Pf Values Research Articles

AGE-RELATED CHANGES IN GLUCOSE DISAPPEARANCE IN RESPONSE TO INSULIN 308

Glucose transporters are developmentally regulated. GLUT1 is predominant in the fetus and newborn. GLUT2 in liver and GLUT4 in muscle and fatty tissue are predominant in the adult. GLUT1 is not insulin-regulatable and GLUT4 is insulin regulatable. Therefore, we hypothesize newborn rats could have a different response to insulin than the adult rat. Materials and Methods Ten day old and adult Sprague-Dawley rats were used after a 4 hour fast and an overnight fast, respectively. Radioactive 2DG with/without 10 unit/kg insulin (INS) was injected by cardiac puncture under pentobarbital anesthesia to evaluate glucose disappearance. 2DG was used because 2DG is not metabolized after phospholyration. Blood was serially collected by cardiac puncture at 0, 5,10,15,30 and 45 minutes after the injection. Plasma glucose(PG) concentration and radioactive 2DG concentration were measured by a oxidase method and scintilation counter, respectively. Glucose disappearance was calculated from plasma glucose concentration. Values for PG and 2DG were calculated as percent of initial values of PF and 2DG at 0 minute, respectively. Results PG and 2DG (%) at 45 minutes are in the Table.

Effect of Light on the Growth of Crop and the pF Value of Bark Bed

Effect of Light on the Growth of Crop and the pF Value of Bark Bed

Developmental changes in rabbit juxtamedullary proximal convoluted tubule water permeability.

The mammalian proximal tubule reabsorbs the bulk of the glomerular filtrate in a nearly isosmotic fashion due to the high osmotic water permeability (Pf) of this segment. Although the characteristics of proximal tubule water transport have been studied in the adult proximal tubule, little is known about the neonatal segment. The present study directly measured the Pf and diffusional water permeability (PDW) of neonatal (10 +/- 2 day old) and adult rabbit juxtamedullary proximal convoluted tubules (PCT) using in vitro microperfusion. The Pf of neonatal juxtamedullary PCT was greater than the Pf of adult juxtamedullary PCT. In contrast, the PDW was not different between the two groups. The Pf and PDW values of both neonatal and adult tubules were inhibited to the same degree by p-chloromercuribenzene sulfonate and had identical activation energies. The transepithelial reflection coefficients of NaCl and NaHCO3 were also found to be similar in both the neonatal and adult proximal tubules. Thus neonatal and adult juxtamedullary PCT have many characteristics of water transport that are identical; however, neonatal Pf is three to five times that of the adult value. This difference in Pf with identical PDW values may give an insight into the transepithelial pathway for water movement in the neonatal tubule.

Peak factors of Mexican accelerograms: Evidence of a non‐Gaussian amplitude distribution

To study statistical properties of the near‐source radiation field, 35 horizontal accelerograms were selected, recorded at distances R < 110 km from five M > 7 Mexican earthquakes. For each one, the maximum‐amplitude segment, assumedly containing direct S waves, was selected by an automatic procedure. Then two parameters were determined for this segment: the peak to rms amplitude ratio, or peak factor PF, and the slope K of the dependence of PF on log duration. These two values were compared to the corresponding values expected for a simplest stochastic record model, of a segment of the stationary Gaussian process. For about 40% of the records, both parameters show clear (above 2σ) deviations from this model. Another record model was also tested, of quasi‐stationary Gaussian process (with time‐varying rms amplitude). With this end, the original records were “stationarized,” that is, modified to attain nearly constant rms amplitude, and then analyzed in the same manner as the original ones. However, for a distinct fraction of records, deviations persisted. The type of the deviations is characteristic for a “heavy‐tailed” amplitude distribution, with the enhanced probability of large peaks. The degree of expression of this phenomenon seems to decay with distance. This observation suggests that source radiation, as it is generated at the fault, often is inherently non‐Gaussian (heavy‐tailed), but because of the scattering and multipathing, it quickly loses this property. One can expect this picture not to be specific for Mexican earthquakes only. From the viewpoint of stochastic strong motion simulation, the results imply that the PF value calculated from the stationary Gaussian model can underestimate average peak acceleration, on the average, by some 25%. In an analysis aimed at the recurrence of rare peaks, the underestimation may be even larger: among 35 records studied, five have peak factors more than 50% above the values expected for the stationary Gaussian model.

Ethanol-induced changes in insulin-like growth factors and IGF gene expression in the fetal brain.

