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There is growing research on carers of people with Parkinson's disease (PD) experiences. However, the impact on carers by PD delusions is not specifically examined. We conducted a qualitative study using semi-structured interviews of spouse carers of PD patients with delusions. Thematic analysis was employed using MAXQDA 2018. Twelve spouse participants (SPs) were interviewed. Four themes emerged: Managing incredulity: trying to make sense of delusion content; Hypervigilance: constant alertness to bizarre and threatening discourse and behavior; Defensive strategizing: anticipating delusions and potential consequences; Concealing and exposing: ambivalence about disclosing the effect of delusions yet wanting support. SPs reported effects on their emotional well-being and marital relationship and challenges to an orderly, predictable life. SPs were reluctant to share their experiences due to delusion content (often infidelity and sexual in nature) and a desire to protect their spouses' image. SPs' awareness of the potential for delusional thought was low prior to their occurrence. Conclusions: education surrounding potential neurobehavioral changes should occur for patients and carers. Clinicians should be aware that the impact of delusions on carers is often greater than disclosed in clinical interviews. Interdisciplinary teams speaking separately to spousal carers may improve disclosure and delivery of appropriate psychological support.

Competing Interests: No conflicts of interest to declare

Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing N ε -thioacetyl-lysine and γ-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N ε -thioacetyl-lysine into proteins expressed in Escherichia coli using amber codon suppression., (© 2020 Wiley Periodicals, Inc.)

Despite major advances in the structure determination of ion channels, the sequence of molecular rearrangements at negative membrane potentials in voltage-gated potassium channels of the Shaker family remains unknown. Four major composite gating states are documented during the gating process: closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI). Although many steps in the gating cycle have been clarified experimentally, the development of steady-state inactivation at negative membrane potentials and mandatory gating transitions for recovery from inactivation have not been elucidated. In this study, we exploit the biophysical properties of Shaker-IR mutants T449A/V474C and T449A/V476C to evaluate the status of the activation and inactivation gates during steady-state inactivation and upon locking the channel open with intracellular Cd2+. We conclude that at negative membrane potentials, the gating scheme of Shaker channels can be refined in two aspects. First, the most likely pathway for the development of steady-state inactivation is C→O→OI⇌CI. Second, the OI→CI transition is a prerequisite for recovery from inactivation. These findings are in accordance with the widely accepted view that tight coupling is present between the activation and C-type inactivation gates in Shaker and underscore the role of steady-state inactivation and recovery from inactivation as determinants of excitability., (© 2020 Szanto et al.)

Background: Squamous cell carcinoma (SCC) is the most common malignancy of the upper limb, and can pose a significant therapeutic challenge. Early treatment needs to be radical whilst maintaining function., Methods: We describe two cases of upper limb squamous cell carcinoma treated with radiotherapy, review the literature on radiotherapy as a primary treatment modality, and discuss the specific difficulties treating SCC in the hand., Results: Radiation therapy was inadequate in tumor clearance in both cases, with recurrence both extensive and distal to the initial focus. Moreover, both patients developed progressive functional loss related to pain, swelling, and stiffness., Conclusion: The evidence basis for radiation therapy as a primary modality is limited, although clearance rates are comparable to surgery. Both radiotherapy and surgery can be utilized to treat SCC. However, we make the case for the hand being especially susceptible to the unwanted side effects of radiotherapy.

