Modeling electrokinetic flows with the discrete ion stochastic continuum overdamped solvent algorithm.

In this article we develop an algorithm for the efficient simulation of electrolytes in the presence of physical boundaries. In previous work the discrete ion stochastic continuum overdamped solvent (DISCOS) algorithm was derived for triply periodic domains, and was validated through ion-ion pair correlation functions and Debye-Hückel-Onsager theory for conductivity, including the Wien effect for strong electric fields. In extending this approach to include an accurate treatment of physical boundaries we must address several important issues. First, the modifications to the spreading and interpolation operators necessary to incorporate interactions of the ions with the boundary are described. Next we discuss the modifications to the electrostatic solver to handle the influence of charges near either a fixed potential or dielectric boundary. An additional short-ranged potential is also introduced to represent interaction of the ions with a solid wall. Finally, the dry diffusion term is modified to account for the reduced mobility of ions near a boundary, which introduces an additional stochastic drift correction. Several validation tests are presented confirming the correct equilibrium distribution of ions in a channel. Additionally, the methodology is demonstrated using electro-osmosis and induced-charge electro-osmosis, with comparison made to theory and other numerical methods. Notably, the DISCOS approach achieves greater accuracy than a continuum electrostatic simulation method. We also examine the effect of under-resolving hydrodynamic effects using a "dry diffusion" approach, and find that considerable computational speedup can be achieved with a negligible impact on accuracy.

Molecular analysis of hybrid dysgenesis-induced derivatives of a P-element allele at the vg locus.

Secondary and tertiary derivatives of a P-element insertion allele at the vestigial (vg) locus were induced by hybrid dysgenesis. The derivatives were characterized by Southern analyses and, in four cases, by DNA sequencing. The alterations found were P-element internal deletions, deletions of the insert and/or adjacent vg region DNA, or novel insertions of P-element sequences into existing P-element inserts. The relatively high frequency of secondary insertions into P-element sequences observed herein is unusual, since secondary insertions have seldom been recovered in other dysgenic screens. The effects of the alleles on vg expression were determined. The results are consistent with a model in which the insertions disrupt vg gene expression by transcriptional interference.

Construction of an opal suppressor by oligonucleotide-directed mutagenesis of a Saccharomyces cerevisiae tRNA(Trp) gene.

In vitro mutagenesis was used to create putative opal suppressor alleles of a tRNA(Trp) gene of Saccharomyces cerevisiae. The construct with the requisite anticodon change did not result in an active suppressor, whereas when a second change was introduced into the portion of the gene encoding the intron, an active and specific opal suppressor was produced. We propose that the secondary structure of transcripts from the first mutant may prevent efficient pre-tRNA processing, whereas normal processing occurs with the double mutant.

A nonspecific inhibitory effect of tRNA on the activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

The inhibitory effect of tRNA on yeast 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) has been reinvestigated. From earlier studies the inhibition by tRNAPhe appeared to be quite specific. This study shows that tRNAPhe is indeed a potent inhibitor but so is unfractionated tRNA, as well as ribosomal RNA and heparin. Complete digestion to mononucleotides relieves the inhibition. Since the enzyme requires a metal ion (Co2+) we suggest that the RNA and heparin are inhibitory by virtue of their capacity to chelate the Co2+.

A P element chimera containing captured genomic sequences was recovered at the vestigial locus in Drosophila following targeted transposition.

A P element carrying the Dopa decarboxylase gene, P[Ddc], was targeted into vg21, a cryptic P element induced mutant allele of the vestigial (vg) locus. The resulting allele, vg28w, contained the expected P[Ddc] plus an additional 9.5 kb of DNA, captured from elsewhere on chromosome II. Reversion of the vg28w mutant allele demonstrated that the entire insert can excise but cannot reinsert at an appreciable frequency. We explain the targeted transposition as the repair of a double stranded gap, created by the excision of the P element at vg21, and suggest that the formation of chimeric elements may be an important component of P element dependent genomic instability.

Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster.

