Time-stretched photonic Doppler velocimetry.

Inertial confinement fusion facilities generate implosions at speeds greater than 100 km/s, and measuring the material velocities is important and challenging. We have developed a new velocimetry technique that uses time-stretched spectral interferometry to increase the measurable velocity range normally limited by the detector bandwidth. In this approach, the signal is encoded on a chirped laser pulse that is stretched in time to reduce the beat frequency before detection. We demonstrate the technique on an imploding liner experiment at the Sandia National Laboratories' Z machine, where beat frequencies in excess of 50 GHz were measured with 20 GHz bandwidth detection.

Surface charge heterogeneity in amphibole cleavage fragments and asbestos fibers.

Aspect ratio and electrophoretic mobility data for amphibole particles reveal that short fibers and blocky cleavage fragments have a smaller net charge than highly elongated particles. Asbestos fibers and cleavage fragments of the same dimensions exhibit the same net negative surface charge but positively charged ends and negatively charged lateral surfaces.

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins.

Cancer cells display an altered distribution of DNA methylation relative to normal cells. Certain tumor suppressor gene promoters are hypermethylated and transcriptionally inactivated, whereas repetitive DNA is hypomethylated and transcriptionally active. Little is understood about how the abnormal DNA methylation patterns of cancer cells are established and maintained. Here, we identify over 20 DNMT3B transcripts from many cancer cell lines and primary acute leukemia cells that contain aberrant splicing at the 5' end of the gene, encoding truncated proteins lacking the C-terminal catalytic domain. Many of these aberrant transcripts retain intron sequences. Although the aberrant transcripts represent a minority of the DNMT3B transcripts present, Western blot analysis demonstrates truncated DNMT3B isoforms in the nuclear protein extracts of cancer cells. To test if expression of a truncated DNMT3B protein could alter the DNA methylation patterns within cells, we expressed DNMT3B7, the most frequently expressed aberrant transcript, in 293 cells. DNMT3B7-expressing 293 cells have altered gene expression as identified by microarray analysis. Some of these changes in gene expression correlate with altered DNA methylation of corresponding CpG islands. These results suggest that truncated DNMT3B proteins could play a role in the abnormal distribution of DNA methylation found in cancer cells.

Characterization and mutational studies of equine infectious anemia virus dUTPase.

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.

The role of retroviral dUTPases in replication and virulence.

Several retroviruses, including equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV) encode dUTPase. The role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dUTPase in virulence and viral mutation rate. Overall, the results of these studies have indicated a central role for dUTPase in facilitating productive viral replication in non-dividing cells. The requirement for dUTPase in EIAV, which replicates exclusively in macrophages, may be the most stringent. Studies of dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major determinant of virulence. In contrast, FIV readily replicates in dividing cell populations such as CD4+ and CD8+ T cells, and B cells as well as in non-dividing macrophages. Thus, the virus burden and disease sequelae are lowered in cats infected with a dUTPase-minus FIV relative to cats infected with wild type FIV, but not totally abrogated. Growth in macrophages is attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G-->A transition mutations in viral integrants present in macrophages. These findings are consistent with an increase in uracil misincorporation in the absence of dUTPase, resulting in transition mutations that cripple the virus. Effects on virus replication and disease production have also been noted for dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase some reports suggest that other viral and host cell factors may substitute for its activity. Betaretroviruses also encode dUTPase and while several of these cause significant disease, the role of dUTPase in their replication and pathogenesis is currently unknown.

Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery.

1. In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), N6-(3-iodo-benzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and 5'-N-ethylcarboxamidoadenosine (NECA), by use of the non-selective antagonist 8-sulphophenyltheophylline (8-SPT) and the A2A selective ligands 2-[p-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxami doadenosine (CGS 21680) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]-triazin-5- ylamino]ethyl) phenol (ZM 241385). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BWA1433). 2. Adenosine, NECA, CPA and R-PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 microM) with the following potency order: NECA > R-PIA > adenosine = CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 microM and IB-MECA elicited relaxant responses which were resistant to blockade by 8-SPT and BWA1433 (100 microM). 3. Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R-PIA in the absence of endothelium. 4. Inhibition of uptake by NBTI (1 microM) had no effect on E/[A] curves to NECA, CPA or R-PIA, but E/[A] curves to adenosine were significantly left-shifted in the presence of NBTI. 5. 8-SPT (10-100 microM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2 = 5.63+/-0.26). The second phase of the concentration-response curve to NECA appeared to be resistant to blockade by 8-SPT, as were E/[A] curves for adenosine, CPA or R-PIA. However, in the presence of NBTI (1 microM), 8-SPT (100 microM) gave significant rightward shifts of E/[A] curves to adenosine. 6. ZM 241385 (0.1-1 microM) produced significant rightward shifts of the high potency phase of NECA E/[A] curves (pA2=7.65+/-0.25 in the presence and 7.20+/-0.12 in the absence of endothelium), while curves to R-PIA were not significantly shifted by 1 microM ZM 241385. In the presence of NBTI E/[A] curves to adenosine were significantly rightward shifted by ZM 241385 (0.1 microM, pA2=7.50+/-0.16). 7. In conclusion, the results suggest activation of A2B receptors located primarily on the smooth muscle by low concentrations of NECA and by adenosine under conditions of uptake blockade, and of another, as yet undefined site which may be intracellular, by higher concentrations of NECA, by CPA, R-PIA and adenosine under conditions where uptake is operational.

