Biomedical subjects

J Johnson

Publications and source records attributed to J Johnson.

At least 55 records · Page 3Linked to original sources

PubMed2001

A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa.

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1 epsilon. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frq Delta PEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frq Delta PEST-1 RNA and FRQ Delta PEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.

Amino Acid Sequence↗

PubMed2001

Effect of aerosolized uridine 5'-triphosphate on mucociliary clearance in mild chronic bronchitis.

Previous studies show that uridine 5'-triphosphate (UTP), a P2Y(2) receptor agonist, is effective at acutely enhancing mucociliary clearance in healthy, nonsmoking adults. UTP solution for inhalation is being developed by Inspire Pharmaceuticals under the compound number INS316. In a double-blind, randomized, crossover, placebo-controlled study we tested the single-dose effect of UTP in chronic smokers with mild chronic bronchitis (n = 15) by measuring the clearance of (99m)Tc-Fe(2)O(3) particles (4.0 microm mass median aerodynamic diameter [MMAD]) after inhalation of nebulized placebo (0.9% saline) and two doses of UTP (20 and 100 mg in the nebulizer). On each study day, gamma camera scanning was performed over a 2-h period. After an initial deposition scan, subjects inhaled placebo or UTP during the first 20 min of scanning. Analysis of whole lung clearance showed that the retention-time curves for each day were biphasic and that the earliest break point in the average curves occurred at 50 min. Mean particle clearance rate (Clr in %/min) through 50 min for placebo treatment was Clr = 0.65 +/- 0.27 whereas treatment with UTP showed Clr significantly increased to 0.95 +/- 0.48 and 0.93 +/- 0.44 for the 20-mg and 100-mg dose respectively, p < 0.005 for both as compared with placebo. These data show that mucociliary clearance associated with mild chronic bronchitis is acutely improved with minimal doses of aerosolized UTP, presumably because of its stimulation of ciliary beating and hydration of airway secretions.

Administration, Inhalation↗

PubMed2001

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies.

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.

Acetophenones↗

PubMed2001

Open-label phase II trial of amprenavir, abacavir, and fixed-dose zidovudine/lamivudine in newly and chronically HIV-1--infected patients.

A Phase II clinical trial was designed to evaluate the efficacy and tolerability of twice-daily abacavir, amprenavir, and zidovudine (ZDV)/lamivudine (3TC) in HIV-1-infected study subjects naive to protease inhibitors and 3TC. Plasma and cerebrospinal fluid (CSF) HIV-1 RNA levels and T-cell subsets were measured. In all, 27 newly diagnosed and 12 chronically HIV-1-infected study subjects are included in the analysis. Week 48 plasma HIV-1 RNA levels were <500 copies/ml in 100% of study subjects, and <50 copies/ml in 80% of chronically infected and 100% of newly infected study subjects. The mean change in CD4 was (+)150 cells/microl (newly infected, p <.001), and (+)155 cells/microl (chronically infected, p <.001). At Week 48, evidence of cellular activation persisted in both cohorts. A twice-daily regimen of amprenavir, abacavir, and ZDV/3TC affords potent viral suppression and significant increases in total CD4(+) cells in HIV-1--infected study subjects. Patient intolerance may limit the efficacy of this combination.

AIDS Dementia Complex↗

PubMed2001

Design, synthesis, and biological evaluation of a series of lavendustin A analogues that inhibit EGFR and Syk tyrosine kinases, as well as tubulin polymerization.

