Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae.

Sequence determinants for the importation of tRNAs into the mitochondrion of Leishmania tarentolae in vivo were investigated. tRNA(Ile)(UAU) is exclusively localized within the mitochondrion and tRNA(Gln)(CUG) exclusively in the cytosol (Lye LF, Chen DHT, Suyama Y, 1993, Mol Biochem Parasitol 58:233-246; Shi X, Chen DHT, Suyama Y, 1994, Mol Biochem Parasitol 65:23-37). L. tarentolae cells were transfected with plasmids encoding either tRNA(Ile) or tRNA (Gln) that were tagged with altered sequences in the D loop, permitting discrimination from the endogenous tRNAs. Primer extension analysis was used to show that the plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar intracellular localization as the endogenous tRNAs. Exchange or deletion of the 5'-flanking genomic sequences had no effect on the expression or mitochondrial localization of the tagged tRNA(Ile) or on the expression or cytosolic localization of the tagged tRNA(Gln), suggesting that the signals for importation are localized within the tRNA itself. Swapping the D loop+stem from the exclusively cytosolic tRNA(Gln) with that from the tRNA(Ile) produced a partial mitochondrial localization of the plasmid-expressed mutated tRNA(Gln). However, D loop exchange did not eliminate the mitochondrial localization of the plasmid-expressed mutated tRNA(Ile), suggesting that tertiary structure or additional sequence elements may be involved in the importation signal.     

Effects of anti-inflammatory drugs on fever and neutrophilia induced by Clostridium difficile toxin B

This study investigated the ability of Clostridium difficile toxin B, isolated from the VPI 10463 strain, to induce fever and neutrophilia in rats. Intravenous injection of toxin B (0.005-0.5 mug/kg) evoked a dose-dependent increase in body temperature. The febrile response to 0.5 mug/kg of the toxin started in 2.5 h, peaked at 5 h, and subsided fully within 24 h. Toxin B also induced a dosedependent neutrophilia. Pretreatment with indomethacin (2 mg/kg, i.p.) did not affect the neutrophilia induced by toxin B, but significantly reduced the febrile response measured 4 to 8 h after toxin B injection. Dexamethasone (0.5 mg/ kg) also markedly diminished the febrile response induced by toxin B. These results show that Clostridium difficile toxin B induced a febrile response susceptible to inhibition by dexamethasone and indomethacin. Furthermore, they suggest that prostaglandins are not involved in the neutrophilia caused by this toxin.     

Oligosaccharides derived from the xyloglucan isolated from the seeds of Hymenaea courbaril var. stilbocarpa

On aqueous extraction, Hymenaea courbaril var. stilbocarpa, known in Brazil as jatobá, furnishes a high yield of viscous xyloglucan (45%) from its seeds. The crude polysaccharide (B1) was hydrolysed and the products, analysed as alditol acetates, were glucose, xylose, galactose and arabinose in the ratio 50:35:13:2. After further fractionation on a DEAE-cellulose column (chloride form), the main fraction (70% yield, B2) was obtained. The basic structure of the xyloglucan was determined as a cellulose-type (1 → 4)-linked β-d-glucan backbone partially substituted with side chains at 06 of -d-xylopyranose, some of which were themselves substituted at 02 by the units of β-d-galactopyranose. Treatment of the xyloglucan (B2) with commercial cellulase from Trichoderma sp. yielded six oligosaccharides. These oligosaccharides were isolated by preparative paper chromatography, and their structures were determined by gas-liquid chromatography-mass spectroscopy of the derived partially O-methylated alditol acetates. These results confirm the structure proposed for jatobá seed xyloglucan.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.     

Infection of Anopheles darlingi fed on patients infected with Plasmodium vivax before and during treatment with chloroquine plus primaquine in Costa Marques, Rond?nia, Brazil.

Five patients with asexual and sexual parasites of Plasmodium vivax were treated orally with 600 mg chloroquine diphosphate (hour 0) followed with 300 mg at 8, 24 and 48 h later. Primaquine phosphate, 15 mg, was administered concurrently at h 0 and at 24 h intervals for 14 days. Anopheles darlingi were fed before the first dose (h -0.5) and 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 60 and 72 h later. Mosquitoes were examined for oocysts on day 8 and for sporozoites on day 15 after infection. Four of the five patients studied were still infective to mosquitoes from 1-5 h after the first dose of chloroquine plus primaquine. One of these and one other patient, who vomited 15 min after the first dose, became infective again at hours 10 and 12, respectively. Once produced, oocysts in mosquitoes fed on patients before, during and after chloroquine plus primaquine treatment appeared normal and produced sporozoite infected salivary glands. In view of these data, it is concluded that primaquine demonstrated rapid gametocytocidal activity and should be administered concurrently with chloroquine to reduce vivax malaria transmission.     

Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.     

Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----GGAG).

A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.