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

Simultaneous quantitation of equine cytokine mRNAs using a multi-probe ribonuclease protection assay.

A rapid multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNA levels in activated equine monocyte-derived macrophages (EMDM) and equine peripheral blood mononuclear cells (EPBMC). Eleven template plasmids specific to 10 equine cytokine genes and the beta-actin gene were generated from which radiolabeled anti-sense RNA probes were produced. The multi-probe RPA simultaneously quantitated mRNA levels of equine IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IFN-gamma, TGF-1beta and TNF-alpha in EPBMC and EMDM with coefficients of variation as low as 0.03-0.08 (3-8%) when normalized to beta-actin expression. This sensitive and rapid assay provides a valuable tool for studies of equine immune responses.

Economic evaluation of lamivudine compared with interferon-alpha in the treatment of chronic hepatitis B in the United States.

OBJECTIVE: To determine whether lamivudine or interferon-alpha (IFN-alpha) is the more successful treatment for chronic hepatitis B given a fixed drug budget. STUDY DESIGN: A decision-tree model of 1 year. PATIENTS AND METHODS: Average wholesale prices were used to estimate drug costs. A fixed drug budget of $558,910, sufficient to treat 100 patients with IFN-alpha, was assumed. Clinical data were taken from randomized controlled trials. The outcome measures used were hepatitis B "e" antigen (HBeAg) seroconversion rates and rates of progression to cirrhosis. RESULTS: The analysis showed that given the fixed drug budget, 353 patients could be treated with lamivudine, resulting in an expected 62 HBeAg seroconversions, with 6 patients progressing to cirrhosis. Given the same drug budget, 100 patients could be treated with IFN-alpha, leaving 253 patients untreated. This treatment scenario would result in an expected 32 HBeAg seroconversions, with 28 patients progressing to cirrhosis. Compared with no treatment, the costs per additional HBeAg seroconversion obtained were $12,703 for lamivudine and $39,922 for IFN-alpha. In addition, each case of cirrhosis avoided through lamivudine treatment resulted in significant annual cost savings. Lamivudine therapy also provided additional clinical benefits (e.g., normalization of alanine transaminase levels, reduction in hepatitis B virus DNA levels, improvement in liver histology) to patients who do not seroconvert. CONCLUSION: From the perspective of a third-party payer with a fixed drug budget, lamivudine is more cost-effective therapy than IFN-alpha for the treatment of chronic hepatitis B.

Differential effects of virulent and avirulent equine infectious anemia virus on macrophage cytokine expression.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia, and viremia. Most infected equid eventually bring the virus under immunological control. We recently reported the development of an equine-specific ribonuclease protection assay (RPA) to quantitate mRNA levels of 10 cytokines. Using this newly developed RPA, we now show significant differences in cytokine induction in equine monocyte-derived macrophages (EMDM) exposed to virulent and avirulent EIAV. Virulent EIAV17 induced significant increases in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, and tumor necrosis factor (TNF)-alpha by 0.5-1 h postinfection (hpi). In contrast, the avirulent virus failed to induce any of the tested cytokines above that of control levels. These data show a direct correlation between cytokine dysregulation and EIAV pathogenesis.

SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties.

Biologic activity of equine infectious anemia virus (EIAV) surface (SU) glycoprotein was assayed in a mouse model. Recombinant SU from virulent EIAV17 (SU17), administered intraperitoneally to mouse pups, induced dose-dependent diarrheal responses similar to those reported for SIV SU (Virology 277 (2000) 250). SU17 caused fluid accumulation without histological lesions in mouse intestinal loops, induced chloride secretory currents in Ussing chambers and increased inositol 1,4,5 triphosphate (IP3) levels in HT29 cells. An SU17 peptide, SU17(299-330), provoked a dose-dependent diarrheal response akin to enterotoxic peptides from SIV. In contrast, SU from an avirulent EIAV strain failed to induce a dose response in mouse pups and produced lower levels of activity than SU17 in Ussing chambers and IP3 assays. These results demonstrate that a mouse pup model is useful to monitor EIAV SU biologic activity, showing clear differences between the activities of SU derived from virulent and avirulent viruses, and may provide a useful screen of EIAV virulence.

