Unusual pressure effects on ligand rebinding to the human myoglobin Leucine 29 mutants.
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Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O(2) rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (DeltaV), as observed for O(2) binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (DeltaV(32)), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step. (+info)
The mechanism of acute renal failure after uranyl nitrate.
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Administration of 25 mg/kg uranyl nitrate (UN) to rats leads to a brief period of polyuria followed by progressive oliguria with death at 5 days. Factors that determine glomerular filtration rate (GFR) were examined in control Munich-Wistar rats (n equals 16) and 2 h after either 15 mg/kg (n equals 8) or 25 mg/kg (n equals 7) of UN (i.v.) utilizing direct measurements of hydrostatic and oncotic pressures and plasma flow. Total kidney GFR was reduced to 47% of control in the low dose group and to 21% in the high dose group. The simultaneous nephron filtration rate (sngfr) was 28.6 plus or minus 0.8 nl/min/g kidney wt in control, 29.1 plus or minus 1.0 in the low dose group, and 18.1 plus or minus 1.2 (P less than 0.001) in the higher dose group. This disparity in UN effect upon GFR and sngfr was due to tubular back-diffusion of solute through damaged epithelia beyond the early proximal tubule as demonstrated by microinjection of inulin and mannitol in the proximal tubule. Inulin "leak" persisted at 6 h after UN when tubular pressure had returned to normal. Comparison of sngfr measured in early vs. late proximal tubule revealed no difference after high dose UN, suggesting no significant leak of inulin from the early proximal tubule, and that the decreased sngfr was due to primary reductions in ultrafiltration. Nephron plasma flow was equal to control at both doses of UN. Also directly measured hydrostatic pressure gradient across the glomerular capillary was not changed. The effective filtration pressure achieved equilibrium in control of animals but became significantly positive at the efferent end of the capillary at both doses of UN and increased. Total glomerular permeability (LpA) was progressively reduced from control (0.089 plus or minus 0.005 nl/s/g kidney wt/mm Hg) at low dose UN (0.047 plus or minus 0.013) and high dose 0.024 plus or minus 0.003 nl/s/g kidney wt/mm Hg). Therefore UN decreases GFR by two mechanisms: (1) tubular damage leading to back-diffusion of solutes and (b) a primary reduction in sngfr due to reduced LpA. (+info)
Renal interstitial hydrostatic pressure and pressure natriuresis in pregnant rats.
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The objective of this study was to test the hypothesis that a decrease in renal interstitial hydrostatic pressure (RIHP) accounts for the blunted pressure natriuresis during pregnancy. RIHP was measured in nonpregnant (NP; n = 9), midterm pregnant (MP; 12-14 days after conception; n = 10), and late-term pregnant (LP; 18-21 days after conception; n = 12) female Sprague-Dawley rats at two renal perfusion pressure (RPP) levels (99 and 120 mmHg). At the lower RPP level, RIHP was 5.9 +/- 0.3 mmHg for NP, 3.4 +/- 0.4 mmHg for MP (P < 0.05 vs. NP), and 2.9 +/- 0.1 mmHg for LP (P < 0.05 vs. NP) rats. The increase in RPP from 99 to 120 mmHg resulted in pressure natriuretic and diuretic responses in all groups; however, the increases in fractional excretion of sodium (DeltaFE(Na)), urine flow rate (DeltaV), and DeltaRIHP were significantly greater (P < 0. 05) in NP compared with both MP and LP rats. DeltaFE(Na), DeltaV, and DeltaRIHP were 2.06 +/- 0.28%, 81.44 +/- 14.10 microl/min, and 3. 0 +/- 0.5 mmHg for NP; 0.67 +/- 0.13%, 28.03 +/- 5.28 microl/min, and 0.5 +/- 0.2 mmHg for MP; and 0.48 +/- 0.12%, 18.14 +/- 4.70 microl/min, and 0.4 +/- 0.1 mmHg for LP rats. In conclusion, RIHP is significantly lower in pregnant compared with nonpregnant rats at similar RPP levels. Also, the ability of pregnant rats to increase RIHP in response to an increase in RPP is blunted. These changes in RIHP may play an important role in the blunted pressure natriuresis and contribute to the conservation of sodium and water that is critical for fetal growth and development during normal pregnancy. (+info)
A new miniature hydrostatic pressure chamber for microscopy. Strain-free optical glass windows facilitate phase-contrast and polarized-light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature.
