The Role of Epigenetic Regulation in Transcriptional Memory in the Immune System.

The immune system is exquisitely poised to identify, respond to, and eradicate pathogens from the body, as well as to produce a more rapid and augmented response to a subsequent encounter with the pathogen. These cellular responses rely on the highly coordinated and rapid activation of gene expression programs as well as the ability of the cell to retain a memory of the initial gene response. It is clear that chromatin structure and epigenetic mechanisms play a crucial role in determining these gene responses, and in fact the immune system has proved an instructive model for investigating the multifaceted mechanisms through which the chromatin landscape contributes to gene expression programs. These mechanisms include modifications to the DNA and histone proteins, the positioning, composition, and remodeling of nucleosomes, as well as the formation of higher-order chromatin structures. Moreover, it is now apparent that epigenetic mechanisms also provide an instrument by which cells can retain memory of the initial transcriptional response, "priming" the genome so that it can respond more quickly to subsequent exposure to the signal. Here, we use the immune system as a model to demonstrate the complex interplay between transcription factors and the chromatin landscape required to orchestrate precise gene responses to external stimuli and further to demonstrate how these interactions can establish memory of past transcriptional events. We focus on what we have learnt from the immune system and how this can inform our understanding of other cellular systems.

Spurious elevation of serum PSA after curative treatment for prostate cancer: clinical consequences and the role of heterophilic antibodies.

BACKGROUND: Various interferences can cause spurious results for common laboratory tests. Although rare, heterophilic antibodies may produce false elevations in PSA that could prompt unnecessary therapy in men previously treated for prostate cancer. The aim of this study was to determine the prevalence of small, spurious PSA elevations, and the role of heterophilic antibodies. METHODS: Phase I: all PSA tests drawn and measured between 27 October 2008 and 26 October 2010 at Vanderbilt University Medical Center were analyzed (n=17 133). Patients who had been treated for prostate cancer with PSA values that changed from undetectable to detectable were evaluated. Phase II: patients with a detectable PSA ≤0.5 ng ml(-1) measured between 24 October 2010 and 19 January 2011 were studied prospectively (n=1288). If any patient had a previously undetectable PSA value, their serum was tested for heterophilic antibody interference. RESULTS: Phase I: 11 men had a spuriously elevated PSA after curative treatment for prostate cancer (0.3%). Mean time to PSA elevation was 3.4±5.5 years, and mean elevation in PSA was 0.33±0.28 ng ml(-1). Each patient's PSA was undetectable after being repeated, and no patient went on to unnecessary treatment. Phase II: 10 men had a newly detectable PSA, 9 of whom had a history of prostate cancer. Each tested negative for interfering heterophilic antibodies when their PSA test was repeated with a heterophilic antibody-blocking reagent. CONCLUSIONS: In a large cohort, we estimate the prevalence of spuriously elevated PSA values in our population to be 0.3%. No patient with a prostate cancer history was subjected to unnecessary diagnostic evaluation or treatment. On prospective evaluation of PSA conversion to low detectable levels, no patient had evidence of interfering heterophilic antibodies. When using PSA for post-treatment surveillance, it is crucial to confirm all concerning values and consider the presence of a spurious elevation in PSA if the value does not correlate with the clinical scenario.

Method for rapid determination of ion gauge sensitivity factors.

In ultrahigh vacuum thin film growth processes using gas phase growth precursors, the pressure of the gas at or near the substrate is a critical parameter since it is directly related to the collision frequency of the precursor with the substrate and ultimately to the growth rate. These pressures are usually measured using a nude Bayrd-Alpert-type ion gauges, which are generally calibrated for nitrogen. Consequently, it is necessary to know the ion gauge sensitivity factor that relates the measured pressure to the actual pressure of the growth precursor. The purpose of this article is to describe a simple method to obtain such sensitivity factors. This method uses a simple gas manifold comprised of equipment commonly found in laboratory settings where ultrahigh vacuum work is performed. Results are reported for dimethyl silane, monomethyl silane, methane, and hydrogen. The gauge sensitivity factors for the latter two gases are known and, therefore, provide a basis for validating the method.

Umbilical cord blood gas analysis at delivery.

