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Journal Article↗
Biomedical subjects
J A Dow
Publications and source records attributed to J A Dow.
CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP.
A cardioacceleratory peptide, CAP2b, identified originally in the lepidopteran Manduca sexta, stimulates fluid secretion by Malpighian tubules of the dipteran Drosophila melanogaster. High-performance liquid chromatography analyses of adult D. melanogaster reveal the presence of a CAP2b-like peptide, that coelutes with M. sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b accelerates fluid secretion in tubules stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) but has no effect on tubules stimulated by guanosine 3',5'-cyclic monophosphate (cGMP), implying that it acts through the latter pathway. By contrast, the action of leucokinin is additive to both cAMP and cGMP but not to thapsigargin, suggesting that leucokinin acts by the elevation of intracellular calcium. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. By contrast, leucokinin has no effect on levels of either cyclic nucleotide. Both CAP2b and cGMP increase transepithelial potential difference, suggesting that stimulation of vacuolar-adenosinetriphosphatase action underlies the corresponding increases in fluid secretion. Overall, the results show that a Drosophila CAP2b-related peptide acts to stimulate fluid secretion by Malpighian tubules through the cGMP-signaling pathway.
Aging↗
Simultaneous multisite recordings and stimulation of single isolated leech neurons using planar extracellular electrode arrays.
Planar extracellular electrode arrays provide a non-toxic, non-invasive method of making long-term, multisite recordings with moderately high spatial frequency (recording sites per unit area). This paper reports advances in the use of this approach to record from and stimulate single identified leech neurons in vitro. A modified enzyme treatment allowed identified neurons to be extracted with very long processes. Multisite extracellular recordings from the processes of such isolated neurons revealed both the velocity and direction of action potential propagation. Propagation in two cell types examined was from the broken stump towards the cell body (antidromic). This was true for spontaneous action potentials, action potentials produced by injecting current into the cell body and extracellular stimulation of the extracted process via a planar extracellular electrode. These results extend previous findings which have shown that the tip of the broken stump of extracted neurons has a high density of voltage-activated sodium channels. Moreover they demonstrate the applicability of extracellular electrode arrays for recording the electrical excitability of single cells.
Action Potentials↗
A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.
The nitric oxide (NO) signaling pathway plays major roles in the vertebrate vascular, nervous, and immune systems. Here we present evidence that all the elements in the NO pathway are present in, and act to control epithelial fluid secretion by, the Malpighian tubules of an insect, Drosophila melanogaster. This finding will allow both a physiological and a molecular genetic dissection of the NO pathway in the same tissue.
3',5'-Cyclic-GMP Phosphodiesterases↗
The malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control.
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Amiloride↗
Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase from Manduca sexta midgut and tubules.
Vacuolar ATPases (V-ATPases), originally characterised as components of endomembranes, have also been implicated in epithelial ion transport, both in vertebrates and in insects. The ATPase is particularly noteworthy in lepidopteran larvae, where it generates large transepithelial potential differences and short-circuit currents across the midgut epithelium. A cDNA library from Manduca sexta larval midguts and Malpighian tubules was screened with a Drosophila melanogaster cDNA encoding the 16-kDa proteolipid subunit of the V-ATPase, and a 1.4-kb cDNA sequenced in its entirety. The sequence contains a long open reading frame, encoding a putative peptide of 156 amino acids (aa) and with an M(r) of 15,967, in close agreement with values previously suggested by sodium dodecyl sulfate-polyacrylamide gels of M. sexta midgut proteins. Correspondence of the deduced aa sequence with those of other species, particularly D. melanogaster, was extremely close. Northern blots of M. sexta midgut mRNA at high stringency revealed two transcripts of 1.4 and 1.9 kb, whereas genomic Southern blots suggest that there is only a single copy of the gene in M. sexta. The possibility that members of the 16-kDa gene family might serve multiple roles in transport and membrane communication is discussed.
Amino Acid Sequence↗
Making real neural nets: design criteria.
