Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.     

Experimental analysis of the annotation of promoters in the public database

The ability to identify and examine promoter elements is important to researchers who wish to understand how gene expression is regulated in normal and pathological states. Unfortunately, the number of human promoters that have been directly experimentally defined is small. In order to determine if promoter sequences can be identified by simply aligning mRNA and genomic sequences, we have used a reporter gene assay to assess the promoter activity of the immediate 5' region flanking 38 mRNAs mapping to chromosome 21. For comparison, we have measured the activities of 19 sequences not thought to be promoters and 39 sequences taken from the Eukaryotic Promoter Database. Our results suggest that alignment of reference mRNAs to genomic sequence allows promoters to be identified for at least 75% of genes. These data provide the first empirical evidence that the current state of annotation of the genome is sufficient to allow molecular geneticists to correctly identify promoter sequences for most genes for which reference mRNA and genomic sequences are available.     

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus

Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of approximately 10(8) plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates > or = 87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates > or = 50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.     

A kinetic analysis of the in vitro sensitization of murine peritoneal mast cells with monoclonal IgE anti-DNP antibody.

Incubation of murine peritoneal cells with monoclonal IgE anti-DNP antibody in vitro led to sensitization of mast cells, measured as release of 5-HT upon challenge with DNP-HSA antigen. Sensitization was maximal at 0.3-3.0 micrograms/ml of IgE anti-DNP and declined above and below this concentration range. In kinetic studies, the time-course of sensitization was clearly divisible into an early slow phase of approximately 4 hr, followed by a more rapid linear phase from 4 to 48 hr. The early slow phase was more pronounced at lower concentrations of IgE anti-DNP (within the range 0.05-5.0 micrograms/ml). The degree of sensitization obtained after incubation of peritoneal cells with IgE anti-DNP for fixed periods (2, 4 and 8 hr) was markedly increased when the cells were washed and recultured in IgE-free medium, thus demonstrating that sensitization proceeds subsequent to an early stage of binding of IgE to receptors. Sensitization with IgE anti-DNP was blocked by addition of excess rat myeloma IgE, but only to a marked extent (greater than 50%) when the blocking immunoglobulin was added during the first 2 hr, thus providing further evidence that the major part of binding of the IgE antibody took place during this early stage, that is, prior to the phase of greatest sensitization. These findings indicate a period of delay between binding of IgE to receptors and functional sensitization, measured as mediator release in response to antigen.     

Measurement of passive membrane parameters with whole-cell recording from neurons in the intact amphibian retina

1. Whole-cell recordings have been obtained from intact, photoactive retinal neurons using patch-clamp electrodes in the amphibian superfused retina eyecup preparation. 2. After removal of the vitreous humor from the surface of the retina, using a collagenase with low tryptic activity, high-resistance seals (1-10 G omega) could be formed between the patch pipette and the cell membrane by applying mild suction to the pipette. Additional suction broke the membrane patch and provided continuity between the low-resistance pipette and the interior of the neuron. 3. Measurements of input resistance and time constant were obtained from bipolar, amacrine, and ganglion cells. Assuming the membrane capacitance was 1 microF/cm2, time constant data were used to derive the specific membrane resistance. The average specific membrane resistance for the inner retinal neurons in our sample was 68,000 omega.cm2. 4. Analysis of the charging curve induced by a brief current pulse applied to the soma was used to analyze the average electrotonic length of dendrites. The charging curves of some ganglion cells were well represented by a single exponential, suggesting that they were essentially isopotential. 5. The voltage decay along an equivalent cylinder model of a ganglion cell was calculated, using the experimentally obtained values of membrane resistance to compute decay of steady-state voltages along the dendritic tree. The calculations indicate that with the high membrane resistance values implied by this study, the electrotonic length of dendritic cables were short, and there may be relatively little attenuation of the synaptic potentials irrespective of their location along the dendritic tree.     

A strain gauge device for measuring feeding or drinking in laboratory rats

The details of mechanical construction and electronic circuitry of a strain gauge system for continuously measuring food and water ingestion in laboratory rats are described. The system has been reliably tested over a number of years. It is eminently suitable for investigating daily rhythms in rat feeding and drinking behavior, where a large volume of data is collected over extended periods of time.     

The effect of ambient temperature cycles upon circadian running and drinking activity in male and female laboratory rats

The effect of a cycle of warm and cool ambient temperature (Ta) upon the free-running circadian running and drinking rhythms of male and female laboratory rats was investigated. Rats free-running in constant darkness and constant cool Ta (21 degrees C +/- 2 degrees C) were exposed to a 12:12 cycle of high (34 degrees C +/- 2 degrees C) and cool (21 degrees C +/- 2 degrees C) Ta. Three male rats and one female rat entrained to the Ta cycle. Ten of 12 male and 9 of 11 female rats exhibited post-Ta cycle phases not predictable from pre-Ta cycle phases. Most rats exhibited positive and negative masking of activity during the Ta cycle. Activity periods shortened for all rats during the Ta cycle, and male free-running periods lengthened upon cessation of the Ta cycle to values significantly greater than precycle periods. It was concluded that Ta acts as a weak zeitgeber in laboratory rats and has greater effects on males compared to females.     

Oligonucleotide fingerprinting of isolates of Candida species other than C. albicans and of atypical Candida species from human immunodeficiency virus-positive and AIDS patients.

Oligonucleotide fingerprinting of genomic DNA from oral isolates of four different Candida species other than C. albicans and atypical chlamydospore-positive isolates from human immunodeficiency virus (HIV)-positive individuals and AIDS patients was investigated as a means for differentiating between isolates within individual species. Oligonucleotides composed of simple repetitive sequence motifs, including (GACA)4, (GATA)4, (GGAT)4, (GTG)5, and (GT)8, all yielded fingerprints suitable for strain segregation of 8 C. tropicalis isolates, 12 Torulopsis (Candida) glabrata isolates, 8 atypical Candida isolates, and, except for (GATA)4, 2 C. krusei probe in turn and so generate several distinct DNA fingerprints of the same DNA sample. However, none of the probes yielded fingerprints suitable for strain segregation with three C. parapsilosis isolates. The (GATA)4 probe was also used to detect restriction fragment length polymorphisms among a genetically closely related group of atypical Candida isolates on primary isolation from an additional HIV-infected patient. These chlamydospore-positive atypical Candida isolates were sucrose positive, were of C. albicans serotype A, hybridized weakly with the C. albicans-specific mid-repeat sequence probe 27A, and yielded fingerprint profiles by random polymorphic DNA analysis that were distinct from those derived from C. albicans isolates. The C. stellatoidea ex-type strain NCPF 3108 was indistinguishable from the atypical Candida isolates in all these tests and also yielded an identical carbohydrate and nitrogen source assimilation profile by using the ID 32C yeast identification system.     

Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID50) in hamsters was determined. The ID50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10(3) cells. The loss of the 27.5- and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID50 from 10(3) to 10(5) cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID50 > 10(3) cells) occurred with cells that lost the 24.7- and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by two-dimensional polyacrylamide gel electrophoresis.