Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae.

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.

Induction by soluble factors of organized axial structures in chick epiblasts.

Inductive action of soluble factors was tested on isolated chick epiblasts. An assay was developed wherein conditioned medium derived from the Xenopus XTC cell line induced the formation of a full-length notochord and rows of bilaterally symmetric somites. Basic fibroblast growth factor, epidermal growth factor, retinoic acid, and transforming growth factor type B1 and B2 were not capable of inducing axial structures. Thus, soluble factors can elicit the development of polarity stored in the epiblast and behave as true morphogens since they can induce the formation of the organized complex structures that constitute the embryonic axis.

Aldosterone and the autocrine modulation of potassium currents and oxidative stress in the diabetic rat heart.

BACKGROUND AND PURPOSE: Aldosterone plays a major role in cardiac pathology. This study was designed to investigate the role of cardiac aldosterone in modulating K(+) currents and oxidative stress in the streptozotocin-induced diabetic rat heart. EXPERIMENTAL APPROACH: Transient and sustained K(+) currents were measured in ventricular myocytes by voltage clamp. Plasma and cellular aldosterone were measured by ELISA. Fluorescent dihydroethidium (DHE) was used to assess superoxide ions as markers of oxidative stress. KEY RESULTS: The mineralocorticoid antagonist spironolactone (1 microM, 5-9 h) significantly augmented both K(+) currents in diabetic males, with a concomitant shortening of the action potential but had no effect in myocytes from control males or from diabetic females. Effects of spironolactone were restored in ovariectomized diabetic females and abolished in orchidectomized diabetic males. The aldosterone synthase inhibitor FAD286 (1 microM, 5-9 h) significantly augmented K(+) currents in cells from diabetic males, but not females. Spironolactone and FAD286 significantly reduced oxidative stress in cells from diabetic males. Plasma aldosterone content was elevated in diabetic males (relative to control), but not in females. Cellular aldosterone was also elevated, but not significantly. The elevation in aldosterone was only partly dependent on a concomitant increase in cellular angiotensin II. CONCLUSIONS AND IMPLICATIONS: A gender-related, sex-hormone-dependent elevation in plasma and cardiac cell aldosterone contributed to oxidative stress and to attenuation of K(+) currents in diabetic male rats. Aldosterone may thus contribute to diabetes-associated cardiac arrhythmias. Aldosterone elevation was partly related to levels of angiotensin II, but residual, angiotensin II-independent, aldosterone maintains functional relevance.

A cellular mechanism for nitric oxide-mediated cholinergic control of mammalian heart rate.

The biochemical signaling pathways involved in nitric oxide (NO)-mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro-arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh-induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Wheat (Triticum aestivum L.) [gamma]-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast.

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat [gamma]-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the [gamma]-gliadin was transported to the yeast vacuoles. When a deletion mutant of the [gamma]-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the [gamma]-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.

Further characterisation of the inotropic effect of a bufodienolide glycoside--an endogenous ouabain like compound.

A ouabain like compound obtained from toad skin and plasma and identified to be a steroidal bufodienolide glycoside was found to displace ouabain from its binding site and to inhibit Na+-K+ ATPase, and have positive inotropic effects on cardiac muscle. The ionic and rate dependence of this positive inotropy was studied in the frog atrium. The effect was dependent on extracellular potassium, sodium, and calcium concentrations and on the rate of stimulation, which is similar to the properties of cardiac glycosides. Occasionally, the compound gave transient or even negative inotropic responses, as do the glycosides. The action potential configuration was also affected by the compound in the same complex pattern as is that of cardiac glycosides. It is concluded that the endogenous bufodienolide compound has the same physiological effects as cardiac glycosides. Since the same compound is present in toad plasma it may serve as an intrinsic humoral regulator of cardiac contractility. This study is the first detailed characterisation of the cardioactive properties of this compound.

Demonstration of a ouabainlike plasma compound in hypertension prone and hypertension resistant rats.

Material extracted and partially purified from plasma of the Sabra hypertension prone rats was found to be capable of 1) inhibiting the binding of 3H-ouabain to rat brain synaptosomes, 2) inhibiting the activity of rat brain microsomal Na, K activated adenosine triphosphatase, and 3) increasing the contractile force of rat heart muscle. The results demonstrate the presence of a ouabainlike compound in the plasma of these rats. The plasma concentration of this compound in Sabra hypertension prone rats was 698 +/- 199 nmol/ml in ouabain equivalents (SEM; n = 11) versus 2543 +/- 1140 nmol/ml (n = 9) in the Sabra normotensive strain. The presence of ouabainlike compound in the plasma is consistent with the hypothesis that this compound functions as a hormone that regulates Na, K activated adenosine triphosphatase activity and the physiological processes in which this enzyme is involved.