Neural nets may be assembled with living nerve cells in vitro to test theories about neural processing and the ways in which patterns develop in the nervous system, and to test ideas about plasticity and learning in processing systems. This may benefit the design of computer systems and prosthetic devices. Extracting information from such nets can be achieved by means of intracellular and extracellular electrodes and fluorescent dyes. Patterning of cells may be achieved using microfabrication techniques, and extracellular electrodes can be combined within the patterned substrate.
Humans↗
Multiphasic diagnostic evaluation of arteriogenic, venogenic, and sinusoidogenic impotency. Value of noninvasive tests compared with penile duplex ultrasonography.
Fifty patients with a mean age of fifty-five years (range 25 to 75 years) in whom vasculogenic impotency was suspected clinically were evaluated to determine the type of vascular lesion involved: arterial insufficiency, venous leak, or sinusoidal dysfunction. All patients underwent first, noninvasive diagnostic tests including penile brachial index, penile brachial subtraction index, and penile plethysmogram, followed by penile duplex ultrasonography with papaverine and phentolamine injection. Patients with abnormal ultrasonography were divided into two groups: One group with suspected sinusoidal dysfunction and those with either arteriogenic or venogenic insufficiency but not considered candidates for surgery; they were not subjected to further studies. Another group with suspected proximal arteriogenic lesions and those with suspected venous leakage considered candidates for surgical correction were subjected to angiography and cavernosometry-cavernosography, respectively. The findings of the noninvasive tests were compared with those provided by the invasive tests. The results indicated that noninvasive tests can predict whether or not impotency is arteriogenic in approximately 90 percent of cases. The noninvasive tests, however, were less accurate in predicting venogenic and sinusoidogenic impotency, for which penile duplex ultrasonography seems to be the choice.
Adult↗
Cell guidance by ultrafine topography in vitro.
Laser holography and microelectronic fabrication techniques have been employed to make grating surfaces in fused quartz with ultrafine period (260 nm) in an attempt to mimic the topography of aligned fibrillar extracellular matrix (ECM), which, in the past, has been shown to affect the behaviour of cells in vitro and in vivo. The alignment of BHK cells, MDCK cells and chick embryo cerebral neurones on 260 nm period grating surfaces (130 nm grooves separated by 130 nm) of various depths (100, 210 and 400 nm) was examined. While all gratings aligned BHK cell populations, the degree of alignment was dependent on depth. The response of single MDCK cells to the grating patterns was both to align precisely to the direction of the gratings, and to elongate; only their elongation was depth-dependent. MDCK cells that were part of epithelial cell islands, and the outgrowth of neurites from chick embryo neurones, were mainly unaffected by the grating surfaces. It is clear that topography on this scale can control cell behaviour, but guidance of this type is strongly dependent on cell type and cell-cell interactions.
Animals↗
Detection and quantitation of HLA class II molecules on keratinocytes by quantitative immunofluorescence.
The paper describes measurements of HLA-DR antigen expression on normal human keratinocytes in culture using anti-HLA-DR antibodies and fluorescent Protein A or fluorescent second antibody methods and a low-light level video camera and image analysis program to quantitate the fluorescence output. The measurements are ultimately quantitated in terms of molecules of Ia antigen reacting material per cell. The method has a sensitivity at a S/N ratio of 10:1 of 180 molecules/micron2. The results show that normal keratinocytes do indeed express class II antigens on their surfaces at levels well above background. We confirm that treatment of the cells with gamma-interferon produces enhanced expression of DR antigens within 4 days. The method of quantitation is applicable to fluorescence and other low light level images.
Calibration↗
An extracellular microelectrode array for monitoring electrogenic cells in culture.
This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.
Action Potentials↗
Topographical control of cell behaviour: II. Multiple grooved substrata.