A series of N-alkylamide analogues of the lavendustin A pharmacophore were synthesized and tested for inhibition of the epidermal growth factor receptor (EGFR) protein tyrosine kinase and the nonreceptor protein tyrosine kinase Syk. Although several compounds in the series were effective inhibitors of both kinases, it seemed questionable whether their inhibitory effects on these kinases were responsible for the cytotoxic properties observed in a variety of human cancer cell cultures. Accordingly, a COMPARE analysis of the cytotoxicity profile of the most cytotoxic member of the series was performed, and the results indicated that its cytotoxicity profile was similar to that of antitubulin agents. This mechanism of action was supported by demonstrating that most compounds in the series were moderately effective as inhibitors of tubulin polymerization. This suggests that the lavendustin A analogues reported here, as well as some of the previously reported lavendustin A analogues, may be acting as cytotoxic agents by a mechanism involving the inhibition of tubulin polymerization.

Animals↗

PubMed2001

Circadian regulation of the light input pathway in Neurospora crassa.

FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These findings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction.

Circadian Rhythm↗

PubMed2001

Fast ignition by intense laser-accelerated proton beams.

The concept of fast ignition with inertial confinement fusion (ICF) is a way to reduce the energy required for ignition and burn and to maximize the gain produced by a single implosion. Based on recent experimental findings at the PETAWATT laser at Lawrence Livermore National Laboratory, an intense proton beam to achieve fast ignition is proposed. It is produced by direct laser acceleration and focused onto the pellet from the rear side of an irradiated target and can be integrated into a hohlraum for indirect drive ICF.

Journal Article↗

PubMed2001

Inhibition of CaT1 channel activity by a noncompetitive IP3 antagonist.

A newly cloned, human epithelial Ca2+ transport protein (CaT1) was expressed in Xenopus laevis oocytes, and its single channel characteristics were examined. The CaT1 channel shows a strong dependence upon hyperpolarizing voltages, being activated by very negative voltages. The probability of channel opening and mean open times increase substantially at more negative voltages in the range of -90 to -160 mV. In addition, CaT1 channel activity was markedly inhibited by micromolar levels of a noncompetitive antagonist of the IP3 receptor originally isolated from a marine sponge, Xestospongin C. This inhibitory effect could be mediated indirectly via the binding of Xestospongin C to the inositol-trisphosphate (IP3) receptor or, alternatively, by a direct action on the CaT1 channel itself. Independent of its mechanism of action in inhibiting CaT1, Xestospongin C will provide a useful tool for elucidating the physiological role(s) of this novel epithelial Ca2+ channel.

Animals↗

PubMed2001

Comparative lipid binding study on the cerebroside sulfate activator (saposin B).

Cerebroside sulfate activator (saposin B) is a small protein involved in glycosphingolipid metabolism. It binds certain membrane lipids, making them available to water-soluble enzymes. Defects in this protein are responsible for a form of metachromatic leukodystropy, a progressive neurodegenerative condition. The protein participates in the catabolism of a number of lipids but does show lipid binding selectivity. However, the basis of this selectivity is unclear. Here we assess the relative binding of a number of lipids compared to cerebroside sulfate (sulfatide). We utilize a competitive binding paradigm, in which the lipids compete for protein under favorable conditions and are then switched to a condition in which the complex is stable. This study is unique in that a single molecular species of the activator is employed, and an expanded selection of natural and semisynthetic membrane lipids is surveyed. No simple "binding rule" can be ascertained from these data, but ligands with longer and/or more complex lipoidal and polar adducts appear to be favored.

Animals↗

PubMed2001

The anterior/posterior polarity of somites is disrupted in paraxis-deficient mice.

Establishing the anterior/posterior (A/P) boundary of individual somites is important for setting up the segmental body plan of all vertebrates. Resegmentation of adjacent sclerotomes to form the vertebrae and selective migration of neural crest cells during the formation of the dorsal root ganglia and peripheral nerves occur in response to differential expression of genes in the anterior and posterior halves of the somite. Recent evidence indicates that the A/P axis is established at the anterior end of the presomitic mesoderm prior to overt somitogenesis in response to both Mesp2 and Notch signaling. Here, we report that mice deficient for paraxis, a gene required for somite epithelialization, also display defects in the axial skeleton and peripheral nerves that are consistent with a failure in A/P patterning. Expression of Mesp2 and genes in the Notch pathway were not altered in the presomitic mesoderm of paraxis(-/-) embryos. Furthermore, downstream targets of Notch activation in the presomitic mesoderm, including EphA4, were transcribed normally, indicating that paraxis was not required for Notch signaling. However, genes that were normally restricted to the posterior half of somites were present in a diffuse pattern in the paraxis(-/-) embryos, suggesting a loss of A/P polarity. Collectively, these data indicate a role for paraxis in maintaining somite polarity that is independent of Notch signaling.

