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Biomedical subjects

M R Lee

Publications and source records attributed to M R Lee.

At least 55 records · Page 3Linked to original sources
PubMed1998

Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle.

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.

Animals↗
PubMed1998

Application of solid-phase microextraction and gas chromatography-mass spectrometry for the determination of chlorophenols in urine.

This study investigated the feasibility of applying solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry to analyze chlorophenols in urine. The SPME experimental procedures to extract chlorophenols in urine were optimized with a polar polyacrylate coated fiber at pH 1, extraction time for 50 min and desorption in GC injector at 290 degrees C for 2 min. The linearity was obtained with a precision below 10% R.S.D. for the studied chlorophenols in a wide range from 0.1 to 100 microg/l. In addition, sample extraction by SPME was used to estimate the detection limits of chlorophenols in urine, with selected ion monitoring of GC-MS operated in the electron impact mode and negative chemical ionization mode. Detection limits were obtained at the low ng/l levels. The application of the methods to the determination of chlorophenols in real samples was tested by analyzing urine samples of sawmill workers. The chlorophenols were found in workers, the urinary concentration ranging from 0.02 microg/l (PCP) to 1.56 microg/l (2,4-DCP) depending on chlorophenols. The results show that trace chlorophenols have been detected with SPME-GC-MS in the workers of sawmill where chlorophenol-containing anti-stain agents had been previously used.

Chlorophenols↗
PubMed1997

Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase.

Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca2+ stores are depleted, we investigated mechanism(s) for the Ca2+ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca2+ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 microM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 microM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 microM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6. 0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca2+ desensitization in vasodilatation.

Animals↗
PubMed1997

Physiological role of nitric oxide in regulation of renal function in humans.

The physiological role of endogenous nitric oxide in regulation of renal function in humans is unclear. Eight healthy men received an inhibitor of nitric oxide synthase, N(G)-monomethyl-L-arginine (L-NMMA, 3 mg/kg), and saline placebo intravenously on two occasions. L-NMMA significantly increased mean arterial pressure (+7%) and total peripheral resistance (+36%). However, because renal plasma flow did not decrease significantly, the increase in renal vascular resistance (+21%) was significantly less than the increase in total peripheral resistance. Glomerular filtration rate (-19%), filtration fraction (-10%), urine flow rate (-18%), sodium (-28%), and free water excretion (-25%) all decreased significantly. Fractional distal, but not proximal, sodium reabsorption increased. L-NMMA also significantly decreased plasma nitrate and urinary excretion of nitrate and dopamine. There were no significant changes in plasma renin activity, plasma endothelin, and aldosterone or in platelet number and ex vivo aggregation. L-NMMA had a plasma half-life of 75 min. Basal generation of nitric oxide appears to contribute less to vascular tone in the kidney than systemically but may alter afferent arteriolar tone. Decreased fractional sodium excretion supports an important physiological role for nitric oxide in inhibition of tubular sodium reabsorption, possibly mediated by the renal dopaminergic system.

Adenosine Diphosphate↗
PubMed1997

Results of lesional vs. nonlesional frontal lobe epilepsy surgery.

Fifty-three seizure focus resections limited to the frontal lobe were performed. Forty-nine had adequate follow-up. Five of 17 (29%) nonlesional (NL) cases and 21 of 32 (66%) lesional (L) cases were seizure free (SF) at 1 year postoperatively. Eight of 9 (89%) L cases < or = 18 years old vs. 13 of 23 (57%) > 18 years old were SF. Eight of 10 (80%) tumor vs. 13 of 22 (59%) nontumor L cases were SF. Sixteen NL cases were localized by invasive recording. Five (31%) were SF. Ictal localization was obtained in 4 of 32 L cases. Three of 4 (75%) were SF. Eighteen of 24 (64%) L cases without ictal localization were SF. Nine of 12 (75%) lateral resections, 7 of 12 (58%) lobectomies, and 2 of 13 (15%) mesial resections were SF. Three of 20 cases with at least 90% reduction in seizures became SD > or = 2 years postresection. No case with < 90% seizure reduction at one year showed improvement with longer follow-up.

Adolescent↗
PubMed1996

Renal metabolism and effects of the glutamyl derivatives of L-dopa and 5-hydroxytryptophan in man.