Many tropical and subtropical species are sensitive to sudden temperature changes, especially drops in temperature. During winters 2009-2010 and 2010-2011, unusually cold temperatures occurred in many parts of Florida, USA, resulting in increased mortality of Florida manatees, sea turtles, fish, corals, and other species. The Florida manatee, in particular, is highly susceptible to cold stress and death when water temperatures drop below 20°C. We sought to characterize the magnitude and timing of reports of cold-related manatee carcasses in relation to fluctuations in water and air temperatures in central-east and central-west Florida during the six winters from 2008 to 2014. We used a generalized linear model to predict counts of manatee carcasses with a cold-related cause of death reported over 7-day bins in relation to various short-term (two weeks or less) and cumulative (incrementally summed from the start of the winter) heating-degree-day effects (HDD; < 20°C) and a categorical winter variable. Using water temperature data, the top-ranked model in both regions included a short-term temperature effect (14-day HDD sum) that preceded increases in reports of cold-related manatee carcasses by 7 days. Cumulative exposure to cold weather over the winter amplified effects on mortality in the central-east region. Quantifying the relationship between cold events and manatee mortality helps us prepare for rescue and salvage operations when extremely cold weather is forecast. This is especially important because anticipated loss or degradation of warm-water refuges due to human activities and sea level rise could potentially impact the manatee population in the future. These methods could also be applied to other species susceptible to cold-related mortality., Competing Interests: The authors have declared that no competing interests exist.

Serum iron concentration is usually decreased in true iron deficiency and with inflammatory disease in man and domestic animals. Serum total iron binding capacity (TIBC) may be increased in true iron deficiency and decreased with inflammatory disease. This prospective study was designed to measure serum iron analytes in healthy free-ranging and housed Florida manatees (Trichechus manatus latirostris) of both sexes and various ages and to evaluate the effects of diseases common to manatees on these analytes. Blood samples were collected without anticoagulant from 137 healthy free-ranging manatees, 90 healthy housed manatees and 74 free-ranging diseased manatees, and serum was prepared by centrifugation. Serum iron concentration and unsaturated iron binding capacity were measured colourimetrically, and TIBC and percent transferrin saturation with iron were calculated. Serum amyloid A (SAA) was measured to assist in the health assessment of manatees and provide evidence of inflammation in diseased manatees. Based on the serum iron analytes, iron availability was lower in immature manatees compared with adults, and it was lower in housed manatees compared with free-ranging manatees. In contrast to other mammals studied, serum iron concentration was elevated rather than depressed in late pregnancy. Serum iron concentrations and transferrin saturation with iron percentages were significantly lower, and SAA concentrations were significantly higher, in diseased (ill and injured) manatees compared with healthy manatees. Serum iron concentration and transferrin saturation with iron values were negatively correlated with SAA concentrations, and manatees with the highest SAA concentrations had lower serum TIBC values. These findings indicate that inflammation is the major factor responsible for alterations in iron analytes in diseased manatees. Consequently, hypoferraemia may be used as supportive evidence of inflammatory disease in manatees (unless haemorrhage is also present). A decision threshold of ≤13.8 μmol/l was determined for hypoferraemia using receiver operating curve analysis. Based on studies in man and domestic animals, iron therapy is unnecessary for manatees with hypoferraemia associated with inflammation and has the potential for causing tissue damage and increased susceptibility to bacterial infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ages of Florida manatees ( Trichechus manatus latirostris ) can be estimated by counting annual growth layer groups (GLGs) in the periotic dome portion of the tympanoperiotic complex of their earbones. The Florida Fish and Wildlife Conservation Commission manages an archive of more than 8,700 Florida manatee earbones collected from salvaged carcasses from 1989 to 2017. Our goal was to comprehensively evaluate techniques used to estimate age, given this large sample size and changes to processing protocols and earbone readers over time. We developed new standards for estimating ages from earbones, involving two independent readers to obtain measurements of within- and between-reader precision. To quantify accuracy, precision, and error, 111 earbones from manatees with approximately known ages (first known as calves: "KAC") and 69 earbones from manatees with minimum known ages ("MKA," based on photo-identification sighting histories) were processed, and their ages were estimated. There was greater precision within readers (coefficient of variation, CV : 2.4-8.5%) than between readers ( CV : 13.1-13.3%). The median of age estimates fell within the true age range for 63.1% of KAC cases and was at least the sighting duration for 75.0% of MKA cases. Age estimates were generally unbiased, as indicated by an average raw error ± SD of -0.05 ± 3.05 years for the KAC group. The absolute error (i.e., absolute value of raw error) of the KAC data set averaged 1.75 ± 2.50 years. Accuracy decreased and error increased with increasing known age, especially for animals over 15 years old, whose ages were mostly underestimated due to increasing levels of resorption (the process of bone turnover that obscures GLGs). Understanding the degree of uncertainty in age estimates will help us assess the utility of age data in manatee population models. We emphasize the importance of standardizing and routinely reviewing age estimation and processing protocols to ensure that age data remain consistent and reliable.

