Manipulation of cellular energy reveals the relationship between ultraweak luminescence and cellular energy during senescence of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis>) fruits

The goal of this study was to investigate the ultraweak luminescence (UWL) of strawberry fruits in relation to mitochondrial functions and energy production during strawberry senescence. Fully ripe strawberry fruits and mitochondria isolated from those fruits were treated with either adenosine triphosphate (ATP) or the respiratory chain uncoupler 2,4-dinitrophenol (DNP). The activities of H⁺-ATPase and Ca²⁺-ATPase, the content of ATP, the free radical O₂^? as well as the UWL intensity were measured. Our results showed that activities of H⁺-ATPase and Ca²⁺-ATPase as well as the ATP content gradually decreased during fruit senescence in all three groups. Compared with the control, DNP treatment exacerbated, while ATP treatment reduced the decrease of H⁺-ATPase and Ca²⁺-ATPase activity, the energy charge and UWL intensity. UWL intensity was positively correlated with mitochondrial function and ATP content. Our results strongly suggest that mitochondria are a major source of UWL of strawberry fruits, and that the cellular energy ATP plays important roles in senescence of strawberry fruits, and in UWL formation. Our study provides convincing evidence of the interrelationship between cellular energy and UWL, which helps researchers to better understand the process of senescence in strawberry fruits.     

Aging: its influence on the intestinal unstirred water layer thickness, surface area, and resistance in the unanesthetized rat

The intestinal absorption of some nutrients changes with aging. As the unstirred water layer (UWL) is an important rate limiting step in the absorption of nutrients in general and of lipid soluble nutrients in particular, we investigated possible changes in the UWL dimensions in the aging rat in vivo. We measured the thickness (d) of the UWL using rapid changes in the luminal sodium concentration to induce changes in the transmucosal potential differences. We assessed the surface area (Sw) and resistance (d/SwD) of the UWL at varying flow rates by using increasingly lipophilic medium chain saturated alcohols as probes. At high UWL resistance, d decreased from 318 to 268 microns between 1 and 29 months of age. As the animals aged, Sw changed from 114 to 106 cm2/100 cm and from 262 to 214 cm2/100 cm at low and high flow rates, respectively, using dodecanol as a probe. The resistance of the UWL (d/SwD) remained relatively stable at all ages studied. These experiments demonstrate that age-related changes in absorption are dependent on the aqueous diffusion coefficient and degree of lipid solubility of the specific nutrients. At low UWL resistance, absorption of compounds with higher diffusion coefficients and greater aqueous solubility is decreased with aging. In contrast, previous studies have demonstrated that the absorption of nutrients with low diffusion coefficients and high lipid solubility increases with aging especially when the resistance of the UWL is high.     

A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer.

A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.     

UWL “Helgoland” - an underwater laboratory for rough sea conditions

Summary 1. The UWL Helgoland system has been built with the rough sea conditions in the Southern North Sea in mind: wave periods up to 8 sec, wave heights up to 8 m, tidal currents up to 1 m/sec.2. The main body can be towed at a velocity of up to 5 knots, and be lowered without the assistance of a floating crane. The system has a high degree of flexibility.3. In order to withstand the oscillating forces caused by sea movements, a horizontal cylinder was chosen as main body. The main body is ballasted to a negative buoyancy of 16 t (more than for other comparable UWL systems). Four swords stabilize the UWL against horizontal forces. The main body acts as a self-righting body.4. Emergency supply of food and energy allows complete surface independence for 10 days.5. Rough surface conditions and poor underwater visibilities do not allow aquanaut activities on the sea bottom to be observed from the surface. A wireless system is used for diver communication on the sea bottom. Three communication systems connect the UWL main body to the supervising station on land: one TV and two radio systems. One radio system is used to transmit alarms to the land base, the other will be used in 1973 as transmission system for oceanological data.6. Aquanauts cannot be transported from the habitat to the deck decompression chamber (DDC) during rough weather. Hence one room in the UWL main body serves as decompression chamber.7. The surface energy station, a floating buoy, is a world first. The station operates automatically; it is controlled by aquanauts in the UWL. Electricity, compressed respiratory gases and fresh water are transported from the buoy to the UWL via an umbilical line. Due to tide and wave motion, the umbilical line is shaped in goose-neck fashion. Other suplies such as food and mail are brought to the UWL by supply containers without assistance from surface divers. Supplementary respiratory gas for the semi-closed diving apparatus is stored on the sea bottom; the same applies for nitrogen.8. The first manned test period in August, 1969, and the subsequent 9-month unmanned period proved that the system is able to withstand rough sea conditions.9. As a research base, UWL Helgoland has met the expectations. It is possible to work and to conduct research in the sea, even under the adverse conditions prevailing in the North Sea. In 1973, UWL Helgoland will operate again on the sea bottom near Helgoland.

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UWL Helgoland - ein Unterwasserlaboratorium für rauhe Seebedingungen

Kurzfassung Die rauhen Seebedingungen in der Deutschen Bucht (südliche Nordsee) (Wellenperiode bis 8 sec, Wellenhöhe bis 8 m, Tidenstrom bis 1 m/sec und Sichtweite um 1 m), haben die technische Konzeption von UWL Helgoland und alle Betriebsabläufe (z. B. Transport, Absenken, Überwachung und Dekompression) maßgeblich beeinflußt. Das UWL unterscheidet sich daher wesentlich von anderen bisher eingesetzten Systemen ähnlicher Größenordnung. Spezielle Probleme und Problemlösungen werden erläutert.

     

杏花开放过程中超弱发光和ATP及活性氧含量的变化

以巴旦水杏 (Prunus armeniaca,Ameniacavulgaris Lam.)为试验材料,研究了杏花开放过程中花朵的超弱发光(ultraweak 1 uminescence,UWL)变化及其与ATP含量、呼吸速率、活性氧产生速率等的关系.结果表明,杏花开放的前半期(盛花期以前),超氧自由基产生速率与丙二醛含量变化平缓,UWL、ATP含量、呼吸速率及蛋白质含量呈上升趋势.盛花期过后,呼吸速率、蛋白质和ATP含量迅速降低,UWL强度缓慢下降,而超氧自由基的产生速率与丙二醛含量迅速升高.分析后认为,杏花的超微弱发光与以ATP含量为标志的能量水平密切相关,同时也受体内活性氧及其它物质代谢的影响;盛花期以前ATP可能是导致UWL升高的主要因素,盛花期过后,活性氧可能对花体的UWL变化起了部分的调节作用.     

水分胁迫下苹果幼苗超弱发光及一些生理特性的变化

以苹果属八棱海棠和平邑田茶当年实生幼苗为试材,研究了在用15％ PEG 6000溶液模拟干旱处理条件下幼苗的超弱发光(ultraweak luminescence,UWL)及ATP含量、呼吸与活性氧(O2^-)产生速率等一些生理特性的变化。结果表明,随水分胁迫时间的延长,幼苗的UWL、ATP含量、蛋白质含量均呈下降趋势,呼吸速率先升后降,而丙二醛(MDA)含量与O2^-的产生速率呈明显上升趋势。品种间各指标比较表明,八棱海棠比平邑田茶具有较强的抗水分胁迫能力,UWL反映幼苗品种抗旱性结果与用其它生理指标测定的结果一致。分析认为,幼苗UWL强度没有随胁迫下氧自由基的增加而增加,说明活性氧不是导致UWL的唯一原因。苹果幼苗UWL的变化是受环境及体内代谢等多种因素的影响,并且与其自身的生长与代谢活性更密切。     

Experimental demonstration of the effect of the unstirred water layer on the kinetic constants of the membrane transport ofd-glucose in rabbit jejunum

Summary The rate of active transport of a probe molecule into the intestinal mucosal cells is determined by the rate of movement of the solute molecule across two barriers, the unstirred water layer and the microvillus membrane of the epithelial cell. Previously a theoretical equation has been derived which describedJ _d, the velocity of unidirectional flux, as a function of the characteristics of the transport carrier in the membrane and of the resistance of the overlying unstirred water layer (UWL). The predictions of these equations have been tested experimentally by studying the effect of the rate of stirring of the bulk phase on thein vitro uptake ofd-glucose by rabbit jejunum. These studies demonstrated that, first, alterations in the UWL have a profound effect on the magnitude of the apparent affinity constant, xK _m ^* , of the active transport process. Second, at bulk phase concentrations in excess of theK _m the passive component of the experimentally determined flux rate becomes of such magnitude as to introduce significant error into the estimate of both the maximal transport rate,J _d ^m , and the trueK _m. Third, as a result of the UWL, the use of double-reciprocal plots to determineJ _d ^m andK _m leads to the overestimation of these constants. Finally, failure to account for the UWL leads to important quantitative errors describing a number of the characteristics of the transport process: these include an underestimation of the Q₁₀ and the effect of sodium ion on the active transport of glucose in the jejunum. The results confirm that the kinetic characteristics of the uptake of an actively transported molecule are a complex function of the resistance of both the UWL and the mucosal cell membrane, and this transport process can be adequately described by a newly-derived equation. It is apparent that there are serious limitations in the interpretation of much of the previously published data dealing with active transport processes in the intestine, since these studies failed to account for the effect of the UWL.     

菊花扦插生根过程中超微弱发光及抗氧化酶活性的动态变化

以菊花品种'神马'和'万盛'为材料,对2品种插穗扦插生根及其过程中超微弱发光(UWL)强度和超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)等抗氧化系统酶活性以及丙二醛(MDA)含量的动态变化进行分析,以探讨UWL产生机理.结果表明:(1)2个菊花品种插穗在扦插第10天后开始生根,15 d后迅速生根,第25天时'神马'的生根数(60.2个)比'万盛'(42.4个)多42.11%.(2)扦插生根过程中,'神马'和'万盛'插穗叶片UWL强度均比对照(母株)显著增加,第15 d时插穗叶片UWL均达到峰值,分别比对照增加125.70%和123.86%,且'神马'比'万盛'始终保持较高水平.(3)2个品种插穗叶片SOD、POD、CAT和APX活性均比对照显著增加,且在扦插第15 d时增加最多,之后缓慢下降,与UWL变化趋势相似;2个品种插穗叶片MDA含量比对照显著增加,但'神马'的MDA含量低于'万盛'.(4)相关性分析表明,菊花扦插生根过程中UWL与生根数相关性不显著,而与SOD、POD、CAT活性呈极显著相关、与APX活性呈显著相关.     

The effect of auxiliary conditions on intestinal unstirred layer diffusion modelled by numerical simulation.

Estimation of intestinal unstirred layer thickness usually involves inducing transmural potential difference changes by altering the content of the solution used to perfuse the small intestine. Osmotically active solutes, such as mannitol, when added to the luminal solution diffuse across the unstirred water layer (UWL) and induce osmotically dependent changes in potential difference. As an alternative procedure, the sodium ion in the luminal fluid can be replaced by another ion. As the sodium ion diffuses out of the UWL, the change in concentration next to the intestinal membrane alters the transmural potential difference. In both cases, UWL thickness is calculated from the time course of the potential difference changes, using a solution to the diffusion equation. The diffusion equation solution which allows the calculation of intestinal unstirred layer thickness was examined by simulation, using the method of numerical solutions. This process readily allows examination of the time course of diffusion under various imposed circumstances. The existing model for diffusion across the unstirred layer is based on auxiliary conditions which are unlikely to be fulfilled in the same intestine. The present simulation additionally incorporated the effects of membrane permeability, fluid absorption and less than instantaneous bulk phase concentration change. Simulation indicated that changes within the physiologically relevant range in the chosen auxiliary conditions (with the real unstirred layer length kept constant) can alter estimates of the apparent half-time. Consequently, changes in parameters unassociated with the unstirred layer would be misconstrued as alterations in unstirred layer thickness.     