1. Equimolar amounts of gamma-L-glutamyl-L-3,4-dihydroxyphenylalanine (gludopa) and gamma-L-glutamyl-5-hydroxy-L-tryptophan were infused separately and together in eight healthy, salt-replete male subjects in a placebo-controlled, cross-over study to investigate whether the administration of one amine precursor affects the renal metabolism of the other and to determine whether dopamine or 5-hydroxytryptamine would be generated preferentially. The overall effect on sodium excretion was also measured when both precursors were administered simultaneously. 2. Administration of gludopa was associated with marked increases in the urinary excretion of L-dopa, dopamine and 3,4-dihydroxyphenylacetic acid, together with a rise in the urinary excretion of sodium. gamma-L-Glutamyl-5-hydroxy-L-tryptophan, on the other hand, produced marked increases in the urinary excretion of 5-hydroxy-L-tryptophan, 5-hydroxy-tryptamine and 5-hydroxyindoleacetic acid, and this was accompanied by a slight, but non-significant, reduction in sodium excretion. About 27% of the infused dose of gludopa (on a molar basis) was recovered in the urine as dopamine whereas 15% of the given dose of gamma-L-glutamyl-5-hydroxy-L-tryptophan was excreted as 5-hydroxytryptamine. 3. The urinary excretion values of L-dopa, dopamine and 3,4-dihydroxyphenylacetic acid after the simultaneous infusion of gludopa and gamma-L-glutamyl-5-hydroxy-L-tryptophan were not significantly different from those observed after infusion of gludopa only. Similarly, the urinary excretion values of 5-hydroxy-L-tryptophan, 5-hydroxytryptamine and 5-hydroxy-indoleacetic acid during the co-infusion were similar to those measured after administration of gamma-L-glutamyl-5-hydroxy-L-tryptophan only. The net effect of the concomitant infusion of both glutamyl derivatives was an increase in urinary sodium excretion. 4. Our study in salt-replete individuals suggests that dopamine rather than 5-hydroxytryptamine was preferentially produced when equimolar amounts of their precursors were provided and that the natriuretic effect of dopamine, generated intrarenally from gludopa, was greater than the sodium retaining action of 5-hydroxytryptamine derived from gamma-L-glutamyl-5-hydroxy-L-tryptophan. Comparison of the urinary metabolite data after the separate and concomitant infusion of the two glutamyl compounds provided no evidence of competitive inhibition of synthesis of either amine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1996

gamma-L-glutamyl-5-hydroxy-L-tryptophan, but not gamma-L-glutamyl-L-tryptophan, causes sodium retention in normal man.

1. This randomized, placebo-controlled, cross-over study compared the relative effectiveness of gamma-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP) and gamma-L-glutamyl-L-tryptophan (glu-TRP) in terms of their ability to act as substrates for renal 5-hydroxytryptamine (5-HT) synthesis and their actions on urinary sodium excretion. 2. Urinary excretion of 5-HT and sodium were determined before, during and after 1 h intravenous infusion of an equimolar amount (45 nmol kg-1 min-1) of glu-5-HTP or glu-TRP or placebo in nine healthy male subjects. 3. Cumulative urinary 5-HT excretion over the 4 h after the start of glu-5-HTP infusion was 350-fold greater than that after placebo, and this was associated with a reduction in the urinary excretion of sodium. 4. In contrast, the urinary excretion values of 5-HT and sodium after administration of glu-TRP were not significantly different from those observed on the placebo day. 5. The marked increase in urinary 5-HT excretion and the retention of sodium after administration of glu-5-HTP have been demonstrated in previous studies and result from increased intrarenal generation of 5-HT. The absence of a rise in urinary excretion of 5-HT after glu-TRP infusion suggests that there was no significant conversion of this glutamyl compound to 5-HT within the kidney. As a result, there was no effect on urinary sodium excretion.

5-Hydroxytryptophan↗
PubMed1996

Effects of frusemide on the urinary excretion of dopamine and 5-hydroxytryptamine in normal man.

The effects of frusemide on the urinary excretion of dopamine and 5-hydroxytryptamine (5-HT) were investigated in eight healthy male subjects in a randomized, placebo-controlled, cross-over study. Frusemide produced the expected rise in urinary dopamine excretion but it did not affect 5-HT excretion when compared with placebo. The lack of an effect on 5-HT excretion in man contrasts with studies in the rat which have reported a marked increase in 5-HT excretion after administration of this loop diuretic.

Adult↗
PubMed1996

The role of L-aromatic amino acid decarboxylase in serotonin-stimulated aldosterone secretion in response to salt intake.