Introduction: Smoke inhalation injury (II) is an independent risk factor for mortality in burns and its management is inherently complex. We aim to make recommendations for best practice in managing II and its sequelae by reviewing all available current evidence in order to provide an evidence-based approach., Methods: We conducted a systematic search of the Cochrane database and Embase using PRISMA guidelines with no patient population exclusion criteria. Published work was reviewed and evidence levels graded., Results: We identified 521 abstracts for inclusion. Of the 84 articles identified for secondary review, 28 papers were excluded leaving 56 papers suitable for final inclusion., Conclusions: We are able to identify a number of strategies in both diagnosis and treatment of II that have support in the published literature, including the role of bronchoscopy, permissive hypercapnia, nebulized heparin and hydroxycobalamin. Other strategies have not been shown to be harmful, but their efficacy is also not firmly established, such as high frequency oscillatory ventilation and exogenous surfactant. Prophylactic antibiotics and corticosteroids are not recommended. In general, published evidence for II is mostly Level 3 or below, due to a noticeable lack of large-scale human studies. This represents a challenge for evidence-based burns practice as a whole., (Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.)

Watercraft pose a threat to endangered Florida manatees (Trichechus manatus latirostris). Mortality from watercraft collisions has adversely impacted the manatee population's growth rate, therefore reducing this threat is an important management goal. To assess factors that contribute to the risk of watercraft strikes to manatees, we studied the diving behavior of nine manatees carrying GPS tags and time-depth recorders in Tampa Bay, Florida, during winters 2002-2006. We applied a Bayesian formulation of generalized linear mixed models to depth data to model the probability (Pt) that manatees would be no deeper than 1.25 m from the water's surface as a function of behavioral and habitat covariates. Manatees above this threshold were considered to be within striking depth of a watercraft. Seventy-eight percent of depth records (individual range 62-86%) were within striking depth (mean = 1.09 m, max = 16.20 m), illustrating how vulnerable manatees are to strikes. In some circumstances manatees made consecutive dives to the bottom while traveling, even in areas >14 m, possibly to conserve energy. This is the first documentation of potential cost-efficient diving behavior in manatees. Manatees were at higher risk of being within striking depth in shallow water (<0.91 m), over seagrass, at night, and while stationary or moving slowly; they were less likely to be within striking depth when ≤50 m from a charted waterway. In shallow water the probability of a manatee being within striking depth was 0.96 (CI = 0.93-0.98) and decreased as water depth increased. The probability was greater over seagrass (Pt = 0.96, CI = 0.93-0.98) than over other substrates (Pt = 0.73, CI = 0.58-0.84). Quantitative approaches to assessing risk can improve the effectiveness of manatee conservation measures by helping identify areas for protection.

