Prolonged activation of mitochondrial conductances during synaptic transmission.

Although ion channels have been detected in mitochondria, scientists have not been able to record ion transport in mitochondria of intact cells. A variation of the patch clamp technique was used to record ion channel activity from intracellular organelles in the presynaptic terminal of the squid. Electron microscopy indicated that mitochondria are numerous in this terminal and are the only organelles compatible with the tips of the pipettes. Before synaptic stimulation, channel activity was infrequent and its conductance was small, although large conductances ( approximately 0.5 to 2.5 nanosiemens) could be detected occasionally. During a train of action potentials, the conductance of the mitochondrial membrane increased up to 60-fold. The conductance increased after a delay of several hundred milliseconds and continued to increase after stimulation had stopped. Recovery occurred over tens of seconds.

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Mitochondrial factors with dual roles in death and survival.

At least in mammals, we have some understanding of how caspases facilitate mitochondria-mediated cell death, but the biochemical mechanisms by which other factors promote or inhibit programmed cell death are not understood. Moreover, most of these factors are only studied after treating cells with a death stimulus. A growing body of new evidence suggests that cell death regulators also have 'day jobs' in healthy cells. Even caspases, mitochondrial fission proteins and pro-death Bcl-2 family proteins appear to have normal cellular functions that promote cell survival. Here, we review some of the supporting evidence and stretch beyond the evidence to seek an understanding of the remaining questions.

Regulation of potassium channels by protein kinases.

Studies of the role of protein phosphorylation in the modulation of neuronal excitability are beginning to identify specific sites on ion channels that are substrates for serine/threonine kinases and that contribute to short-term and long-term regulation of current amplitude and kinetics. In addition, it is becoming apparent that phosphorylation of tyrosine residues may produce acute changes in the characteristics of ion channels. These recent findings are best illustrated by examining the Shaker superfamily of potassium channels.

Treatment of nortriptyline's side effects in elderly patients: a double-blind study of bethanechol.

A double-blind, placebo-controlled study of bethanechol was conducted in 26 elderly depressed patients being treated with nortriptyline. Patients receiving bethanechol had reduced subjective complaints of anticholinergic side effects and showed a trend toward improvement on an objective measure of salivary flow. The potential use of bethanechol in older patients to reduce morbidity and improve compliance with medication regimens is discussed.

Platelet aggregability in rats with early atherosclerotic changes induced by parenterally-administered lipid emulsions.

The present study is the first work to evaluate thrombin-, ADP-, and collagen-induced platelet aggregation in laboratory rats receiving alimentation with the parenterally-administered lipid emulsion, Lipofundin-S, in doses sufficient to induce early atherosclerotic changes in the aorta. The aggregometry parameters of percent maximum aggregation, slope, and b2 or b20 almost uniformly indicate that such lipid treatments result in a statistically significant increased sensitivity of the platelets to ADP and collagen, while no change is noted with thrombin as the aggregating agent. By varying the amounts of ADP and collagen during aggregometry, we also demonstrate that the concentrations of these reagents necessary for equivalent platelet aggregation is substantially lower in lipid-infused rats than in controls. We conclude from this study that such lipid infusions can cause increased platelet aggregation, and that these lipids probably act in a synergistic fashion by affecting a variety of components which comprise the atherogenic process and its clinical endpoint. In addition, we believe that this experimental approach is of interest in that infusions of clinically-useful lipid emulsions are easily controlled, while alterations in platelet physiology and aortic structure occur concurrently and rapidly.

Evidence of subclinical heart muscle dysfunction in acromegaly.

A group of ten acromegalic patients, who had no history of heart failure, was studied to determine whether subtle carciac impairment may also be common. None had clinical evidence of coronary artery disease or severe hypertension. Systolic time intervals were recorded in each patient and compared with normal values predicted for sex and heart rate by our own controls and published data. The results indicate that measurable abnormalities in left ventricular performance are common in this sampling. Known duration and activity of disease (growth hormone levels at time of study) did not correlate with the time intervals. The results are consistent with cardiomyopathic effect of excessive growth hormone.

Cardiogenic shock due to huge right atrial thrombus.

An unusual case is reported of right atrial thrombus associated with cardiogenic shock. The role of two-dimensional echocardiography in detection of blood clots is highlighted.

