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.     

Design and synthesis of bridged gamma-lactams as analogues of beta-lactam antibiotics

Anti-Bredt bridged bicyclo[3.2.1] gamma-lactams were designed as inhibitors of penicillin binding proteins (PBPs). The compounds were prepared by a carbenoid insertion into a lactam N-H bond. Their weak antibacterial activity could either be explained by a poor chemical stability or by unfavorable steric interactions of the methylene bridge of the gamma-lactam with the targeted enzymes.     

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.     

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.