Objective: To report our experience of sinogenic intracranial abscesses in the paediatric population and to guide medical and surgical management., Methods: All children with sinogenic intracranial abscesses presenting to a large university teaching hospital over a five-year period were included in the study. Data on clinical presentation, radiological findings, microbiology, medical and surgical management and follow-up were recorded and analysed., Results: We identified 27 children aged 12.9 ± 3.4 years of which 56% were male. Fourteen (52%) children had extradural abscesses, nine (33%) subdural abscesses and four (15%) parenchymal abscesses. Early sinus drainage procedures were performed on 24 (89%) patients, and the same number required neurosurgical drainage. Streptococcus milleri was isolated in 18 (67%) cases. An initial conservative neurosurgical approach failed in 50% of cases where trialled, and was associated with longer length of stay (p = 0.025). In comparison to extradural abscesses, subdural abscesses were more likely to present with neurological deficits (p < 0.001) and reduced consciousness (p = 0.018), and required multiple neurosurgical procedures (p < 0.001), longer stays (p = 0.017), and had greater morbidity at six months (p = 0.017). A third of children had significant morbidity at six months, which included cognitive and behavioural problems (25%), residual hemiparesis (19%) and expressive dysphasia (7%). There were no mortalities., Conclusion: Sinusitis complicated by intracranial abscess remains a contemporary problem. We demonstrate good outcomes with an early combined rhinological and neurosurgical approach. S. milleri is identified as the causative organism in the majority of cases, and empirical antimicrobial treatments should reflect this., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate., (© 2010 Wiley-Liss, Inc.)

Background: Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals., Objectives: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender., Methods: Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations., Results: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals., Conclusions: Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

The objective was to validate a high-sensitivity chemiluminescent assay of serum progesterone concentrations for pregnancy diagnosis in manatees. Assay analytical sensitivity was 0.1 ng/mL, with mean intra- and inter-assay coefficients of variation of 9.7 and 9.2%, respectively, and accuracy had a mean adjusted R(2) of 0.98. Methods comparison (relative to Siemen's Coat-A-Count RIA) demonstrated r=0.98, Deming regression slope of 0.95, and an intercept of 0.01. Based on ROC analysis, a progesterone concentration >or=0.4 ng/mL was indicative of pregnancy. Assay results were not significantly altered by two freeze-thaw cycles of samples. Characteristic progesterone concentrations during pregnancy were Months 1-4 (1.7-4.7 ng/mL), 5-8 ( approximately 1.0 ng/mL), and 10 and 11 (0.3-0.5 ng/mL), whereas two late-pregnant females with impending abortion had progesterone concentrations of 0.1 ng/mL. Among pregnant females, maximum progesterone concentrations occurred in autumn (3.9+/-1.8 ng/mL), and were greater during all seasons than concentrations in non-pregnant females (0.1-0.2 ng/mL). Progesterone concentrations were also significantly higher in pregnant females than in non-pregnant females and males. This highly sensitive, specific, and diagnostic assay will be valuable for monitoring pregnancy and abortion in manatees.

Florida manatees (Trichechus manatus latirostris) are endangered aquatic mammals living in coastal and riverine waterways of Florida and adjacent states. Serum or plasma biochemical analyses are important tools in evaluating the health of free-ranging and captive manatees. The purpose of this study was to measure diagnostically important analytes in the plasma of healthy manatees and to determine whether there was significant variation with respect to location (free-ranging versus captive), age class (small calves, large calves, subadults, adults), and gender. No significant differences in plasma sodium, potassium, bilirubin, glucose, alanine aminotransferase, or creatine kinase were found among these classes of animals. Compared to free-ranging manatees, captive animals had significantly lower mean concentrations of plasma chloride, phosphate, magnesium, triglycerides, anion gap, and lactate. Captive manatees had significantly higher mean values of total CO2, calcium, urea, creatinine, alkaline phosphatase, gamma-glutamyltransferase, total protein, albumin, and albumin/globulin ratio than did free-ranging animals. Differences in the environments of these two groups, including diet, temperature, salinity, and stress, might account for some of these results. The higher plasma lactate and anion gap concentrations and lower total CO2 concentrations of free-ranging manatees were probably due to greater exertion during capture, but the lack of elevated plasma creatine kinase activity relative to captive animals indicates that there was no serious muscle injury associated with capture. Plasma phosphate decreased and total globulins increased with age. Plasma cholesterol and triglyceride concentrations were highest in small calves. Plasma aspartate aminotransferase was higher in large calves than in adults and subadults, and the albumin/ globulin ratio was higher in subadults than in adults. Plasma total CO2 was higher and chloride was slightly lower in females than in males.