P-element transposition is thought to occur by a cut-and paste mechanism that generates a double-strand break at the donor site, the repair of which can lead to internally deleted elements. We have generated a series of both phenotypically stronger and weaker allelic derivatives of vg21, a vestigial mutant caused by a P-element insertion in the 5' region of the gene. Virtually all of the new alleles arose by internal deletion of the parental element in vg21, and we have characterized a number of these internally deleted P elements. Depending upon the selection scheme used, we see a very different spectrum of amount and source of P-element sequences in the resultant derivatives. Strikingly, most of the breakpoints occur within the inverted-repeats such that the last 15-17 bp of the termini are retained. This sequence is known to bind the inverted-repeat-binding protein (IRBP). We propose that the IRBP may act to preserve the P-element ends when transposition produces a double-strand gap. This allows the terminus to serve as a template upon which DNA synthesis can act to repair the gap. Filler sequences found at the breakpoints of the internally deleted P elements resemble short stretches, often in tandem arrays, of these terminal sequences. The structure of the filler sequences suggests replication slippage may occur during the process of gap repair.

Genetic and molecular analysis of vgU and vgW: two dominant vg alleles associated with gene fusions in Drosophila.

In the absence of a vg+ gene, extensive cell death occurs in third instar imaginal discs, which results in a complete loss of adult wing margin structures. Essentially all molecularly characterized vg alleles are associated with deletions or insertions of DNA into the vg locus. These alterations reduce or eliminate a 3.8-kb vg-specific transcript, resulting in recessive loss of function alleles. We report here the analysis of two dominant vg alleles which have been identified (vgU and vgW). The vgU allele is associated with a chromosomal inversion which splits the vg locus, resulting in a gene fusion between vg and the mastermind (mam) neurogenic locus. Reversion analysis of vgU indicates that sequences from the mam locus are required for vgU dominance. The vgW allele is also the result of a chromosomal inversion, in this case resulting in a gene fusion between vg and the homeobox-containing invected (inv) gene. It is also associated with novel dominant homeotic transformations. Revertant analysis indicates that sequences from inv are required for the dominant wing and dominant homeotic effects of vgW. The vg dominance does not appear to be mediated through a reduction of vg expression or a novel fusion transcript in either vgU or vgW. The results are consistent with a model in which inappropriate expression of inv causes the dominant homeotic effects seen in vgW.

Identification of a regulatory allele of teashirt (tsh) in Drosophila melanogaster that affects wing hinge development. An adult-specific tsh enhancer in Drosophila.

A cis-acting regulatory element defined herein is required to drive teashirt (tsh) expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. This represents the first description of a viable adult-specific regulatory allele of tsh with a visible phenotype, and it enlarges our understanding of the expression of tsh and its function during the development of the adult.

Comparison of interleukin-6 levels in the bone marrow of multiple myeloma patients with disease severity and clonogenicity in vitro.

Fifteen of 25 bone marrow aspirates from 23 patients who presented or had been treated for multiple myeloma at the Royal Marsden Hospital produced myeloma colonies (MY-CFUc) in vitro. There was no correlation between disease severity and the level of interleukin-6 (IL-6) in bone marrow plasma nor was there any evidence that the level of IL-6 was higher in bone marrow aspirates from patients whose tumour produced MY-CFUc in vitro compared with those who did not. The mean level of IL-6 in the whole group of patients was 0.41 ng/ml (range 0.1-0.66 ng/ml), a value similar to that found in plasma from normal donor bone marrow, 0.42 ng/ml (range 0.14-0.62 ng/ml). Separation of peripheral blood cells from serum 24 h after collection, compared with 2 h after collection, resulted in a substantial increase of IL-6 in the serum. The results suggest that levels of IL-6 in bone marrow plasma is not a monitor of disease severity in multiple myeloma (MM) and that the collection and separation of blood and/or bone marrow samples into the cellular and aqueous components should be performed using standardized conditions to minimize inter-sample variation resulting from the release of IL-6 from the cellular components.

G-CSF is a major component of colony-stimulating activity (CSA) in the plasma of patients with multiple myeloma after treatment with high-dose melphalan (HDM).

