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Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation.

Disulfide trapping studies have revealed that the pore-lining (P) segments of voltage-dependent sodium channels undergo sizable motions on a subsecond time scale. Such motions of the pore may be necessary for selective ion translocation. Although traditionally viewed as separable properties, gating and permeation are now known to interact extensively in various classes of channels. We have investigated the interaction of pore motions and voltage-dependent gating in micro1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation (kSS) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the kSS, which first increased to a maximum at 200 msec and then decreased with longer depolarizations. This result suggested that occupancy of an intermediate inactivation state (IM) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, kSS varied inversely with external [Na+]o. We examined the converse relationship, namely the effect of pore flexibility on gating, by measuring recovery from inactivation in Y401C+E758C (YC/EC) channels. Under oxidative conditions, recovery from inactivation was slower than in a reduced environment in which the spontaneous YC/EC cross-link is disrupted. The most prominent effects were slowing of a component with intermediate recovery kinetics, with diminution of its relative amplitude. We conclude that occupancy of an intermediate inactivation state facilitates motions of the P segments; conversely, flexibility of the P segments alters an intermediate component of inactivation.

Animals↗

EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins.

Exon trapping from a bacterial artificial chromosome (BAC 78138) mapping to the 19q13.3 glioma tumor suppressor candidate region yielded two exons that recognized a 3.6-kb transcript on Northern blot. Screening of a human fetal brain cDNA library with these exons identified three novel genes, designated EHD2, EHD3, and EHD4, which are homologous to the recently characterized human EHD1 (testilin/HPAST) and its mouse homolog Ehd1, as well as to homologs in Drosophila (Past1) and Caenorhabditis elegans. Alignment of the predicted peptide sequences revealed striking similarities, with multiple conserved regions that include a nucleotide-binding consensus site at the N-terminus, a bipartite nuclear localization signal, and an eps15 homology (EH) protein-binding domain with an EF-hand motif at the C-terminus. The genes are specifically expressed, with EHD2 highly expressed in heart, EHD3 in brain and heart, and EHD4 in heart and pancreas. EHD2 was confirmed to originate from BAC 78138 at 19q13.3; radiation hybrid mapping localized EHD3 and EHD4 to 2p21 and 15q11.1, respectively; EHD1 has been previously mapped to 11q13. The three EHD1 paralogs therefore represent novel members of a family of human EH domain-containing proteins that may play a role in endocytosis and signaling. Mutation analysis of the five coding exons of EHD2 in gliomas failed to detect any tumor-specific alterations, thus indicating that EHD2 is an unlikely candidate for the 19q tumor suppressor gene.

Amino Acid Sequence↗

Recipes for reconstituting skin.

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF beta 1 increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGF beta 1 present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 micrograms/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 micrograms/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent (LDE) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Contraction of collagen lattice by peritubular cells from rat testis.

Peritubular cells from 15- and 25-day-old rat testis trapped in collagen lattices caused those lattices to contract. Contraction proceeded more rapidly and to a greater extent using cells from younger rats. When 36,000 cells from 15- and 25-day-old rats were trapped in 800 mm2 lattices, the areas were reduced to 28 mm2 and 170 mm2, respectively, within 24 h. The cells from older rats were less effective at contracting the lattice than cells from younger rats. Cytochalasin B (5 micrograms ml-1) inhibited lattice contraction and caused disruption of actin filaments as seen by fluorescent staining with Rh-phalloidin. Cholera toxin (10 micrograms ml-1), and 1 mM-dibutyryl cAMP inhibited lattice contraction, as did 10 microM-trifluoperazine, commonly an inhibitor of calmodulin. The total intracellular concentration of cAMP was greater in peritubular cells from 25-day-old rats than in those from 15-day-old rats: 427 +/- 34 and 120 +/- 16 pmol mg-1 cell protein, respectively. When peritubular cells in monolayer were permeabilized with glycerol, the addition of ATP caused the cells to contract. Cell contraction was greater in cells from 15-day-old rats than 25-day-old rats. When cells were grown on silicone rubber, they caused that surface to wrinkle. Peritubular cells from 15-day-old rats caused the onset of wrinkling at 4 h. At the same time, no wrinkling was observed with cells from 25-day-old rats. Studies of lattice contraction and cell contraction were also made using cells from 20-day-old rats. In each case, contraction was intermediate between that of cells from 15-and 25-day-old rats. The possibility exists that lattice contraction, cell contraction and wrinkling of silicone film result from a mechanism of actin filament sliding, generated by myosin ATPase activity, and is inhibited by cAMP. The reduced rate of contraction in cells from 25-day-old rats may be related to their higher intracellular levels of cAMP. Evidence exists to show that cAMP blocks myosin ATPase activity by inhibiting the phosphorylation of its regulatory peptide, myosin light chain.

Animals↗

Influencing cellular transformation by modulating the rates of GTP hydrolysis by Cdc42.

