Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Effect of several uncouplers of cell-to-cell communication on gap junction morphology in mammalian heart

Summary Electrical conduction in sheep Purkinje fibers has been blocked by three different procedures: (I) 1mm 2–4-dinitrophenol, (II) 3.5mm n-Heptan-1-ol (heptanol), and (III) treatment by a hypotonic (120 mOsmoles) Ca²⁺-free solution for half an hour, followed by return to normal conditions. The gap junction morphology was analyzed quantitatively in freezefracture replicas and compared in electrically conducting and nonconducting fibers. It is found that the three uncouplers of cell-to-cell conduction induce consistent and statistically significant alterations of the gap junction structure. The investigated morphological criteria: (a) P-face junctional particle diameter, control value 8.18±0.70 nm (mean±sd), (b) P-face junctional particles center-to-center spacing, control value 10.23±1.57 nm, and (c) E-face pits spacing, control value 9.45±0.98 nm, are, respectively, decreased to 7.46±0.62 nm, 9.25±1.34 nm and 8.67±1.13 nm in Purkinje fibers with complete conduction blocks. All three gap junctional dimensions are seen to decline progressively with time from the onset of an uncoupling treatment towards stable minima reached in half an hour. The observed morphological transitions appear related to the electrical uncoupling for the following reasons: partial electrical uncoupling results in values of the gap junctional dimensions that are intermediate between those measured in electrically coupled and uncoupled preparations, and the three morphological indices are seen to increase again towards control values very soon after electrical conduction has been re-established. It is concluded that the junctional channels closure on electrical uncoupling correlates with a measurable (−0.72±0.01 nm, difference of the means±se) decrease of the junctional particle diameters.     

Permeance of novikoff hepatoma gap junctions: Quantitative video analysis of dye transfer

Summary Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined. In the present study, junctional permeance (PA, i.e., the product of the junctional permeability coefficient,P, times the junctional area,A) to Lucifer Yellow CH (LY) has been obtained for pairs of Novikoff hepatoma cells. Dye was microinjected into one cell and the subsequent transfer monitored by a SIT camera and recorded on video tape. The intensities of fluorescence in the injected and recipient cell were measured using a Digisector (Microworks) digitizing board and an Apple II Plus computer to analyze the video records. These changes in intensity, along with an estimate of volume of the spherical cells, were used to calculate the junctional permeance (PA) of cell pairs according to Fick's diffusion equation. Junctional permeances show considerable variation ranging from 0.08×10^–11 to 27.0×10^–11 cm³/sec. Using the meanPA and a previous estimate of the mean number of junctional channels per interface in the Novikoff cultures, a value for diffusion coefficient of LY through gap junctions is calculated to be about 1.4×10^–6 cm²/sec. There is a general proportionality between meanPA and cell volume for hepatoma cell pairs of a certain size range. Such a relationship between cell volume and junctional capacity suggests one source of variation ofPA. Other possible sources, e.g., related to position in the cell cycle, are discussed.     

Connexin32 gap junction channels in stably transfected cells: unitary conductance.

Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.     

Effect of Membrane Tension on Gap Junctional Conductance of Supporting Cells in Corti's Organ

The effects of turgor pressure-induced membrane tension on junctional coupling of Hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques. Turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions. Both positive pipette pressure and extracellular applications of hypotonic solutions, which caused cell size to concomitantly increase, uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance. These changes were, in many cases, reversible and repeatable. Intracellular application of 50 μM H-7, a broad-based protein kinase inhibitor, and 10 mM BAPTA did not block the uncoupling effect of positive turgor pressure on inner ear gap junctions. The transjunctional conductance at a holding potential of −80 mV was 53.6 ± 5.8 nS (mean ± SEM, n = 9) and decreased ∼40% at a turgor pressure of 1.41 ± 0.05 kPa. Considering the coincident kinetics of cell deformation and uncoupling, we speculate that mechanical forces work directly on gap junctions of the inner ear. These results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

ATP activates P2X receptors to mediate gap junctional coupling in the cochlea

ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or oxidized ATP (oATP, 0.1 mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca²⁺ influxing, thereby increasing intracellular Ca²⁺ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K⁺-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.     

Effects of Epidermal Growth Factor and Phorbol Ester on Thyroid Epithelial Integrity

The effects of epidermal growth factor (EGF) and phorbol ester (tetra-O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance (R_TE > 6 · 10³ Ω · cm²) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [³H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained (R_TE 1-3 · 10³ Ω · cm²; impermeability to [³H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca²⁺ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 μM) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [³H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca²⁺-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.     

Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide,vasoactive intestinal peptide (VIP) and a VIP-antagonist

Cholecystokinin-octapeptide (CCK-OP) induces a time- and dose-dependent decrease of gap Junctional conductance in isolated pairs of pancreatic acinar cells. In double whole-cell experiments, the time course could be described by the latency and the half-life time (t _1/2 ) of cell-to-cell uncoupling. The latency shows a biphasic dependence on [CCK-OP] with a minimum of about 50 sec at 10^–9 m CCK-OP. In the presence of vasoactive intestinal peptide (VIP), the biphasic relationship is shifted to lower CCK-OP concentrations. The increase of latency at high concentrations of CCK-OP (> 10⁰⁹ m) was blocked by addition of a VIP-antagonist. t _1/2 decreases monophasically with increasing [CCKOP]. Addition of GTPS to the pipette solution suppresses the [CCK-OP] dependence of the latency and potentiates the uncoupling phase. The kinetic data are discussed in terms of CCK binding to receptors of high and low affinity. Evidence is presented that secretion and cell-to-cell coupling are not related by an all-ornone process, but that for physiological CCK-OP concentrations, gap junctional uncoupling follows secretion.The authors would like to thank Dipl. Biol. F. Mendez for his support in software development for analysis of gap junctional conductance. The work was supported by the Graduiertenkolleg Biochemische Pharmakologie, the Herrmann und Lilly Schilling Stiftung and the Sonderforschungsbereich 156 of the Deutsche Forschungsgemeinschaft.     

HIF-1α/ERK通路活化调控微波长期辐射致大鼠海马神经元线粒体损伤

本文旨在探讨微波辐射致大鼠海马神经元线粒体损伤中HIF-1α和ERK通路分子表达的改变及意义,为深入研究微波辐射损伤机制和防治提供新靶标.2.5,5和10mW/cm2的微波辐射100只雄性Wistar大鼠,辐射时间为6min/次,5次/周,连续辐射1月,于辐射后6h,7d,14d,1周和2月,采用Real-timePCR,Westernblot和免疫组织化学检测海马中hif-1αmRNA,HIF-1α,ERK1/2和p-ERK1/2表达.结果发现,大鼠海马hif-1αmRNA和HIF-1α蛋白分别在2.5和5mW/cm2组于辐射后14d和1月明显增加,10mW/cm2组辐射后14d～2月降低.但海马ERK1/2未见明显改变.假辐射组p-ERK1/2于海马神经元胞浆中呈弱阳性,2.5mW/cm2组p-ERK1/2表达无明显变化,5和10mW/cm2辐射后7d～1月,p-ERK1/2于海马神经元胞浆和胞核中呈阳性或强阳性.2.5,5和10mW/cm2微波长期辐射后大鼠海马HIF-1α和p-ERK1/2表达的改变,表明HIF-1α和ERK通路活化参与微波辐射致海马线粒体损伤的过程,并可能发挥修复线粒体损伤的作用.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Combination of high‐frequency ultrasound and UV radiation of excilamp for surface disinfection

The combination of high‐frequency ultrasound (HFUS) and UV represents a new approach to disinfecting surfaces. This study aimed to examine the inactivation efficiency of HFUS (1.7 MHz) and monochromatic UV radiation of KrCl excilamp (222 nm) in a single and a sequential mode against Bacillus cereus cells and spores added to glass surfaces. When treated by UV only, cells at populations of 10³, 10⁴, and 10⁵ colony‐forming units (CFU)/cm² showed 100% disinfection at high doses up to 1760 mJ/cm². Spores at 10⁴ CFU/cm² were completely inactivated at a dose of 1170 mJ/cm². Treatment with aqueous aerosol (produced by HFUS) reduced cell counts by 100% within a 40‐min exposure, whereas it was ineffective in inactivating spores under these conditions. In a sequential mode, the contaminated surface was pretreated with the sonicated aqueous aerosol and subsequently irradiated with the excilamp. It was found that HFUS exposure times and UV doses for complete inactivation decreased by a factor of 2 and 6–7, respectively, compared to sole HFUS or UV. A portable apparatus for surface disinfection was designed. The combined HFUS/UV method may be a promising technique for rapid disinfection of microbially contaminated surfaces.     

Changes in the human nuclear chromatin induced by ultra wideband pulse irradiation

The effects of ultra wideband pulse radiation on human cells were investigated. The density of the flow of energy on the surface of irradiated object varied from 10⁻⁶ to 10⁻² W/cm² with exposure of 10 s. It was shown that heterochromatin granule quantity in cell nuclei increased under the influence of radiation from 10⁻⁴ to 10⁻² W/cm². In some intervals the effect increased with irradiation dose. At irradiation intensity 10⁻³ W/cm² the process of heterochromatin granule formation was fully reversible after 2 h of recovery; at intensity 10⁻² W/cm² the reversion of irradiation effects was not full. The data obtained indicated the strong biological activity of ultra wideband ultra short pulse radiation.     

Temperature Characteristics of Excitation in Space-Clamped Squid Axons

Temperature characteristics of excitability in the squid giant axon were measured for the space-clamped axon with the double sucrose gap technique. Threshold strength-duration curves were obtained for square wave current pulses from 10 µsec to 10 msec and at temperatures from 5°C to 35°C. The threshold change of potential, at which an action potential separated from a subthreshold response, averaged 17 mv at 20°C with a Q¹⁰ of 1.15. The average threshold current density at rheobase was 12 µa/cm² at 20°C with a Q¹⁰ of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10^-8 coul/cm². This was relatively independent of temperature and occasionally showed a minimum in the temperature range. At intermediate times and all temperatures the threshold currents were less than for both the single time constant model and the two factor excitation process as developed by Hill. FitzHugh has made computer investigations of the effect of temperature on the excitation of the squid axon membrane as represented by the Hodgkin-Huxley equations. These are in general in good agreement with our experimental results.     