The membrane potential of human PBMC was modulated in culture by isotonic high extracellular K+ (K+e), or the K+ channel blocker, charybdotoxin (ChTX), to determine the effect of depolarization on stimulated proliferation, IL-2 elaboration, and gene expression. In serum-free cultures, ChTX and high [K+]e induced a specific dose-dependent decrease in IL-2 production. ChTX inhibited proliferation of PBMC and purified T cells, decreased IL-2 elaboration 15 h after stimulation by 78.4 +/- 5.3% (n = 5), and decreased IL-2 mRNA steady-state levels by 80% between 8 and 10 h after stimulation. The IC50 for ChTX-inhibition of IL-2 elaboration and IL-2 mRNA were both 1 nM. Similarly, high [K+]e inhibited proliferation with an IC50 of 38.9 +/- 1.1 mM (n = 13), decreased IL-2 elaboration with an IC50 of 21.3 +/- 1.2 mM (n = 6), and decreased IL-2 mRNA steady-state levels with an IC50 of 18 mM. The sensitivities of both IL-2 production and proliferation to depolarization were substantially reduced by calcium, serum, and exogenous rIL-2. From these findings we conclude that membrane potential may contribute to the control of immune responsiveness in vivo.

Transmembrane electrical and pH gradients have been measured across human erythrocytes and peripheral blood lymphocytes using equilibrium distributions of radioactively labelled lipophilic ions, and of weak acids and weak bases, respectively. The distributions of methylamine, trimethylamine, acetic acid and trimethylacetic acid give calculated transmembrane pH gradients (pHe-pHi) for erythrocytes of between 0.14-0.21 for extracellular pH values of 7.28-7.16. The distributions of trimethylacetic acid. DMO and trimethylamine were determined for lymphocytes, establishing upper and lower limits of the calculated pH gradient over the external pH range of 6.7 to 7.7. Tritiated triphenylmethyl phosphonium ion (TPMP) and 14C-thiocyanate ion (SCN) equilibrium distributions were measured in order to calculate transmembrane electrical potentials, using tetraphenylboron as a catalyst to facilitate TPMP equilibrium. Transmembrane potentials of -7 to -10 mV were calculated from SCN and TPMP, respectively for red cells, and -35 to -52 mV respectively, in the case of lymphocytes. Distributions of TPMP and potassium ions were determined in the presence of valinomycin over a wide range of extracellular potassium concentrations for red cells and the calculated Nernst potentials for TPMP compared to the calculated potential using the Goldman equation for chloride and potassium ions. Distributions of TPMP, SCN and potassium ions were also determined for lymphocyte suspensions as a function of extracellular potassium and the calculated Nernst potentials for TPMP and SCN compared to the calculated potassium diffusion potential.

19F NMR was used to measure intracellular [H+] of hepatocytes before and after incubation with glucagon and adrenergic agonists at their concentrations which give maximal stimulation of both glucose and urea production. Intracellular and extracellular pH was determined from the chemical shifts in resonances of alpha-difluoromethylalanine. The alterations in intracellular [H+] with agonist treatment were, in all cases, found to be less than 0.1 pH unit in the pH range 6.7-7.8. It is concluded that changes in concentration of the intracellular [H+] do not play a significant role in the stimulation of urea and glucose production caused by these hormonal effectors.

The pH dependence of the 19F chemical shift has been characterized for a number of fluorine-substituted aniline derivatives. These compounds constitute a new class of 19F nuclear magnetic resonance (NMR) pH indicators, characterized by single 19F resonance lines with sensitivities ranging from 2 to 7 ppm/pH unit near the aniline pKa; total shifts between conjugate acid and base of 5-15 ppm; and pKas ranging from 1 to 7. One compound, N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline, has a pKa of 6.8 and a sensitivity of 5 ppm/pH unit. This compound displays significant broadening of its 19F resonance near the aniline pKa (6.8), due to a decreased rate of exchange between conjugate acid and base species. Our results are consistent with slow dissociation of an intramolecular hydrogen bond in the zwitterionic species that limits the exchange rate between protonated and unprotonated forms for N,N-(methyl-2-carboxyisopropyl)-4-fluoroaniline.