Acute myocardial infarction with normal coronary artery: a case report and review of literature.

A 17-year-old girl with no risk factors for coronary artery disease had acute myocardial infarction. She received thrombolytic therapy with tissue-type plasminogen activator. An extensive workup for the cause of myocardial infarction revealed protein S deficiency. Angiography showed normal coronary arteries. We speculate that the cause of myocardial infarction was coronary spasm or thrombus formation, which was successfully dissolved by thrombolytic therapy. This is the eighth case report of acute myocardial infarction in a patient with normal coronaries and protein S deficiency. We reviewed the literature concerning myocardial infarction and normal coronaries and protein S deficiency. This case report and review of the literature suggest the need to extend the concept of classic risk factors for coronary artery disease in young patients with myocardial infarction and normal coronary arteries.

Educational and training systems in Sweden for prehospital response to acts of terrorism.

Sweden has a long tradition in planning for disaster situations in which the National Board of Health and Welfare has a key responsibilty within the health sector. One important part of this disaster preparedness is education and training. Since 11 September 2001, much focus has been placed on the acts of terrorism with special reference to the effects of the use of chemical, biological, or nuclear/radiological (CBNR) agents. In the health sector, the preparedness for such situations is much the same as for other castastrophic events. The National Board of Health and Welfare of Sweden is a national authority under the government, and one of its responsibilities is planning and the provision of supplies for health and medical services, environmental health, and social services in case of war or crises. "Joint Central Disaster Committees" in each County Council/Region in the country are responsible for overseeing major incident planning for their respective counties/regions. The "Disaster Committee" is responsible for ensuring that: (1) plans are established and revised; (2) all personnel involved in planning receive adequate information and training; (3) equipment and supplies are available; and (4) maintenance arrangements are in place. Sweden adopts a "Total Defense" strategy, which means that it places a high value in preparing for peacetime and wartime major incidents. The Swedish Emergency Management Agency coordinates the civilian Total Defense strategy, and provides funding to the relevant responsible authority to this end. The National Board of Health and Welfare takes responsibility in this process. In this area, the main activities of the National Board of Health and Welfare are: (1) the establishment of national guidelines and supervision of standards in emergency and disaster medicine, social welfare, public health, and prevention of infectious diseases; (2) the introduction of new principles, standards, and equipment; (3) the conducting education and training programmes; and (4) the provision of financial support. The budget for National Board of Health and Welfare in this area is approximately 160 million SEK (US dollar 18 million). The National Board of Health and Welfare also provides funding to the County Councils/Regions for the training of healthcare professionals in disaster medicine and crises management by arranging (and financing) courses primarily for teachers and by providing financial support to the County Councils/Regions for providing their own educational and training programmes. The National Board of Health and Welfare provides funding of approximately 20 million SEK (US dollar 2.4 million) to the County Councils/Regions for this training of healthcare professionals in disaster medicine and crises.

Four conventional soybean [Glycine max (L.) Merrill] seeds exhibit different protein profiles as revealed by proteomic analysis.

Soybeans have several functional properties due to their composition and may exert beneficial health effects that are attributed to proteins and their derivative peptides. The present study aimed to analyze the protein profiles of four new conventional soybean seeds (BRS 257, BRS 258, BRS 267, and Embrapa 48) with the use of proteomic tools. Two-dimensional (2D) and one-dimensional (1D) gel electrophoreses were performed, followed by MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analyses, respectively. These two different experimental approaches allowed the identification of 117 proteins from 1D gels and 46 differentially expressed protein spots in 2D gels. BRS 267 showed the greatest diversity of identified spots in the 2D gel analyses. In the 1D gels, the major groups were storage (25-40%) and lipid metabolism (11-25%) proteins. The differences in protein composition between cultivars could indicate functional and nutritional differences and could direct the development of new cultivars.

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage.

Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1-Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer.

Induction of early atherosclerosis in rats using parenterally-administered lipid emulsions.