Brain growth retardation is a major feature of the fetal alcohol syndrome (FAS). Insulin-like growth factors (IGF-I and IGF-II) have been shown to exert significant metabolic and growth-promoting effects. Previously, we showed that circulating levels of IGF-I as well as hepatic gene expression of both IGFs were decreased in newborn offspring of rats fed ethanol during pregnancy. This study investigated the effects of maternal ethanol ingestion on fetal rat brain growth and on levels of IGF-I and IGF-II, as well as their mRNAs, in fetal brain. IGF-binding protein (IGFBP) levels also were determined. Rats were fed 5% w/v ethanol in a liquid diet during gestation (EF group). Weight-matched animals were pair-fed equicaloric control diet (PF group) or were fed ad libitum (AF group). The mean fetal brain weight of EF offspring was 13% and 16% lower (P < 0.01) than that of PF and AF offspring, respectively. Body weight of EF pups was decreased to a greater extent, resulting in higher brain to body weight ratios in EF pups than in either control group (P < 0.05). IGF-I levels in EF pups decreased by 33% and 41% compared with the corresponding PF and AF values (P < 0.01). IGF-I mRNA levels decreased by 27% and 40% compared with PF and AF values, respectively. A positive correlation was observed between brain IGF-I level and brain weight (r = 0.561, P < 0.01). IGF-II levels were not affected despite a 50% decrease in IGF-II expression. In PF animals, the fetal brain IGF-I and IGF-II mRNA levels were reduced by 28% and 21%, apparently in response to undernutrition. IGF-binding proteins levels were low in the EF group but not statistically significant compared with control values. The diminished fetal brain concentration of IGF-I and decreased gene expression of IGFs may play a role in brain growth retardation associated with FAS.

Identification of a chloride conductance in basal membrane of human placental syneytiotrophoblast

Identification of a chloride conductance in basal membrane of human placental syneytiotrophoblast

Phosphorylation of Aquaporin-2 Does Not Alter the Membrane Water Permeability of Rat Papillary Water Channel-containing Vesicles

Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.

Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC/AQP-4.

The mercurial-insensitive water channel (MIWC or AQP-4) is a 30-32 kDA integral membrane protein expressed widely in fluid-transporting epithelia [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500]. To investigate the mercurial insensitivity and key residues involved in MIWC-mediated water transport, amino acids just proximal to the conserved NPA motifs (residues 69-74 and 187-190) were mutated individually to cysteine. Complementary RNAs were expressed in Xenopus oocytes for assay of osmotic water permeability (Pf) and HgCl2 inhibition dose-response. Oocytes expressing the cysteine mutants were highly water permeable, with Pf values (24-33 x 10(-3) cm/s) not different from that of wild-type (WT) MIWC. Pf was reversibly inhibited by HgCl2 in mutants S70C, G71C, G72C, H73C, and S189C but insensitive to HgCl2 in the other mutants. K1/2 values for 50% inhibition of Pf by HgCl2 were as follows (in millimolar): 0.40 (S70C), 0.36 (G71C), 0.14 (G72C), 0.45 (H73C), 0.24 (S189C), and > 1 for WT MIWC and the other mutants. To test the hypothesis that these residues are near the MIWC aqueous pore, residues 72 and 188 were mutated individually to the larger amino acid tryptophan. Pf in oocytes expressing mutants G72W or A188W (1.3-1.4 x 10(-3) cm/s) was not greater than that in water-injected oocytes even though these proteins were expressed at the oocyte plasma membrane as shown by quantitative immunofluorescence. Coinjection of cRNAs encoding WT MIWC and G72W or A188W indicated a dominant negative effect; Pf (x 10(-3) cm/s) was 22 (0.25 ng of WT), 10 (0.25 ng of WT + 0.25 ng of G72W), and 12 (0.25 ng of WT + 0.25 ng of A188W). Taken together, these results suggest the MIWC is mercurial-insensitive because of absence of a cysteine residue near the NPA motifs and that residues 70-73 and 189 are located at or near the MIWC aqueous pore. In contrast to previous data for the channel-forming integral protein of 28kDa (CHIP28), the finding of a dominant negative phenotype for mutants G72W and A188W indicates that MIWC monomers interact at a functional level.

Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes.

