DNA binding by the Xis protein of the conjugative transposon Tn916

We purified the Xis protein of the conjugative transposon Tn916 and showed by nuclease protection experiments that Xis bound specifically to sites close to each end of Tn916. These specific binding sites are close to, and in the same relative orientation to, binding sites for the N-terminal domain of Tn916 integrase protein. These results suggest that Xis is involved in the formation of nucleoprotein structures at the ends of Tn916 that help to correctly align the ends so that excision can occur.     

NMR solution structure of the oxidized form of MerP, a mercuric ion binding protein involved in bacterial mercuric ion resistance

Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.     

Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ

The 6.3 kb Clostridium perfringens transposon Tn4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RSA site, and an RSA-like sequence has been identified upstream of the tnpZ gene. In Escherichia coli, in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RSA site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens. Site-directed mutagenesis of two bases within the RSA site resulted in a significant reduction in mobilization frequency, demonstrating that the RSA site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn4451 is the first mobilizable but non-self-transmissible transposon to be identified from a gram-positive bacterium.     

Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds

We have isolated beta-trace protein from cerebrospinal fluid, serum, plasma, and urine samples of normal volunteers and sera and hemofiltrate of patients with chronic renal failure. Blood-derived and urinary beta-trace have significantly higher molecular weights than their cerebrospinal fluid counterpart, the amino acid sequences being identical. Oligosaccharide structural analysis revealed these molecular weight differences to be due to different N-glycosylation. beta-Trace from hemofiltrate and urine has larger sugar chains and concurrently significantly higher sialylation than cerebrospinal fluid-beta-trace which bears truncated "brain-type" oligosaccharide chains (published previously). beta-Trace concentrations were about 40 ng/ml for normal sera and plasma. 2000-6000 ng/ml were measured in sera of dialysis patients whereas in normal human cerebrospinal fluid, beta-trace concentration was about 8000 ng/ml. A reduced amount of 900 ng/ml was found in a single case of hydrocephalus cerebri. The sialylated glycoforms of beta-trace detected in the blood are presumably derived from resorbed cerebrospinal fluid protein whereas beta-TP-molecules bearing asialo-oligosaccharides are absent due to their hepatic clearance. The residual, sialylated beta-TP-species are probably eliminated from the blood via the kidney. This physiological clearance mechanism for the sialylated glycoforms is disturbed in hemodialysis patients resulting in about 100-fold elevated serum concentrations. These results let us suggest beta-trace may become a useful novel diagnostic protein in renal diseases.     

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.     

Participation of mercuric conjugates of cysteine, homocysteine, and N-acetylcysteine in mechanisms involved in the renal tubular uptake of inorganic mercury

Mechanisms involved in the renal uptake of inorganic mercury were studied in rats administered a nontoxic 0.5 mumol/kg intravenous dose of inorganic mercury with or without 2.0 mumol/kg cysteine, homocysteine, or N-acetylcysteine. The renal disposition of mercury was studied 1 h after treatment in normal rats and rats that had undergone bilateral ureteral ligation. In addition, the disposition of mercury (including the urinary and fecal excretion of mercury) was evaluated 24 h after treatment. In normal rats, coadministering inorganic mercury plus cysteine or homocysteine caused a significant increase in the renal uptake of mercury 1 h after treatment. The enhanced renal uptake of mercury was due to increased uptake of mercury in the renal outer stripe of the outer medulla and/or renal cortex. Ureteral ligation caused reductions in the renal uptake of mercury in all groups except for the one treated with inorganic mercury plus N-acetylcysteine. Thus, it appears that virtually all of the mercury taken up by the kidneys of the normal rats treated with inorganic mercury plus N-acetylcysteine occurred at the basolateral membrane. Urinary excretory data also support this notion, in that the rate of excretion of inorganic mercury was greatest in the rats treated with inorganic mercury plus N-acetylcysteine. Our data also indicate that uptake of inorganic mercury in the kidneys of rats treated with inorganic mercury plus cysteine occurred equally at both luminal and basolateral membranes. In addition, the renal uptake of mercury in rats treated with inorganic mercury plus homocysteine occurred predominantly at the basolateral membrane with some component of luminal uptake. The findings of the present study confirm that there are at least two distinct mechanisms involved in the renal uptake of inorganic mercury, with one mechanism located on the luminal membrane and the other located on the basolateral membrane. Our findings also show that cysteine and homologs of cysteine, when coadministered with inorganic mercury, greatly influence the magnitude and/or site of uptake of mercuric ions in the kidney.     

Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Intestinal ion transport: effect of norepinephrine, pilocarpine, and atropine

The effects of parenteral pilocarpine, atropine, and norepinephrine on salt and water transport were studied in jejunum and ileum of anesthetized rats. Pilocarpine increased jejunal transmural PD, reduced absorption of Na, K, HCO3, and H2O, and increased secretion of Cl; in ileum, it caused secretion of Na and H2O, elicited secretion of K, and reduced the absorption of Cl. In both segments, perfusate became more akaline, and there was less of a rise in PCO2. Atropine prevented all changes caused by pilocarpine. Atropine alone increased jejunal absorption of Na and HCO3 and acidity of perfusate, implying that cholinergic nerves influence transport. Norepinephrine augmented jejunal absorption of Na, Cl, and H2O but caused no change in PD. In ileum, norepinephrine increased absorption of Na and Cl, reduced the rise in pH, increased the rise in PCO2 of perfusate, but did not affect net HCO3 movement. With all agents, when Na absorption increased, perfusate became more acidic in jejunum and less alkaline in ileum, evidence of an association between Na and H transport.     

Basolateral uptake of mercuric conjugates of N-acetylcysteine and cysteine in the kidney involves the organic anion transport system

Methodology used for the development of anti-Alzheimer's disease (AD) drugs raises specific problems which are rarely examined in the literature. While the general development scheme is similar to that required for most drugs, some specific aspects must be analyzed, highly dominated by the dual goal of pharmacology, i.e., to obtain both symptomatic and etiopathogenic drugs. During preclinical studies, aged or lesioned animals are mainly useful for symptomatic drugs, whereas transgenic models or neurodegeneration-induced techniques would probably lead to etiopathogenic drugs potentially slowing down the process of AD. The first administrations of a new compound to human beings raise the question of the activity measurement techniques. Psychometry remains the most informative procedure to detect and analyze the activity of the drugs on the different components of cognition. Electrophysiology and neuroimaging need some complementary studies before they can be proposed as surrogate criteria in phase III trials. At this stage of development, American and the recently published European guidelines are of great help while insisting on long-term (6 months) placebo controlled trials with the use of the triple efficacy criterion: an objective cognition scale, a global assessment, and the opinion of the caregiver. In the long term, pharmacoepidemiology and pharmacoeconomy will have to confirm the rationale of this recent progress in the methodology of anti-AD drug development.     

Characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62: instability of transposon Tn1545 delta3 in gonococci and evidence that multiple genetic loci are essential for lipooligosaccharide sialylation

Neisseria gonorrhoeae strain JB1 was previously shown to be defective in the sialylation of lipoologosaccharide (LOS) by exogenous CMP-NANA. The LOS components synthesized by the mutant now have been shown by mass spectrometry to be similar to those in the parental strain, F62, and to include the 4.5 kDa widely conserved lacto-N-neotetraose component that can be sialylated. The same two LOS components could be sialylated on the surface of the mutant and parental strains. One major component was sialylatable after chemical extraction of the LOS from either strain. These data confirm that the mutant, JB1, retains the ability to synthesize the LOS target required for the conversion by sialylation of serum-sensitive gonococci to serum resistance. A single base frame-shift mutation was found in the lst gene from the mutant, resulting in the replacement of the final 61 amino acids at the C-terminus of the sialyltransferase by four residues. Seventeen independent clones of the lst gene were isolated from the parental strain, but none of them complemented the sialyltransferase defect of the mutant and no sialyltransferase activity expressed from the clones could be detected in Escherichia coli. Although the data suggest that the mutant might be defective in genes at more than one chromosomal locus and that multiple loci are essential for sialyltransferase synthesis and activity, the alternative possibility, that DNA adjacent to the lst gene encodes a product which is toxic to E. coli, cannot be excluded. The site of insertion of the transposon Tn1545-Delta3 in strain JB1 was cloned and sequenced. The transposon is located in an intergenic region adjacent to genes for a putative ATP-dependent transport protein, but encoding no recognizable function relevant to LOS sialylation. Evidence that transposon Tn1545-Delta3 is unstable in gonococci is presented.     