The small GTPase Cdc42 has been implicated in a number of cellular responses ranging from the regulation of the actin cytoskeletal architecture to intracellular trafficking and cell cycle progression. Cdc42 mutants that constitutively exchange GDP for GTP but still hydrolyze GTP (called 'fast-cycling' mutants) promote cellular transformation, whereas Cdc42 mutants that are unable to hydrolyze GTP and are irreversibly trapped in the GTP-bound state often inhibit cell growth. In this work, we have set out to further establish that Cdc42 needs to cycle between its 'on' and 'off' states to stimulate cell growth, by examining the consequences of manipulating its GTP-binding/GTP hydrolytic cycle in two different ways. One approach was to examine whether substitutions that act in a manner opposite to the 'fast cyclers', and extend the lifetime of the activated GTP-bound state by slowing the GTP hydrolytic reaction (i.e., 'slow-cycling' mutations), positively influence cell growth. Indeed we show that one such slow-cycling mutant, Cdc42[Y32A], which is insensitive to Cdc42GAP but still exhibits a measurable intrinsic GTP hydrolytic activity, gives rise to increased levels of activated Cdc42 in NIH 3T3 cells. We go on to show that the Y32A mutant stimulates the actin cytoskeletal changes that lead to filopodia formation, confer growth advantages to fibroblasts under low serum conditions, and enable cells to grow to high densities when exposed to normal levels of serum. The second approach was to determine whether the transforming activity of the fast-cycling Cdc42[F28L] mutant can be reversed by compensating for its accelerated nucleotide exchange reaction through the expression of the GTPase-activating protein (Cdc42GAP) and the ensuing stimulation of GTP hydrolytic activity. We showed that expression of the limit functional domain of Cdc42GAP inhibited Cdc42[F28L]-induced transformation, as well as selectively reversed the transformed phenotypes caused by the hyperactivation of wild-type Cdc42 in cells expressing the oncogenic version of Dbl (for Diffuse B cell lymphoma), a guanine nucleotide exchange factor for Cdc42 and the related Rac and Rho GTPases. Overall, the results reported here establish the requirement for Cdc42 to cycle between its signaling-on and -off states in order to positively influence cell growth and highlight how the Cdc42GAP can play an important role in regulating cell proliferation.

Animals↗

Upflow anaerobic sludge blanket reactor--a review.