Animals↗

PubMed2001

Virologic and immunologic effect of antiretroviral therapy on HIV-1 in gut-associated lymphoid tissue.

OBJECTIVES: We evaluated virologic and immunologic responses to antiretroviral therapy in gut-associated lymphoid tissue (GALT) compared with those found in peripheral blood. METHODS: Eight HIV-1-infected individuals were treated with three reverse transcriptase inhibitors and one protease inhibitor. Endoscopic biopsies were performed at baseline, and at months 1, 2, and 6. We measured the level of cell-associated multiply spliced and unspliced HIV-1 mRNA in GALT and in peripheral blood mononuclear cells. Immunologic responses were assessed by flow cytometry. RESULTS: Levels of multiply spliced HIV-1 mRNA declined in parallel fashion both in peripheral blood and GALT. After 6 months of therapy, unspliced HIV-1 mRNA in the GALT was below assay detection although it persisted in peripheral blood mononuclear cells in 4 study subjects. Although the percentage of CD4+ lymphocytes increased significantly in peripheral blood, only modest increases occurred in GALT. The percentage of activated CD8+ T cells decreased significantly in peripheral blood whereas only modest reductions occurred in GALT. CONCLUSIONS: Antiretroviral therapy effectively suppressed HIV-1 replication in GALT. The percentage of CD4+ T cells in peripheral blood uniformly increased in all study subjects, whereas it was more variable in the GALT.

Adult↗

PubMed2001

The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.

The identification and characterization of DNA sequences necessary for proper gene expression have provided insights into gene regulation and generated tools useful for further experimentation. Studies of developmentally regulated genes have demonstrated how transcription factors interact at enhancers to generate restricted patterns of expression during embryogenesis. In vertebrates, the pursuit of such studies has relied on the generation of transgenic mice and thus has been limited by the time and expense required generating and characterizing these mice. The recently developed technique of in ovo electroporation allows the rapid introduction of exogenous DNA into developing chicken embryos. Here we have used this technique to introduce DNA containing murine enhancer/reporter constructs into cells of the chicken neural tube, resulting in appropriate expression of the reporter. This technique has the potential to greatly reduce the effort involved in the study of vertebrate enhancers. Furthermore, we have characterized factors such as timing of electroporation, concentration of DNA, and choice of basal promoters and found that they can influence the degree to which expression of enhancer constructs reflects endogenous gene expression.

Animals↗

PubMed2001

Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii.

The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M. IgA. IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3-12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.

Acute Disease↗

PubMed2001

Urinary tract infection in adults: research priorities and strategies.

Waning interest in urinary tract infection (UTI) research has limited clinical advances during the past two decades. Although care has improved for some specific UTI syndromes, there is limited evidence for most of the decisions made each day in the management of these infections. Additional clinical research is necessary to improve UTI prevention and care strategies.

Adult↗

PubMed2001

Notch pathway genes are expressed in mammalian ovarian follicles.

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation. This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch signaling pathway in the regulation of mammalian folliculogenesis.

Animals↗

PubMed2001

Protein S levels are lower in women receiving desogestrel-containing combined oral contraceptives (COCs) than in women receiving levonorgestrel-containing COCs at steady state and on cross-over.