Periodic selection, infectious gene exchange and the genetic structure of E. coli populations

As a consequence of sequential replacements by clones of higher fitness (periodic selection), bacterial populations would be continually purged of genetic variability, and the fate of selectively neutral alleles in very large populations of bacteria would be similar to that in demes of sexually reproducing organisms with small genetically effective population sizes. The significance of periodic selection in reducing genetic variability in these clonally reproducing species is dependent on the amount of genetic exchange between clones (recombination). In an effort to determine the relationship between the rates of periodic selection, recombination and the genetically effective sizes of bacterial populations, a model for periodic selection and infectious gene exchange has been developed and its properties analyzed. It shows that, for a given periodic selection regime, genetically effective population size increases exponentially with the rate of recombination.--With the parameters of this model in the range anticipated for natural populations of E. coli, the purging effects of periodic selection on genetic variability are significant; individual populations or lineages of this bacterial species would have very small genetically effective population sizes.--Based on this result, some other a priori considerations and a review of the results of epidemiological and genetic variability studies, it is postulated that E. coli is composed of a relatively limited number of geographically widespread and genetically nearly isolated and monomorphic lineages. The implications of these considerations of the genetic structure of E. coli populations on the interpretation of protein variation and the neutral gene hypothesis are discussed.     

Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1

The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e. cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa. Recently we have cloned a gene encoding a 68 kDa (p68) protein product, which is associated with chromosomes during interphase. The N-terminal 190 amino acids share 43% sequence identity with the second half of the GTPase activating domain (residues 210-397) of GAP3m. The N-terminal fragment of 209 amino acids of Spa-1 (called Span-N) was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein and affinity purified. Rap1 and Rsr1 GTPase stimulatory activity of Spa-1 was tested and compared with GAP3m. Spa-1 preferentially stimulates Rsr1 GTPase rather than Rap1 GTPase, while GAP3m has a preference for Rap1 GTPase. This suggests that although Spa-1 and GAP3m stimulate GTPase of Rap1 family members, they differ in affinity for them. By mutational analysis it was also found that amino acid residues 10-183 are enough for Rap GAP activity of Spa-1.     

Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs

The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.     

Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii

We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).     

The design, synthesis and transmembrane transport studies of a biomimetic sterol-based ion channel

A model sterol-based ion channel was rationally designed and synthesized. The potential ion channel is comprised of a tartrate-derived crown ether to which six steroids are appended. Macromolecule 1a was incorporated into phospholipid vesicles and shown to facilitate the transmembrane transport of sodium and lithium ions using alkali metal NMR spectroscopy.     

Rapid transepithelial antigen transport in rat jejunum: impact of sensitization and the hypersensitivity reaction

BACKGROUND & AIMS: Intestine from sensitized rats develops a rapid secretory response to luminal antigen challenge that depends on activation of subepithelial mast cells. The aim of this study was to determine the timing and route of the transepithelial protein antigen transport. METHODS: Rats were sensitized to horseradish peroxidase (HRP). After 10-14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. RESULTS: Electron microscopy of tissue specimens fixed at 2 minutes (before mast cell activation) showed enhanced endocytic uptake of HRP in enterocytes of HRP-sensitized rats compared with ovalbumin-sensitized or saline-injected controls. At this time, HRP was distributed throughout epithelial cells and was already evident in the lamina propria. In contrast, HRP was restricted to the apical region of enterocytes in controls. At 30 minutes (after mast cell activation), in HRP-sensitized rats only, HRP was also located within tight junctions and the paracellular region between epithelial cells. Tissue conductance was increased in HRP-sensitized rats beginning 30 minutes after HRP addition and correlated with the overall flux of HRP across the tissue. CONCLUSIONS: The results show that specific sensitization enhances the initial uptake and transcytosis of antigen across intestinal epithelium. Subsequent to activation of mast cells, antigen transport is further enhanced by penetration through the paracellular pathway.     

Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the postprandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.