Copulation behaviour of Glossina pallidipes (Diptera: Muscidae) outside and inside the female, with a discussion of genitalic evolution

If species-specific male genitalia are courtship devices under sexual selection by cryptic female choice, then species-specific aspects of the morphology and behaviour of male genitalia should often function to stimulate the female during copulation. The morphology and behaviour of the complex, species-specific male genitalia of the tsetse fly, Glossina pallidipes Austen, were determined from both direct observations and dissections of flash-frozen copulating pairs; we found that some male genitalic traits probably function to stimulate the female, while others function to restrain her. The male clamps the ventral surface of the female's abdomen tightly with his powerful cerci. Clamping does not always result in intromission. Clamping bends the female's body wall and her internal reproductive tract sharply, posteriorly and dorsally, and pinches them tightly. The male performed sustained, complex, stereotyped, rhythmic squeezing movements with his cerci that were not necessary to mechanically restrain the female and appeared instead to have a stimulatory function. Six different groups of modified setae on and near the male's genitalia rub directly against particular sites on the female during squeezing. The designs of these setae correlate with the force with which they press on the female and the probable sensitivity of the female surfaces that they contact. As expected under the hypothesis that these structures are under sexual selection by female choice, several traits suspected to have stimulatory functions have diverged in G. pallidipes and its close relative, G. longipalpis. Additional male non-genitalic behaviour during copulation, redescribed more precisely than in previous publications, is also likely to have a courtship function. The elaborate copulatory courtship behaviour and male genitalia may provide the stimuli that previous studies showed to induce female ovulation and resistance to remating.     

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.     

Scaffolding as an organizing principle in trans-translation. The roles of small protein B and ribosomal protein S1

A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger RNA, small protein B, and ribosome protein S1. By means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein B, which appears near the decoding center. This result suggests that, when lacking a codon, the A-site on the small subunit is a target for small protein B. To investigate the role of S1 played in trans-translation, we obtained a cryo-electron microscopic map, including a stalled ribosome, transfer-messenger RNA, and small protein Bs but in the absence of S1. In this complex, several connections between the 30 S subunit and transfer-messenger RNA that appear in the +S1 complex are no longer found. We propose the unifying concept of scaffolding for the roles of small protein B and S1 in binding of transfer-messenger RNA to the ribosome during trans-translation, and we infer a pathway of sequential binding events in the initial phase of trans-translation.     

Effect of temperature on enzymatic and physiological factors related to chilling injury in carambola fruit (Averrhoa carambola L.)

Three groups of carambola fruits (Averrhoa carambola L.) were stored at 2 and 10 degrees C (85-90% relative humidity). The major physicochemical, physiological, and enzymatic responses of fruit were measured in each group over a 30-day period: chilling injury index (CII), decay (%), intracuticular waxes, cuticle permeability, pulp firmness, weight loss, sucrose, fructose and glucose contents, ion electrolyte leakage in pulp (%), ethylene and carbon dioxide production rates, and the activities of peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes. CII values were statistically different at 2 and 10 degrees C, showing high significance with respect to sucrose content and weight loss (P < 0.05). Chilling injury included darkened ribs and skin desiccation. According to the CI symptom development, a possible relationship of POD and PPO activities was found at 2 degrees C. A significant sucrose content increase was observed at 10 degrees C. CI symptoms were associated with POD and PAL activities.     

Polyamine derivatives as selective RNaseA mimics

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it’s binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.     

Changes in GAD67 mRNA expression evidenced by in situ hybridization in the brain of R6/2 transgenic mice

Huntington's disease is an autosomal dominant disorder with degeneration of medium size striatal neurones. As the disease evolves, other neuronal populations are also progressively affected. A transgenic mouse model of the disease (R6/2) that expresses exon 1 of the human Huntington gene with approximately 150 CAG repeats has been developed, but GABA concentrations are reported to be normal in the striatum of these animals. In the present study, we analysed the status of GABAergic systems by means of glutamic acid decarboxylase (GAD)67 mRNA in situ hybridization in the brain of R6/2 transgenic mice and wild-type littermates. We show that GAD67 expression is normal in the striatum, cerebellum and septum but decreased in the frontal cortex, parietal cortex, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata of R6/2 mice. These data, which may, in part, account for the behavioural changes seen in these animals, indicate that at 12.5 weeks of age the pathological features seen in the mice differ from those seen in humans with Huntington's disease.     

Inhibition of transfer messenger RNA aminoacylation and trans-translation by aminoglycoside antibiotics

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.