This study aimed to identify specific haemostatic changes that might account for previous observations of higher venous thromboembolic risk among users of combined oral contraceptives (COCs) containing desogestrel (DSG) than levonorgestrel (LNG). Sixty-three current users of monophasic 30 microg oestrogen COCs containing either LNG or DSG omitted one pill-free interval (PFI), switching immediately either to the opposite formulation for one cycle or continuing with the same pill. Venesection followed the initial PFI after one cycle (21 tablets) and two cycles (42 tablets) of continuous pill taking, and after the following PFI. Protein S was lower in users of DSG than LNG formulations after the first PFI (mean +/- SD, 0.67 +/- 0.09 vs 0.76 +/- 0.10, P < 0.001) and after one cycle (0.61 +/- 0.09 vs 0.76 +/- 0.09, P < 0.0001). Protein S decreased when switching from LNG to DSG pills (0.77 +/- 0.07-0.65 +/- 0.06, P < 0.0001), mirrored by an increase at switching from DSG to LNG formulations (0.61 +/- 0.08-0.73 +/- 0.10, P < 0.005). Mean protein S levels remained within the normal range. Three different markers of thrombin generation remained unaltered. Potential explanations for COC-related thrombotic events are 'acquired resistance to activated protein C' or inhibition of fibrinolysis. A potential role has been described for protein S deficiency in both. A further triggering factor is a probable prerequisite for actual thrombosis, but pill-takers whose levels of protein S were in the lowest percentiles may be at greatest risk.

Adult↗

PubMed2001

Personal view. Is it reality or an illusion that liquid-based cytology is better than conventional cervical smears?

Liquid-based cytology (LBC) has been heralded as the way forward for cervical screening, and as the answer to many of its problems. It is already used as a sole method of cell preparation in many private clinics in the UK. It is being used for colposcopy smears in many NHS clinics and is now being piloted for primary screening in three screening centres in England, as well as one in Scotland and one in Wales. LBC has been welcomed as a new technology because it deals with the problem of specimen adequacy at source, removing responsibility for slide preparation and fixation from the clinician or nurse. It provides uniformly well-fixed preparations that are free of inflammatory exudate and blood, and seem easier to screen than conventional smears. There are many articles in the world literature suggesting that LBC is more accurate than conventional screening, and it is thought likely to reduce the number of false negative tests. The main reasons for piloting LBC in the NHS Cervical Screening Programme (NHSCSP) lie in its potential for reducing screening times and for reducing the numbers of repeats for inadequate tests. LBC is expensive in terms of equipment, capital costs, maintenance, consumables, training, technical preparation time, transportation and disposal of liquid media. Its costs could be justified if they were offset by the money saved from reduced screening time and repeat tests, but only if its accuracy in terms of sensitivity and specificity were proven to be equal to or better than conventional cytology. Although that is generally held to be true by the public and medical profession alike, there is very little hard evidence to support it.

Female↗

PubMed2001

Inappropriate fosphenytoin use in the ED.

The objective of the study was to evaluate how often intravenous (IV) fosphenytoin is used when oral phenytoin loading is possible. The methods included a retrospective chart review of all patients receiving IV fosphenytoin in the emergency department. We prospectively derived criteria that identify patients with seizures who could receive oral phosphenytoin loading (awake on arrival, alert, no emesis, and lack of endotracheal intubation, repeated seizures, or status epilepticus after arrival). The setting of the study was at an urban, university hospital emergency department with an annual census of 55,000 patients. The outcomes included the number of patients receiving IV fosphenytoin who could have received oral phenytoin loading. From February 1997 to June 1999, 55 patients received IV fosphenytoin. Thirty of these patients (55%, 95% confidence interval 41%-68%) were felt to have received fosphenytoin appropriately. The remaining 25 (45%, 95% confidence interval 32%-59%) patients could have been loaded orally with phenytoin. In a single institution, fosphenytoin administration is inappropriate almost half the time. Oral phenytoin loading is less expensive and safe.

Administration, Oral↗