Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

A MACROMUTATION IN TRIPSACUM DACTYLOIDES (POACEAE): CONSEQUENCES FOR SEED SIZE,GERMINATION, AND SEEDLING ESTABLISHMENT

A recessive allele of a gene in Tripsacum dactyloides L. (eastern gamagrass) changes staminate florets to pistillate or hermaphrodite, and restores fertility to suppressed florets. There were ten to 25 times more seeds in the mutant pistillate form, and these were 0.32 to 0.59 times smaller than seeds from the normal form. Seeds from pistillate plants had significantly lower germination rates (22% vs. 50%), and seedlings grew 20% slower than those of normal plants in a greenhouse experiment. Pistillate seedling survival rates were lower in both high- (18.8% vs. 62.6%) and low- (52.8% vs. 72.6%) competition environments in a field experiment, and surviving seedlings were smaller. The maternal parent of volunteer seedlings next to a plantation of normal and pistillate plants was determined by dissecting the attached fruitcases of 1,313 seedlings. Pistillate plants in the plantation produced 90% of all seeds falling on the site but only 29% of the volunteer seedlings. The pistillate macromutation is not likely to spread in the wild due to morphological constraints on seed size and packaging.     

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.     

Isolation for bacteria and fungi for the hydrolysis of phthalate and terephthalate esters

Bacterial and fungal strains were isolated from enrichment cultures using diethylphthalate, diethylterephthalate, or ethylene glycol dibenzoate as sole carbon sources.Aureobacterium, Flavobacterium, andMicrococcus species were isolated from diethylphthalate enrichments;Rhodococcus andXanthomonas species were isolated from diethylterephthalate enrichments;Rhodococcus andFusarium species were isolated from ethylene glycol dibenzoate enrichments.     

Opposite effects of interleukin-4 and interleukin-10 on nitric oxide production in murine macrophages

Interleukin-4 (IL-4) and interleukin-10 (IL-10) were evaluated for their ability to inhibit the production of nitric oxide (NO) by interferon-gamma (IFN-gamma)- or lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 and J774.2). Macrophages pre-treated with IL-4 and then stimulated with IFN-gamma or LPS showed significant inhibition in their ability to produce NO as measured by nitrite production. Simultaneous treatment of IL-4 pre-incubated cells with IFN-gamma and LPS together augmented nitrite accumulation. On the other hand, similar exposures of the macrophages to IL-10 followed by IFN-gamma or LPS treatments resulted in significantly increased NO production. Thus IL-10 failed to suppress IFN-gamma or LPS-induced NO production and showed opposite effects in these experiments to IL-4. We conclude that the two lymphokines have differing roles in the control of production of NO and might act to control the secretion of nitric oxide in vivo.     

Partitioning of water resources among plants of a lowland tropical forest

Source water used by plants of several species in a semi-evergreen lowland tropical forest on Barro Colorado Island, Panama, was assessed by comparing the relative abundance of deuterium, D, versus hydrogen, H (stable hydrogen isotope composition, D) in xylem sap and in soil water at different depths, during the dry season of 1992. Ecological correlates of source water were examined by comparing xylem water D values with leaf phenology, leaf water status determined with a pressure chamber, and rates of water use determined as mass flow of sap using the stem heat balance method. Soil water D values decreased sharply to 30 cm, then remained relatively constant with increasing depth. Average D values were-13, for 0–30 cm depth and-36.7 for 30–100 cm depth. Soil water D values were negatively associated with soil water content and soil water potential. Concurrent analyses of xylem water revealed a high degree of partitioning of water resources among species of this tropical forest. Xylem water D of deciduous trees (average=-25.3±1.4) was higher than that of evergreen trees (average=-36.3±3.5), indicating that evergreen species had access to the more abundant soil water at greater depth than deciduous species. In evergreen shade-tolerant and high-light requiring shrubs and small trees, D of xylem water was negatively correlated with transpiration rate and leaf water potential indicating that species using deeper, more abundant water resources had both higher rates of water use and more favorable leaf water status.     

Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter.

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.     

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of the cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRAPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type I turn involving the same amino acids in both glycosylated and unglycosylated peptides. The GalNAc attached to the threonine (T³) and serine (S⁴) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.     

Unusual distribution of tubulin isoforms in the snail Lymnaea stagnalis

Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against -tubulin and -tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome -tubulin, however, labelled -tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total -tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated -tubulin and acetylated -tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.