It has been demonstrated by us and other workers that rats receiving I.V. infusions of Lipofundin-S will develop aortic changes indicative of early atherosclerosis. However, different lipid emulsions which are used in the clinical setting for parenteral nutrition vary substantially in chylomicron size and fatty acid composition. Therefore, in an attempt to better understand the mechanism by which a lipid emulsion might induce vessel lesions, we compared the nature of potential aortic changes resulting from infusions of Liposyn, Intralipid, or Lipofundin-S into the tail veins of Sprague-Dawley rats. Three groups of animals received either Liposyn (N = 10), Intralipid (N = 5), or Lipofundin-S (N = 9) at the rate of 6 g fat/kg body wt/day for 10 consecutive days. A fourth group (N = 5) received saline in equivalent dose to evaluate the effect of injection volume on vessel lesion formation. The other controls (N = 6) received no injections. Rats were sacrificed 24 hrs after the last infusion, and 1 mm rings from the top of the aortic arch and proximal third of the thoracic aorta were prepared for transmission electron microscopy (TEM). Examination by TEM allows two main conclusions to be drawn for both segments of the aorta. First, all three emulsions are capable of inducing early vessel changes which include endothelial damage, platelet adherence to damaged endothelium or subendothelial collagen, intimal phagocytic cells, and intimal smooth muscle cells surrounded by collagen bundles and elastin plates. Saline-infused rats only show occasional subendothelial swelling. None of the above-described changes are seen in any of the uninjected controls. Second, Lipofundin-S induces smooth muscle penetration of the intima in 7 of 9 rats, while Liposyn causes such changes in 2 of 10 animals. This difference in the efficiency with which the two emulsions induce the most advanced changes is statistically significantly by Chi Square (p less than 0.05). Intralipid produces smooth muscle penetration of the intima in 2 of 5 rats. The composition of the three emulsions suggests that the lower percent of linoleic acid and larger chylomicron size in Lipofundin-S may account for these differences, at least in part.

Transcriptional regulation of a Xenopus embryonic epidermal keratin gene.

XK81A1 is a type I epidermal keratin gene expressed in early developmental stages of Xenopus (Jonas et al. 1985). Fusion of the keratin promoter (-5900 to +26) to a human beta globin gene led to fully epidermis-specific accumulation of human globin mRNA and protein when this DNA was injected into fertilized eggs. Further localization of regulatory sequences was accomplished by injecting marked, 5'-deleted keratin gene DNA into fertilized eggs and evaluating tissue specificity of expression. All 5' flanking DNA upstream from -487 could be removed without interfering with keratin gene expression or regulation. These results suggest that the primary mode of regulation of epidermis-specific keratin gene expression is at the level of transcription, and that sequence elements in the 5' flanking region of the keratin gene, between -487 and +26, are responsible for this regulation.

KTF-1, a transcriptional activator of Xenopus embryonic keratin expression.

Nuclear extracts from embryos of Xenopus laevis were shown to contain a protein activity, KTF-1, which binds in vitro to the promoter of the embryonic, epidermis-specific keratin gene, XK81A1. Mobility shift assays, methylation interference and footprinting analysis were used to show that the KTF-1 binding site contains an imperfect, palindromic sequence, ACCCTGAGGCT. This sequence occurs once in the XK81A1 promoter, 152-162 base pairs upstream of the transcription start site. A construct of the keratin gene in which this sequence was altered so that it no longer binds KTF-1 in vitro showed severely reduced transcription levels upon injection into Xenopus embryos, but retained epidermal specificity. Addition of KTF-1 binding sites also enhanced epidermal and non-epidermal activity of a heterologous promoter, Xenopus beta-globin, in embryos. These results suggest that KTF-1 is a general activator of embryonic keratin transcription, which acts in concert with other factors to produce high levels of epidermis-specific expression.

The effect of atherogenic infusions of the triglyceride-rich, lipid emulsion, Lipofundin-S, on the in vitro growth characteristics of rat aortic smooth muscle cells.

This study shows that arterial smooth muscle cells (SMC) isolated from rats receiving atherogenic doses of the lipid emulsion, Lipofundin-S, alter their in vitro growth properties. Compared to cells from control animals, SMC isolated from Lipofundin-S-infused rats show a reduction in both saturation density and response to increasing serum concentrations, without a change in the baseline proliferation. Also, SMC isolated from lipid-treated animals and grown for five days in the presence of 30, 150, or 300 pg/ml estradiol show a 30% increase in growth vs. cells from controls. Epinephrine at 1 microM stimulates growth in SMC from control rats, while causing no growth enhancement over five days in cells from lipid-infused animals. Thus, atherogenic infusions of Lipofundin-S into rats cause phenotypic changes in arterial SMC which can be passed to successive cell generations in vitro.

