Molecular and morphological examination of Cyrtobagous sp. collected from Argentina, Paraguay, Brazil, Australia, and Florida

Two members of the floating fern genus Salvinia (Salviniaceae), S. minima Baker and S. molesta Mitchell, have established in the United States. Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), long established on Florida S. minima, was released in Texas and Louisiana as a biocontrol agent for both species. Subsequently, sequence analysis of the 28S rRNA D2 expansion domain suggested that the Florida and Brazilian populations (used worldwide for biocontrol) of C. salviniae might constitute two cryptic species. In response, the Brazilian weevil was imported from Australia and released instead onto S. molesta. We sampled C. singularis Hustache and C. salviniae from their native ranges in Brazil, Argentina, and Paraguay and sequenced them (D2) along with Australian and Florida samples. The genetic distance between C. singularis and C. salviniae samples is much greater (almost 5×) than the distance between either the Florida and Brazilian samples or the Brazilian and Argentinean/ Paraguayan C. salviniae samples. Since C. singularis and C. salviniae are cryptic species, the Florida and Brazilian populations (or for that matter Brazilian and Argentinean/Paraguayan) could reasonably be described as demes or ecotypes. Occurrence data indicates that, in parts of their ranges, C. salviniae and C. singularis are not only sympatric but also feed on the same plant species at the same site. While host adaptation (species preferences) likely occurs within local demes, both species seem capable of adapting to the available resource (Salvinia species). Finally, a polymerase chain reaction (PCR) primer was developed to distinguish the Florida and Brazilian/Australian types.     

The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.     

Temperature modification of respiratory metabolism and caloric intake of Pandalus borealis (Krøyer) first zoeae

Oxygen consumption rates (Q_O2) of laboratory reared stage one zoeae of Pandalus borealis (Krøyer) at 1.5, 3, 4.5, 6, and 9°C were 1.5, 2.2, 2.6, 3.6 and 4.1μ O₂ · mg^?1 · h^?1, respectively. These values of Q_O2 correspond to 0.26, 0.38, 0.44, 0.60, and 0.70 μl O₂ · individual^?1 · h^?1. At 10.5 °C oxygen consumption rates decreased suggesting thermally induced respiratory stress.The equation log₁₀Q_O2 = 0.55 log₁₀T°C + 0.086 describes the relationship between Q_O2 (μl O₂ · mg^?1 · h^?1) and sea-water temperature between 1.5 and 9°C. Corresponding values of Q_O2 for an individual (μl O₂ · h^?1) exhibited the relationship log₁₀Q_O2 = 0.55 log₁₀T°C ?0.686.The minimum daily metabolic caloric requirements for an individual zoea ranged from 0.04 at 3 °C to 0.07 calories per day at 8 °C. The number of calories ingested daily ranged from 0.4 to 0.5 at 3 to 8 °C.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

Recent results on how aminoacyl transfer RNA synthetases recognize specific transfer RNAs

Summary Aminoacyl tRNA synthetases discriminate between tRNA species by a highly specific mechanism. Physical and chemical studies indicate that the synthetases bind along and around the inside of the three-dimensional L-shaped tRNA structure. Studies of mutant tRNAs that affect synthetase interaction tend to confirm this conclusion. However, in contrast to proteins that recognize a specific block of contiguous nucleotide units (e.g., repressors, restriction enzymes, etc.), synthetases appear to interact with spatially disperse elements of the structure. Available evidence suggests that tRNA binding clefts on various synthetases may be roughly similar, with specificity being achieved by the choice of amino acid residues in a few critical positions in the tRNA binding clefts. With this idea in mind, it should be possible to introduce amino acid substitutions into the binding clefts and thereby change tRNA recognition specificity. This has been attempted (by genetic manipulations) and a mutant alanine tRNA synthetase with altered tRNA recognition has been isolated. This enzyme can attach alanine to isoleucine specific tRNA. When presented with valine specific tRNA, a tRNA similar in some structural features to the isoleucine specific tRNA, or with the structurally quite different tyrosine specific tRNA, no significant aminoacylation occurs. Thus, a precise specificity alteration can occur through mutation; this result supports the idea of similarities in synthetase binding clefts, with specificity being achieved by the positioning of amino acids at critical positions in these clefts. Finally, further data have been obtained on the issue of possible transient covalent bond formation between synthetases and tRNAs, as a critical part of the interaction.Abbreviations tRNA^x a tRNA specific for the amino acid - x where x is given the standard 3 letter abbreviation     

Cross-adaptation and bitterness inhibition of L-tryptophan, L-phenylalanine and urea: further support for shared peripheral physiology

A previous study investigating individuals' bitterness sensitivities found a close association among three compounds: L-tryptophan (L-trp), L-phenylalanine (L-phe) and urea (Delwiche et al., 2001, Percept. Psychophys. 63, 761-776). In the present experiment, psychophysical cross-adaptation and bitterness inhibition experiments were performed on these three compounds to determine whether the bitterness could be differentially affected by either technique. If the two experimental approaches failed to differentiate L-trp, L-phe and urea's bitterness, then we may infer they share peripheral physiological mechanisms involved in bitter taste. All compounds were intensity matched in each of 13 subjects, so the judgments of adaptation or bitterness inhibition would be based on equal initial magnitudes and, therefore, directly comparable. In the first experiment, cross-adaptation of bitterness between the amino acids was high (>80%) and reciprocal. Urea and quinine-HCl (control) did not cross-adapt with the amino acids symmetrically. In a second experiment, the sodium salts, NaCl and Na gluconate, did not differentially inhibit the bitterness of L-trp, L-phe and urea, but the control compound, MgSO(4), was differentially affected. The bitter inhibition experiment supports the hypothesis that L-trp, L-phe and urea share peripheral bitter taste mechanisms, while the adaptation experiment revealed subtle differences between urea and the amino acids indicating that urea and the amino acids activate only partially overlapping bitter taste mechanisms.     

A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) is a purine salvage enzyme that catalyses the conversion of hypoxanthine and guanine to their respective mononucleotides. Partial deficiency of this enzyme can result in the overproduction of uric acid leading to a severe form of gout, whilst a virtual absence of HPRT activity causes the Lesch-Nyhan syndrome which is characterised by hyperuricaemia, mental retardation, choreoathetosis and compulsive self-mutilation. The HPRT-encoding gene is located on the X chromosome in the region q₂₆–q₂₇ and consists of nine exons and eight introns totalling 57 kb. This gene is transcribed to produce an mRNA of 1.6 kb, which contains a protein encoding region of 654 nucleotides. With the advent of increasingly refined techniques of molecular biology, it has been possible to study the HPRT gene of individuals with a deficiency in HPRT activity to determine the genetic basis of the enzyme deficiency. Many different mutations throughout the coding region have been described, but in the absence of precise information on the three-dimensional structure of the HPRT protein, it remains difficult to determine any consistent correlation between the structure and function of the enzyme.