BACKGROUND: Umbilical cord blood gas values are better indicators of perinatal asphyxia than Apgar scores. However, the reported normal range of umbilical blood gas values vary greatly in the literature. The aim of this prospective study was to establish the normal range of umbilical cord blood gas values in our labour ward. METHODS AND RESULTS: Umbilical cord blood gas from 153 vaginal deliveries and 52 Caesarean sections for indication other than fetal distress were evaluated. In our labour ward, the mean and standard deviation of umbilical artery pH were 7.21 and 0.08 for vaginal deliveries and 7.22 and 0.07 for Caesarean sections respectively. The mean and standard deviation for umbilical artery base deficit were 5.08 and 3.85 for vaginal deliveries and 4.09 and 3.07 for Caesarean sections respectively. CONCLUSION: In conclusion, pH of 7.05 is the statistical lower limit of umbilical artery pH in our labour ward and is a good cut-off value to indicate perinatal asphyxia. Five cases with abnormal umbilical artery pH (< 7.05) were also analysed and discussed.

Molecular cloning and sequencing of cDNAs encoding insecticidal peptides from the primitive hunting spider, Plectreurys tristis (Simon).

Plectreurys tristis cephalothorax mRNA was isolated and amplified by PCR using degenerate primers corresponding to reverse translated mature Plt-VI toxin. An oligonucleotide corresponding to a portion of the amplified product was then used to screen a P. tristis cDNA library. The cDNAs from 10 positive clones were sequenced. Eight of these cDNAs corresponded to Plt-VI toxin, one to Plt-XI toxin, and one was very similar to Plt-VIII toxin, with the exception of a single amino acid substitution. Analysis of these cDNAs indicated that these toxins are initially synthesized as prepro-forms which undergo signal cleavage followed by additional processing at both their N- and C-termini to produce the mature products.

A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase.

Acetolactate synthase (ALS) inhibitors are among the most commonly used herbicides. They fall into four distinct families of compounds: sulfonylureas, imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We have investigated the molecular basis of imidazolinone tolerance of two field isolates of cocklebur (Xanthium sp.) from Mississippi and Missouri. In both cases, tolerance was conferred by a form of ALS that was less sensitive to inhibitors than the wild type. The insensitivity pattern of the Mississippi isolate was similar to that of a commercial mutant of corn generated in the laboratory: ICI 8532 IT. Sequencing revealed that the same residue (Ala57-->Thr) was mutated in both Mississippi cocklebur and ICI 8532 IT corn. ALS from the Missouri isolate was highly insensitive to all the ALS herbicide families, similar in this respect to another commercial corn mutant: Pioneer 3180 IR corn. Sequencing of ALS from both plants revealed a common mutation that changed Trp552 to Leu. The sensitive cocklebur ALS cDNA, fused with a glutathione S-transferase, was functionally expressed in Escherichia coli. The recombinant protein had enzymatic properties similar to those of the plant enzyme. All the possible point mutations affecting Trp552 were investigated by site-directed mutagenesis. Only the Trp-->Leu mutation yielded an active enzyme. This mutation conferred a dramatically reduced sensitivity toward representatives of all four chemical families, demonstrating its role in herbicide tolerance. This study indicates that mutations conferring herbicide tolerance, obtained in an artificial environment, also occur in nature, where the selection pressure is much lower. Thus, this study validates the use of laboratory models to predict mutations that may develop in natural populations.

Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.

Characterization of the precursor for Manduca sexta diuretic hormone Mas-DH.

We have isolated a cDNA clone encoding a precursor form of the diuretic hormone from the tobacco hornworm Manduca sexta (Mas-DH). Translation of the cDNA revealed a 138-amino acid precursor consisting of the Mas-DH amino acid sequence bounded by dibasic amino acid processing sites, a putative signal sequence, and additional peptide sequence on either side of the Mas-DH coding sequence. The region of the precursor upstream of the mature Mas-DH sequence shows limited (28%) homology to the cryptic region of the ovine corticotropin-releasing factor precursor. The Mas-DH RNA is 1.5-1.6 kilobases long; it is present in both the heads and bodies of adult and larval insects. In prewandering fifth stadium larvae, Mas-DH mRNA is expressed in brain, nerve cord, gut, and Malpighian tubules, but not in the fat body. There is a single genomic copy of the Mas-DH gene; the message is multiply spliced.

Chlorine dioxide gas sterilization of oxygenators in an industrial scale sterilizer: a successful model.

Artificial organ oxygenators, with large surface areas and complicated structures, were sterilized using chlorine dioxide gas in an industrial scale sterilizer. Bacillus subtilis var. niger ATCC 9372 biological indicators (BI) (10(6) spores/BI) planted in the artificial organs were reproducibly sterilized in a designed cycle schedule with a 30-min dwell time with a chlorine dioxide gas concentration of approximately 30 mg/L in 80 to 85% relative humidity at 30 degrees C. The D value (time required for 90% spore inactivation under specific conditions) was estimated to be 4.4 min. Chlorine dioxide was not detected after post-sterilization aeration. The intravenous median lethal dose (LD50) of chlorine dioxide derivatives, chlorite and chlorate, in rats was found to be 112.8 and 2,228.6 mg/kg, respectively. In an immediate hypersensitivity test, chlorine dioxide gas-treated ovalbumin and bovine serum albumin, unlike ethylene oxide gas-treated proteins, did not cause sterilant-specific IgE-mediated hypersensitivity reactions in rats. Results of an Ames mutagenicity test on chlorine dioxide and on the extracts of the chlorine dioxide gas-exposed oxygenators were negative.