Parenteral lipid emulsion-induced atherosclerosis in the obese Zucker rat and its lean littermate.

We have shown previously that parenterally-administered lipid emulsions can be utilized to induce early atherosclerosis in the aortas of Sprague-Dawley rats. In order to evaluate the effect of obesity on lipid-induced atherogenesis, we have utilized this same approach in the present study to demonstrate that i.v. infusions of the parenteral lipid emulsion, Lipofundin-S, will induce in the genetically obese Zucker rat and its lean littermate aortic endothelial and myofibroelastic changes indicative of early atherogenesis. Four groups of rats were used: 1) obese controls, 2) obese lipid-infused, 3) lean littermate controls, and 4) lean littermate lipid-infused. Observations were made with light and transmission electron microscopy (TEM), using qualitative morphological criteria to evaluate the results. Based on the fact that both untreated control and Lipofundin-S-induced atherosclerosis was more frequent and generally more advanced in the obese animals than in their respective lean counterparts, it appears that the obese Zucker rat is more susceptible to both spontaneous and hyperlipidemia-induced atherosclerosis than its respective lean littermate. Thus, obesity in these animals, as might be the case in humans, could potentiate an atherogenic process already enhanced by hyperlipidemia.

Intracellular ionized calcium ([Ca++]i) mobilization in platelets from rats receiving atherogenic lipids. Modulation by nifedipine.

Atherosclerosis is a progressive disease in which its clinical sequelae are manifest with increasing frequency as individual age. The present study seeks to better understand the mechanisms underlying this process by utilizing our previously-characterized rat model of early atherosclerosis induction to evaluate the effect of atherogenic plasma lipids on intracellular ionized calcium levels in rat platelets. Sprague-Dawley male rats were infused i.v. with 20% Lipofundin-S, a triglyceride-rich emulsion shown by us in previous studies to induce early athero-sclerosis and platelet hyperactivity. Twenty four hrs after the last infusion, blood was obtained by cardiac puncture. Washed platelets were loaded with aequorin, stimulated with ADP, and [Ca++]i was determined by measuring luminescence in platelets from lipid-infused vs. control rats. In platelets isolated from lipid-infused rats, [Ca++]i levels were 34% higher (p < or = 0.05) than in platelets from control animals. In addition, the mean, median, and mode diameters of platelets from lipid-treated rats were significantly greater (p < or = 0.001) than those of platelets from controls. With ADP as the aggregating agent, nifedipine at 1 microgram/ml caused a 27% (p < or = 0.05) inhibition of [Ca++]i release in platelets from lipid-treated rats, but showed no inhibitory action in platelets isolated from control animals. Hyperlipidemia results in elevated platelet [Ca++]i levels, with a concomitant increase in cell size, both indicating enhanced platelet function. Nifedipine modulates this increased activity in platelets isolated from lipid-infused rats, but not in cells from control animals.

The effect of parenteral lipid emulsion-induced hyperlipidemia on prostaglandin E1 modulation of platelet function.

The purpose of this study is to better understand how hyperlipidemia alters the modulating action of prostaglandin E1 (PGE1) on platelet function. Using our previously characterized rat model of atherogenesis, we demonstrate that the parenteral lipid emulsions, Lipofundin-S and Liposyn, significantly (p < or = 0.05) enhance baseline platelet aggregation. In addition, dose response curves show that in all animals, PGE1 substantially inhibits platelet aggregation at 10(-7) to 10(-6) M, while significantly stimulating platelet function at lower doses. However, at all PGE1 concentrations, aggregation values are higher in platelets from lipid-treated vs. control rats, showing that hyperlipidemia significantly reduces the ability of high concentrations of PGE1 to inhibit platelet activity, based on the absolute values of the controls. Also, dose response curves for PGE1 on platelet aggregation show a marked similarity in shape for control ratsvs. normal humans. Thus, this study demonstrates that hyperlipidemia significantly alters the platelet modulating action of prostaglandin E1, and it shows that PGE1 can either inhibit or stimulate platelet activity in both rat and human platelets.