In this study we tested a new hypothesis namely that serotonin (5-hydroxytryptamine, 5-HT) could be synthesised within the zona glomerulosa of the rat adrenal gland from exogenous 5-hydroxytryptophan (5-HTP) by the enzyme L-aromatic amino acid decarboxylase (L-AAAD). A specific monoclonal antibody against L-AAAD showed that the enzyme was present predominantly in the adrenal medulla but also in the zona glomerulosa and zona fasciculata. Wistar rats, maintained on a normal (NS), low (LS) or high (HS) salt diet for one week, were sacrificed by decapitation, blood samples taken and the adrenal glands removed. Plasma aldosterone concentrations were significantly higher in the LS diet group (2.91 +/- 0.35 nM) and significantly lower in the HS diet group (0.261 +/- 0.55 nM) compared with the NS diet group (1.025 +/- 0.133 nM) (p < 0.001). Capsules from the LS diet group synthesised significantly higher maximal levels of 5-HT (2615.463 +/- 480.88 nM/mg protein) than capsules from the NS (1219.117 +/- 150.259 nM/mg protein) and the HS (968.477 +/- 214.485 nM/mg protein) salt diet groups (p < 0.05). Maximal aldosterone secretion in adrenal capsules obtained from rats on the LS diet (73.428 +/- 4.053 nM/mg protein) was significantly higher than in those obtained from rats on the NS diet (41.658 +/- 1.87 nM/mg protein) (p < 0.05). Maximal aldosterone secretion in adrenal capsules from the HS diet group (30.624 +/- 2.114 nM/mg protein) was significantly lower than in the capsules from both the LS and NS groups (p < 0.05). Carbidopa (10(-4) M), a specific inhibitor of L-AAAD, markedly attenuated the secretion of aldosterone when adrenal capsules from all three salt diet groups were incubated with 10(-4) M 5-HTP (p < 0.05), but had no significant effect on basal aldosterone secretion. These results clearly demonstrate that L-AAAD is not only present in the medulla, but also in the zona glomerulosa and zona fasciculata of the rat adrenal gland. In addition, 5-HT can be synthesised in the zona glomerulosa/capsular region of the rat adrenal gland and both its biosynthesis and its ability to stimulate aldosterone secretion is increased by sodium depletion and attenuated by sodium loading. This raises the interesting possibility that L-AAAD could play a role in the regulation of aldosterone secretion during sodium deficiency in the rat by converting circulating 5-HTP (which is present in blood at concentrations exceeding 1 micromolar) into 5-HT within the adrenal cortex.

5-Hydroxytryptophan↗
PubMed1995

Isolation of ice-nucleating active bacteria from the freeze-tolerant frog, Rana sylvatica.

Ice-nucleating active (INA) bacteria were isolated from the gut of field-collected freeze-tolerant wood frogs (Rana sylvatica) collected in winter. Thirteen strains of Pseudomonas fluorescens, four strains of Pseudomonas putida, and two strains of Enterobacter agglomerans had ice-nucleating activity. Each of the INA pseudomonad strains was psychrophilic. P. putida strains were differentiated from P. fluorescens strains by gelatinase, lecithinase, and lipase production. The maximum nucleation temperatures (Tmax) of aqueous suspensions (10(9) bacteria/ml) of the four INA P. putida strains ranged from -1.6 to -3.0 degrees C, which places this INA species among the most potent known biological nucleators. Ingestion of INA P. putida isolated from R. sylvatica by another freeze-tolerant frog. Pseudacris crucifer, decreased the capacity of this frog to supercool and remain unfrozen at -2 degrees C. This is the first report of INA bacteria isolated from a vertebrate, and suggests that, as part of the gut flora in some posthibernation freeze-tolerant wood frogs, these bacteria may play a role in enhancing winter survival by promoting ice nucleation at high subzero temperatures (ca. -2 degrees C).

Animals↗
PubMed1995

Analysis of the neuronal promoter of the rat aromatic L-amino acid decarboxylase gene.

The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.

Animals↗
PubMed1995

Blood and urine 5-hydroxytryptophan and 5-hydroxytryptamine levels after administration of two 5-hydroxytryptamine precursors in normal man.

Six healthy male subjects received equimolar amounts of two 5-hydroxytryptamine (5-HT) precursors, 5-hydroxy-L-tryptophan (5-HTP) and gamma-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP), on two occasions in a randomised cross-over study. There were marked increases in urinary 5-HTP and 5-HT excretion after infusion of both compounds. Mean urinary excretion rate of 5-HT, which was < 0.7 nmol min-1 before dosing, rose to a peak value of 412 +/- 92 nmol min-1 at the end of 5-HTP infusion and 303 +/- 29 nmol min-1 after administration of glu-5-HTP. This occurred without significant changes in blood 5-HT levels measured in platelet-rich plasma. These findings provide further evidence that the increase in urine 5-HT after administration of both 5-HT precursors is largely due to 5-HT synthesised within the kidney.

5-Hydroxytryptophan↗