Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.     

A group contribution method for the estimation of equilibrium constants for biochemical reactions

Using Gibbs Energies of compounds, as well as Gibbs Energy changes and equilibrium constants of biochemical reactions, the contributions of functional groups to the Gibbs Energy (in aqueous solution, temperature 25°C, and pH=7) have been estimated. These contributions allow the estimation of the Gibbs Free Energy and the equilibrium constant of a biochemical reaction, given the structure of the reactants and products.     

Differences in learning by foraging juvenile pumpkinseed and bluegill sunfish in a structured habitat

Synopsis We investigated the ability of two congeneric species of sunfish to learn to forage on a novel prey item in feeding arenas containing structured habitats. Eight bluegill sunfish and eight pumpkinseed sunfish were given the opportunity to forage on whiteworms daily for 10 days. Each day, several behavioural measures were recorded for each fish. Both species of sunfish learned to feed over the 10-day period but the bluegill sunfish learned to feed more quickly than the pumpkinseed sunfish. Pumpkinseeds, however, attained a higher level of foraging efficiency. The differences in learning and foraging efficiency were related to body morphology.     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

Outer membranes of Gram-negative bacteria are permeable to steroid probes

The permeability of bacterial outer membranes was assayed by coupling the influx of highly hydrophobic probes, 3-oxosteroids, with their subsequent oxidation catalysed by 3-oxosteroid delta 1-dehydrogenase, expressed from a gene cloned from Pseudomonas testosteroni. In Salmonella typhimurium producing wild-type lipopolysaccharide, the permeability coefficients for uncharged steroids were 0.45 to 1 x 10(-5) cm s-1, and the diffusion appeared to occur mainly through the lipid bilayer domains of the outer membrane. These rates are one or two magnitudes lower than that expected for their diffusion through the usual biological membranes. The permeation rates were markedly increased (up to 100 times) when the lipopolysaccharide leaflet was perturbed either by adding deacylpolymyxin or by introducing mutations leading to the production of deep rough lipopolysaccharides. An amphiphilic, negatively charged probe, testosterone hemisuccinate, penetrated much more slowly than the uncharged steroids. Study of various Gram-negative species revealed that P. testosteroni, Pseudomonas acidovorans, and Acinetobacter calcoaceticus showed higher outer membrane permeability to steroid probes and higher susceptibility to hydrophobic agents such as fusidic acid, novobiocin and crystal violet relative to S. typhimurium and Escherichia coli.