Colony-stimulating activity (CSA) was measured by the production of granulocyte-macrophage colony-forming units (GM-CFU) from normal donor bone marrow in the plasma of 29 patients with multiple myeloma (MM) after intensive treatment with high-dose melphalan (HDM) with or without autologous bone marrow rescue (ABMR). Although patients who received ABMR had an earlier recovery of circulating neutrophils compared with those who received HDM alone, the time at which CSA reached a maximum was similar in both groups (10 to 11 days) after therapy. The decline in CSA correlated with the recovery of the neutrophil count. In plasma from patients who received recombinant human granulocyte colony-stimulating factor (rhG-CSF), in addition to an autograft, CSA reached a maximum earlier (7 days). Furthermore, neutrophil recovery was earlier in these patients. Platelet recovery was not increased by rhG-CSF. The time at which CSA was maximum in four patients who were undergoing intensive therapy for the second time occurred 9 days after treatment with HDM. Although the period without neutrophils was longer in three (of four) patients who survived long term, one patient who received rhG-CSF had a shorter period of neutropenia than the two who had not had the cytokine. G-CSF was detected in plasma from seven of seven patients but not at all times after treatment. In plasma samples that contained G-CSF, colony numbers were increased by recombinant interleukin-4 (rIL-4) in vitro. Neither IL-3 nor GM-CSF was detected in plasma; however, antibody to GM-CSF reduced CSA in all samples after intensive therapy. The data suggest that CSA is a consistent physiologic response to intensive therapy, even in previously treated patients, but that hematologic recovery is dependent on the availability of viable progenitor cells.

Colony-stimulating activity in the serum of patients with hemopoietic malignancies after intensive chemotherapy/radiotherapy: its augmentation by GM-CSF in vivo and interleukin 4 in vitro.

Colony-stimulating activity (CSA) in the serum of patients with hematological malignancies increased substantially after intensive therapy with cyclophosphamide/busulfan, cyclophosphamide/total body irradiation, or melphalan/total body irradiation. This was not dependent on patients receiving allogeneic bone marrow transplantation (ABMT) or autologous bone marrow rescue (ABMR). In 44 of 62 patients CSA was maximum approximately 7 days after chemotherapy/radiotherapy, whereas in 18 of 62 patients CSA was maximum between 9 and 20 days after therapy and decreased thereafter. The time course of CSA was not dependent on disease and was not affected by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) given as a continuous infusion for 14 days after therapy; however, serum from patients receiving rhGM-CSF produced significantly more colonies from donor bone marrow than serum from patients who did not receive the cytokine (p = 0.013). Despite the early peak in CSA in the majority of patients, there was no correlation between the time at which CSA was maximum and the return of patients' neutrophils to 500/microliters. Recombinant human interleukin 4 (IL-4) increased the number of granulocyte-macrophage colony-forming unit colonies, principally granulocyte colony-forming unit colonies, from normal bone marrow exposed to patients' serum after intensive therapy and antibody to GM-CSF reduced colony numbers. The results suggest that after intensive therapy granulocyte colony-stimulating factor (G-CSF) as well as GM-CSF is released into the serum and, in addition to acting directly with G-CSF, IL-4 may stimulate mononuclear cells to produce and/or release G-CSF.

Drosophila melanogaster tRNA(Ser) suppressor genes function with strict codon specificity when introduced into Saccharomyces cerevisiae.

The anticodon of the wild-type tRNA(7Ser) gene of Drosophila melanogaster was mutated using oligodeoxyribonucleotide-directed, site-specific mutagenesis, and all three nonsense suppressor derivatives of the gene were constructed. These constructs were cloned into an Escherichia coli-yeast shuttle vector (YRp7), and used to transform a Saccharomyces cerevisiae strain [JG 369-3B(alpha)] containing an array of nonsense alleles. When tested on appropriate omission media, the D. melanogaster suppressor genes were found to function in the yeast with strict codon specificity. Subsequent Northern hybridization analyses revealed that the D. melanogaster suppressor genes were transcribed and processed well, when in S. cerevisiae.