Tactual speech perception by minimally trained deaf subjects.

Research on tactual perception of speech has shown that many phonetic contrasts can be transmitted to the deaf through artificial hearing devices that stimulate the sense of touch. Past research has emphasized long-term training with tactual reception for the achievement of maximal perceptual performance. The present study demonstrates that, with a brief training period, deaf adolescents can attain a high level of perceptual performance with a tactual speech system in discrimination of certain hard-to-lipread word pairs pronounced by both a male and female speaker. Thus some speech sounds previously indistinguishable by the deaf people can be immediately available for speech comprehension through the tactual vocoder; and other speech sounds will be recognized with further training. The reason that some contrasts are learned quickly and others require extensive training may be found in a pattern perception postulate proposed by Gavin (1979): word patterns that result in stimulation across a greater area of skin tend to be more discriminable than word patterns which stimulate only small areas of the skin.

Heat injury and recovery of vegetative cells of Clostridium botulinum type E.

Vegetative cells of Clostridium botulinum type E (Tenno) were heated at 40.5 C in a prereduced peptone-yeast extract broth (PY). During the heating period, cell numbers remained essentially constant for 3 h as indicated by roll tube counts in PY agar (PYA); however, injury and recovery from injury were observed when the cells were enumerated using PYA containing either 0.06 or 0.07% bile salts.

A comparison of fluticasone propionate 200 micrograms/day with beclomethasone dipropionate 400 micrograms/day in adult asthma.

A total of 261 patients with symptomatic, mild to moderate asthma were randomized to treatment in this 4-week, double-blind, parallel-group comparison of fluticasone propionate 200 micrograms/d with beclomethasone dipropionate 400 micrograms/d. Improvements from both treatments were seen in diary card data. Morning peak expiratory flow rate (PEFR) improved from 375 to 390 and 371 to 382 l/min with fluticasone propionate and beclomethasone dipropionate, respectively. Symptom scores, percentage of symptom-free days and nights, and use of rescue beta 2-agonist medication also improved, as did clinical lung function. With the exception of percentage of rescue-free days, which was greater for beclomethasone dipropionate, none of the differences between the groups were statistically significant. There was a significant difference between treatments in the number of rescue-free days over days 1-28; however, there was no difference between treatments in the number of rescue-free days over days 1-14, nor was there any difference in the number of inhalations of rescue medication used throughout the study. Very few adverse effects were reported. Although all mean plasma cortisol values were within the normal range, they were significantly different between treatments, rising from 402 to 429 nmol/l with fluticasone propionate, and falling from 435 to 394 nmol/l with beclomethasone dipropionate (P = 0.006). Mean stimulated cortisol levels 30 min after tetracosactin injection were also significantly greater with fluticasone propionate (P = 0.024). In conclusion, fluticasone propionate 200 micrograms/d is as effective as beclomethasone dipropionate 400 micrograms/d with less effect on plasma cortisol levels.

EIAV genomic organization: further characterization by sequencing of purified glycoproteins and cDNA.

Nucleotide sequence analyses of two different proviral clones of equine infectious anemia virus (EIAV), designated lambda 12 (K. Rushlow et al., 1986, Virology 155, 309-321) and 1369 (T. Kawakami et al., 1987, Virology 158, 300-312), indicate significant differences in the organization of two critical regions of the viral genome, i.e., in the short open reading frames in the pol-env intergenic region and in the 5'-end of the env gene. To determine the correct structure of the EIAV genome, we have performed nucleotide sequence analyses of cDNA clones produced from viral RNA and direct sequencing of purified EIAV envelope glycoproteins (gp90 and gp45). The results of the cDNA sequencing confirm the presence of two short open reading frames in the pol-env intergenic region, as reported previously for the lambda 12 clone. The protein sequencing data correlated exactly with the amino-terminal sequences of gp90 and gp45 deduced from lambda 12 nucleotide sequences. However, the protein sequencing also revealed that the putative signal sequence of EIAV gp90 is not removed during processing. Thus, EIAV apparently contains short open reading frames analogous to human immunodeficiency virus, but differs in its mode of env polyprotein processing.

Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli.

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions. Other gp90 segments, both from conserved and variable env sequences, displayed variable reactivities with the panel of equine sera. A panel of MAbs was also used in Western blot assays with the recombinant protein fragments for physical localization of previously identified MAb epitopes. The binding sites of two neutralizing MAbs were localized to a highly variable region of gp90, while nonneutralizing epitopes were localized to conserved and variable regions of the envelope glycoproteins. These results, in addition to localizing important antigenic sites on EIAV glycoproteins, indicate that previously defined conserved and variable env nucleotide sequences indeed encode protein sequences constituting conserved and variable immunogens during persistent infection by EIAV.