Cooperative properties of hormone receptors in cell membranes

The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady–state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.     

On the origin of molecular “handedness” in living systems

Elementary particle effects (beta-decay) provide at best only a weakly handed radiation in the biologically effective energy ranges. Global magnetic effects coupled to sunlight are randomized by paleomagnetic reversals. Hence a persistent terrestrial handed bias at possible local biopoetic sites offers a more promising explanation for the origin of the "handedness" of the molecules found among living systems on earth. Magnetite in lava flows maintains a handed bias for surface catalysis through many magnetic reversals. Magnetite contaminated with sulfur has already been proposed by Granick as a biopoetic site because it provides a weak source of chemical energy derived by photochemical conversion. Indirect evidence for this hypothesis has been provided by the molecular structure of ferredoxin - a single strand of the 14 primordial amino acids wrapped around an FeS core. Lava flows have been suggested as biopoetic sites by Fox, since their temperature and chemical composition might allow for the rapid synthesis of prebiotic compounds at the surface of the primitive earth. The additional fact that magnetite in lave flows also provides a persistent handed site for surface catalysis offers a further argument for the experimental investigation of this specific biopoetic environment.     

Assessment of the stream invertebrate β‐diversity along an elevation gradient using a bidimensional null model analysis

β‐Diversity, commonly defined as the compositional variation among localities that links local diversity (α‐diversity) and regional diversity (γ‐diversity), can arise from two different ecological phenomena, namely the spatial species turnover (i.e., species replacement) and the nestedness of assemblages (i.e., species loss). However, any assessment that does not account for stochasticity in community assembly could be biased and misinform conservation management. In this study, we aimed to provide a better understanding of the overall ecological phenomena underlying stream β‐diversity along elevation gradients and to contribute to the rich debate on null model approaches to identify nonrandom patterns in the distribution of taxa. Based on presence‐absence data of 78 stream invertebrate families from 309 sites located in the Swiss Alpine region, we analyzed the effect size of nonrandom spatial distribution of stream invertebrates on the β‐diversity and its two components (i.e., turnover and nestedness). We used a modeling framework that allows exploring the complete range of existing algorithms used in null model analysis and assessing how distribution patterns vary according to an array of possible ecological assumptions. Overall, the turnover of stream invertebrates and the nestedness of assemblages were significantly lower and higher, respectively, than the ones expected by chance. This pattern increased with elevation, and the consistent trend observed along the altitudinal gradient, even in the most conservative analysis, strengthened our findings. Our study suggests that deterministic distribution of stream invertebrates in the Swiss Alpine region is significantly driven by differential dispersal capacity and environmental stress gradients. As long as the ecological assumptions for constructing the null models and their implications are acknowledged, we believe that they still represent useful tools to measure the effect size of nonrandom spatial distribution of taxa on β‐diversity.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R? P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin. 1994 John Wiley & Sons, Inc.     

Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination

Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.