1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by Ca2+; Pf) were determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamate activated Ca2+ inflow in the voltage range -60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than -30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8.2% (NR1-NR2C subunits) and 11% (NR1-NR2A subunits) in normal extracellular solution (1.8 mM Ca2+) at -60 mV membrane potential. Pf values increased with increasing [Ca2+]o in the range of 0.5-10mM [Ca2+]o in a saturating fashion. 4. In cells expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamate-evoked Ca2+ inflow increased from 20 to -80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. Pf values of Q-form homomeric channels at -60 mV and 1.8 mM [Ca2+]o were between 3.2 and 3.9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1.8-10mM. Pf values in cells co-expressing Q- and R-form subunits were almost one order of magnitude smaller (0.54%). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS. Pf decreased with increasing relative concentration of R-form encoding CDNAs from 3.4 to 1.4%, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2+ inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments. Pf values were largest for the Q-form (1.55-2.0%) and lowest for R-form channels (< 0.2%), suggesting that Q/R site editing also decreases Ca2+ inflow through KAR channels. Cells co-expressing both subunit forms showed an intermediate value (0.58%).(ABSTRACT TRUNCATED AT 400 WORDS)

Application of technetium-99m hexamethylpropylene amine oxime single-photon emission tomography to neurologic prognosis in patients undergoing urgent carotid surgery.

In this study we aimed to work out a quantitative prognostic index for preoperative assessment of brain technetium-99m hexamethylpropylene amine oxime (HMPAO) single-photon emission tomography (SPET) in patients referred for urgent carotid endarterectomy due to acute obstructive disease of the internal carotid artery (ICA) and neurological deficit. To this end we compared data from preoperative SPET studies with the postinterventional changes in neurological status in 20 patients (17 males, three females; mean age 53 years, SD 4 years) with acute ischaemic cerebral disorders induced by obstruction of the ICA. Carotid obstruction was diagnosed by ultrasound B-mode study. All patients underwent urgent carotid endarterectomy from the ICA. Patients were divided into two groups in accordance with the results of postoperative follow-up: group A comprised patients with significant (more than 3 points) postoperative improvement in neurological condition as quantified by the Canadian Neurological Scale (11 patients); group B consisted of patients with minimal improvement or deterioration (nine, three of whom died). All patients were studied preoperatively by 99mTc-HMPAO SPET. The volume of nonperfused tissue (VS, cm3) was quantified using the Mountz technique. Hypoperfused volume (Vhypoperf, cm3) in the affected hemisphere was calculated as the total volume of voxels with 99mTc-HMPAO uptake < 90% of the contralateral symmetric voxels. Discriminant prognostic function was calculated by discriminant analysis as: PF = 0.072 x VS + 29.46x(VS/Vhypoperf). Patients with preoperative PF values < 8.20 demonstrated postoperative improvement in neurological status, while the group with PF > 8.90 comprised patients who demonstrated minimal improvement or deterioration. PF values in the range 8.20-8.90 carried an indefinite prognosis. We conclude that the preoperative 99mTc-HMPAO SPET can be used for the selection of patients in whom improvement in neurological status may be expected after urgent surgical correction of acute extracranial obstruction of the ICA.

Synthesis and thermal characterization of chemically modified phenolic resins

AbstractPhenolic resins with improved/altered thermal stability and flame retardancy were prepared by introducing flame retardant elements such as phosphorus and bromine at the monomer stage, followed by condensation with formaldehyde or hexamethylene tetramine (HMTA). Thus, monophenyl phosphoric acid (MPPA), monophenyl phosphoric acid–formaldehyde resin (MPPAF), brominated phenol–formaldehyde resin (BrPF) and 2,4,6‐tribromophenyl‐phosphoric acid–formaldehyde resin (BrMPPAF) were prepared and characterised. Thermal stability and flammability of these polymers were evaluated by TGA and LOI respectively and compared with those of phenol–formaldehyde resin (PF) and phosphorylated phenol–formaldehyde resin (PPF). The TGA (in air) of MPPAF showed enhanced thermal stability (char yield 66%) over that of PF (char yield 52%) and PPF (char yield 58%) indicating the role of phosphorus in imparting thermo‐oxidative resistance to PF. The enhancement is better when phosphorus is introduced at the monomer stage. The incorporation of bromine, however, brought about an unexpected reduction in the thermal stability of PF (char yield: BrPF, 2% and BrMPPAF, 26%). Both phosphorus and bromine are found to enhance the LOI values of PF (cured) from 35 to 50, 48, and 56, respectively, for MPPAF, BrPF, and BrMPAFF. A comparison of LOI values of these resins with their char yield suggests a condensed phase mechanism for phosphorus and vapor phase mechanism for bromine operating during thermal decomposition. © 1994 John Wiley &amp; Sons, Inc.

