Persistent confusion of total entropy and chemical system entropy in chemical thermodynamics.

The change in free energy with temperature at constant pressure of a chemical reaction is determined by the sum (dS) of changes in entropy of the system of reagents, dS(i), and the additional entropy change of the surroundings, dS(H), that results from the enthalpy change, W. A faulty identification of the total entropy change on reaction with dS(i) has been responsible for the attribution of general validity to the expressions (d deltaG/dT)p = -deltaS(i) and d(deltaG/T)/d(1/T)= deltaH, which are found in most textbooks and in innumerable papers.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

RNA-peptide fusions for the in vitro selection of peptides and proteins

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.

The estimation of statistical parameters for local alignment score distributions

The distribution of optimal local alignment scores of random sequences plays a vital role in evaluating the statistical significance of sequence alignments. These scores can be well described by an extreme-value distribution. The distribution’s parameters depend upon the scoring system employed and the random letter frequencies; in general they cannot be derived analytically, but must be estimated by curve fitting. For obtaining accurate parameter estimates, a form of the recently described ‘island’ method has several advantages. We describe this method in detail, and use it to investigate the functional dependence of these parameters on finite-length edge effects.

A host–guest system to study structure–function relationships of membrane fusion peptides

We designed a host–guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted (≈33° from the surface) α-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n = 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is −7.6 kcal/mol (1 cal = 4.18 J). This energy is sufficient to stabilize a “stalk” intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.

A yeast genetic system for selecting small molecule inhibitors of protein–protein interactions in nanodroplets

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.

Antioxidative Defense System, Pigment Composition, and Photosynthetic Efficiency in Two Wheat Cultivars Subjected to Drought1

We analyzed antioxidative defenses, photosynthesis, and pigments (especially xanthophyll-cycle components) in two wheat (Triticum durum Desf.) cultivars, Adamello and Ofanto, during dehydration and rehydration to determine the difference in their sensitivities to drought and to elucidate the role of different protective mechanisms against oxidative stress. Drought caused a more pronounced inhibition in growth and photosynthetic rates in the more sensitive cv Adamello compared with the relatively tolerant cv Ofanto. During dehydration the glutathione content decreased in both wheat cultivars, but only cv Adamello showed a significant increase in glutathione reductase and hydrogen peroxide-glutathione peroxidase activities. The activation states of two sulfhydryl-containing chloroplast enzymes, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase, were maintained at control levels during dehydration and rehydration in both cultivars. This indicates that the defense systems involved are efficient in the protection of sulfhydryl groups against oxidation. Drought did not cause significant effects on lipid peroxidation. Upon dehydration, a decline in chlorophyll a, lutein, neoxanthin, and β-carotene contents, and an increase in the pool of de-epoxidized xanthophyll-cycle components (i.e. zeaxanthin and antheraxanthin), were evident only in cv Adamello. Accordingly, after exposure to drought, cv Adamello showed a larger reduction in the actual photosystem II photochemical efficiency and a higher increase in nonradiative energy dissipation than cv Ofanto. Although differences in zeaxanthin content were not sufficient to explain the difference in drought tolerance between the two cultivars, zeaxanthin formation may be relevant in avoiding irreversible damage to photosystem II in the more sensitive cultivar.

Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.