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J T Fleming

Publications and source records attributed to J T Fleming.

At least 19 recordsLinked to original sources
PubMed2003

Two week nicotine treatment selectively increases bone vascular constriction in response to norepinephrine.

This study was designed to determine if nicotine treatment alters the constrictor and/or dilator function of the vessels which regulate blood flow to intact bone. Nicotine (1.7 mg/kg/day) or nicotine-free, phosphate-buffered saline was administered subcutaneously to mature male rats for 2 weeks via osmotic mini-pumps. On the 14th day, the rats were anesthetized and in vivo experiments were performed to quantitate the changes in arterial blood pressure and perfusion of the intact tibia (measured by laser Doppler flowmetry) in response to two constrictor agonists (norepinephrine, NE and arginine vasopressin, AVP) and two vasodilator agents (acetylcholine, ACh and sodium nitroprusside, SNP). Dose-response curves were generated by plotting the change in the bone vascular resistance index (mmHg/bone perfusion units) evoked by each dose of agonist. In addition, bone arteriolar expression of endothelial nitric oxide synthase protein was quantitated by Western blot analysis. Nicotine treatment significantly enhanced the constriction of the bone vasculature in response to NE, but not to AVP. Vascular dilation in response to ACh and SNP was not changed by nicotine. These results indicate that nicotine selectively accentuates the constrictor response of the bone vasculature to exogenous NE. This enhanced constriction to NE is not due to impaired endothelial cell release of nitric oxide or diminished smooth muscle response to nitric oxide. Since NE and AVP activate similar cell signaling mechanisms to induce constriction, the selective enhancement of NE-induced constriction suggests that nicotine alters a mechanism unique to NE signaling; possibly the number or binding affinity of alpha adrenergic receptors. Since endogenous NE regulates basal blood flow to bone, the effect of nicotine to augment NE-induced constriction could lead to a chronic reduction in blood flow to bone.

Acetylcholine↗
PubMed2002

Endogenous norepinephrine regulates blood flow to the intact rat tibia.

The goal of our study was to determine if endogenous norepinephrine (NE) has a role in the regulation of basal blood flow to intact bone. The experimental plan was to measure bone blood flow before and after pharmacological blockade of alpha-adrenergic receptors. A significant increase in blood flow after receptor blockade would suggest that endogenous norepinephrine exerts a tonic constrictor effect on the vessels supplying blood to the bone. Mature, male rats were anesthetized with Inactin. Arterial blood pressure and left tibia blood flow (laser Doppler flowmetry) were measured. A cannula was inserted into the right iliac artery and advanced to the aortic bifurcation to deliver drugs into the left hindlimb circulation, including the left tibia vasculature. Bolus injection of norepinephrine caused a dose-dependent decrease in bone blood flow (30-40%). Blockade of alpha-adrenergic receptors with phentolamine or phenoxybenzamine attenuated by more than 50% the norepinephrine-induced decrease in bone blood flow. In separate rats that had not received exogenous norepinephrine, injection of phentolamine alone decreased bone vascular resistance by 34+/-3%. Similarly, phenoxybenzamine decreased resistance by 25+/-4%. These results are consistent with the conclusion that alpha-adrenergic receptors mediate a significant constriction of blood vessels which participate in the partial control of basal blood flow to the intact rat tibia.

Adrenergic alpha-Antagonists↗
PubMed2001

Whole-cell biocomputing.

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

Computational Biology↗
PubMed2001

Gene expression monitoring in soils by mRNA analysis and gene lux fusions.

Two methods recently developed to monitor the gene expression of microbial communities in soil are the extraction and detection of messenger RNA from soil microorganisms and the construction and use of lux-based bioreporter strains. The goal of these approaches is to assess microbial activity in natural and impacted soil environments.

Artificial Gene Fusion↗
PubMed2001

Bone blood flow and vascular reactivity.

