Protamine alters structure and conductance ofNecturus gallbladder tight junctions without major electrical effects on the apical cell membrane

Summary Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 m), induces about a twofold increase in transepithelial resistance inNecturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity.In this leaky epithelium, where >90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular actiona priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50mm) caused changes in apical membrane voltage not different from control.To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixedin situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork.Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.     

Noise analysis of the K+ current through the apical membrane ofNecturus gallbladder

Summary Current noise power spectra of the voltage-clamped (V=0)Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f noise component, with being about 2. Substitution of all Na⁺ by K⁺ on either the serosal or mucosal side increased the relaxation as well as the 1/f noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm 2,4,6-triaminopyrimidine (TAP) or 5mm of tetraethylammonium (TEA⁺) added to ification of the mucosal solution to pH 5 and lower. Fivemm of tetraethylammonium (TEA⁺) added to the mucosal solution, abolished K⁺ relaxation noise and decreased the 1/f noise component. Applying a Cs⁺ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb⁺ was substituted for all Na⁺ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K⁺ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K⁺>Rb⁺>Sc⁺. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.     

The formation of intercellular junctions in insect stem cell progeny (cockroach intestinal epithelium)

Summary The stages in the development of intercellular junctions have been followed in the mesenteric caecal cells of the cockroach midgut, where two types of mature cell, the columnar and the secretory, exist. Nests of undifferentiated replacement cells occur at intervals along the basal lamina, consisting of central, dividing cells and peripheral semi-lunar cells; the former act as proliferative stem cells to give rise to either pre-columnar or pre-secretory cells. The semi-lunar cells are pre-columnar and produce an attenuated process which gradually projects up to the luminal surface, producing microvilli and a dense extracellular substance en route. Intercellular gap junctions appear between these maturing columnar cell borders first, while septate junctions differentiate later; these are assembled from two different sets of intramembranous particle which become organized into either plaques or rows in parallel alignment, possibly mediated by actin filaments and microtubules. The pre-secretory cells, which are much fewer in number, remain associated only with the basal lamina and never reach the lumen; they develop into one of three distinct mature secretory cell types which release their secretory product in different ways. Offprint requests to: N.J. Lane     

Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

The duct system of the avian salt gland as a transporting epithelium: structure and morphometry in the duck Anas platyrhynchos

Summary The duct system of the nasal salt gland of the duck comprises central canals, secondary ducts and main ducts. The secondary and main ducts consist of a layer of columnar cells overlying a layer of small cuboidal cells. The columnar cells have complex intercellular spaces showing evidence of Na⁺ K⁺ -ATPase at the apical regions. Approximately 70% of surface area of the duct system is external to the gland. During adaptation to salt water the duct system increases in size as does the gland. Although the components of the gland of adapted ducks, including the duct system within the gland, increase in size compared with normal ducks, the percentage volume densities of the components remain similar in both categories of ducks, i.e. the duct system increases in size in proportion to the glandular tissue. The volume of the duct system external to the gland is six to seven times larger than the volume within the gland. Thus, if ductal modification of secreted fluid occurs, it will be most likely to take place in the ducts external to the gland.Total surface areas of the duct system were measured from serial sections of glands and ducts from one normal and one adapted duck. These were used to calculate possible flux rates of water and sodium across the duct epithelium, assuming the occurrence of either water reabsorption or sodium secretion. Although these flux rates are high it is shown that they are similar to calculated flux rates across the luminal surface of the secretory tubules.     

Glutamatergic synapses of Drosophila neuromuscular junctions: a high-resolution model for the analysis of experience-dependent potentiation

The glutamatergic synapses of developing neuromuscular junctions (NMJ) of Drosophila larvae are readily accessible, morphologically simple, and physiologically well-characterized. They therefore have a long and highly successful tradition as a model system for the discovery of genetic and molecular mechanisms of target recognition, synaptogenesis, NMJ development, and synaptic plasticity. However, since the development and the activity-dependent refinement of NMJs are concurrent processes, they cannot easily be separated by the widely applied genetic manipulations that mostly have chronic effects. Recent studies have therefore begun systematically to incorporate larval foraging behavior into the physiological and genetic analysis of NMJ function in order to analyze potential experience-dependent changes of glutamatergic transmission. These studies have revealed that recent crawling experience is a potent modulator of glutamatergic transmission at NMJs, because high crawling activities result after an initial lag-phase in several subsequent phases of experience-dependent synaptic potentiation. Depending on the time window of occurrence, four distinct phases of experience-dependent potentiation have been defined. These phases of potentiation can be followed from their initial induction (phase-I) up to the morphological consolidation (phase-III/IV) of previously established functional changes (phase-II). This therefore establishes, for the first time, a temporal hierarchy of mechanisms involved in the use-dependent modification of glutamatergic synapses.     

Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Mechanisms of fluid and ion secretion by the parotid gland of the kangaroo,Macropus rufus,assessed by administration of transport-inhibiting drugs

Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05–0.5 mmol·l^-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0±0.04 to 0.22±0.024 ml·min^-1 (mean±SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135±1.6 to 67±1.7 mmol·l^-1 and 272±7.6 to 15±2.6 mol·min^-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11±1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol·l^-1), bumetanide (up to 0.2 mmol·l^-1) and ethacrynate (1 mmol·l^-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25–1.0 mmol·l^-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid at 0.1 mmol·l^-1 was without effect, whereas at 0.5 mmol·l^-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (>90%) by secretion of bicarbonate which is formed in the endpiece cells from CO₂ delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na⁺-K⁺-2Cl^- symports, Na⁺-Cl^- symports or Cl^-/HCO ₃ ^- antiports.Abbreviations CA carbonic anhydrase - CAI carbonic anhydrase inhibitors - MAP mean arterial blood pressure - PAH p-aminohippurate - SITS 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid     

The absence of cytoplasm ribosomes on the envelopes of chloroplasts and mitochondria in plants: Implications for the mechanism of transport of proteins into these organelles

Summary Chloroplasts and mitochondria ofChlamydomonas were examined by electron microscopy to determine if cytoplasmic ribosomes were associated with the envelopes of these organelles. Cells were treated with cycloheximide to prevent polypeptide chain completion, and resultant dissociation of envelope-ribosome associations. No extensive association of cytoplasm ribosomes with envelopes of chloroplasts, or mitochondria was detected in intact cells or in damaged cells in which cytoplasm was partly removed. Our results indicate that association of cytoplasm ribosomes with envelopes of chloroplasts or mitochondria is not an essential requirement for transport of polypeptides from cytoplasm to organelle.     

Photoinhibition of photosystem II in vivo is preceded by down-regulation through light-induced acidification of the lumen: Consequences for the mechanism of photoinhibition in vivo

The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (q_N) and photochemical quenching (q_p) of chlorophyll fluorescence were monitored. The ‘energy’ and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5–20° C), [CO₂] and [O₂] at different PFDs. A linear or curvilinear relationship between q_p and photoinhibition of PSII was observed. When [CO₂] and [O₂] were both low (30 μl · l^?1 and 2%, respectively), PSII was less susceptible at a given q_p than at ambient or higher [CO₂] and photoinhibition became only substantial when q_p decreased below 0.3. When high levels of energy-dependent quenching (q_E) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from q_E to photoinhibitory quenching (q_I). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by q_E. The shift from q_E to q_I occurred concomitant with q_P decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between q_P, q_E and photoinhibition of PSII at low temperature.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase     

Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents 7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole system inP. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems.     

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: A new method of testing for diffusion-limited ion flow

The kinetics of single K⁺ channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the tilt effect model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092–8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca²⁺ binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter^–1 vacuolar or 1 mmol liter^–1 cytoplasmic Ca²⁺; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca²⁺ at membrane PDs more negative than –100 mV; and (3) the effect of Mg²⁺ on channel conductance when added to solutions initially containing 3 mmol liter^–1 KCl.The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg²⁺ on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg²⁺ effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than –70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg²⁺ within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg²⁺ of surface potentials arising from diffusion-limited flow of K⁺.     

Differential glycosylation of N-POMC regulates the production of γ3-MSH by purified pro-opiomelanocortin converting enzyme A possible mechanism for tissue-specific processing

The amino terminus of bovine pro-opiomelanocortin (N-POMC^1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC^1–49 and lys-γ₃ -melanotropin. Two pools of N-POMC^1–77 were isolated which were differentially glycosylated at threonine⁴⁵, while N-POMC^1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC^1–77 may regulate cleavage at the Arg⁴⁹-Lys⁵⁰ processing site. We tested this hypothesis by incubating N-POMC^1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC^1–77 and O-glycosylated N-POMC^1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ₃ -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.     

A novel method for the quantitative assessment of the percolation of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> conidia through horticultural growing media

Incorporation of fungal biological control agents (BCAs) into plant growing media has considerable ergonomic and economic benefits for growers. These agents usually give prophylactic control of target pests and diseases. However, their efficacy is dose dependent and loss of inoculum through leaching could influence the degree of protection they provide. At present there are no protocols to determine the loss of inoculum in the disparate growing media used in horticulture. We describe a method based on a nutrient leaching column to quantify leaching of conidia of the insect-pathogenic fungus Metarhizium anisopliae in a range of growing media. Conidia of this biocontrol agent were applied as a drench or premixed into the medium. Both the application method and growth medium influenced conidial leaching. Inoculum losses were greater following drench application than premixing (95% vs. 15%) irrespective of media type. Comparatively more inoculum was lost from bark and coir following drench application whereas losses were relatively high in peat following premixed application. The leaching column assay provided a simple and accurate method to quantify inoculum loss in real time. This assay could help determine leaching of other fungal BCAs in growing media. It could help in improving pest and disease control by optimizing the rate and frequency of conidial application as well in the design of more efficacious formulations.