Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.     

Prefracture and cold-fracture images of yeast plasma membranes

Fracture-temperature related differences in the ultrastructure of plasmalemma P faces of freeze-fractured baker's yeast (Saccharomyces cerevisiae) have been observed in high-resolution replicas prepared in freeze-etch systems pumped to 2 X 10(-7) torr in which the specimens were protected from contamination by use of liquid nitrogen-cooled shrouds. Two major P-face images were observed regardless of the source of the yeast, the age of the culture, the growth temperature, the physiological condition, or the suspending medium used: (a) a "cold- fracture image" with many strands closely associuated with tubelike particles (essentially the same image as those previously published for yeast freeze-fractured at 77 degrees K), and (b) a "prefracture image" characterized by the presence of more distinct tubelike particles with few or no associated strands (for aging cultures, the image recently referred to as "paracrystalline arrays" of "craterlike particles"). Both types of P-face image can be found in separate areas of single replicas and occasionally even within a single plasma membrane. Whereas portions of replicas known to be fractured at any temperature colder than 218 degrees K reveal only the cold-fracture image, prefracture images are found in cells intentionally fractured at 243 degrees K and in cracks or fissures which develop during the freezing of other specimens. These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens at some temperature between 173 degrees and 77 degrees K, and not from the use of "starved" yeast cells.     

Decoration of specific sites on freeze-fractured membranes

Fracturing under ultrahigh vacuum (UHV, P less than or equal to 10(-9) Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite for studies of interactions between condensing gases and distinct regions of a surface. For the study of water condensation, a device has been developed which enables production of pure water vapor and controlled variation of its partial pressure in an UHV freeze-fracture apparatus. Experiments with yeast plasmalemma fracture faces, produced at -196 degrees C and exposed to pure water vapor before replication, resulted in a "specific decoration" with ice crystals of those pits in the extraplasmic face where the corresponding particles of the plasmic face had been removed. Because water condenses as discrete ice crystals which resemble intramembrane particles, ice crystals might easily be misinterpreted as actual membrane structures. At low specimen temperature (T less than or equal to 110 degrees C) the structural features of membrane fracture faces produced under high vacuum (P approximately 10(-6) Torr) should, therefore, be interpreted with caution.     

Production and Cleaning of Freeze-Etch Replicas which Show Complementary Surfaces of Fractured Fungus Spores and Hyphae

London finder grids (H-2) and 3 mm copper discs were flared on opposite sides by use of a die and punch. The grids were registered on the discs and attached with vaseline. The specimen was squeezed through the grid apertures by the 2 copper discs. The complete sandwich was frozen in freon 22 and placed in a hinged specimen holder which fractured the layer of the specimen between the 2 finder grids. Platinum-carbon and carbon replicas were made immediately after fracturing. After drying the replicas, the vaseline was removed by chloroform, and replicas were cleaned with 70% H₂SO₄ for 1-18 hr at 52 C, followed by 6% commercial sodium hypochlorite (Chlorox) for 1-2 hr at 23-25 C.     

Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.     

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4-5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1 degrees C/min to -120 degrees C, followed by storage in liquid nitrogen at -196 degrees C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100 degrees C/min) or gradual (1 degrees C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.     

Behavioral thermoregulation by hypoxic rats.

To address whether a shift in hypothalamic thermal setpoint might be a significant factor in induction of hypoxic hypothermia, behavioral thermoregulation was examined in 7 female Sprague-Dawley rats implanted with radiotelethermometers for deep body temperature (Tb) measurement in a thermocline during normoxia (PO2 = 125 torr) and hypoxia (PO2 = 60 torr). Normoxic rats (TNox) selected a mean ambient temperature of 19.7 +/- 1.4 (SE) degrees C and maintained Tb at 37.0 +/- 0.2 degrees C. Hypoxic rats selected a significantly higher ambient temperature (THox = 28.6 +/- 2.2 degrees C) but maintained Tb significantly lower at 35.5 +/- 0.3 degrees C. Without a thermal gradient (ambient temperature = 25 degrees C), Tb during hypoxia was 35.4 +/- 0.4 degrees C. The maintenance of a lower body temperature during hypoxia through behavioral thermoregulation despite having warmer temperatures available supports the hypothesis that the thermoregulatory setpoint of hypoxic rats is shifted to promote thermoregulation at a lower Tb, effectively reducing oxygen demand when oxygen supply is limited.     

Differentiation of food vacuolar membranes during endocytosis in Tetrahymena

The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.     

Functional status of cryopreserved rat liver mitochondria

Mitochondria from the rat liver have been frozen down to -196 degrees C under protection of dimethylsulfoxide, after which they were subjected to periodical increase and decrease in temperature (cycling) in the ranges of -105 degrees C to -196 degrees C or -135 degrees to -196 degrees C. There have been observed some non-lethal damages of mitochondria which were revealed in the decrease in the respiratory control and in the ADP/O ratio after the cycling. The damage rate increased with the increase of the number of temperature decrease-increase cycles. The damages were also greater in the case when the upper limit of cycling temperature was higher than the glass transition temperature of the freezing medium (Tg = -126 degrees C). The non-lethal damages of mitochondria are explained by the influence of electric fields, appearing in the frozen sample during the temperature increase or decrease.     

