ramipril in young prehypertensive stroke-prone spontaneously hypertensive rats (SHR-SP) and age-matched normotensive Wistar-Kyoto (WKY) rats. Ramipril was given in an antihypertensive and subantihypertensive dose. In addition to the primary end point, lifespan, surrogate parameters such as cardiac left ventricular hypertrophy, cardiac function and metabolism, and endothelial function were studied. METHODS AND RESULTS: One-month-old SHR-SP and WKY rats, 135 of each, were randomized into 3 groups. Each group was treated via drinking water with an antihypertensive high dose of ramipril (HRA, 1 mg x kg(-1) x d(-1)), a nonantihypertensive low dose of ramipril (LRA, 10 microg x kg(-1) x d(-1)), or placebo. Body weight and blood pressure were determined every 3 months. Molecular, biochemical, and functional data were assessed in SHR-SP and WKY rats after 15 and 30 months, respectively. These were the times when approximately 80% of the corresponding placebo group had died. Early-onset long-term ACE inhibition with HRA doubled lifespan to 30 months in SHR-SP, which was identical to the lifespan of placebo-treated normotensive WKY rats. LRA treatment prolonged lifespan from 15 to 18 months. In SHR-SP, left ventricular hypertrophy was completely prevented by HRA but not by LRA treatment. Cardiac function and metabolism as well as endothelial function were significantly improved by both doses of ramipril. Carotid expression of endothelial NO synthase was moderately enhanced, whereas cardiac ACE expression and activity were decreased to values of placebo-treated WKY rats. CONCLUSIONS: Lifelong ACE inhibition doubles lifespan in SHR-SP, matching that of normotensive WKY rats. This effect correlated with preservation of endothelial function, cardiac function/size, and metabolism. Thus, these data predict a beneficial outcome on survival in high-risk patients with hypertension and associated cardiovascular diseases by ACE inhibition.

Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2.

A network of interacting proteins controls the activity of cyclin-dependent kinase 2 (Cdk2) (refs 1,2) and governs the entry of higher eukaryotic cells into S phase. Analysis of this and other genetic regulatory networks would be facilitated by intracellular reagents that recognize specific targets and inhibit specific network connections. We report here the expression of a combinatorial library of constrained 20-residue peptides displayed by the active-site loop of Escherichia coli thioredoxin, and the use of a two-hybrid system to select those that bind human Cdk2. These peptide aptamers were designed to mimic the recognition function of the complementarity-determining regions of immunoglobulins. The aptamers recognized different epitopes on the Cdk2 surface with equilibrium dissociation constant in the nanomolar range; those tested inhibited Cdk2 activity. Our results show that peptide aptamers bear some analogies with monoclonal antibodies, with the advantages that they are isolated together with their coding genes, that their small size should allow their structures to be solved, and that they are designated to function inside cells.

False negative anti-HIV-1/HIV-2 ELISAs in acute HIV-2 infection.

Since HIV-2 was isolated in 1986, only 1 case of acute HIV-2 infection has been reported. We have identified another patient with primary HIV-2 infection. Follow-up samples were requested from the patient due to discrepant results. The HIV-2 infection was confirmed with HIV-2-specific proviral DNA amplification by PCR. The HIV-2 seroconversion panel obtained was used to evaluate the sensitivity of both combined and specific ELISAs currently in use in Europe, and to investigate the Western-blot patterns on both HIV-1-and HIV-2-specific Western blots. The window period was determined to be less than 37 days with the most sensitive assays. A remarkable difference in sensitivity to HIV-2 antibodies in acute HIV-2 infection was found in combined HIV-1/HIV-2 ELISAs. Three out of the 4 combined sandwich ELISAs appeared to be less sensitive than the indirect ELISAs in HIV-2 seroconversion, leading to a prolonged window period. One HIV-2-specific ELISA was also negative on the first sample, but positive on the second sample. In the HIV-2 Western blot, early reaction with HIV-2-specific env and gag proteins was seen, whereas the HIV-1 Western blot on the first sample revealed gag (p24, p55) reactivity only.

Crystal structure of the U1A spliceosomal protein complexed with its cognate RNA hairpin.

We have determined the crystal structure of the RNA binding domain of the U1A spliceosomal protein bound to a 21-nucleotide RNA hairpin at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the four-stranded beta-sheet of the RNP domain as an open structure. The AUUGCAC hexanucleotide sequence interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. The stacking interaction between RNA bases and aromatic protein side chains and the extensive hydrogen bonding network involving RNA bases, protein side chains and protein mainchain amide and carbonyl groups are crucial for the sequence specific recognition of RNA.

In vivo binding of human inter-alpha-trypsin inhibitor free heavy chains to hyaluronic acid.

In vivo binding of human inter-alpha-trypsin inhibitor to hyaluronate was investigated by immunoelectrophoretic techniques. Pathological synovial fluids and follicular fluids both contain high concentrations of soluble hyaluronate. Heavy chain epitopes of inter-alpha-trypsin inhibitor were firmly associated with the hyaluronate in synovial fluid and follicular fluid. The hyaluronate-bound inter-alpha-trypsin inhibitor epitopes did not cross-react immunologically with bikunin. Several hyaluronate-bound inter-alpha-trypsin inhibitor fragments with molecular masses in the range 120,000-30,000 Da were demonstrated by immunoblotting. Heavy chain 1 of inter-alpha-trypsin inhibitor was shown to associate with hyaluronate by amino acid sequence analysis of isolated hyaluronate-bound proteins. These data indicate that in vivo metabolism of inter-alpha-trypsin inhibitor takes place in pathological synovial fluid and in ovarian follicular fluid shortly before ovulation.

Mutant hemoglobins (alpha 119-Ala and beta 55-Ser): functions related to high-altitude respiration in geese.

The unusually high blood-O2 affinity in the bar-headed and Andean geese is a necessary adaptation for migration across high mountain ranges. The amino acid residues alpha-119 and beta-55, which form an alpha 1 beta 1 contact in human hemoglobin (Hb), are altered in bar-headed and Andean geese, respectively, which suggests that loss of this contact increases O2 affinity. Two mutant human Hbs with equivalent mutations at these sites prepared by site-directed mutagenesis show the same increase in O2 affinity compared with Hb A, which indicates that these mutations are responsible for the changes in the protein. The intrinsic affinity difference compared with native Hb A is amplified by organic phosphates. Whereas the recombinant and native Hbs displayed similar sensitivities to pH, chloride, and 2,3-diphosphoglycerate, the oxygenation heat of the alpha-chain mutant decreased in the presence of 2,3-diphosphoglycerate. O2 association constants for the deoxygenated state of the alpha-mutant were about three times those for Hb A. The mutant Hb analogously exhibited higher affinity constants for binding the first three O2 molecules. Calculated heme-heme interaction energies indicated that loss of a single contact, resulting in destabilization of the deoxy (tense) structure, underlies the increased O2 affinity. Adaptations securing Hb-O2 binding at extreme altitude are discussed.