Characterization of the tRNA(Trp) genes of Saccharomyces cerevisiae.

The purpose of this work was to examine the tRNA(Trp)-encoding genes (tRNA(Trp)) of Saccharomyces cerevisiae to gain insight as to why tRNA(Trp) amber suppressors, isolated by conventional genetic techniques, have not been reported. The results herein indicate that the haploid yeast genome contains six tRNA(Trp) genes which map to five or six chromosomes. Not only do the six genes have identical coding sequences but their introns are also identical. Gene replacement experiments indicate that five copies of tRNA(Trp) are sufficient for cell viability. Thus, mutation of one tRNA(Trp) gene to a suppressor in vivo, lowering the functional number of tRNA(Trp) genes, would not be expected to be lethal.

The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA(Trp) genes.

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA(Trp) processing difficulties could help to explain the lack of classical tRNA(Trp)-based suppressors. It is clear that inefficient processing of introns from precursor tRNA(Trp), or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA(Trp)-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA(Trp). Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA(Trp) ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.

Suppression of carbamazepine-induced rash with prednisone.

We report our experience with 20 patients who developed a rash shortly after the introduction of carbamazepine and were treated with prednisone and an antihistamine. Sixteen patients were successfully continued on carbamazepine while 4 had to discontinue the drug.

Preparation and steroidogenic properties of purified zona fasciculata and zona reticularis cells from the guinea-pig adrenal gland.

Mixtures of zona fasciculata (ZF) and zona reticularis (ZR) cells, obtained by enzyme dispersion of decapsulated guinea-pig adrenal glands, were separated either by unit gravity sedimentation or by equilibrium density sedimentation. There was no evidence of deleterious effects on ultrastructural integrity or the ability of cells to respond to (1-24)ACTH (Synacthen) after either separation technique. Unit gravity sedimentation gave one fraction in which 90% of the cells were from the ZR and another fraction in which 70% of the cells were from the ZF. Equilibrium density sedimentation of cell mixtures on Percoll gradients gave fractions containing either 90% pure ZR or 95% pure ZF cells. Cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 11 beta-hydroxyandrostenedione and androstenedione were all formed from [14C]pregnenolone on incubation with purified preparations of both types of cell. No product was seen to be unique to either cell type although ZR cells appeared deficient in 11 beta-hydroxylase activity relative to ZF cells. The ratio of androstenedione to cortisol (formed either from labelled pregnenolone or from endogenous precursors) was higher for ZR cells than for ZF cells. When the purer cells obtained by equilibrium density sedimentation were studied, it was found that (1-24)ACTH stimulated greater steroid production (both androstenedione and cortisol) by the ZF cells compared with the ZR cells.

Properties of rat adrenal zona reticularis cells: production and stimulation of certain steroids.

The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay. Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone : corticosterone ratio when compared with zona fasciculata cells. Adrencorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output. In every cell pool, ACTH stimulated the output corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 beta-hydroxylation. Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available. Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells. The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.

Properties of rat adrenal zona reticularis cells: preparation by gravitational sedimentation.