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K+]. At a cell concentration of 30 X 10(6) lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24-27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K+ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K+] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.

Paracoccus denitrificans suspended in media containing 20-300 mM NaCl swelled progressively as the salt concentration was decreased. The increase in intracellular water volume was accompanied by an enhancement of respiration and a stimulation of the rates of net potassium and alpha-aminoisobutyric acid accumulation. It is postulated that influx of water and consequent lowering of intracellular solute concentration trigger transport mechanisms which are destined to restore the original ion and metabolite balance. Since a number of transport reactions operate against the electrochemical gradient of their substrates, energy utilization increases. The increased ATP usage and lowering of [ATP] stimulates the activity of the respiratory chain and increases oxygen uptake and energy production.

The relationship of intracellular pH to extracellular pH has been measured in suspensions of isolated hepatocytes at 25 degrees C. The internal pH was found to be a linear function of external pH and it changed by 0.45 pH unit per 1.0 unit change in external pH. The internal [H+] was equal to the external [H+] at approximately pH 7.1. Gluconeogenesis, urea synthesis, and oxidative phosphorylation showed different dependencies on the intracellular pH. Gluconeogenesis was the most sensitive to changes in [H+] and it declined by 80% when the intracellular pH decreased from 7.1 to 6.9. Urea synthesis was less pH-dependent, decreasing by about 30% for the same change in the intracellular [H+] whereas respiratory rate showed very little dependence on pH at this temperature. Intracellular [ATP]/[ADP] decreased linearly from 8.5 to 1.5 as the intracellular pH increased from 6.8 to 7.6, while intracellular [Pi] was essentially constant at 3.2 nmol/mg of cells, wet weight. Cytochrome c became more reduced with increasing intracellular pH, from less than 10% at pH 6.8 to 35% at pH 7.7. The calculated free energy of hydrolysis of ATP was nearly independent of pH as was the free energy of electron transfer from the intramitochondrial NAD couple (calculated from the [acetoacetate]/[3-OH-butyrate] ratio) to cytochrome c.

19F NMR pH measurements with the fluorinated pH indicators we have described are rapid and sensitive, work with readily available commercial instruments, and extend the applicability of nondestructive NMR measurements to systems for which 31P NMR measurements are presently impractical. The family of 19F pH indicators is useful for independent confirmation of pHi values obtained by 31P NMR, distribution of radioactive weak acids, or other methods. The necessity for using exogenous indicators, which at first appears as a liability, can be turned to advantage also. Our future goal is to direct our measurements unambiguously to compartments (in cells or in tissues) of particular interest, by matching the indicator precursor molecule to the hydrolytic enzyme activities inherent in the target cell or compartment, so that the pH indicator is generated in situ.

Paracoccus denitrificans requires potassium for normal growth and transports this cation by at least two systems, one with low (Km approximately 1 to 2 mM) and another with high (Km approximately 0.1 microM) affinity. Neither of the two systems seems to be dependent on periplasmic components since each retains full activity in cells subjected to an osmotic shock. P. denitrificans accumulates potassium at high velocity (270 nmol of K+/min/mg dry weight of cells) and against a large concentration gradient. The intracellular concentration of K+ in media of high osmolarity (about 320 mosmol) is 0.4 M; this gives a concentration gradient [K+]i/[K+]e of greater than or equal to 2 X 10(4). The uptake of potassium against its concentration gradient requires a source of energy and is eliminated by the addition of uncouplers. The increased rate of energy usage for potassium transport results in an increased rate of ATP synthesis by the respiratory chain and is expressed in enhanced rates of respiration and substrate utilization. The stimulation of respiration is accompanied by increased steady state reduction level of the components of the respiratory chain. The calculations show that two K+ are most likely to be transported per one ATP hydrolyzed.