Blood flow is essential for normal bone growth and bone repair. Like other organs, the regulation of blood flow to bone is complex and involves numerous physiologic mechanisms including the sympathetic nervous system, circulating hormones, and local metabolic factors. Our studies addressed the following questions: (1) Which endogenous vasoconstrictor agents regulate in vivo blood flow to bone? (2) Does a decrease in bone vascular reactivity to vasoconstrictor hormones account for the increase in blood flow during bone healing? (3) Does the endothelium influence bone arteriolar function? An intact bone model was developed in the rat to assess hormonal regulation of in vivo bone blood flow and in vivo bone vascular reactivity. An isolated, perfused bone arteriole preparation was employed to characterize the responsiveness of small resistance-size arterioles (diameter < 100 microm) to vasoconstrictor hormones and to evaluate the role of the vascular endothelium to modulate vascular smooth muscle reactivity. Our results indicate that: (1) though exogenous endothelin is a potent constrictor of the in vivo bone vasculature, endogenous endothelin does not actively regulate in vivo blood flow; (2) the increase in blood flow to a bone injury site is not due to a decrease in bone vascular sensitivity to norepinephrine, and (3) isolated bone arterioles of young rats are very sensitive to vasoconstrictor hormones but exhibit only modest endothelium-mediated vasodilation.

Acetylcholine↗
PubMed2001

Screening for ribosomal-based false positives following prokaryotic mRNA differential display.

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.

DNA Probes↗
PubMed2000

Dietary chloride does not correlate with urinary thromboxane in deoxycorticosterone acetate-treated rats.

Renal vascular resistance in deoxycorticosterone acetate (DOCA) salt-treated uninephrectomized rats is increased by high dietary chloride. Because DOCA salt-hypertensive rats exhibit an increased urinary excretion of thromboxane B2 (TXB2), a metabolite of thromboxane A2 (TXA2), the increased TXB2 excretion by DOCA salt-treated rats could relate to elevated dietary chloride, increased blood pressure, and/or the presence of intact renal tubules. We hypothesized that high NaCl intake, resulting in an elevated tubular chloride excretion, stimulates TXA2 production. A result of that production could be renal vasoconstriction. Baseline blood pressures were measured for 10 days, and then the rats were treated with DOCA (30 mg/kg) and fed (1) normal NaCl, (2) normal sodium with high chloride, or (3) high sodium chloride (NaCl) for 4.5 weeks. Next, the rats were uninephrectomized (1K) or unihydronephrectomized (1KHK) to yield one kidney without an intact tubular system and therefore no macula densa. Two and a half weeks later, urinary excretion of TXB2 was determined. DOCA-high NaCl-fed 1KHK or 1K rats had significant increases in systemic blood pressure to 172 +/- 12 and 190 +/- 5 mm Hg, respectively, compared with no significant increase in blood pressure among the other groups. Urinary TXB2 excretion was increased to 29 +/- 4 pg per 24 hours per gram of body weight in all DOCA-treated 1KHK and 1K animals regardless of diet compared with DOCA-treated animals with two intact kidneys (13 +/- 2 pg per 24 hours per gram of body weight). DOCA treatment in rats with one functional kidney results in the excretion of high levels of urinary TXB2 unrelated to dietary chloride load, blood pressure, or intact renal tubules.

Animals↗
PubMed2000

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Acetylcholine↗
PubMed1998

Optimization of differential display of prokaryotic mRNA: application to pure culture and soil microcosms.

The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction of todC1 in Pseudomonas putida F1 was used to optimize the procedure. At 24-h tod induction was determined to be approximately 8 x 10(7) transcripts/microg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2 concentrations were varied to optimize amplification of a todC1 fragment. The limit of detection of todC1 by DD was found to be 0.015 ng of total RNA template or approximately 10(3) transcripts. Once optimized, a todC1C2 gene fragment from P. putida F1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase in Burkholderia cepacia JS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.

Gene Expression Regulation, Bacterial↗
PubMed1997

Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits.

We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.

Amino Acid Sequence↗
PubMed1997

Lack of nitric oxide contributes to vasospasm during ischemia/reperfusion injury.