Electronics miniaturization techniques have been used to fabricate substrata to study contact guidance of cells. Topographical guidance of three cell types (BHK, MDCK and chick embryo cerebral neurones) was examined on grooved substrata of varying dimensions (4-24 microns repeat, 0.2-1.9 microns depth). Alignment to within 10 degrees of groove direction was used as our criterion for guidance. It was found that repeat spacing had a small effect (alignment is inversely proportional to spacing) but that groove depth proved to be much more important in determining cell alignment, which increased with depth. Measurements of cell alignment and examination by scanning electron microscopy showed that BHK cells and MDCK cells interacted differently with grooved substrata, and also that the response of MDCK cells depended on whether or not the cells were isolated or part of an epithelial cell island. Guidance by a multiple topographical cue is greater than could be predicted from cells' reactions to a single cue (Clark et al. Development 99: 439-448, 1987). Substratum topography is considered to be an important cue in many developmental processes. Cellular properties such as cytoskeletal organisation, cell adhesion and the interaction with other cells are discussed as being factors determining a cells susceptibility to topography.
Animals↗
Expression of blood group precursor T antigen as a prognostic marker for human bladder cancer treated by bacillus Calmette-Guerin and interleukin-2.
The relationship between T antigen expression in transitional cell carcinoma of the bladder and response to bacillus Calmette-Guerin plus interleukin-2 treatment was studied. A total of 25 patients received combined treatment with bacillus Calmette-Guerin and interleukin-2 at weekly instillations for 6 consecutive weeks and then monthly for 1 year. T antigen expression in all patients was studied before treatment. Of the patients 16 had positive T antigen expression in the tumors: 13 (81%) remained free of tumor for an average of 28 months, 3 had a noninvasive recurrence each at 8 to 27 months (mean 18 months) and all responded well to a repeated treatment cycle. Nine patients were negative for T antigen: 1 (11%) remained free of tumor for 11 months, while 8 had recurrence within 12 months. Of the latter 8 patients 2 had noninvasive recurrence each at 4 to 5 months but they responded to a repeated treatment cycle and remained free of tumor for 4 to 22 months (mean 13 months), 4 had subsequent repeated recurrences at 12 to 41 months (mean 22 months) and 2 had progression to deep invasion resulting in cystectomy at 11 to 12 months. Thus, the disease-free rate (continuous complete response) in patients with tumors positive for T antigen was 81%, while that in patients with tumors negative for T antigen was 11% (p less than 0.005, chi-square test). The over-all response rate in patients with positive and negative tumors to bacillus Calmette-Guerin and interleukin-2 treatment was 100 and 33%, respectively, and that for the total patients was 76%. T antigen may serve as a target recognition structure for effector cells generated by bacillus Calmette-Guerin plus interleukin-2 treatment. This study suggests that T antigen is useful to predict the response of bladder tumors to treatment with bacillus Calmette-Guerin and interleukin-2.
Aged↗
Characterization of specific binding of atrial natriuretic peptide (ANP) to rat PC12 phaeochromocytoma cells.
Specific binding sites for atrial natriuretic peptide have been identified in membrane of the phaeochromocytoma cell line PC12. Scatchard analysis of binding studies revealed a Kd of 794 pM and a density (Bmax) of 254 fmol/mg protein. Hormones unrelated to ANP such as angiotensin II, bradykinin and arginine-8-vasopressin did not complete for the binding sites. Of the ANP-related peptides which competed for the binding sites, the following order of affinity was established; rANP (8-33) greater than rANP (28 amino acid) greater than rat atrial peptide fragment (13-28) greater than a-hANP (28 amino acid) greater than atrial peptide fragment (1-11) greater than atriopeptin I.
Adrenal Gland Neoplasms↗
Role of midgut electrogenic K+ pump potential difference in regulating lumen K+ and pH in larval lepidoptera.
Larvae of Manduca sexta were fed on tobacco leaves or synthetic diets with differing potassium content. For all diets tested, midgut lumen K+ activity coefficients were low, and midgut lumen K+ activities, K+ concentrations and pH values were as closely regulated as in blood. The transmidgut electrochemical potential difference for potassium based on activities was lower than had previously been estimated from concentrations, but was dominated by the electrical gradient established by the electrogenic K+ ATPase. Midgut K+ activities and pH profiles were similar, as would be suggested by a model in which the generation of high pH was linked to electrogenic K+ transport.