Effects of aluminium on electrical and mechanical properties of frog atrial muscle.

1. The effects of aluminium on membrane ionic currents were studied in single cardiac myocytes. Most of the work was done on frog atrial cells, but some experiments were also carried out on single cells isolated from rabbit ventricles and atria. 2. The effects of aluminium on the force of contraction of frog atrial trabeculae were also investigated. 3. Aluminium was prepared from AlCl3 as a stock 0.5 M solution which has a pH of 3.5. Before each experiment, this solution was added to the control solution, to give a final concentration of 20-100 micrograms ml-1 aluminium (0.75-3.75 mM AlCl3). The solutions were brought to a pH of 7.4 or 7.6. at which they consist of a mixture of amorphous aluminium hydroxides and a very small amount of soluble ionic aluminium complexes: free aluminium cations (less than 10 pM), aluminohydroxide anions (less than 8 microM). The addition of this suspension reduced the peak inward calcium currents in single rabbit atrial and ventricular cells and in frog atrial cells. In the latter, the peak current was reduced (at + 10 mV) to 45% of control (mean of 9 cells). This effect was reversible upon washout, and was obtained at all membrane potentials, with no shift of the calcium current voltage relationship along the voltage axis. 4. Aluminium also reduced the time-dependent potassium current IK. This reduction was observed at all membrane potentials. For example, at + 10 mV, the mean reduction of IK (n = 9) was to 69% of the control amplitude. This effect, which was very difficult to reverse, was not due to IK rundown. The fully activated current-voltage relationships (obtained by standard 'tail' analysis) showed that the effect of aluminium was due mainly to a decrease in conductance and not to a shift in the activation range of IK. The mean voltage of half activation was shifted by 8 mV in the depolarizing direction (n = 5). 5. The background potassium current IK1 was also slightly but consistently changed in a complex fashion, with an outward shift at membrane potentials positive to -60 mV. For example, at a membrane potential of -40mV, the mean shift was by 22 + 4pA. At more negative potentials, there was an inward shift in the current amplitudes. For example, for steps to -I00 mV the current elicited was larger (more inward) by 53 pA (mean value, n = 10). The reversal potential was slightly shifted (<10 mV) in the hyperpolarizing direction. 6. The force of contraction of frog atrial trabeculae was altered by aluminium in a complex manner, which showed marked seasonal variation. During most of the year, 50-100,ug ml-1 aluminium caused a biphasic change, with an early small and consistent decrease, followed by a large increase in twitch amplitude. For a short period corresponding to the (local) winter months the sensitivity to aluminium was greatly enhanced. Aluminium lOOupgml-1 totally abolished contraction (n = 5), while a lower concentration (20,ug ml- 1) produced a sustained reduction in the force of contraction. Similar biphasic and seasonal responses have been reported to be induced by lanthanum. 7. The biphasic changes in twitch amplitude were independent of the transmembrane sodium gradient. Aluminium produced the same effects when 90% of the extracellular sodium was replaced by lithium. Caffeine (5 mM) attenuated or even inverted the positive inotropic effect of aluminium. These results imply that aluminium alters the release of calcium from intracellular, caffeine-sensitive stores. This could be effected either by augmenting the amount released during each activation, and/or by increasing the loading of stores prior to release.

Mediation by nitric oxide of the indirect effects of adenosine on calcium current in rabbit heart pacemaker cells.

1. Adenosine (ADO) is a potent negative chronotropic agent in the mammalian myocardium. We have used single myocytes from rabbit sino-atrial node (SAN) to examine whether nitric oxide (NO) is a significant mediator of the effects of ADO on the pacemaker activity, or the underlying Ca2+ and K+ currents. 2. SAN pacemaker cells were isolated from rabbit hearts by enzymatic dispersion, and Ca2+ and K+ currents were recorded by the nystatin-perforated patch voltage clamp method. ADO was applied in the presence of the beta-adrenoceptor agonist, isopremaline (Iso) to mimic the adrenergic tone which the SAN is subjected to in vivo. 3. Control experiments confirmed that isolated SAN cells responded to ADO (10-100 microM) with the expected (i) small increase in background inwardly rectifying K+ current, IK-ADOi and (ii) pronounced decrease in L-type Ca2+ current, ICa-L. These effects were mimicked by a selective A1 purinoceptor agonist, N6-cyclopentyladenosine (CPA, 10 microM); and were inhibited following bath application of the antagonist, DPCPX (10 microM), which selectively blocks A1 purinoceptors. DMPX (10 microM), a blocker of A2 purinoceptor, had no effect on the actions of ADO. 4. A nitric oxide synthase inhibitor, L-NMMA (100 microM), abolished the inhibitory effect of ADO on ICa-L but did not alter activation of IK-ADO. After L-NMMA washoff, it was possible to obtain the normal response (inhibition) of ICa-L to ADO in the same cell. 5. To evaluate whether the observed effect of nitric oxide (NO) on ICa-L was mediated by an increase in guanylyl cyclase (GC) activity and cyclic GMP formation, the guanylyl cyclase inhibitor, LY 83583 (40 microM) was applied prior to ADO. Under these conditions, the inhibitory effect of ADO on ICa-L was abolished, but the activation of IK-ADO was still observed. 6. In combination, these findings strongly suggest that in mammalian primary pacemaker tissue which is under adrenergic tone, the effects of ADO on ICa-L are mediated by NO.