Vasospasm can be a complication after free tissue transfer and replant operations. Recent studies suggest that vasospasm may be due to endothelium dysfunction, resulting in impairment of nitric oxide production. The present experiment was designed to investigate acute responses of the microcirculation of skeletal muscle to local interarterial infusion of sodium nitroprusside (a direct donor of nitric oxide and thus an endothelium-independent vasodilator) or acetylcholine chloride (which stimulates endothelium release of endogenous nitric oxide) during reperfusion after 4 hours of warm ischemia. Male Sprague-Dawley rats, each weighing 100 to 120 gm, were anesthetized with sodium pentobarbitone and were surgically prepared with vascular isolated and denervated cremaster muscles that were subjected to 4 hours warm ischemia and 2 hours of reperfusion. Sodium nitroprusside (10(-3) M), acetylcholine chloride (10(-4) M), or normal saline (eight rats for each group) were administered by local infusion (0.1 ml/hour) through the femoral artery into the natural blood flow of the cremaster. The arterial tree in the cremaster was observed and arteriole diameters (A1-A4) were measured using intravital microscopy. The number of arteriole branches having temporary stoppage of flow were counted in each cremaster. The results from this study show that local infusion of sodium nitroprusside, but not acetylcholine chloride, prevents ischemia/reperfusion vasoconstriction in A3 and A4 arterioles and thus improves microvascular blood flow. Generalized vasoconstriction caused by topically applied norepinephrine (10(-6) M) to sham ischemia cremasters could be completely reversed by the local infusion of 10(-4) M acetylcholine chloride. These results indicate that vasospasm after ischemia/reperfusion may be related to temporary endothelial cell dysfunction, resulting in the inability to produce sufficient nitric oxide during early reperfusion. Vascular smooth muscle, however, is responsive to locally administered sodium nitroprusside infusion (which is thought to provide exogenous nitric oxide).

Acetylcholine↗
PubMed1997

ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression.

The molecular cloning and functional co-expression of a novel nicotinic acetylcholine receptor (nAChR) non-alpha subunit gene, acr-3, is described. Previously we determined the sequence and demonstrated the functional co-expression of acr-2, a nAChR non-alpha subunit gene from Caenorhabditis elegans. Analysis of the acr-2 genomic DNA revealed the existence of another potential nAChR subunit gene, acr-3, in the same orientation, only 281 bp downstream of acr-2. A cDNA containing the entire acr-3 coding sequence was isolated by RT-PCR and sequenced. The predicted protein contains the conserved features typical of nAChR non-alpha subunits and most closely resembles other invertebrate nAChR non-alpha polypeptides. Unusually, the highly conserved glycine residue (equivalent to residue 240 in the Torpedo alpha subunit) upstream of transmembrane domain 2 (m2) is replaced by a serine residue in ACR-3. When acr-3 cDNA was injected alone into Xenopus oocytes no levamisole-gated channel activity was observed. However when co-expressed with a C. elegans alpha subunit (UNC-38), ACR-3 contributed to the formation of levamisole-gated channels. The response of this hetero-oligomer to levamisole (100 microM) was reduced by the nAChR antagonists mecamylamine (1 microM) and d-tubocurarine (10 microM).

Amino Acid Sequence↗
PubMed1996

Molecular cloning and in vitro expression of C. elegans and parasitic nematode ionotropic receptors.

The free living nematode, C. elegans is understood at a level of detail equalled by few other organisms, and much of the cell biology and sequence information is proving of considerable utility in the study of parasitic nematodes. Already, C. elegans provides a convenient vehicle for investigating anthelmintic drug action and resistance mechanisms. Among the ionotropic receptors, with their important roles in the behaviour and development of the organism, are targets for anthelmintics. The subunits of nicotinic acetylcholine receptors of C. elegans form a large and diverse multigene family. Members of this family are among the 11 genes associated with resistance to the anthelmintic drug levamisole.

Amino Acid Sequence↗
PubMed1996

20-HETE is an endogenous inhibitor of the large-conductance Ca(2+)-activated K+ channel in renal arterioles.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

Animals↗
PubMed1996

Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K(+)-channel activity.

The present study examined the effects of 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on the diameter of small renal arteries of the rat and assessed their action on K(+)-channel activity in vascular smooth muscle (VSM) cells isolated from these vessels. The R,S-isomer of 11,12-EET (1, 10, and 100 nM) increased the diameter of small renal arteries preconstricted with phenylephrine; however, the S,R-isomer was inactive. Both the R,S- and S,R-isomers of 14,15-EET had little effect on the diameter of these vessels even at a high concentration (100 nM). The vasodilator effect of 11(R),12(S)-EET was attenuated by tetraethylammonium (TEA, 1 mM) and iberiotoxin (100 nM), selective inhibitors of the large-conductance Ca(2+)-activated K+ (KCa) channel. In contrast, apamin (100 nM) and 4-aminopyridine (2 mM), which are inhibitors of other types of K+ channels, had no effect on the vasodilatory effect of 11,12-EET. In patch-clamp experiments, 100 nM racemic 11,12-EET increased outward K+ currents in VSM cells. Addition of the R,S-isomer or racemic 11,12-EET (1-100 nM), but not the S,R-isomer, increased the activity of KCa channel recorded from renal VSM cells with cell-attached patches. However, racemic EET had no effect on this channel when added to the internal (inside-out) or external (outside-out) face of excised membrane patches. These results suggest that 11,12-EET is a potent dilator of small renal arteries and that the R,S-isomer is the active enantiomer. The vasodilator effect of 11,12-EET appears to involve activation of KCa channel.

