Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans

There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an α-1,2-mannosyl transferase, which adds the second mannose residue in a tri-mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Kinetic equilibrium of forces and molecular events in muscle contraction

In biomolecular systems, the mechanical transfer of free energy occurs with both high efficiency and high speed. It is shown here that such a transfer can be achieved only if the participating free-energy-storing elements exhibit opposing relationships between their content of free energy and the force they exert in the transfer direction. A kinetic equilibrium of forces (KEF) results, in which the transfer of free energy is mediated essentially by thermal molecular motion. On the basis of present evidence, KEF is used as a guiding principle in developing a mechanical model of the crossbridge cycle in muscle contraction. The model allows the basic features of molecular events to be visualized in terms of plausible structures. Real understanding of the process will require identification of the elements that perform the functions described here. Besides chemomechanical energy transduction, KEF may have a role in other biomolecular processes in which free energy is transferred mechanically over large distances.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Molecular mechanisms of sound amplification in the mammalian cochlea

Mammalian hearing depends on the enhanced mechanical properties of the basilar membrane within the cochlear duct. The enhancement arises through the action of outer hair cells that act like force generators within the organ of Corti. Simple considerations show that underlying mechanism of somatic motility depends on local area changes within the lateral membrane of the cell. The molecular basis for this phenomenon is a dense array of particles that are inserted into the basolateral membrane and that are capable of sensing membrane potential field. We show here that outer hair cells selectively take up fructose, at rates high enough to suggest that a sugar transporter may be part of the motor complex. The relation of these findings to a recent candidate for the molecular motor is also discussed.

Arabidopsis thaliana Responses to Mechanical Stimulation Do Not Require ETR1 or EIN21

Plants exposed to repetitive touch or wind are generally shorter and stockier than sheltered plants. These mechanostimulus-induced developmental changes are termed thigmomorphogenesis and may confer resistance to subsequent stresses. An early response of Arabidopsis thaliana to touch or wind is the up-regulation of TCH (touch) gene expression. The signal transduction pathway that leads to mechanostimulus responses is not well defined. A role for ethylene has been proposed based on the observation that mechanostimulation of plants leads to ethylene evolution and exogenous ethylene leads to thigmomorphogenetic-like changes. To determine whether ethylene has a role in plant responses to mechanostimulation, we assessed the ability of two ethylene-insensitive mutants, etr1–3 and ein2–1, to undergo thigmomorphogenesis and TCH gene up-regulation of expression. The ethylene-insensitive mutants responded to wind similarly to the wild type, with a delay in flowering, decrease in inflorescence elongation rate, shorter mature primary inflorescences, more rosette paraclades, and appropriate TCH gene expression changes. Also, wild-type and mutant Arabidopsis responded to vibrational stimulation, with an increase in hypocotyl elongation and up-regulation of TCH gene expression. We conclude that the ETR1 and EIN2 protein functions are not required for the developmental and molecular responses to mechanical stimulation.