Acetylcholine-induced metabolic changes in the perfused rabbit mandibular salivary gland studied by 31P-NMR spectroscopy

Changes in the content of high-energy phosphates, intracellular pH (pHi) and the ratio of MgATP to total ATP ([MgATP]/[ATP]t) resulting from continuous stimulation with acetylcholine (10(-9) to 10(-4) M) were measured by 31P-NMR spectroscopy in the isolated, perfused rabbit mandibular gland at 37 degrees C. With 10(-9) to 10(-7) M acetylcholine, no significant changes in these parameters were observed. On stimulation with 10(-6) M acetylcholine, the optimal concentration for sustained secretion, the content of ATP decreased by 28 +/- 10% (mean +/- S.E.; n = 8) of its control value. pHi decreased initially by approx. 0.05 pH unit, then showed an alkalinization of 0.09 +/- 0.02 pH unit (n = 8). With 10(-5) and 10(-4) M acetylcholine, changes in ATP and pHi were similar to those induced by 10(-6) M acetylcholine: the total content of high-energy phosphates remained at approx. 70% of the control value and no decrease in [MgATP]/[ATP]t was observed. As possible causes of the reduced secretory rate observed with higher concentrations of acetylcholine (10(-5) to 10(-3) M), we can exclude depletion of high-energy phosphates, inhibition of metabolism caused by intracellular acidosis, and inhibition of ATP usage caused by a decrease in MgATP availability.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

Study on the interaction of bovine serum albumin with acid cyanine 5R and its application in analysis.

A supramolecular complex of bovine serum albumin (BSA) with acid cyanine 5R (AC 5R, C.I. acid blue 113, C.I.: 26360) has been shown to form in Tris-HCl buffer solution (pH 7.42) by linear sweep voltammetry (LSV), fluorimetry, and spectrophotometry. The binding ratio and binding constant of BSA with AC 5R have been detected by LSV and fluorimetry. The binding mechanism is also preliminarily discussed. In Tris-HCl buffer solution (pH 7.42), AC 5R can easily be reduced on the mercury electrode, and it has a well-defined LSV peak current (Ip) and peak potential (Ep) at -0.65 V (vs. SCE). In the presence of BSA, the Ip of AC 5R decreases, and the peak potential (Ep) shifts to a more positive potential. The decrease of the second-order derivative of reductive peak current (deltaIp') of AC 5R is proportional to the logarithm of BSA concentration in the range of 1.54 x 10(-8) mol x L(-1)-1.54 x 10(-5) mol x L(-1) (r = 0.9931-0.9977). The limit of detection of BSA is 9.0 x 10(-9) mol x L(-1). The relative standard deviation is 1.83% (n = 10), and the standard recovery is 97.5%-104.8%. This method can be used to determine BSA concentration on the basis of the interaction of BSA with AC 5R.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.     

No effects of oral ribose supplementation on repeated maximal exercise and de novo ATP resynthesis.

A double-blind randomized study was performed to evaluate the effect of oral ribose supplementation on repeated maximal exercise and ATP recovery after intermittent maximal muscle contractions. Muscle power output was measured during dynamic knee extensions with the right leg on an isokinetic dynamometer before (pretest) and after (posttest) a 6-day training period in conjunction with ribose (R, 4 doses/day at 4 g/dose, n = 10) or placebo (P, n = 9) intake. The exercise protocol consisted of two bouts (A and B) of maximal contractions, separated by 15 s of rest. Bouts A and B consisted of 15 series of 12 contractions each, separated by a 60-min rest period. During the training period, the subjects performed the same exercise protocol twice per day, with 3-5 h of rest between exercise sessions. Blood samples were collected before and after bouts A and B and 24 h after bout B. Knee-extension power outputs were approximately 10% higher in the posttest than in the pretest but were similar between P and R for all contraction series. The exercise increased blood lactate and plasma ammonia concentrations (P < 0.05), with no significant differences between P and R at any time. After a 6-wk washout period, in a subgroup of subjects (n = 8), needle-biopsy samples were taken from the vastus lateralis before, immediately after, and 24 h after an exercise bout similar to the pretest. ATP and total adenine nucleotide content were decreased by approximately 25 and 20% immediately after and 24 h after exercise in P and R. Oral ribose supplementation with 4-g doses four times a day does not beneficially impact on postexercise muscle ATP recovery and maximal intermittent exercise performance.     

