Sodium currents in toad cardiac pacemaker cells

Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nm to 1 m). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (I_f) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nm), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than –40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.We thank the Australian National Heart Foundation for their support. D.A.S. is an NHMRC Senior Research Officer.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

脂质泡中的菌紫质旋转运动的介质依赖性

用闪光诱导瞬间二向色性方法测量了蔗糖和甘油对菌紫质（ＢＲ）分子在脂质泡中的旋转扩散运动的影响。结果表明脂质囊泡本身在悬浮液中的运动并不会影响ＢＲ分子在膜中的旋转运动的测量。蔗糖和甘油对ＢＲ旋转运动的影响在相变温度以上和相变温度以下是不同的,相变温度以下的主要作用是相分离,使运动减慢,相变温度以上的作用可能是脂的分散,使运动加快。     

A proposed model for rhodopsin in photoreceptor membranes

Transient electric birefringence studies have been made on bovine rhodopsin solubilized in the detergent lauryldimethylamine oxide from glutaraldehyde fixed rod outer segment (ROS) membranes. It was found that fixation caused no appreciable differences in the measured relaxation times when compared with unfixed ROS. On the basis of these findings a model for the orientation of rhodopsin in photoreceptor membranes is proposed which accounts for translational diffusion and two modes of rotational diffusion. The proposed model is related to a number of experimentally determined biophysical properties reported in the literature.     

Regional and Temporal Alterations in Ca2+/Calmodulin-Dependent Protein Kinase II and Calcineurin in the Hippocampus of Rat Brain After Transient Forebrain Ischemia

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.     

Regulation of the utilization of 4-hydroxybenzoate and vanillate in batch and continuous cultures of Pseudomonas acidovorans

In Pseudomonas acidovorans, the pathways of 4-hydroxybenzoate and vanillate metabolism converge on the early intermediate, protocatechuate, which undergoes meta-cleavage. The methoxyl group of vanillate is almost completely oxidized, as shown by an experiment with (¹⁴C-methoxyl) vanillate. In batch cultures, 4-hydroxybenzoate and vanillate are simultaneously oxidized. Simultaneous oxidation was explained above all by the fact that both substrates mutually repress the ability of the cells to utilize the partner substrate.If P. acidovorans is growing in a turbidostat on one of the two substrates and is suddenly exposed to an equimolar mixture of both substrates, the respiration rates for the two substrates reciprocate, the for the substrate utilized first passing through a transient minimum, that for the added substrate passing through a transient maximum. Finally, a balance appears to be established, the for 4-hydroxybenzoate being slightly above that for vanillate. Transient phenomena also occur if a chemostat culture with both substrates is suddenly operated as a turbidostat culture or if cells not adapted to either substrate are suddenly exposed to a mixture of both substrates in the turbidostat.If a chemostat culture of P. acidovorans, growing at the expense of an equimolar mixture of 4-hydroxybenzoate and vanillate, is operated under conditions of increasing oxygen deficiency, the utilization ratio of the two substrates increases in favour of 4-hydroxybenzoate. However, if the culture is operated under conditions of increasing nitrogen deficiency, the utilization ratio increases in favour of vanillate.Abbreviations 4HB 4-hydroxybenzoate - VA vanillate - OD optical density     

Satiation time and predatory behaviour of the dragonfly nymph Mesogomphus lineatus

Summary The dragonfly nymph Mesogomphus lineatus satiated predating 21 larvae of Culex fatigans in 50 minutes; number of attack and predatory efficiency decreased precipitously from 2 attacks/min and 70% during the first 10 minutes of feeding to 0.04 attack/min and 0.01% efficiency respectively during the sixth 10 minutes interval. The nymphs fed after 6, 12, 18, 24, 36 or 42 hours of deprivation consumed 4, 13, 15, 20, 21 or 21 larvae; apparently, the maximum appetite is returned after about 36 hours of deprivation. Satiation time, which was 50 minutes at the density of 15 larvae/aquarium, decreased to 30 minutes in aquaria containing 200 larvae. The nymphs predated increased the number of prey when they were exposed to higher densities of C. fatigans and Anopheles stephansi larvae, and pupa of C. fatigans. They consumed equal weight (but different number) of Culex and Anopheles larvae at all the tested prey densities and selectively selected Culex larva over the pupa or Anopheles larva. Comparative analyses suggest that the dragonfly nymphs deserve serious consideration as larvivorous predators.     

Anticoagulation for the Acute Management of Ischemic Stroke

Few prospective studies support the use of anticoagulation during the acute phase of ischemic stroke, though observational data suggest a role in certain populations. Depending on the mechanism of stroke, systemic anticoagulation may prevent recurrent cerebral infarction, but concomitantly carries a risk of hemorrhagic transformation. In this article, we describe a case where anticoagulation shows promise for ischemic stroke and review the evidence that has discredited its use in some circumstances while showing its potential in others.     

Synthesis of Novel Aryloxyethylamine Derivatives and Evaluation of Their in Vitro and in Vivo Neuroprotective Activities

A series of aryloxyethylamine derivatives were designed, synthesized and evaluated for their biological activity. Their structures were confirmed by ¹H‐NMR, ¹³C‐NMR, FT‐IR and HR‐ESI‐MS. The preliminary screening of neuroprotection of compounds in vitro was detected by MTT, and the anti‐ischemic activity in vivo was tested using bilateral common carotid artery occlusion in mice. Most of these compounds showed potential neuroprotective effects against the glutamate‐induced cell death in differentiated rat pheochromocytoma cells (PC12 cells), especially for (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone, (4‐bromophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone, (4‐chlorophenyl)(1‐{2‐[(naphthalen‐2‐yl)oxy]ethyl}piperidin‐4‐yl)methanone, (4‐chlorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone and {1‐[2‐(4‐bromophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone, which exhibited potent protection of PC12 cells at three doses (0.1, 1.0, 10 μM). Compounds (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, (4‐fluorophenyl){1‐[2‐(naphthalen‐2‐yloxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone and {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone possessed the significant prolongation of the survival time of mice subjected to acute cerebral ischemia and decreased the mortality rate at all five doses tested (200, 100, 50, 25, 12.5 mg/kg) and had significant neuroprotective activity. In addition, (4‐fluorophenyl){1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}methanone, {1‐[2‐(4‐methoxyphenoxy)ethyl]piperidin‐4‐yl}(4‐methoxyphenyl)methanone and {1‐[2‐(4‐chlorophenoxy)ethyl]piperidin‐4‐yl}(4‐chlorophenyl)methanone possessed outstanding neuroprotection in vitro and in vivo. These compounds can be used as a promising neuroprotective agents for future development of new anti‐ischemic stroke agents. Basic structure–activity relationships are also presented.