Chlorine Dioxide Gas Sterilization under Square-Wave Conditions.

Experiments were designed to study chlorine dioxide (CD) gas sterilization under square-wave conditions. By using controlled humidity, gas concentration, and temperature at atmospheric pressure, standard biological indicators (BIs) and spore disks of environmental isolates were exposed to CD gas. The sporicidal activity of CD gas was found to be concentration dependent. Prehumidification enhanced the CD activity. The D values (time required for 90% inactivation) of Bacillus subtilis subsp. niger ATCC 9372 BIs were estimated to be 1.5, 2.5, and 4.2 min when exposed to CD concentrations of 30, 15, and 7 mg/liter, respectively, at 23 degrees C and ambient (20 to 40%) relative humidity (RH). Survivor tailings were observed. Prehumidification of BIs to 70 to 75% RH in an environmental chamber for 30 min resulted in a D value of 1.6 min after exposure to a concentration of 6 to 7 mg of CD per liter at 23 degrees C and eliminated survivor tailing. Prolonging prehumidification at 70 to 75% RH for up to 16 h did not further improve the inactivation rate. Prehumidification by ultrasonic nebulization was found to be more effective than prehumidification in the environmental chamber, improving the D value to 0.55 min at a CD concentration of 6 to 7 mg/liter. Based on the current observations, CD gas is estimated, on a molar concentration basis, to be 1,075 times more potent than ethylene oxide as a sterilant at 30 degrees C. A comparative study showed B. subtilis var. niger BIs were more resistant than other types of BIs and most of the tested bacterial spores of environmental isolates.

Mechanism of microwave sterilization in the dry state.

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.

Relationship of glutathione depletion and inhibition of glutathione-S-transferase activity to the antimitotic properties of estramustine.

A depletion of intracellular glutathione (GSH) is accompanied by a subsequent inhibition of GSH-S-transferase activity in a DU145 human prostatic carcinoma cell line treated with cytotoxic concentrations of estramustine. When GSH depletion reached approximately 50% of normal (approximately 2 hours after 10 microM estramustine), the mitotic index of logarithmically dividing cultures began to increase. A linear increase from 3.14% to 22.5% occurred during the period of 2-24 hours following 10 microM estramustine. Morphological studies showed that mitotic figures accumulated in metaphase and had abnormalities consistent with spindle malformation. Nocodazole and cytochalasin B also possess anticytoskeletal properties, but had little effect upon the intracellular levels of GSH or its associated transferase enzymes. The constituent moieties of estramustine, estradiol, and nor-mechlorethamine had effects on thiol metabolism which were dissimilar from estramustine, confirming previous findings that the unmetabolized parent drug is responsible for pharmacological activity. Estramustine had no effect upon GSH reductase activity, suggesting that drug toxicity was not a general thiol phenomenon. Buthionine sulfoximine decreased intracellular GSH in DU145 cells and enhanced the cytotoxic potential of estramustine in this cell line. The anticytoskeletal and antimitotic properties of estramustine may be enhanced by the drug-induced GSH imbalance and subsequent effects on GSH-S-transferase enzymes.

Effects of sodium on chemotactic peptide binding to polymorphonuclear leukocytes.

The affinity of binding of the chemotactic peptide N-formylnorleucylleucylphenylalanine to rabbit peritoneal polymorphonuclear leukocytes is increased when sodium ions are removed from the medium. In Hanks' balanced salt solution, the dissociation constant of the binding is about 2 X 10(-8) M, while in Na+-free medium, the dissociation constant is between 3 and 6 X 10(-9) M. Removal of Na+ appears to cause little or no change in receptor number. The change in affinity is rapid and reversible, occurs at 4 degrees C as well as 37 degrees C, and occurs when the Na+ is replaced by K+, choline, or sucrose. The increased binding of low concentrations of peptide is seen on broken as well as whole cells and therefore does not depend on an ion gradient across the membrane. The high affinity receptors are functional in mediating peptide uptake and lysosomal enzyme release. The receptors undergo down-regulation in Na+-free medium, and the dose dependence of the receptor loss is shifted to lower concentrations consistent with the higher affinity of the binding.