Regulation of oxidative phosphorylation in the inner membrane of rat liver mitochondria by calcium ions

The effect of accumulation of Ca2+ at physiological concentrations (10(-8)-10(-6) M) on the rates of ATP synthesis and hydrolysis in rat liver mitochondria was studied. An addition of 5 x 10(-7) M Ca2+ resulted in the maximal rates of synthesis and hydrolysis of ATP. Decrease in the concentration of Ca2+ to 10-8 M or its increase to 5 x 10(-6) M inhibited oxidative phosphorylation and ATP hydrolysis. It was found that the rate of oxidative phosphorylation correlated with the phosphorylation level of a 3.5-kD peptide in the mitochondrial inner membrane on varying the Ca2+ concentration. The possible regulation of oxidative phosphorylation in mitochondria by Ca2+ is discussed.     

Myometrial cells induce angiogenesis and salvage damaged myocardium

Characteristically, uterine myometrial cells (MCs) are proliferative, inducing angiogenesis within the female reproductive organ. We evaluated whether MCs implanted into myocardium could also induce angiogenesis and restore heart function after injury. MCs were isolated from the adult rat uterus and cultured for three studies: 1) Intracellular VEGF levels were measured in MCs cultured with progesterone (10(-11), 10(-9), and 10(-7) M) (n = 6 tests per group). 2) Blood vessel density was evaluated 8 days after MCs (3 x 10(6) or 6 x 10(6)), smooth muscle cells (SMCs), or endothelial cells (n = 6 rats per group) were injected with matrigel into the subcutaneous tissue of adult rats. 3) MCs, SMCs (5 x 10(6)/rat), or media were injected into a transmural scar 3 wk after cryoinjury in rat hearts (n = 12 rats per group), and heart function, blood vessel density, and myocardial scar size and thickness were evaluated 5 wk later. In study 1, cultured MCs expressed VEGF, with levels significantly (P < 0.05) upregulated by progesterone at an optimal dose of 10(-11) M. In study 2, MCs injected into the subcutaneous tissue with matrigel induced significantly more blood vessels, especially large-diameter vessels, than did SMCs or endothelial cells (P < 0.01 for all groups). This angiogenic effect was greatest (P < 0.01) at higher doses of MCs and was enhanced by progesterone (10(-11) M). In study 3, MCs implanted into the injured myocardium increased blood vessel density at the implant area, reduced scar size, and improved cardiac function relative to SMCs and media. Overall, MCs induced angiogenesis in vitro and in vivo, prevented cardiac remodeling, and improved heart functional recovery after cardiac injury.     

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells

The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 M final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 ± 0.9 mol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 ± 0.7 umol/min), compared to the heart with control treatment (8.2 ± 0.5 and 6.7 ± 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with u.-phenyl-tert-butylnitrone (PBN, 3 mM final) were used to monitor free radical generation during reperfusion. The vascular concentration of PBN-radical adducts (untreated: 6.4 ±1.0 nM, at 10 min) decreased in the presence of DNP (1.7 ± 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 ± 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 ± 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation. (Mol Cell Biochem 160/161: 167–177, 1996)     

Mutagenicity of bisphenol A and its suppression by interferon-alpha in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1x10(-7) to 1x10(-5)M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1x10(-8)M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua(R)) phenotypic mutation was also found in cells treated with 1x10(-7) and 1x10(-5)M of bisphenol A. The induction of K-ras codon 12 mutations and Oua(R) mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-alpha prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1x10(-6)M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Insulin-like growth factor I (IGF I) induces cortisol production in bovine adrenocortical cells in primary culture

The effects of a physiological dose of IGF I (40 ng/ml approximately 5 x 10(-9) M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml approximately 10(-10) M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 +/- 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 +/- 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 +/- 0.79, n = 5). The cortisol/corticosterone ratio increased in the presence of IGF I from 1 +/- 0.19 to 1.76 +/- 0.45 (n = 7, P less than 0.02), although less so than in the presence of ACTH (5.50 +/- 0.98). Moreover, cortisol production was accompanied by androstenedione production (2.36 ng/10(6) cells, n = 3) similar to that induced by ACTH (2.10 ng/10(6) cells, n = 3). These findings together suggest stimulation of 17 alpha-hydroxylase activity. Cell multiplication was unaffected by IGF I. [3H]Thymidine incorporation into DNA reached only 193% +/- 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% +/- 5. Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.     

