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.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum.

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.     

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.     

Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund's incomplete adjuvant. Immunized mares ovulated a significantly larger (P < 0.01) number of follicles per estrous cycle (2.8 +/- 1.1; range 1 to 8 ovulations) than 14 nonimmunized control mares (1.1 +/- 0.1; range 1 to 2 ovulations). Day-7 embryo recovery rates tended to be higher (P < 0.1) in immunized mares (1.6 +/- 0.5 embryos per flush) than in control mares (0.7 +/- 0.2 embryos per flush). No differences in interovulatory intervals were found between the 2 groups. These results indicate that immunization against inhibin may be useful in inducing development and ovulation of multiple follicles for embryo transfer in the mare.     

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.     

Liquid chromatographic determination of caffeine in biologic fluids

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 μg/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.