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This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase-contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardation (BR) og 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20 times UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40 times phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chambers, as well as methods for shifting and controlling the temperature within the chamber, are included. (+info)
Oncotic pressure in solid tumors is elevated.
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Oncotic and hydrostatic pressure differences control the movement of fluid and large molecules across the microvascular wall of normal and tumor tissues. Recent studies have shown that the interstitial fluid pressure in tumors is elevated and is approximately equal to the microvascular pressure. Whereas oncotic pressure in blood plasma of various species is known, no data are available on the oncotic pressure in the interstitial space of tumors. We hypothesize that because of the leaky nature of tumor vessels, oncotic pressure in tumor interstitium should be close to that in plasma. To this end, we first developed a chronic wick method for the direct measurement of oncotic pressures in the interstitial fluid of tumors grown in mice. We found interstitial oncotic pressures in four human tumor xenografts to be higher than in s.c. tissue and comparable to that in plasma [rhabdomyosarcoma (RD), 24.2+/-4.7; squamous cell carcinoma (FaDu), 19.9+/-1.9; small cell lung carcinoma (54A), 21.1+/-2.8; colon adenocarcinoma (LIS174T), 16.7+/-3.0 mm Hg; s.c. tissue, 8.2+/-2.3; plasma, 20.0+/-1.6 mm Hg]. These results support our hypothesis that the oncotic pressure difference across the tumor microvascular wall is low. The high oncotic pressure in tumors is consistent with the elevated interstitial fluid pressure, and it contributes to the suboptimal delivery of large therapeutic agents to neoplastic cells. (+info)
Lung thermal volume in pulmonary edema: effect of positive end expiratory pressure.
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Effects of intermittent (IPPB) and positive eng-expiratory pressure (PEEP) ventilation on accumulation of pulmonary edema were compared, in dogs, after infusion of oleic acid. Pulmonary extravascular water was approximated as lung thermal volume (LTV), a double indicator method based on differential transit time for simultaneously injected right-to-left conductivity and thermal pulses. LTV was found to be decreased in animals treated with PEEP. The possibility that observed LTV changes reflect only the effect of PEEP on flow distribution, not lung water, was examined by alternating PEEP and IPPB; short-term changes in LTV did not occur. Mean values of other factors influencing pulmonary water transfer, e.g., pulmonary capillary wedge pressure, serum protein, arterial blood gasses, were not significantly different with or without PEEP. It was concluded that, for the oleic acid lesion, PEEP effects a small reduction in the rate of accumulation of pulmonary edema. (+info)
Ontogenetic scaling of burrowing forces in the earthworm Lumbricus terrestris.
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In hydrostatic skeletons, it is the internal fluid under pressure surrounded by a body wall in tension (rather than a rigid lever) that enables the stiffening of the organism, the antagonism of muscles and the transmission of force from the muscles to the environment. This study examined the ontogenetic effects of body size on force production by an organism supported with a hydrostatic skeleton. The earthworm Lumbricus terrestris burrows by forcefully enlarging crevices in the soil. I built a force-measuring apparatus that measured the radial forces as earthworms of different sizes crawled through and enlarged pre-formed soil burrows. I also built an apparatus that measured the radial and axial forces as earthworms of different sizes attempted to elongate a dead-end burrow. Earthworms ranging in body mass m(b) from hatchlings (0.012 g) to adults (8.9 g) exerted maximum forces (F, in N) during active radial expansion of their burrows (F=0.32 m(b)(0.43)) and comparable forces during axial elongation of the burrow (F=0.26 m(b)(0.47)). Both these forces were almost an order of magnitude greater than the radial anchoring forces during normal peristalsis within burrows (F=0.04 m(b)(0.45)). All radial and axial forces scaled as body mass raised to the 2/5 power rather than to the 2/3 power expected by geometric similarity, indicating that large worms exert greater forces than small worms on an absolute scale, but the difference was less than predicted by scaling considerations. When forces were normalized by body weight, hatchlings could push 500 times their own body weight, while large adults could push only 10 times their own body weight. (+info)
Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460.
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The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions. (+info)