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

ATP-induced reverse temperature effect in isohemoglobins from the endothermic porbeagle shark (Lamna nasus)

The evolutionary convergence of endothermic tunas and lamnid sharks is unique. Their heat exchanger-mediated endothermy represents an interesting example of the evolutionary pressure associated with this specific characteristic. To assess the implications of endothermy for gas transport and the possible contribution of hemoglobin (Hb), we investigated the effect of temperature on the oxygen equilibria of purified isohemoglobin components V and III from the porbeagle shark (Lamna nasus). In the absence of ATP the effect of temperature on oxygen affinity is normal in both Hb III (P50 = 0.9 and 2.2 torr at 10 and 26 degrees C, respectively) and Hb V (P50 = 1.5 and 2.5 torr at 10 and 26 degrees C, respectively). In the presence of this effector P50 decreases with increasing temperature in both components (P50 at 10 and 26 degrees C = 9.9 and 8.4 torr (Hb III), respectively, and 9.6 and 7.4 torr (Hb V), respectively. The reverse temperature effect in the presence of ATP will reduce the risk of oxygen loss from the arterial to the venous blood by lowering the oxygen tension gradient between the blood vessels. The mechanism behind the reverse temperature effect resembles that found in the bluefin tuna (Thunnus thynnus), an endothermic teleost, thus evidencing further convergent evolution.     

Ultrastructure of Saccharomyces cerevisiae strain AG1-7 and its responses to changes in environment

Asynchronous populations of the budding yeast Saccharomyces cerevisiae strain AG1-7 were examined by freeze-fracture electron microscopy for ultrastructural changes occurring in response to changes in the environment, specifically the following: temperature (23 or 37 degrees C); cell density (exponential, early stationary, and stationary phases); various periods of nitrogen starvation at low cell density, and return of nitrogen-starved cells to nitrogen-replete medium. This information has been gathered in preparation for ultrastructural examination of comparable responses of temperature-sensitive cell-cycle mutants. The plasma membrane was found to be particularly responsive to changes in environment. A high proportion (75%) of cells in exponential phase populations at 37 degrees C displayed paracrystalline arrays of plasma membrane particles, whereas this proportion was much lower (20%) at 23 degrees C in the same medium; plasma membrane grooves were longer at 37 than at 23 degrees C. In budded cells, the mother cell displayed paracrystalline arrays more frequently than the bud. Entry of cells into stationary phase, either through permitting population growth or by limiting nitrogen supply, resulted in increases in numbers of paracrystalline arrays and grooves. Groove depth also increased. The paracrystalline-array and groove-density responses were independent, both during entry into stationary phase and during the subsequent lag phase. Unusual groove forms appeared during stationary phase in high cell density populations, but not in low cell density nitrogen-starved populations. "Aggregate" and "geometric" tonoplast forms, previously described in strain A364A when grown under some of the conditions used here, were not found in AG1-7 under any of the conditions used here. It was demonstrated that particle-free patches can arise rapidly on the tonoplast of AG1-7 in response to temperature change from 37 to 23 degrees C. During stationary phase, spherosomes (lipid droplets) increased in size, particularly in response to nitrogen depletion. After 72 h of nitrogen starvation, about 10% of cell volume consisted of spherosomes. Changes in vacuolar content and mitochondrial form were also noted during entry into stationary phase.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.     

No evidence for brain stem cooling during face fanning in humans.

The interpeak latencies (IPLs) of the acoustically evoked brain stem potentials depend on brain stem temperature. This was used to see whether face fanning during hyperthermia lowers brain stem temperature. In 15 subjects, three thermally stable conditions were maintained by a water bath. In each condition the IPLs were determined in 10 separate trials. In condition A esophageal temperature (Tes) was 36.9 +/- 0.3 degrees C and increased to 38.6 +/- 0.2 degrees C in condition B. In conditions A and B the head was enclosed in a ventilated hood (air temperature 38 degrees C, relative humidity 100%) to suppress any direct heat loss from the head. From conditions A to B the IPL at peaks I-V decreased by 0.146 ms/degrees C change in Tes, reflecting a change in brain stem temperature. In condition C the hood was removed and the face was fanned by a cold air-stream (8-15 degrees C, 4-10 m/s) to maximize direct heat loss from the head. Skin temperature at the sweating forehead decreased from 38 to 23 degrees C, whereas Tes in condition C was maintained at the same level as in condition B (38.5 +/- 0.2 degrees C). The IPL at peaks I-V showed no difference between conditions B and C. It is concluded that face fanning in hyperthermic subjects does not dissociate brain stem temperature from Tes.     

Acid-base regulation during thermal panting in the fowl (Gallus domesticus): comparison between breeds

1. The effects of high ambient temperatures on blood acid base status were studied in four breeds of fowl. 2. All breeds efficiently regulated body temperature below ambient temperature at 45 degrees C (Tb = 38.521 + 0.110Ta, at 25-45 degrees C). 3. A slight hypocapnia was partly compensated for by a decreased HCO3 concentration. This resulted in only a slight respiratory alkalosis at extreme temperatures (+0.021 and +0.042 pH units at 42 and 45 degrees C, respectively). 4. Changes in Paco2 were negatively correlated with tidal volume: Paco2 (torr) = 33.10390 - 1.17493 VT(ml); r = -0.925, P much less than 0.001. 5. The present findings are consistent with an hypothesis that modulation of tidal volume during thermal panting might play a major role in acid-base regulation.     

Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction.

The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule.