Impaired Membrane Function Research Articles

The clinical relevance of the new criteria for cirrhotic cardiomyopathy and future directions.

Cardiac dysfunction in patients with liver disease has been recognized since the 1950s. Initially attributed to shared risk factors, it is now evident that cardiac dysfunction in patients with cirrhosis can occur in the absence of known cardiac, i.e., coronary artery and valvular heart disease, and across all etiologies for cirrhosis. In 1996 this myocardial dysfunction was termed cirrhotic cardiomyopathy (CCM). The pathophysiologic mechanisms underlying CCM include impaired beta-adrenergic membrane function and circulating proinflammatory and cardiotoxic substances. In 2005, the first diagnostic criteria for CCM were introduced enabling greater sensitivity and accuracy of diagnosis. Since 2005, advancements in echocardiographic methods and better understanding of the pathophysiology of cardiac dysfunction in patients with cirrhosis necessitated revision of CCM criteria. Changes in CCM criteria included removal of blunted contractile or heart rate response on stress testing and addition of global longitudinal systolic strain. Refinement of criteria for diastolic dysfunction were also incorporated into the new diagnostic approach. Since 2020, the prevalence of the disorder and clinical considerations for pre-transplant, peritransplant, and post-transplant patients with cirrhosis have been further evaluated and CCM was found to adversely impact clinical outcomes during all three phases of care. Future research considerations should address the timing of universal echocardiographic screening for patients with cirrhosis, utility of biomarkers in aiding CCM diagnosis, impact of CCM on right heart function, and role of anti-remodeling agents post-liver transplant (post-LT).

Abstract 238: Serum Opacity Factor Normalizes Dysfunctional High Density Lipoproteins In Srbi -/- Female Mice And Rescues Fertility

Although plasma HDL-C levels negatively correlate with atherosclerotic cardiovascular disease (ACVD), attempts to reduce ACVD risk by raising plasma HDL have disappointed. Thus, hypotheses about salutary HDL effects have shifted from higher-is-better to function-is-more-important. The SRBI-/- mouse is an extreme model of HDL dsyfunctionality; compared to WT mice, SRB1-/- mice have higher plasma HDL levels and an HDL surface that is free cholesterol (FC)-rich (60 vs. 15 mol%). This would be expected to increase HDL-FC bioavailability to cytotoxic levels. HDL dysfunctionality among SRB1-/- mice is associated with multiple metabolic abnormalities—impaired cell membrane structure and function and atherosusceptibility, despite having high plasma HDL-C levels, and infertility among female SRB1-/- mice. Dietary probucol, an HDL-lowering drug, partially reverses infertility in SR-B1-/- mice. The mechanisms underlying ovaritoxicity is not known and interventions that fully reverse this state has not been identified. We hypothesized that a bacterial protein, serum opacity factor (SOF), which acts on HDL and lowers cholesterol in WT mice by 43% in 3 hours, would normalize HDL functionality and rescue infertility in SRBI-/- female mice. SOF DNA was cloned into a TBG-AAV8 plasmid and virus produced by UPENN Vector Lab. The AAV-SOF was delivered by IP injection leading to the endogenous expression of SOF. Probucol-fed and AAV-GFP mice were used as controls. SOF expression drastically decreased plasma cholesterol and normalized the size of HDL in SRBI-/- females comparable to that of WT mice. Fertility was restored in female mice injected with SOF while GFP controls remained infertile. Compared to Probucol-fed mice, AAV-SOF females had a higher percentage of fertile females and shorter time to first litter drop. We concluded that SOF performed equally or better than probucol in rescuing fertility and normalizing HDL.

The relationship between glycosylated hemoglobin level and red blood cell storage lesion in blood donors.

Glycosylated hemoglobin (HbA1c), not routinely screened in blood donors, is associated with morphological, biochemical, and functional abnormalities of red blood cells (RBCs) and with enhanced oxidative stress. We aimed to explore HbA1c levels in blood donors and their effect on RBC storage. An analytical cross-sectional study was conducted on 875 eligible blood donors aged 18-60 years from May 1, 2021, to August 30, 2021. Two selected groups of donors (HbA1c <6.5%, n=10; HbA1c ≥ 6.5%, n=10) exhibiting as similar as possible baseline values (such as age, sex, and living habits, etc.) were recruited for blood donation in leukoreduced CPDA-1units. RBC morphological, biochemical, structural, and oxidative stress states were measured during 5-35 days of storage. Elevated HbA1c prevalence was 37%, including 31.7% (277/875) in the prediabetes range (HbA1c 5.7%-6.4%) and 5.4% (47/875) in the diabetes range (HbA1c ≥ 6.5%). Age, body mass index (BMI), smoking, and alcohol consumption were the main factors influencing the HbA1c levels. During storage, high-HbA1c group had abnormal RBC morphology, impaired membrane function, and ion imbalance (higher mean corpuscular volume, distribution width, hemolysis rate, potassium ion efflux, and phosphatidylserine exposure) as compared with low HbA1c group. Additionally, RBC oxidative stress was significantly increased in donors with high HbA1c levels during 21-35 days. Blood donors proportion with abnormal HbA1c levels was relatively high, and donor HbA1c levels may be associated with stored RBCs capacity. Our study provides new insights into the different effects of donor HbA1c levels on RBC storage lesions.

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons.

SummaryAdvances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

The Role of Metabolic Lipases in the Pathogenesis and Management of Liver Disease.

Intracellular lipolysis is an enzymatic pathway responsible for the catabolism of triglycerides (TGs) that is complemented by lipophagy as the autophagic breakdown of lipid droplets. The hydrolytic cleavage of TGs generates free fatty acids (FFAs), which can serve as energy substrates, precursors for lipid synthesis, and mediators in cell signaling. Despite the fundamental and physiological importance of FFAs, an oversupply can trigger lipotoxicity with impaired membrane function, endoplasmic reticulum stress, mitochondrial dysfunction, cell death, and inflammation. Conversely, impaired release of FFAs and other lipid mediators can also disrupt key cellular signaling functions that regulate metabolism and inflammatory processes. This review will focus on specific functions of intracellular lipases in lipid partitioning, covering basic and translational findings in the context of liver disease. In addition, the clinical relevance of genetic mutations in human disease and potential therapeutic opportunities will be discussed.

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of &lt;i&gt;SNCA-A53T&lt;/i&gt; Human Dopamine Neurons

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional read out of cell activity and membrane structure. Using liquid extraction surface analysis (LESA) coupled with high-resolution mass spectrometry we have developed a new high-throughput method for untargeted single cell lipid profiling. This technological advance revealed new insights into the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein ( SNCA ) mutant neurons have impaired membrane function. These results demonstrate that this novel single cell lipid profiling platform can provide robust data that will open new frontiers in biomedical research.

Abstract 394: Bacterial Serum Opacity Factor Rescues HDL Functionality in SR-B1 -/- Mice

Although plasma HDL-C levels negatively correlate with atherosclerotic cardiovascular disease (ACVD), attempts to reduce ACVD risk by raising plasma HDL have disappointed. Thus, hypotheses about salutary HDL effects have shifted from higher-is-better to function-is-more-important. The SR-B1 -/- mouse is an extreme model of HDL dsyfunctionality; compared to WT mice, SR-B1 -/- mice have higher plasma HDL levels and an HDL surface that is free cholesterol (FC)-rich (60 vs. 15 mol%). This would be expected to increase HDL-FC bioavailability to cytotoxic levels. HDL dysfunctionality among SR-B1 -/- mice is associated with multiple metabolic abnormalities—impaired cell membrane structure and function and atherosusceptibility, despite having high plasma HDL-C levels; moreover, female SR-B1 -/- mice are infertile. Liver-specific SR-B1 expression in SR-B1 -/- mice normalizes HDL size and FC content. Thus, the SR-B1 -/- mouse phenotype is due to lack of hepatic clearance of lipids from dysfunctional HDL. Serum opacity factor (SOF) is a bacterial protein that catalyzes the quantitative disproportionation of HDL into a cholesteryl ester-rich micro emulsion (CERM), neo HDL, and lipid-free apo AI. The CERM contains apo E and all HDL-CE. Injection of SOF (4 μg) into WT mice lowers plasma cholesterol by diverting the CERM to hepatic LDLR. Thus, we began testing whether adeno-viral delivery of SOF (AAV SOF ) to SR-B1 -/- mice rescues HDL functionality. A plasmid encoding the SOF gene was synthesized; SOF DNA was isolated by restriction enzyme digestion and cloned into a pAAV-TBG-mcs plasmid that was submitted to the PENN Vector Lab for virus production and isolation. AAV GFP plasmid (UPENN) was used as control. Good SOF production and secretion was confirmed by transfection of Huh7 hepatocytes. AAV SOF injection into SR-B1 -/- mice induced constitutive plasma SOF activity and reduced HDL-C levels to nil. Superposition of high plasma HDL levels and a high mol% FC in SR-B1 -/- is expected to increase HDL-FC bioavailablity that contributes to whole-body FC-toxicity and the observed metabolic abnormalities. Future tests will determine whether ablation of dysfunctional HDL in SR-B1 -/- mice rescues their pathological phenotype, especially atherosclerosis.

Frailty Syndrome: Visceral Adipose Tissue and Frailty in Patients with Symptomatic Severe Aortic Stenosis.

In patients with severe aortic stenosis (AS), frailty is a clinically relevant measure of increased vulnerability that should be included in the preoperative risk assessment. Bioelectrical impedance analysis (BIA) derived phase angle (PA) reflects cell membrane integrity and function. Few studies are available on the relative contribution of adiposity distribution on frailty, and about the influences of frailty and visceral obesity in PA value. Therefore, we aimed to evaluate associations among frailty, visceral fat depots and PA in patients with symptomatic severe AS. In a cohort of patients with symptomatic severe AS and preserved ejection fraction, we examined the associations between frailty, visceral fat depots and bioelectrical impedance analysis (BIA) derived phase angle (PA); and between visceral fat and PA. Frailty was defined according the Fried et al. scale criteria and the body fat distribution was determined by multidetector computed tomography and by BIA. Of the fifty-five included patients, 26 were frail (47%). Adjusting for age and gender, frailty was associated with indexed epicardial adipose tissue volume (EATVi) (the odds of frailty increased 4.1-fold per additional 100 cm3/m2 of EAT [95% confidence interval (CI) of 1.03 to 16.40, p=0.04] and with PA (OR of 0.50, 95% CI, 0.26 to 0.97, p=0.04), but not with body mass index (BMI), waist circumference (WC), indexed total, visceral and subcutaneous abdominal fat areas (TAFAi, VAFAi and SAFAi) nor with indexed mediastinal adipose tissue volume (MATVi). In an age and gender adjusted linear model, PA was inversely correlated with EATVi (β=-0.008, 95% CI, -0.016 to -0.001, p=0.03), but not with BMI, WC, nor with MATVi, VAFAi, SAFAi and TAFAi. In patients with symptomatic severe AS, EATVi is associated with frailty, independently of age and gender, but not with MAFVi or VAFAi. Moreover, frailty and EATVi are associated with impaired cell membrane integrity and function assessed by PA.

PROTEIN PROFILE AND MDA SPERMATOZOA LEVELS OF HYPERGLYCEMIC MICE FED BY BEAN SPROUTS EXTRACT

Hyperglycemic conditions lead increased lipid peroxide and MDA which would damage structure and fluidity membrane so that impaired membrane function. Vitamin E on sprouts is an antioxidant that is capable of maintaining the integrity of cell membranes. Twenty eight mice which were grouped into 4 groups: control , P1, P2 and P3. Hyperglycemic treatment is by injecting of monohidrate alloxan dose of 150 mg/kgbw in group (P1, P2 and P3). Preparation and injecting of bean sprouts extract dose of 50 mg/20gbw to P2 and 100 mg/20gbw to P3 on 30 days.Taking of sperm to sperm protein isolation, and then analysis of protein molecular weight, testing MDA TBARS method. Based on the research results of the analysis protein profiles found in the control and P3 to be 8 protein bands is 72, 60, 49, 42, 35, 30, 28, and 23 kDa. While the P1 and P2 protein band 42 kDa are not found. Results of the analysis showed MDA significant different, control group is 2.43 nmol/ml, P1 7.89 nmol/ml, P2 5.19 nmol/ml and P3 3.29 nmol/ml. Concluded that the bean sprouts extract dois 100 mg/20ggw shows a significance influence on recovery protein profiles and MDA levels spermatozoa of hyperglycemic mice. Keywords: Protein profile, MDA, Hyperglycemic, Bean sprouts

Abstract 5422: Inhibition of oxidosqualene cyclase blocks proliferation and survival of prostate cancer cells

Abstract Despite concerted efforts to develop new strategies for preventing and treating prostate cancer, almost 240,000 new cases of the disease are reported each year in the United States, and more than 28,000 men die of the disease annually. Most deaths occur due to complications following metastasis from the primary prostate tumor to other tissues and organs, a process dependent upon increased angiogenesis. Human prostate cancer cells are often dependent on endogenous or exogenous androgens and estrogens, which stimulate cell proliferation and metastasis and inhibit cell death. Standard treatment for primary prostate cancer often includes systemic exposure to chemotherapeutic drugs or antihormones (chemical castration); however, drug-resistant cancer cells often emerge during treatment, limiting the continued use of systemic chemotherapy. Therefore, novel, less toxic and more effective treatments for prostate cancer are urgently needed. The cholesterol biosynthetic pathway is an attractive therapeutic target for endocrine dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as being the metabolic precursor of endogenous steroid hormones. Due to certain undesirable side-effects associated with statins, such as reduced levels of isoprenoids, defective post-translational modification of membrane proteins, and impaired membrane structure and function, alternative approaches are under consideration. 2, 3-oxidosqualene cyclase (OSC) is a key enzyme in the cholesterol biosynthetic pathway downstream of HMGCoA-reductase, the target of statins. While studying anti-cancer properties of Ro 48-8071 [4′-[6-(Allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate]) (RO), an OSC inhibitor, we found that as well as reducing tumor cell viability, RO simultaneously induced the anti-proliferative protein estrogen receptor β (ERβ). Moreover, RO also elevated ERβ in hormone-insensitive cells that are difficult to treat clinically. Initial studies suggest that combining RO with an ERβ agonist increases the antiproliferative effects of RO in prostate cancer cells. RO also induced apoptosis in prostate cancer cells, determined by FACS analysis. In addition, RO effectively reduced the growth of prostate cancer xenografts (PC3 cells) in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells (RWPE-1). Our study is the first to demonstrate that disrupting cholesterol biosynthesis through OSC inhibition is a novel and potent means by which to destroy human prostate cancer cells. Further studies are necessary to determine the mechanism of RO-mediated loss of prostate cancer cell viability. Support: Department of Defense Prostate Cancer Pgm grant W81XWH-14-1-0246, NIH grant R21 GM088517, and by a COR grant from the University of Missouri, Columbia. Note: This abstract was not presented at the meeting. Citation Format: Yayun Liang, Benford Mafuvadze, Xiaoqin Zou, Cynthia Besch-Williford, Salman M. Hyder. Inhibition of oxidosqualene cyclase blocks proliferation and survival of prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2015-5422

PAR-1 Mediates the Thrombin-induced Mesothelial Cell Overproduction of VEGF and PAI-1

Thrombin mediates an excess in the production of neoangiogenetic (VEGF) and profibrotic (PAI-1) factors in human peritoneal mesothelial cells (HMC). The mechanisms leading to this overproduction have not been elucidated so far; in the context of peritoneal dialysis it can result in impaired peritoneal membrane function. This study was performed to evaluate the presence of the thrombin receptor protease-activated receptor-1 (PAR-1) in HMC and to characterize its function in the thrombin-dependent effects mentioned above. All experiments were performed using cultured primary HMC. Real-Time PCR and Western Blot were used to evaluate PAR-1; ELISA and Real-Time PCR were employed to examine PAR-1 effects on target mediators. We found that cultivated primary HMC show a basal presence of PAR-1. Stimulation with IL-1β induced an increase of the mesothelial PAR-1 expression whereas stimulation with glycosilated human serum albumin or the ligand thrombin itself resulted in decreased PAR-1 expression. Stimulation with the specific PAR-1 ligand TFLLR-NH(2) caused increased VEGF and PAI-1 levels similar to stimulation with thrombin, whereas preincubation with PAR-1 blocking antibodies ATAP2 and WEDE15 attenuated the thrombin-induced overproduction of VEGF and PAI-1. HMC express PAR-1 and the receptor is involved in thrombin effects on these cells. These findings may be a basis for pharmacological prevention of neoangiogenesis and adhesions in the context of peritoneal dialysis and peritonitis.

Biocompatible peritoneal dialysis solutions: do they indeed affect the outcome?

Numerous studies have confirmed beneficial effects of polyglucose dialysis solution (PG-DS), an amino acid dialysis solution (AA-DS), and bicarbonate (bic) or bicarbonate/lactate (bic/lac) buffered solutions on selected components of peritoneal bioavailability or clinical parameters of peritoneal dialysis (PD) patients. Few adverse effects have also been described. A question arises whether these solutions affect the PD outcome. A better controlled fluid status of PD patients associated with the use of PG-DS has been shown in a double-blind randomized controlled trial. Continuous cyclic PD patients treated with the PG-DS did not show changes in solute kinetics and peritoneal membrane markers remained unaltered. The use of PG-DS in anuric automated PD patients was associated with less impaired membrane function. A prospective, randomized, controlled study on the AA-DS in malnourished continuous ambulatory PD patients did not show significant effects of the AA-DS on patient survival, hospitalization rate, C-reactive protein levels, total urea Kt/V, ultrafiltration and drop-out rates, but nutritional status improved or was stable. Improved acid-base balance with bic-buffered solutions was shown in patients treated with automated PD or continuous PD. A registry-based study suggests better survival of patients treated with a neutral pH, low glucose degradation product solution, but there were no differences in dialysis technique survival, peritonitis-free survival, or peritonitis rates. However, reduced peritonitis rate was also reported with the use of bic/lac solutions. Current concepts of PD solutions involve efforts to use fluids which combine the advantages of PG-DS, AA-DS and bic-buffered solutions. Large-scale studies should be continued to improve biocompatibility of peritoneal solutions and to establish their effect on the clinical outcome.

Hepatoprotective Activity of Aqueous Ethanolic Extract of Chamomile capitula in Paracetamol Intoxicated Albino Rats

Hepatoprotective activity of aqueous ethanolic extract of Chamomile recutita capitula against paracetamol induced hepatic damage in albino rats was observed. In the present study the effect of aqueous ethanolic extract of Chamomile recutita capitula on blood and liver glutathione, Na+ K+- ATPase activity, serum marker enzymes, serum bilirubin, glycogen and thiobarbutiric acid reactive substances against paracetamol induced damage in rats have been studied to find out the possible mechanism of hepatoprotection. It was observed that extract of chamomile has reversal effects on the levels of above-mentioned parameters in paracetamol hepatotoxicity. The extract of capitula of chamomile functions as a hepatoprotective agent and this hepatoprotective activity of chamomile may be due normalization of impaired membrane function activity.

Identification of Intra- and Intermolecular Disulfide Bridges in the Multidrug Resistance Transporter ABCG2

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

Lung injury in uncomplicated and severe falciparum malaria: a longitudinal study in papua, Indonesia.

In patients with severe malaria, acute respiratory distress syndrome usually develops after the start of drug treatment and is a major cause of death. Its pathogenesis is not well understood. Respiratory symptom, spirometry, and gas transfer analyses were performed longitudinally in adults in Papua, Indonesia, with uncomplicated (n=50) and severe (n=30) falciparum malaria; normal values were derived from 109 control subjects. Gas transfer was partitioned into its alveolar-capillary membrane (D(M)) and pulmonary vascular (Vc) components, to characterize the site of impaired gas transfer. Cough was frequent in both patients with uncomplicated malaria (50%) and those with severe malaria (30%) and resolved by day 14. Reduced midexpiratory flow indicated obstruction of the small airways. Gas transfer was significantly impaired in patients with severe malaria. D(M) was reduced in patients with severe malaria but not in those with uncomplicated malaria and only returned to normal levels after 2 weeks. In patients with uncomplicated malaria, Vc was reduced at presentation but improved thereafter. In patients with severe malaria, Vc decreased with treatment and was lowest at day 7. Our results suggest that pulmonary vascular occlusion occurs in both patients with uncomplicated malaria and those with severe malaria, likely from sequestration of both red blood cells (RBCs) and white blood cells. There was also impaired alveolar-capillary membrane function in patients with severe malaria but not in those with uncomplicated malaria. Persistent impairment long after clearance of parasitized RBCs suggests prolonged posttreatment inflammatory alveolar-capillary injury.

Partial Reversal of Methylprednisolone-Induced Opacity in Isolated Rat Lenses

We examined whether opacity is reversed in isolated rat lenses with opacities of different grades induced by methylprednisolone treatment by withdrawal of the steroid treatment. Transparent lenses isolated from male Wistar rats aged 6 weeks were incubated in TC-199 medium containing methylprednisolone hemisuccinate (1.5 mg/ml) for 24 or 48 h to induce opacities of two different grades (one was a slight cortical opacity around the equator and the other a heavy cortical opacity spreading from the equator to the cortex). In the slightly opaque lenses, water content was preserved at the level of transparent lenses untreated with steroid, although a 3.2-fold increase in Na⁺/K⁺ ratio and a 20% decrease in Na⁺,K⁺-ATPase activity were observed. In contrast, in the heavily opaque lenses, a significant increase in water content was accompanied by a 31.0-fold increase in Na⁺/K⁺ ratio and a 27% decrease in Na⁺,K⁺-ATPase activity. When the slightly and heavily opaque lenses were further incubated in steroid-free medium for 24 or 48 h, a partial opacity reversal was found in the slightly opaque lenses, but not in the heavily opaque lenses. In the slightly opaque lenses incubated in steroid-free medium for 48 h, increased Na⁺/K⁺ ratio and decreased Na⁺,K⁺-ATPase activity were restored to nearly the levels of untreated transparent lenses. Incubation of the heavily opaque lenses in steroid-free medium for 48 h caused further slight increases in water content and Na⁺/K⁺ ratio and further slight decrease in Na⁺,K⁺-ATPase activity. These results indicate that opacity is partially reversed in rat lenses with a slight opacity induced by methylprednisolone hemisuccinate when the steroid treatment is withdrawn, and suggest that recovery of impaired lens membrane function could be involved in this in vitro partial opacity reversal.

Hypoxia-induced inhibition of the response to nitroxidergic nerve stimulation in canine cerebral arteries.

In isolated canine middle cerebral arteries contracted with prostaglandin F2 alpha, transmural electrical stimulation (TES), nicotine, and substance P produced relaxations. Transmural electrical stimulation- and nicotine-induced endothelium-independent responses are mediated by nitric oxide (NO) liberated from perivascular nerve, whereas substance P-induced relaxations are mediated by endothelium-derived NO. These responses were attenuated by replacement of 95% O2 and 5% CO2 gas (about 550 mm Hg of partial O2 pressure) with 95% N2 and 5% CO2 gas (about 40 mm Hg); inhibition of the response to TES was stabilized 30 minutes later. Reoxygenation partially reversed the response. Relaxations caused by exogenous NO were not influenced by hypoxia. Inhibition by hypoxia of the response to TES was not affected by superoxide dismutase. However, the inhibitory effect was prevented by amiloride and dimethyl-amiloride, Na(+)-H+ exchange inhibitors, or acidosis caused by the addition of HCl. The inhibition by hypoxia was reversed by amiloride. It is concluded that depression by hypoxia of the response mediated by endogenous NO is associated with impaired membrane function caused by restoration of normal intracellular pH by Na(+)-H+ exchanger.

Prevention of aluminium toxicity with supplemental boron. I. Maintenance of root elongation and cellular structure

ABSTRACTAluminium toxicity is an important factor limiting plant growth mi acid soils. Symptoms of B deficiency and Al toxicity are very similar and generally associated with impaired membrane Function and root growth. Thus the objective of this study was to determine whether supplemental B prevents Al inhibition of root growth and development. Squash (Cucurbita pepo L. cv. Sunbar) was grown in hydroponic nutrient media with 44 mmol m−3 free Al and B concentrations extending from 5 to 100 mmol m−3. Our results establish that B protects against Al inhibition of root growth. Protection was apparent at all levels of organization examined: primary root and lateral root lengths; primary root cell elongation, cell production rate, tissue organization and cell structure; primary root morphology and maturation. Protection against Al inhibition was also apparent for shoot growth. These studies were undertaken in solution culture to limit the variables examined; however, the underlying motivation for this study is the problem of worldwide Al toxicity in soils. Therefore, the effect of adding additional B to a high‐Al soil was also investigated and is the subject of the companion paper (Le Noble. Blevins &amp; Miles 1996, Plant, Cell and Environment 19, 1143–1148).

Intrahepatic cholestasis: a review of biochemical-pathological mechanisms.

Intrahepatic cholestasis involves impaired excretion of bile via the hepatobiliary system as a consequence of one or more lesions within the liver. In humans, intrahepatic cholestasis most often results as a side-effect of drug therapy and the clinical manifestation of this condition, jaundice, has been estimated to account for hospitalization in 2 to 5% of the cases for the general population and approaches as much as 20% in the elderly. With the aging of the population and the common occurrence of poly-drug therapy in geriatric patients, it is to be expected that jaundice due to drug-induced intrahepatic cholestasis will become even more prevalent, and accordingly the need to understand the basic mechanisms of this disease condition will become more urgent. The list of culprit agents implicated in the induction of intrahepatic cholestasis in humans is continually expanding. These include various steroid hormones, bile acids, drugs and other chemicals. Experimentally, a wide spectrum of agents has been shown to precipitate intrahepatic cholestasis. Over the years, a number of hypotheses on the biochemical and pathological mechanisms of intrahepatic cholestasis has emerged, including the following: impaired sinusoidal membrane function; interference with the distribution and binding of cytoplasmic endogenous carrier proteins; interference with mitochondrial energy supply; defects in the canalicular membrane including altered Na+/K+ -ATP-ase activity; impairment of microfilament and microtubule functions; interference with bile secretion involving bile acid dependent and independent fractions, and altered bile acid metabolism due to "hypoactive hypertrophic smooth endoplasmic reticulum". In partial agreement with the latter hypothesis, our studies indicated that impairment of the endoplasmic reticulum might represent one of the early stages in the development of intrahepatic cholestasis. Various experimental conditions that induce intrahepatic cholestasis to different degrees resulted in an interference of the synthesis of microsomal phospholipids and altered microsomal function. The conditions included the administration of various hepatotoxic compounds or steroids, pregnancy, delayed development of the endoplasmic reticulum in neonates, and dietary methyl donor or choline deficiency. This review reports the biochemical-pathological mechanisms postulated to be involved in the genesis of intrahepatic cholestasis with specific reference to experimental models of drug-induced intrahepatic cholestasis. The important practical implications of cholestasis are also briefly surveyed.

Immediate decrease in antigen-presenting function and delayed enhancement of interleukin-1 production in human epidermal cells after in vivo UVB irradiation.

Human skin was irradiated in vivo with a single UVB dose (100 mJ/cm2 or 200 mJ/cm2) to examine simultaneously the antigen-presenting function and interleukin-1 (IL-1) production capacity of irradiated epidermal cells (EC). Suction blisters were produced on irradiated areas on days 0, 3 and 7 after UVB. Irradiated EC were harvested and co-cultured with autologous T lymphocytes in the presence of antigens (PPD, HSV) or mitogen (ConA). Culture supernatants were tested for IL-1 activity using the thymocyte comitogenity assay. We found that a single 200 mJ/cm2 dose of UVB caused an immediate suppression of the antigen-presenting function of EC, but no alteration in their IL-1 production capacity or surface marker expression (ATPase, CDI). PPD- and HSV-induced lymphocyte proliferation was decreased 70-80% and ConA-driven proliferation 30% when compared to non-irradiated EC. However, this suppression was restored on days 3 and 7 after UVB irradiation, this being coexistent with an increased capacity of EC to produce IL-1. It remains to be elucidated whether the immediate UVB-induced photoimmunosuppression observed in the present study is due to inhibitory mediators or impaired membrane function of EC or both.