An enriched fraction of zona reticularis cells was obtained by unit gravity sedimentation of decapsulated adrenal glands from female rats. From light microscopic and ultrastructural studies of the whole gland and the isolated cell fractions, the zona reticularis cells of the adrenal gland can be classified mainly on the bases of size, position and mitochondrial morphology. This cell population consists of two types of cell, the 'true' zoma reticularis cells (Type I, modal diameter 9 micrometer), which usually constitute 90% of the isolated reticularis fraction and 80% of the intact reticularis tissue, and cells (Type II, modal diameter 13 micrometer) with fasciculata-like properties (rich in lipid and spherical mitochondria with vesicular cristae). Staining of the cell preparation for 3beta-hydroxysteroid dehydrogenase activity also demonstrates the existence of two types of cell in the zona reticularis. The zona reticularis cell fraction, like the zona fasciculata cell fraction, was capable of producing the subsequent steroids from radioactive pregnenolone: corticosterone, deoxycorticosterone, 18-hydroxydeoxycorticosterone, 11-dehydrocorticosterone, progesterone and androstenedione. However, the pattern of steroid production differed markedly between the zona reticularis and zona fasciculata cells, particularly with respect to the production of deoxycorticosterone and corticosterone (and its correlated steroids, 11-dehydrocorticosterone and 18-hydroxydeoxycorticosterone). When R (the ratio of deoxycorticosterone : corticosterone plus 11-dehydrocorticosterone) for the purest preparation of reticularis cells was compared with R for the corresponding preparation of fasciculata cells, the normalized ratio was found to be 6.4, 16.4 and 20.1 in three experiments. The pattern of production of androstenedione per cell was similar in the reticularis and fasciculata cell fractions. The exact mechanism for the altered pattern of steroid metabolism remains to be elucidated. However, these results establish that the corticosteroids produced by the cells of the zona reticularis may be quantitatively, if not qualitatively, different from those produced by the zona fasciculata cells.

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone.

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.

Further studies on the stimulation of rat adrenal capsular cells: four types of response.

Preparations of capsular rat adrenal cells consisting mainly of zona glomerulosa with less than 5% zona fasciculata contamination are described. The responses of the aldosterone and corticosterone outputs of these preparations to various stimuli were of four types. (1) Variations in K+ concentration gave a maximum aldosterone response at 5.9-8.4 mM-K+, about sixfold greater than the control output at 3.6 mmol/l. At higher K+ concentrations such as 13 mmol/l, the response decreased. (2) Serotonin (at a concentration of about 10(-4) mol/l) gave only a slightly lower maximal aldosterone response than did K+ but this did not decrease significantly at higher concentrations. Serotonin gave significant steroidogenic response at 10(-8) mol/l. (3) [Asp1,Val5]-Angiotensin II (10(-10) mol/l) with 3.6 mM-K+ gave a significant response and a constant maximal response at 2.5 x 10(-8) mol/l. This maximum response was about half that found for both aldosterone and corticosterone when stimulated maximally by K+ or serotonin: [des-Asp1,Ile5]- and [des-Asp1,Val5]-angiotensin II (angiotensin III) gave similar response characteristics but had a lower potency in this cell preparation. The initial maximum response could be further increased at a higher concentration (from 2.5 x 10(-5) mol/l) of a preparation of [Asn1,Val5]-amide angiotensin II (Hypertensin-Ciba) and might eventually be greater than with K+. This additional response was, to a major extent, due to stimulation of the contaminating zona fasciculata cells and was not seen with high concentrations of the free acid, angiotensin II. It was also not seen in two experiments with pure [Asn1]-amide angiotensin II and therefore it could have been due to some impurity in Hypertensin-Ciba. (4) Adrenocorticotrophin (Synacthen) at 3 x 10(-11) mol/l gave a significant steroidogenic response. Higher concentrations (3 x 10(-10) to 7.5 x 10(-9) mol/l) gave no constant maximum but the response could be much greater than for other stimuli such as K+, serotonin and [Asp1]-angiotensin II. This additional response was again due to steroid precursors, e.g. deoxycorticosterone and corticosterone from contaminating zona fasciculata cells. Similar results were obtained with ACTH (ACTHAR) in three experiments. Threshold sensitivity (a significant increase in steroidogenesis) for ACTH (Synacthen) was, in two experiments, greater for zona fasciculata-reticularis cells (3 x 10(-12) mol/l) than for zona glomerulosa cells (3 x 10(-11) mol/l). The data show that aldosterone output was approximately a function of the square of the corresponding corticosterone value. Specific effects on this pathway can be shown by values of aldosterone/corticosterone2 greater than one. Of all stimuli used, only K+ concentrations of 5.3, 5.9 and 13 mmol/l gave such effects. However, because of several considerations, only positive results with other stimuli may be meaningful. Calculation of this parameter might be useful as a screening test in bioassays for substances with aldosterone-stimulating activity.