The effects of prenylamine on single ventricular myocytes of guinea-pig.

1. The action of prenylamine, an antianginal drug, was studied in single ventricular guinea-pig myocytes. In concentrations of 10-50 microM, prenylamine significantly (P less than 0.01) shortened action potentials, and significantly (P less than 0.001) reduced the inward calcium current by 29% to 76% (n = 7). This effect was also present in the presence of adrenoceptor-blockade (with phentolamine and propranolol), and was thus not due to indirect changes in endogenous catecholamine action. 2. Prenylamine did not affect the steady state level of current at the end of long pulses, and does therefore not act by changing time-dependent outward currents. Since the resting potential in the unclamped mode is unchanged during gross changes in action potential duration, it is also unlikely that there are any changes in the background, time-independent potassium conductance. 3. It is concluded that prenylamine has a direct effect on cardiac calcium channels, not mediated by adrenoceptor activation.

Heat acclimation: cardiac performance of isolated rat heart.

Cardiac performance was studied in the isolated perfused hearts of rats heat acclimated at 34 degrees C (AC) and their age-matched controls (C). The pressure-volume curves during isovolumetric conditions showed a shift to the right in AC compared with C hearts. At similar left ventricular (LV) volumes end-diastolic and peak systolic pressures of AC hearts were lower, but no difference was observed in the maximal pressure developed at the highest LV volumes measured. In both C and AC hearts the developed force decreased as pacing rate increased. AC and C heart responses were the same up to 250 pulses/min. At higher frequencies the amplitude of the developed force of AC hearts was smaller than that of the controls. In accordance the tension produced by very early premature beat reduced in AC compared with C hearts. Since no hypertrophy was observed in AC hearts, it is concluded that heat acclimation results in a change in the intrinsic properties of the AC hearts exhibited by increased compliance, reduced chamber stiffness, and a decrease in the tension developed for each volume load. It is also suggested that at a high beating rate AC hearts fail to restitute its contractility as quickly as C hearts.

Ultrasound controlled operative hysteroscopy.

BACKGROUND: Uterine perforation is one of the risks of operative hysteroscopy. Although usually performed alone, laparoscopy has been recommended to aid the surgeon in preventing uterine perforation at the time of operative hysteroscopy. STUDY DESIGN: Since women suffering from infertility or habitual abortion with known or suspected intrauterine pathologic factors are at low risk for secondary pelvic abnormalities, we have been using ultrasound for control during operative hysteroscopy in these women. One hundred twenty-eight women underwent ultrasound-guided operative hysteroscopy. RESULTS: There were no complications, such as uterine perforation, during or after any of the procedures. CONCLUSIONS: Women with known intrauterine pathologic factors should be offered operative hysteroscopy controlled by ultrasound, avoiding the use of unnecessary laparoscopy.

Data predictability for compression of digital fluorography images.

Images obtained by digital fluorography were checked for compressability. These images include images of coronary vessels and images of peripheral vessels. These images have a very low signal-to-noise ratio compared to the optical images usually used for developing compression methods. Configurational entropy was used to represent the information content of these images. Reversible prediction algorithms were extensively checked in a search for minimal residual information, enabling more efficient reversible compression. Optimal results were obtained for algorithms based on two or three neighboring pixels and a semiempirical rule, based on the noise level, was found which decides on the best approach. It was found that raw data images are more predictable than subtracted images although the latter are visually preferred.

Nonlinear filters applied on computerized axial tomography: theory and phantom images.