8,11,14-Eicosatrienoic Acid↗
PubMed1996

Lophotoxin-insensitive nematode nicotinic acetylcholine receptors.

Nematode nicotinic acetylcholine receptors (nAChRs) are molecular targets of several anthelmintic drugs. Studies to date on Caenorhabditis elegans and Ascaris suum have demonstrated atypical pharmacology with respect to nAChR antagonists, including the finding that kappa-bungarotoxin is a more effective antagonist than alpha-bungarotoxin on Ascaris muscle nAChRs. Lophotoxin and its naturally occurring analogue bipinnatin B block all vertebrate and invertebrate nAChRs so far examined. In the present study, the effects on nematode nAChRs of bipinnatin B have been examined. The Ascaris suum muscle cell nAChR was found to be insensitive to 30 mumol l-1 bipinnatin B, a concentration that is highly effective on other nAChRs. To our knowledge, this is the first demonstration of a nAChR that is insensitive to one of the lophotoxins. Xenopus laevis oocytes injected with C. elegans polyadenylated, poly(A+), mRNA also expressed bipinnatin-B-insensitive levamisole responses, which were, however, blocked by the nAChR antagonist mecamylamine (10 mumol l-1). In contrast to the findings for nematode receptors, bipinnatin B (30 mumol l-1) was effective in blocking mouse muscle nAChRs expressed in Xenopus laevis oocytes and native insect nAChRs. A possible explanation for insensitivity of certain nematode nAChRs to lophotoxins is advanced based on the sequence of an alpha-like C. elegans nAChR subunit in which tyrosine-190 (numbering based on the Torpedo californica sequence), a residue known to be critical for lophotoxin binding in vertebrate nAChRs, is replaced by a proline residue.

Amino Acid Sequence↗
PubMed1996

Dietary myo-inositol restores diabetic renal arteriolar reactivity to angiotensin II but not to norepinephrine.

OBJECTIVE: This study addresses the hypothesis that the diminished constriction of renal arterioles to angiotensin II (Ang II) and norepinephrine (NE) in diabetic rats is due to elevated activity in the polyol pathway. This activity results in reduced incorporation of myo-inositol into membrane phospholipids and impaired signal transduction. METHODS: The left ureter of female Wistar rats (140-160 g) was surgically ligated. Four to six weeks later, streptozotocin (50 mg/kg, i.p.) was injected in half of the rats in induce diabetes. Beginning on the day of streptozotocin injection, diabetic and nondiabetic rats were fed either a standard diet or a diet enriched with 1% myo-inositol. Seven to 10 days later, all rats were anesthetized and the hydronephrotic kidney was bisected and exteriorized in a bath for direct visualization of the renal microvasculature. The constrictor responses of interlobular, afferent, and efferent arterioles to Ang II or NE (applied to the bath) were directly quantitated by in vivo microscopy. RESULTS: Among diabetic rats, the myo-inositol-enriched diet significantly enhanced the constriction of interlobular, afferent, and efferent arterioles in response to Ang II, so that the responses to the peptide were almost completely restored to normal. Constriction to NE by interlobular arteries and afferent arterioles (but not efferent arterioles) was also significantly attenuated among diabetic rats fed the standard diet. However, unlike what was observed for Ang II, the myo-inositol-enriched diet did not enhance constriction to NE among diabetic rats. CONCLUSIONS: These data indicate that different mechanisms are responsible for decreased renal arteriolar constriction due to Ang II and NE in early diabetes. Diminished arteriolar constriction due to Ang II, but not to NE, may be linked to altered myo-inositol metabolism.

Acetylcholine↗