Synmetamorphic veining: origin of andalusite‐bearing veins in the Vedrette di Ries contact aureole, Eastern Alps, Italy

ABSTRACTDiscordant andalusite‐biotite‐quartz‐bearing veins occur in the contact aureole of the Vedrette di Ries pluton (Italian Eastern Alps), never outside the area of contact metamorphic andalusite development. Andalusite veins are found only within andalusite‐bearing hornfelses, and vein biotite occurs wherever host‐rock garnet is partially replaced by biotite. Veins formed during contact metamorphism, synchronously with the crystallization of andalusite and biotite within host rocks. Their pegmatitic structure and their orientation suggest that vein parageneses crystallized within fluid‐filled cavities that opened by hydraulic fracturing.A mechanism of synmetamorphic veining is proposed to explain rock failure and subsequent mineral deposition within veins. During hydrofracturing induced by dehydration reactions in response to heating in the aureole, fissures were immediately filled with locally derived fluids. The lack of large‐scale flux, together with high fluid pressures required by hydrofracturing, suggest fluid in the cavities was a virtually stagnant, passive medium, and that mass‐transport toward fractures was driven by intergranular diffusion. Because temperature and Pf values within veins are similar to those in the host rock, vein assemblages are interpreted as the stable, high‐T side of reactions taking place within pelitic schists, at the time when fractures opened. Once nucleation of product phases occurred, chemical components released by dissolution of reactant minerals were driven to precipitation sites by chemical potential gradients. Since nucleation was favoured at the strained grains of vein walls, andalusite and biotite simultaneously grew in vein and host rock.The proposed genetic model contrasts with generally adopted metasomatic mechanisms for the genesis of Al2SiO5‐bearing veins, in not requiring large fluid/rock ratios or a highly ‘aggressive’ fluid composition. The mechanism of synmetamorphic veining may be particularly useful in the interpretation of vein occurrences in medium‐ and deep‐crustal rocks which have undergone extensive devolatilization.

ON THE LIMITATIONS OF NEUTRINO EMISSIVITY FORMULA OF IWAMOTO

The neutrino emissivity from two- and three-flavor quark matter is numerically calculated and compared with Iwamoto’s formula. We find that the calculated emissivity is smaller than Iwamoto’s result by orders of magnitude when pf(u)+pf(e)−pf(d(s)) is comparable with the temperature. We attribute it to the severe restriction imposed by momentum conservation on the phase space integral. We obtain an alternative formula for the neutrino emissivity which is valid for any value of pf(u)+Pf(e)−Pf(d(s)) when the quarks and electrons are degenerate.

Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.

CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels.

Computerized equipment to control soil water matric potential and soil temperature in inoculum potential assays of soil-borne pathogens

Computerized equipment to control soil temperature and soil water matric potential, at high soil hydric conditions was developed and evaluated. A series of experiments demonstrated the accuracy and reproducibility of the equipment's performance and its adequacy for the assessment of the inoculum potential of soil-borne pathogens in soils with different characteristics. Control of soil water potential is achieved by variation in the height of the water table in a medium with high water conductivity supporting the soil. The equipment consists of double-walled tanks, permitting the adjustment of soil temperature. It is provided with sensors, control software and valves for automatic operation. in a growth chamber at 24°C. with RH 70% and irradiation of 90 W.m−2, was maintained in dynamic equilibrium for pF values ranging from 1 (−1 kPa) to 2 (−10 kPa) in various arable soil samples during the four to five weeks period of the bioassays. During the fourth week of pea or iris growth at pF=2, the system controlled within an amplitude of 0.4 pF-units. Between replications, variance was approximately 0.1 at F=2, decreasing with increasing. Soil temperature in the system could be maintained at a constant level with a variance below 0.1, within an amplitude of 0.3°C.

Verification of absorbed doses determined with thimble and parallel-plate ionization chambers in clinical electron beams using ferrous sulphate dosimetry.

Absorbed dose values determined with the commonly applied NACP and PTW/Markus parallel-plate chambers and the cylindrical NE2571 Farmer chamber were compared to values obtained with ferrous sulphate dosimetry in a number of electron beams. For the ionometry with the parallel-plate chambers the dose-to-air chamber factor ND (or Ngas) was derived from a 60Co beam calibration free in air with an additional buildup layer of 0.54 g cm-2 graphite as proposed by the protocol for electron dosimetry published by the Netherlands Commission on Radiation Dosimetry. For the product kattkm in this calibration geometry values of 0.980 +/- 0.003 [1 standard deviation (s.d.)] and 0.993 +/- 0.004 (1 s.d.) were obtained for the parallel-plate NACP and PTW/Markus chambers, respectively. The behavior of the fluence perturbation correction factor pf versus the mean electron energy at depth was deduced for the flat PTW/Markus and cylindrical NE2571 chamber by comparison with the NACP chamber, for which pf was assumed unity. Our results show a small but significant energy dependence of pf for the PTW/Markus chamber. The absorbed dose values, determined ionometrically with the different chambers considered in the study using the experimentally determined kattkm and pf values, are systematically 0.5% higher than those obtained with ferrous sulphate dosimetry adopting 352 x 10(-6) m-2 kg-1 Gy-1 for epsilon mG. The performed comparative study confirms also that for the NACP chamber pf is unity independent of the electron energy down to a mean energy at depth of 2 MeV.