New nonlinear image processing techniques, in particular smoothing based on the understanding of the image, may create computerized tomography (CT) images of good quality using less radiation. Such techniques may be applied before the reconstruction and particularly after it. Current CT scanners use strong linear low-pass filters applied to the CT projections, reducing noise but also deteriorating the resolution of the image. The method in this study was to apply a weak low-pass filter on the projections, to perform the reconstruction, and only then to apply a nonlinear filter on the image. Various kinds of nonlinear filters were investigated based on the fact that the image is approximately piecewise constant. The filters were applied with many values of several parameters and the effects on the spatial resolution and the noise reduction were evaluated. The signal-to-noise ratio of a high-contrast phantom image processed were compared with the nonlinear filter, with the SNR of the phantom images obtained with the built-in CT linear filters in two scanning modes, the normal and the ultra high resolution modes. It was found that the nonlinear filters improve the SNR of the image, compared to the built-in filters, about three times for the normal mode and twice for the UHR scanning mode. The most successful filter on low-contrast phantom image was applied and it also seems to lead to promising results. These results seem to show that applying nonlinear filters on CT images might lead to better image quality than using the current linear filters.(ABSTRACT TRUNCATED AT 250 WORDS)

T-2 toxin effect on cultured myocardial cells.

Beat rate, contractility and viability of cultured myocardial cells perfused with solutions containing various concentrations of T-2 toxin were studied. While doses below 50 micrograms/ml had no immediate effect, those above 250 micrograms/ml decreased beat rate and amplitude. After 10-30 min of perfusion most cells stopped beating and did not restart after withdrawal of toxin. Nevertheless, most cells remained viable as judged by morphology and trypan blue exclusion. A 24-h exposure to doses of 5 or 2.5 micrograms/ml of toxin decreased the beat rate and inotropic responses of the myocytes. After 48 h cell death ensued. Thus T-2 toxin has some direct toxicity to myocardial cells but the lethal dose seems too high to make this the cause of cardiovascular failure.

Thyroid hormone induces earlier onset of auditory function in neonatal rats.

The effect of thyroid hormone injection on the development of auditory function in neonatal rats was evaluated using auditory nerve-brainstem evoked responses (ABR). The hormone induced earlier onset of auditory function. In order to differentiate between conductive and sensorineural factors, both air-conducted (AC) and bone-conducted (BC) ABR responses were recorded. Neonatal rats were injected with thyroxine (T4), or with saline (control animals), from day of birth (post-natal day-PND-0), daily, until PND 9. AC- and BC-ABRs were recorded from PND 6 up to PND 20. It was found that both AC- and BC-ABR thresholds were lower in the T4-injected rats up to PND 15, after which no difference was found between the two groups. This indicated earlier maturity of both conductive (external and middle ears) and sensorineural (inner ear) factors and is probably due to the earlier appearance in the blood of higher T4 levels, following injection, than that occurring naturally during the neonatal period in these animals.

Pregnancy and complicated familial Mediterranean fever.

Familial Mediterranean Fever (FMF) is an inherited disease, closely following the pattern of autosomal recessive inheritance. Amyloidosis is the most severe complication of the disease. The prevalence of pregnancy loss in women with FMF is considered to be high. There is no information to support the possibility of increase risk of late pregnancy complications or change in the natural course of the disease. Two cases are presented with complicated FMF. One case with proved amyloidosis and the second patient with ascites. Pregnancy and neonatal outcome were uneventful in both. No further deterioration in the systemic disease occurred.

Propagation of the cardiac impulse in the diabetic rat heart: reduced conduction reserve.

Diabetes mellitus is a growing epidemic with severe cardiovascular complications. Although much is known about mechanical and electrical cardiac dysfunction in diabetes, few studies have investigated propagation of the electrical signal in the diabetic heart and the associated changes in intercellular gap junctions. This study was designed to investigate these issues, using hearts from control and diabetic rats. Diabetic conditions were induced by streptozotocin (STZ), given i.v. 7-14 days before experiments. Optical mapping with the voltage-sensitive dye di-4-ANEPPS, using hearts perfused on a Langendorff apparatus, showed little change in baseline conduction velocity in diabetic hearts, reflecting the large reserve of function. However, both the gap junction uncoupler heptanol (0.5-1 mM) and elevated potassium (9 mM, to reduce cell excitability) produced a significantly greater slowing of impulse propagation in diabetic hearts than in controls. The maximal action potential upstroke velocity (an index of the sodium current) and resting potential was similar in single ventricular myocytes from control and diabetic rats, suggesting similar electrical excitability. Immunoblotting of connexin 43 (Cx43), a major gap junction component, showed no change in total expression. However, immunofluorescence labelling of Cx43 showed a significant redistribution, apparent as enhanced Cx43 lateralization. This was quantified and found to be significantly larger than in control myocytes. Labelling of two other gap junction proteins, N-cadherin and beta-catenin, showed a (partial) loss of co-localization with Cx43, indicating that enhancement of lateralized Cx43 is associated with non-functional gap junctions. In conclusion, conduction reserve is smaller in the diabetic heart, priming it for impaired conduction upon further challenges. This can desynchronize contraction and contribute to arrhythmogenesis.