Combining simple blood tests to identify primary care patients with unexpected weight loss for cancer investigation: Clinical risk score development,internal validation,and net benefit analysis

BackgroundUnexpected weight loss (UWL) is a presenting feature of cancer in primary care. Existing research proposes simple combinations of clinical features (risk factors, symptoms, signs, and blood test data) that, when present, warrant cancer investigation. More complex combinations may modify cancer risk to sufficiently rule-out the need for investigation. We aimed to identify which clinical features can be used together to stratify patients with UWL based on their risk of cancer.Methods and findingsWe used data from 63,973 adults (age: mean 59 years, standard deviation 21 years; 42% male) to predict cancer in patients with UWL recorded in a large representative United Kingdom primary care electronic health record between January 1, 2000 and December 31, 2012. We derived 3 clinical prediction models using logistic regression and backwards stepwise covariate selection: Sm, symptoms-only model; STm, symptoms and tests model; Tm, tests-only model. Fifty imputations replaced missing data. Estimates of discrimination and calibration were derived using 10-fold internal cross-validation. Simple clinical risk scores are presented for models with the greatest clinical utility in decision curve analysis. The STm and Tm showed improved discrimination (area under the curve ≥ 0.91), calibration, and greater clinical utility than the Sm. The Tm was simplest including age-group, sex, albumin, alkaline phosphatase, liver enzymes, C-reactive protein, haemoglobin, platelets, and total white cell count. A Tm score of 5 balanced ruling-in (sensitivity 84.0%, positive likelihood ratio 5.36) and ruling-out (specificity 84.3%, negative likelihood ratio 0.19) further cancer investigation. A Tm score of 1 prioritised ruling-out (sensitivity 97.5%). At this threshold, 35 people presenting with UWL in primary care would be referred for investigation for each person with cancer referred, and 1,730 people would be spared referral for each person with cancer not referred. Study limitations include using a retrospective routinely collected dataset, a reliance on coding to identify UWL, and missing data for some predictors.ConclusionsOur findings suggest that combinations of simple blood test abnormalities could be used to identify patients with UWL who warrant referral for investigation, while people with combinations of normal results could be exempted from referral.

Dr. Brian D Nicholson and colleagues investigate whether combinations of routine blood tests could be used to stratify patients in UK with unexpected weight loss based on their risk of cancer.     

Lactose hydrate can increase the transcellular permeability of β-naphthol in rat jejunum and ileum

Molecular Biology Reports - The unstirred water layer (UWL) is an integral part of the apical surface of mucosal epithelia and comprises mucins (MUC), for which there are many molecular species....     

Oral Delivery of Lipophilic Drugs: The Tradeoff between Solubility Increase and Permeability Decrease When Using Cyclodextrin-Based Formulations

The purpose of this study was to investigate the impact of oral cyclodextrin-based formulation on both the apparent solubility and intestinal permeability of lipophilic drugs. The apparent solubility of the lipophilic drug dexamethasone was measured in the presence of various HPβCD levels. The drug’s permeability was measured in the absence vs. presence of HPβCD in the rat intestinal perfusion model, and across Caco-2 cell monolayers. The role of the unstirred water layer (UWL) in dexamethasone’s absorption was studied, and a simplified mass-transport analysis was developed to describe the solubility-permeability interplay. The PAMPA permeability of dexamethasone was measured in the presence of various HPβCD levels, and the correlation with the theoretical predictions was evaluated. While the solubility of dexamethasone was greatly enhanced by the presence of HPβCD (K_1∶1 = 2311 M⁻¹), all experimental models showed that the drug’s permeability was significantly reduced following the cyclodextrin complexation. The UWL was found to have no impact on the absorption of dexamethasone. A mass transport analysis was employed to describe the solubility-permeability interplay. The model enabled excellent quantitative prediction of dexamethasone’s permeability as a function of the HPβCD level. This work demonstrates that when using cyclodextrins in solubility-enabling formulations, a tradeoff exists between solubility increase and permeability decrease that must not be overlooked. This tradeoff was found to be independent of the unstirred water layer. The transport model presented here can aid in striking the appropriate solubility-permeability balance in order to achieve optimal overall absorption.     

甜菜碱对干旱胁迫下湖北海棠超微弱发光及抗氧化能力的影响

以盆栽4年生的苹果砧木湖北海棠（Malus hupehensis）为试材,叶面喷施100 mmol·L^-1的甜菜碱,研究外源甜菜碱对干旱胁迫下湖北海棠叶片超微弱发光（UWL）、丙二醛(MDA)、超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢（H₂O₂）、脯氨酸（Pro）及甜菜碱（GB）含量的影响.结果表明,轻度干旱胁迫下,叶片UWL强度提高,但随着干旱胁迫程度的加重而下降,至严重干旱胁迫时UWL明显低于正常供水处理;H₂O₂、MDA含量随干旱胁迫加重而升高;SOD和POD活性随胁迫加重而升高,但至重度干旱时下降.与单独干旱处理相比,叶片喷施甜菜碱显著提高了轻度和中度干旱胁迫叶片的UWL（分别提高35.27%和43.95%）、SOD和POD活性,降低了H₂O₂和MDA含量;促进了Pro和GB的积累.表明甜菜碱及通过甜菜碱诱导的脯氨酸积累,能够提高干旱胁迫下叶片的抗氧化能力,减轻过氧化损伤,对叶片细胞起到保护作用.     

A molecular mechanism of the energetic coupling of a sequence of electron transfer reactions to endergonic reactions.

A molecular mechanism of the energetic coupling of a sequence of electron transfer reactions to endergonic reactions is proposed and discussed from a physical point of view. The scheme represents a synthesis of concepts of electron transfer by tunneling and the conformational and chemiosmotic aspects of energy coupling processes. Its relation to existing experimental information and theoretical models is discussed, and further experimental tests are suggested.     

The Unstirrable Water Layer Is Unstirrable because It Does Not Exist

A number of membrane‐permeation models require the incorporation of an unstirred or unstirrable water layer (UWL). An example occurs in PAMPA models when the effective permeation rate of lipophilic acids and bases, P_e, falls behind the expected permeation rate, P_m, at pH values providing a high concentration of unionized species in the donor phase. In such cases, the compound has an apparent pK_a of a weaker acid or base. The explanation is that an UWL adjacent to the membrane provides a rate‐limiting diffusion barrier for such compounds. The thickness of the UWL is correlated with the difference between the aqueous pK_a and the apparent pK_a (pK ). Here, we provide an explanation for the pK term that requires no UWL. It comes from the fact that, in the process of passing into a membrane, an ionizable compound undergoes a change in pK_a. At some point along its path into the membrane, the compound attains a maximum free energy, at which point it is as likely to continue into the membrane, as it is to return to the donor phase. This is the transition state for absorption. The pK is the pK_a of the compound at the transition state. This is a testable hypothesis (see text). The relevance of absorption to permeation depends on the rate‐limiting step of permeation.     

Enzymatic reactions in biotechnology (48th Bach lecture)]

The paper is the 48th Bach Lecture presented under the same title. It covers the biochemical mechanisms of the biogenesis of microbial biosynthetic products, role of acetyl-CoA, function of the succinate-glycine cycle, reactions of the hexose-monophosphate pathway of carbon metabolism. The reversible action of hydrolases in enzymatic catalysis and degradation of xenobiotics are discussed. The data on redox reactions are pooled. Such modern biotechnological processes as epoxidation, synthesis of acrylamide and some monomers involved in chemical syntheses of polymers, synthesis of oligosaccharide and fluorine-containing amino acids are considered. Promising commercial applications of biocatalysis are discussed.     

Mass spectrometry-based proteomics strategies for protease cleavage site identification

Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.     

The measurement of free radical reactions in humans. Some thoughts for future experimentation

The question as to whether free radical reactions are a major cause of tissue injury in human disease, or merely an accompaniment to such injury, is very difficult to answer because of lack of adequate experimental techniques. New techniques that are becoming available are discussed, with specific reference to their use in humans.     

Embryogenesis of the pea (Pisum sativum)

Summary The most striking internal feature of the suspensor cells inPisum is the abundant occurrence of a plastid containing spherical bodies consisting of intertwined bundles of tubules. These tubular complexes are not typical prolamellar bodies and they are not converted into grana. Cytochemical reactions indicate that they are proteinaneous. The participation of this plastid in the possible nutritional function of the suspensor is discussed but it is pointed out that critical experimental evidence is needed before the role of the suspensor and its contents in embryogenesis can be understood.Supported by a grant from the Australian Research Grants Committee.     

On the role of subunit III in proton translocation in cytochromec oxidase

Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,M _r 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.