Enantioselective plasma protein binding of bimoclomol

The binding of bimoclomol enantiomers to human plasma, its components, as well as to plasma from monkey, dog, rat, and mouse was investigated by ultrafiltration and equilibrium dialysis. The considerably stronger binding of the (-)-(S)-enantiomer found in human plasma is due to the alpha(1)-acid glycoprotein (AAG) component. The binding parameters for AAG (n(R)K(R) = 1.3 x 10(4) M(-1) and n(S)K(S) = 1.0 x 10(5) M(-1)) revealed high enantioselectivity, while the binding to human serum albumin was found to be weak (nK = 5 x 10(3) M(-1)) and not stereoselective. (-)-(S)-Bimoclomol was extensively displaced in the presence of specific marker ligands for the "FIS" subfraction of human AAG. Comparative binding studies indicated considerable differences between plasma of the five species investigated.     

慢性缺氧对大鼠肺动胍内皮依赖性舒张反应及CGMP含量的影响

This experiment was designed to investigate whether chronic hypoxia affect rat pulmonary artery (PA) endothelium-dependent relaxation and the content of cGMP in PA. Both ACh and ATP could induce endothelium-dependent relaxation of PA, not prevented by indomethacin, but completely abolished by methylene blue. These results indicated that vasodilatation of PA induced by both ACh and ATP is mediated by EDRF (endothelium-derived relaxing factor). Chronic hypoxia significantly depressed PA endothelium-dependent relaxation. The percent relaxation of IPPA and EPPA by 10(-6) mol/L ACh was 61.3% and 59.2% of those in control, and the percent relaxation of IPPA and EPPA by 1.8 x 10(-5) mol/L ATP was 64.9% and 55.3% respectively of the control. Chronic hypoxia also depressed SNP-induced endothelium-independent relaxation. Chronic hypoxia significantly decreased the content of cGMP in PA. The basic level of cGMP was 51.9 +/- 5.7 (n = 14) in hypoxia group and 84.9 +/- 9.7 (n = 14) pmol/g wet wt. in control group (P less than 0.01). After treatment of PA with ACh (10(-7) mol/L), the content of cGMP was 91.4 +/- 7.3 (n = 5) pmol/g wet wt. in hypoxic group and 240.8 +/- 30.6 (n = 5) pmol/g wet wt. in control group (P less than 0.01). Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of soluble guanylate cyclase in vascular smooth muscle cells.     

Study and application of a new adenosine electrode with thymus tissue.

A new adenosine-selective membrane electrode using rabbit thymus tissue as catalyst is described. A typical response slope of 51.2 mV per concentration decade is observed over a linear range which extends from 3.16 x 10(-5) M to 5.62 x 10(-3) M. Detection limits of 2.99 x 10(-5) M have been established. Measured response times are 7 min. The coefficient of variation ranged from 1 to 5.62% (n = 7, m = 5). Fourteen compounds were specifically tested as possible interferents, but no significant response was observed. The standard recoveries of adenosine were from 95.3 to 104.0% (m = 5, n = 5), and the recoveries of adenosine in rabbit blood ranged from 94.0 to 108.4% (n = 3, m = 5) over the linear range. This tissue-based biosensor has excellent sensitivity and selectivity, and has additional advantages of simplicity and low cost. The biosensor can be used to measure directly the concentration of adenosine in body fluid samples without sample processing.     

Probing actomyosin interactions with 2,4-dinitrophenol

Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.     

Molecular Sieving by the Bacillus megaterium Cell Wall and Protoplast

Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values (R(w)) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of R(w) for intact cells as a function of number-average molecular weight ( M(n)) or Einstein-Stokes hydrodynamic radius ( r(ES)) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of M(n) = 0.6 x 10(3) to 1.1 x 10(3), r(ES) = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of M(n) = 0.7 x 10(5) to 1.2 x 10(5), r(ES) congruent with 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples ( M(n) = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to M(n) = 1,200, r(ES) = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm.     

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.