Junctional membrane permeability

Summary Substitution of extracellular Na⁺ by Li⁺ causes depression of junctional membrane permeability inChironomus salivary gland cells; within 3 hr, permeability falls to so low a level that neither fluorescein nor the smaller inorganic ions any longer traverse the junctional membrane in detectable amounts (uncoupling). The effect is Li-specific: if choline⁺ is the Na⁺ substitute, coupling is unchanged. The Li-produced uncoupling is not reversed by restitution of Na⁺. Long-term exposure (>1 hr) of the cells to Ca, Mg-free medium leads also to uncoupling. This uncoupling is fully reversible by early restitution of Ca⁺⁺ or Mg⁺⁺. Coupling is maintained in the presence of either Ca⁺⁺ or Mg⁺⁺, so long as the total divalent concentration is about 12mm. The uncoupling in Ca, Mg-free medium ensues regardless of whether the main monovalent cation is Na, Li or choline.The uncouplings are accompanied by cell depolarization. Repolarization of the cells by inward current causes restoration of coupling; the junctional conductance rises again to its normal level. The effect was shown for Li-produced uncoupling, for uncoupling by prolonged absence of external Ca⁺⁺ and Mg⁺⁺, and for uncoupling produced by dinitrophenol. In all cases, the recoupling has the same features: (1) it develops rapidly upon application of the polarizing current; (2) it is cumulative; (3) it is transient, but outlasts the current; and (4) it appears not to depend on the particular ions carrying the current from the electrodes to the cell. The recoupling is due to repolarization of nonjunctional cell membrane; recoupling can be produced at zero net currernt through the junctional membrane. Recoupling takes place also as a result of chemically produced repolarization; restoration of theK gradients in uncoupled cells causes partial recoupling during the repolarization phase.An explanation of the results on coupling is proposed in terms of known mechanisms of regulation of Ca⁺⁺ flux in cells. The uncouplings are explained by actions raising the Ca⁺⁺ level in the cytoplasmic environment of the junctional membranes; the recoupling is explained by actions lowering this Ca⁺⁺ level.     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.     

Variation in sessile microflora during biofilm formation on AISI-304 stainless steel coupons

Coupons of stainless steel type AISI-304 were exposed to the industrial cooling system of a petrochemical plant fed by seawater from the Guanabara Bay, Rio de Janeiro, Brazil, in order to study thein situ formation of biofilms. Bacteria, microalgae and fungi were detected on the coupons as soon as 48 h after exposure. Their respective numbers were determined at times 48, 96 and 192 h and over the following 8 weeks. Aerobic, anaerobic and sulfate-reducing bacteria were quantified according to the technique of the most probable number, and fungi by the pour plate technique. The number of microorganisms present in the forming biofilm varied over the experimental period, reaching maximal levels of 14×10¹¹ cells cm^–2, 30×10¹³ cells cm^–2, 38×10¹¹ cells cm^–2 and 63×10⁵ cells cm^–2, respectively, for aerobic bacteria, anaerobic bacteria, sulfate-reducing bacteria and fungi, and the dynamics of this variation depended on the group of microorganisms.Bacillus sp,Escherichia coli, Serratia sp andPseudomonas putrefaciens were identified among the aerobic bacteria isolated. Additionally, microalgae and bacteria of the genusGallionella were also detected. Nonetheless, no evidence of corrosion was found on the stainless steel type AISI-304 coupons over the experimental period.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

New Insights on the Burstein-Moss Shift and Band Gap Narrowing in Indium-Doped Zinc Oxide Thin Films

The Burstein-Moss shift and band gap narrowing of sputtered indium-doped zinc oxide (IZO) thin films are investigated as a function of carrier concentrations. The optical band gap shifts below the carrier concentration of 5.61 × 10¹⁹ cm^-3 are well-described by the Burstein-Moss model. For carrier concentrations higher than 8.71 × 10¹⁹ cm^-3 the shift decreases, indicating that band gap narrowing mechanisms are increasingly significant and are competing with the Burstein-Moss effect. The incorporation of In causes the resistivity to decrease three orders of magnitude. As the mean-free path of carriers is less than the crystallite size, the resistivity is probably affected by ionized impurities as well as defect scattering mechanisms, but not grain boundary scattering. The c lattice constant as well as film stress is observed to increase in stages with increasing carrier concentration. The asymmetric XPS Zn 2p_3/2 peak in the film with the highest carrier concentration of 7.02 × 10²⁰ cm^-3 suggests the presence of stacking defects in the ZnO lattice. The Raman peak at 274 cm^-1 is attributed to lattice defects introduced by In dopants.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.