Sensitivity of metal frames reliability to semirigid connections parameters

The present study deals with the sensitivity of metal frames reliability to the randomness of the characteristics of both the structural members and the connections. The random variables involved are the geometrical dimensions, the mechanical properties of the constitutive materials and the applied loads. The reliability analysis must be, in principle, defined through the probability of failure, denoted Pf, by either buckling or plastic mechanisms events. Monte Carlo simulations could be run to assess the Pf values. However, as a first step in the comprehension of the phenomena that cause the structural failure, the authors restrict their present study to the analysis of the evolution of the critical values of the applied loads. The results show that, with an adequate numerical procedure, an acceptable precision of these values may be reached for a reduced number of simulations. The multimodal failure of the structure is also demonstrated. Conclusions can be made about the attention that must be devoted to the random character of either the structural members or the connections.

Cultured bovine corneal endothelial cells express CHIP28 water channels

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic water permeabilities of human placental microvillous and basal membranes.

Literature data suggest that water accumulation by the human fetus is driven by osmotic gradients of small solutes. However, the existence of such gradients has not been supported by prior measurements. Attempts to estimate the size of the gradient necessary to drive net water movement have been seriously hampered by the lack of permeability data for the syncytiotrophoblast membranes. Stopped-flow light scattering techniques were employed to measure the osmotic water permeability (Pf) of microvillous (MVM) and basal membrane (BM) vesicles isolated from human term placenta. At 37 degrees C, the Pf was determined to be 1.9 +/- 0.06 x 10(-3) cm/sec for MVM and 3.1 +/- 0.20 x 10(-3) cm/sec for BM (mean +/- SD, n = 6). At 23 degrees C, Pf was reduced to 0.7 +/- 0.04 x 10(-3) cm/sec in MVM and 1.6 +/- 0.05 x 10(-3) cm/sec in BM. These Pf values are comparable to those observed in membranes where water has been shown to permeate via a lipid diffusive mechanism. Arrhenius plots of Pf over the range 20-40 degrees C were linear, with activation energies of 13.6 +/- 0.6 kcal/mol for MVM and 12.9 +/- 1.0 kcal/mol for BM. Water permeation was not affected by mercurial sulfhydryl agents and glucose transport inhibitors. These data clearly suggest that water movement across human syncytiotrophoblast membranes occurs by a lipid diffusion pathway. As noted in several other epithelial tissues, the basal membrane has a higher water permeability than the microvillous membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k.

CHIP28k is an important water-transporting protein in the kidney proximal tubule and the thin descending limb of Henle [Zhang, Skach, Hasegawa, Van Hoek, & Verkman (1993) J. Cell Biol. 120, 359-369] that is homologous to human erythrocyte CHIP28 [Preston & Agre (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11110-11114]. Oligonucleotide-directed mutagenesis was used to identify the cysteine(s) involved in inhibition of the water-transporting function of CHIP28k by the mercurial HgCl2. Each of the four cysteines (at positions 87, 102, 152, and 189) were mutated to serine individually, or in combinations. In vitro transcribed cRNA was expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf) in the absence or presence of 0.3 mM HgCl2. Pf (in cm/s x 10(-4) measured at 10 degrees C) was 7 +/- 1 in water-injected oocytes. In wild-type CHIP28k, Pf was 58 +/- 7 (-HgCl2) and 12 +/- 1 (+HgCl2). Mutation of cysteine 87, 102, or 152, individually or in combinations, had little effect on oocyte Pf or on the inhibition by HgCl2. Mutation of cysteine 189 to serine or glycine gave similar Pf values of 49-56 (-HgCl2); however, Pf was not inhibited up to 1 mM HgCl2. Mutation of cysteine 189 to the larger amino acid tryptophan gave a low Pf of 9 +/- 1; coexpression with wild-type CHIP28k indicated that the tryptophan mutation was not dominant negative. Mutation of the asparagine 42 and 205 glycosylation sites to threonine had little effect on Pf.(ABSTRACT TRUNCATED AT 250 WORDS)