Biological treatment of wastewater basically reduces the pollutant concentration through microbial coagulation and removal of non-settleable organic colloidal solids. Organic matter is biologically stabilized so that no further oxygen demand is exerted by it. The biological treatment requires contact of the biomass with the substrate. Various advances and improvements in anaerobic reactors to achieve variations in contact time and method of contact have resulted in development of in suspended growth systems, attached growth or fixed film systems or combinations thereof. Although anaerobic systems for waste treatment have been used since late 19th century, they were considered to have limited treatment efficiencies and were too slow to serve the needs of a quickly expanding wastewater volume, especially in industrialized and densely populated areas. At present aerobic treatment is the most commonly used process to reduce the organic pollution level of both domestic and industrial wastewaters. Aerobic techniques, such as activated sludge process, trickling filters, oxidation ponds and aerated lagoons, with more or less intense mixing devices, have been successfully installed for domestic wastewater as well as industrial wastewater treatment. Anaerobic digestion systems have undergone modifications in the last two decades, mainly as a result of the energy crisis. Major developments have been made with regard to anaerobic metabolism, physiological interactions among different microbial species, effects of toxic compounds and biomass accumulation. Recent developments however, have demonstrated that anaerobic processes might be an economically attractive alternative for the treatment of different types of industrial wastewaters and in (semi-) tropical areas also for domestic wastewaters. The anaerobic degradation of complex, particulate organic matter has been described as a multistep process of series and parallel reactions. It involves the decomposition of organic and inorganic matter in the absence of molecular oxygen. Complex polymeric materials such as polysaccharides, proteins, and lipids (fat and grease) are first hydrolyzed to soluble products by extracellular enzymes, secreted by microorganisms, so as to facilitate their transport or diffusion across the cell membrane. These relatively simple, soluble compounds are fermented or anaerobically oxidized, further to short-chain fatty acids, alcohols, carbon dioxide, hydrogen, and ammonia. The short-chain fatty acids (other than acetate) are converted to acetate, hydrogen gas, and carbon dioxide. Methanogenesis finally occurs from the reduction of carbon dioxide and acetate by hydrogen. The initial stage of anaerobic degradation, i.e. acid fermentation is essentially a constant BOD stage because the organic molecules are only rearranged. The first stage does not stabilize the organics in the waste. However this step is essential for the initiation of second stage methane fermentation as it converts the organic material to a form, usable by the methane producing bacteria. The second reaction is initiated when anaerobic methane forming bacteria act upon the short chain organic acids produced in the 1st stage. Here these acids undergo methane fermentation with carbon dioxide acting as hydrogen acceptor and getting reduced to methane. The methane formed, being insoluble in water, escapes from the system and can be tapped and used as an energy source. The production and subsequent escape of methane causes the stabilization of the organic material. The methane-producing bacteria consist of several different groups. Each group has the ability to ferment only specific compounds. Therefore, the bacterial consortia in a methane producing system should include a number of different groups. When the rate of bacterial growth is considered, then the retention time of the solids becomes important parameter. The acid fermentation stage is faster as compared to the methane fermentation stage. This means that a sudden increase in the easily degradable organics will result in increased acid production with subsequent accumulation of acids. This inhibits the methanogenesis step. Acclimatization of the microorganisms to a substrate has been reported to take more than five weeks. Sufficiently acclimated bacteria have shown greater stability towards stress-inducing events such as hydraulic overloads, fluctuations in temperature, fluctuations in volatile acid and ammonia concentrations etc. Several environmental factors can affect anaerobic digestion, by altering the parameters such as specific growth rate, decay rate, gas production, substrate utilization, start-up and response to changes in input. It has long been recognized that an anaerobic process is in many ways ideal for wastewater treatment and has following merits: A high degree of waste stabilization A low production of excess A low nutrient requirements No oxygen requirement Production of methane gas Anaerobic microorganisms, especially methanogens have a slow growth rate. At lower HRTs, the possibility of washout of biomass is more prominent. This makes it difficult to maintain the effective number of useful microorganisms in the system. To maintain the population of anaerobes, large reactor volumes or higher HRTs are required. This may ultimately provide longer SRTs upto 20 days for high rate systems. Thus, provision of larger reactor volumes or higher HRTs ultimately lead to higher capital cost. Among notable disadvantages, it has low synthesis/reaction rate hence long start up periods and difficulty in recovery from upset conditions. Special attention is, therefore, warranted towards, controlling the factors that affect process adversely; important among them being environmental factors such as temperature, pH and concentration of toxic substances. The conventional anaerobic treatment process consists of a reactor containing waste and biological solids (bacteria) responsible for the digestion process. Concentrated waste (usually sewage sludge) can be added continuously or periodically (semi-batch operation), where it is mixed with the contents of the reactor. Theoretically, the conventional digester is operated as a once-through, completely mixed, reactor. In this particular mode of operation the hydraulic retention time (HRT) is equal to the solids retention time (SRT). Basically, the required process efficiency is related to the sludge retention time (SRT), and hence longer SRT provided, results in satisfactory population (by reproduction) for further waste stabilization. By reducing the hydraulic retention time (HRT) in the conventional mode reactor, the quantity of biological solids within the reactor is also decreased as the solids escape with the effluent. The limiting HRT is reached when the bacteria are removed from the reactor faster than they can grow. Methanogenic bacteria are slow growers and are considered the rate-limiting component in the anaerobic digestion process. The first anaerobic process developed, which separated the SRT from the HRT was the anaerobic contact process. In 1963, Young and McCarty (1968) began work, which eventually led to the development of the anaerobic upflow filter (AF) process. The anaerobic filter represented a significant advance in anaerobic waste treatment, since the filter can trap and maintain a high concentration of biological solids. By trapping these solids, long SRT's could be obtained at large waste flows, necessary to anaerobically treat low strength wastes at nominal temperatures economically. Another anaerobic process which relies on the development of biomass on the surfaces of a media is an expanded bed upflow reactor. The primary concept of the process consists of passing wastewater up through a bed of inert sand sized particles at sufficient velocities to fluidize and partially expand the sand bed. One of the more interesting new processes is the upflow anaerobic sludge blanket process (UASB), which was developed by Lettinga and his co-workers in Holland in the early 1970's. The key to the process was the discovery that anaerobic sludge inherently has superior flocculation and settling characteristics, provided the physical and chemical conditions for sludge flocculation are favorable. When these conditions are met, a high solids retention time (at high HRT loadings) can be achieved, with separation of the gas from the sludge solids. The UASB reactor is one of the reactor types with high loading capacity. It differs from other processes by the simplicity of its design. UASB process is a combination of physical & biological processes. The main feature of physical process is separation of solids and gases from the liquid and that of biological process is degradation of decomposable organic matter under anaerobic conditions. No separate settler with sludge return pump is required, as in the anaerobic contact process. There is no loss of reactor volume through filter or carrier material, as in the case with the anaerobic filter and fixed film reactor types, and there is no need for high rate effluent recirculation and concomitant pumping energy, as in the case with fluidized bed reactor. Anaerobic sludge inherently possesses good settling properties, provided the sludge is not exposed to heavy mechanical agitation. For this reason mechanical mixing is generally omitted in UASB-reactors. At high organic loading rates, the biogas production guarantees sufficient contact between substrate and biomass. Regarding the dynamic behaviour of the water phase UASB reactor approaches the completely mixed reactor. For achieving the required sufficient contact between sludge and wastewater, the UASB-system relies on the agitation brought about by the natural gas production and on an even feed inlet distribution at the bottom of the reactor. (ABSTRACT TRUNCATED)

Bacteria, Anaerobic↗