Structure-function relationships in the integument of Salamandra salamandra during ontogenetic development

Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post-metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria-rich (MR)-like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR-like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride-sensitive short-circuit current (I(SC)) indicating that functional Na+ uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl- conductance, and band 3 was not detected in its MR cells. The functional properties of MR-like and MR cells remain to be established.     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl(-) conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca(2+)-activated Cl(-) conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl(-)/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca(2+) signal in response to mucosal nucleotides that may contribute to the increased Cl(-)/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca(2+) signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

Transport of 1-kestose across the tonoplast of Jerusalem artichoke tubers

The capacity for 1-kestose uptake into the vacuole of fructan storing Jerusalem artichoke tubers was investigated. 1-kestose serves both as building block for fructan initiation and as a fructose donor for chain elongation. Tonoplast vesicles were isolated from actively storing tubers, and their vesicles were capable of transporting sucrose in a manner indicative of a sucrose/H(+) antiport. Under similar conditions, 1-kestose was not taken up by vesicles energized by either a pH jump or in the presence of ATP. When added together at 2 mM, sucrose uptake was not affected by the presence of 1-kestose. The data argues against the possible synthesis of 1-kestose in the cytosol and subsequent transport to the vacuole. The data also presents definite evidence for the existence a mechanism for sucrose accumulation in fructan storing vacuoles.     

Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells

Arf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP-fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co-expressed GFP-tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP- and GDP-fixed mutants, respectively, changed the localization of a cis-Golgi marker, AtErd2-GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP-AtRer1B or GFP-AtSed5. GFP-AtRer1B and GFP-AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin-GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.     

The β-Amyloid Precursor Protein (APP) and Alzheimer's Disease: Does the Tail Wag the Dog?

The β-amyloid precursor protein has been the focus of much attention from the Alzheimer's disease community for the past decade and a half. The β-amyloid precursor protein holds a pivotal position in Alzheimer's disease research because it is the precursor to the amyloid β-protein which many believe plays a central role in Alzheimer's disease pathogenesis. It was also the first gene in which mutations associated with inherited Alzheimer's disease were found. Although the molecular details of the generation of amyloid β-protein from β-amyloid precursor protein are being unraveled, the actual physiological functions of β-amyloid precursor protein are far from clear. This situation is changing as accumulating new evidence suggests that the C-terminal cytosolic tail of β-amyloid precursor protein may have multiple biological activities, ranging from axonal transport to nuclear signaling. This article reviews the current state of knowledge about the biological functions of β-amyloid precursor protein .     

Isolation and functional analysis of a Brassica juncea gene encoding a com-ponent of auxin efflux carrier

Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (G(?)lweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpin1), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPINl and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPINl and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpin1 was expressed in most of the tissues tested, with a relatively h     

Intramanchette transport (IMT): managing the making of the spermatid head,centrosome, and tail

Intramanchette transport (IMT) and intraflagellar transport (IFT) share similar molecular components: a raft protein complex transporting cargo proteins mobilized along microtubules by molecular motors. IFT, initially discovered in flagella of Chlamydomonas, has been also observed in cilia of the worm Caenorhabditis elegans and in mouse ciliated and flagellated cells. IFT has been defined as the mechanism by which protein raft components (also called IFT particles) are displaced between the flagellum and the plasma membrane in the anterograde direction by kinesin-II and in the retrograde direction by cytoplasmic dynein 1b. Mutation of the gene Tg737, encoding one of the components of the raft protein complex, designated Polaris in the mouse and IFT88 in both Chlamydomonas and mouse, results in defective ciliogenesis and flagellar development as well as asymmetry in left-right axis determination. Polaris/IFT88 is detected in the manchette of mouse and rat spermatids. Indications of an IMT mechanism originated from the finding that two proteins associated with the manchette (Sak57/K5 and TBP-1, the latter a component of the 26S proteasome) repositioned to the centrosome and sperm tail once the manchette disassembled. IMT has the features of the IFT machinery but, in addition, facilitates nucleocytoplasmic exchange activities during spermiogenesis. An example is Ran, a small GTPase present in the nucleus and cytoplasm of round spermatids and in the manchette of elongating spermatids. Upon disassembly of the manchette, Ran GTPase is found in the centrosome region of elongating spermatids. Because defective molecular motors and raft proteins result in defective flagella, cilia, and cilia-containing photoreceptor cells in the retina, IMT and IFT are emerging as essential mechanisms for managing critical aspects of sperm development. Details of specific role of Ran GTPase in nucleocytoplasmic transport and its relocation from the manchette to the centrosome to the sperm tail await elucidation.