Animals↗
Topographical control of cell behaviour. I. Simple step cues.
The photolithographic techniques of the microelectronics industry have allowed us to fabricate patterned plastic substrata to investigate contact guidance of animal tissue cells. The reactions of cells to single steps on a substratum were examined using time-lapse videorecording and scanning electron microscopy. BHK cells and chick embryonic neural cell processes exhibited gradual inhibition of crossing steps with a concomitant increase in alignment at steps dependent on increasing step height. Comparison of these cells' reactions, with those of chick heart fibroblasts and rabbit neutrophils, at a 5 micron step revealed that the influence of topography is also dependent on cell type, the neutrophils being relatively unaffected. The presence of an adhesive difference at a series of steps altered BHK cells' reactions such that the frequency of crossing was dependent on the direction of approach to a step. Although our data are consistent with Dunn & Heath's proposal (1976) that the inflexibility of the cytoskeleton of a moving cell's protrusion is the cellular property determining such reactions to topography, we have found that, on encountering a topographical feature, the response of a cell may be predictable on a probabilistic basis, i.e. the topographical feature reduces the probability of a cell making a successful protrusion and contact in a given direction, that even the largest features tested did not act as absolute barriers to cell locomotion since a small proportion of a population of cells were able to overcome them, and that other guidance cues could significantly alter a cell's response. Even in situations where it is not the primary cue in directing cell locomotion, topographical control may be an important factor during morphogenesis since it must, at the very least, influence the efficiency of other cues.
Animals↗
Novel methods for the guidance and monitoring of single cells and simple networks in culture.
The effects of the topography, adhesiveness and chemistry of surfaces in modulating the behaviour of cells in vivo and in vitro have been extensively researched. However, few natural systems are simple enough to allow straightforward conclusions to be drawn, as many different cues are likely to be present at one time. Microelectronic fabrication, normally employed in making integrated circuits, can produce substrates patterned on scales highly relevant to studies of cell behaviour. In this paper, we describe progress in fabricating simple artificial substrata both at the micrometre and sub-micrometre scales. The former can be considered as models for contact guidance along other cells or axonal processes: the latter, models for guidance along aligned collagen matrices. We have systematically studied the reactions of different cell types to simple cues (steps and grooves). Additionally, it may be possible to produce fine-resolution patterns with differential adhesiveness, or with other cell-specific surface-chemical properties, such as the differential deposition of proteins, e.g. cell adhesion molecules. We also describe early results in using topographic and other cues to guide cells onto patterned metal electrodes, forming simple electrically active networks of controlled design, from which long-term recordings can conveniently be made.
Animals↗
A simple microcomputer-based system for real-time analysis of cell behaviour.
An image analysis package based on a BBC microcomputer has been developed, which can simultaneously track many moving cells in vitro. Cells (rabbit neutrophil leucocytes, BHK C13 fibroblasts, or PC12 phaeochromocytoma cells) are viewed under phase optics with a monochrome TV camera, and the signal digitized. Successive frames are acquired by the computer as a 640 X 256 pixel array. Under controlled lighting conditions, cells can readily be isolated from the background by binary filtering. In real-time tracking, the positions of a given cell in successive frames are obtained by searching the area around the cell's centroid in the previous frame. A simple box-search algorithm is described, which proves highly successful at low cell densities. The resilience of different search algorithms to various exceptional conditions (such as collisions) is discussed. The success of this system in real-time tracking is largely dependent upon the leisurely speed of movement of cells, and on obtaining a clean, high quality optical image to analyse. The limitations of this technique for different cell types, and the possible configurations of more sophisticated hardware, are outlined. This system provides a versatile and automated solution to the problem of studying the movement of tissue cells.
Algorithms↗