Correlation of open cell-attached and excised patch clamp techniques

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na⁺ channel, located on the apical surface of A₆ cells, to determine whether the nystatininduced open cell-attached patch could serve as an alternative configuration.Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and E _rev was zero in symmetrical Na⁺ solutions, indicating similar Na⁺ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3–4pS) and E _rev (75 mV), when the bath was perfused with a high K:low Na solution (E _Na=80 mV), were nearly equal in both patch configurations.Our results therefore indicate that the nystatininduced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.We thank Dr. Olaf S. Andersen for his suggestions in the development of the open cell-attached recording technique. This work was supported by a National Institutes of Health grant (DK-18061)     

A random fatigue of mechanize titanium abutment studied with Markoff chain and stochastic finite element formulation

To measure fatigue in dental implants and in its components, it is necessary to use a probabilistic analysis since the randomness in the output depends on a number of parameters (such as fatigue properties of titanium and applied loads, unknown beforehand as they depend on mastication habits). The purpose is to apply a probabilistic approximation in order to predict fatigue life, taking into account the randomness of variables. More accuracy on the results has been obtained by taking into account different load blocks with different amplitudes, as happens with bite forces during the day and allowing us to know how effects have different type of bruxism on the piece analysed.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

The reconciliation behavior of golden monkeys (Rhinopithecus roxellanae roxellanae) in small breeding groups

Observations were made following 130 spontaneous aggressive incidents in two small breeding groups of captive golden monkeys (Rhinopithecus roxellanae roxellanae). Participants were observed both during the first 10 min following these incidents and during matched control observations. An increased contact rate was observed between opponents following the aggressive incident. Post-conflict contacts were characterized by a number of behavior patterns: open mouth, rapid grooming, embrace, and crouching. Adult males played an important role as mediator in agonistic disputes among females: males intervened in 93.6% of female fights. It is speculated that this intervention behavior is related to the species' organization into one-male units.     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.     

Lipid enrichment of thylakoid membranes. I. Using soybean phospholipids

(1) Using asolectin (mixed soybean phospholipids) liposomes, extra lipid, with or without additional plastoquinone, has been introduced into isolated thylakoid membranes of pea chloroplasts. (2) Evidence for this lipid enrichment was obtained from freeze-fracture which indicated that a decrease in the numbers of EF and PF particles per unit area of membrane occurred with increasing lipid incorporation. The decrease was not due to loss of integral membrane polypeptides as judged by assay of cytochrome present or SDS-polyacrylamide gel electrophoresis of lipid-enriched membrane fractions. Moreover, the enrichment procedure did not lead to extraction of low molecular weight lipophilic membrane components or of thylakoid membrane lipids. (3) The introduction of phospholipids into the membrane affected steady-state electron transport. Inhibition of electron transport was observed when either water (Photosystem (PS) II + PS I) or duroquinol (PS I) was used as electron donor with methyl viologen as electron acceptor, and the degree of inhibition increased with higher enrichment levels. Introduction of exogenous plastoquinone with the additional lipid had little effect on whole-chain electron transport, but caused an increase in the 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-sensitive rate of PS I electron transport. The inhibition was also detected by flash-induced oxidation-reduction changes of cytochrome f.     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

Sequence and characterization of the Escherichia coli genome between the ndk and gcpE genes

Abstract The region of the chromosome immediately upstream of the Escherichia coli gene gcpE has been cloned and sequenced. This region contains two functional open reading frames, orf 384 and orf 337, encoding proteins of 43082 and 36189 Da, respectively. Sequencing analysis (this paper) and the isolation of a DNA fragment containing a functional promoter (Talukder, A.A., Yanai, S., and Yamada, M. (1994) Biosci. Biotech. Biochem. 58, 117–120) indicate that orf 337 is in an operon with gcpE . The gene orf 384 is immediately downstream of the gene ndk , which encodes nucleoside diphosphate kinase.