Localization of the cotyledon reserves of Theobroma grandiflorum (Willd. ex Spreng.) K. Schum., T. subincanum Mart., T. bicolor Bonpl. and their analogies with T. cacao L.

Cotyledon mesophyll cell morphology and lipid and protein synthesis of T. grandiflorum, T. subincanum and T. bicolor were analyzed and compared with T. cacao. These species possess foliar cotyledons folded around the hypocotyl radicle axis, typical of Sterculiaceae. Fruit size, morphology and weight are very distinct amongst the four species and so are the respective seeds. The main axis of the T. grandiflorum and T. bicolor seeds measured about 30 mm, while T. subincanum and T. cacao seeds measured 17 mm and 26 mm respectively. The seed weights of T. grandiflorum, T. bicolor, T. subincanum and T. cacao were 11.6 g, 9.4 g, 2.1 g and 3.0 g, respectively. The cotyledon mesophylls of the four species contained mainly polysaccharides and lipid-protein reserve cells. Theobroma cacao, T. grandiflorum and T. subincanum were composed of greater than 50% lipids. For the four species, lipid globules gradually accumulated adjacent to the cell wall, and these globules measured from 1 to 3 µm. TEM showed low-density proteins inside the central vacuole of the young mesophyll cells of T. cacao. The protein reserves of the mature cells were densely scattered amongst the lipid bodies, and a few starch granules occurred together with the cotyledon mesophyll of the four species. Polyphenolic cells were found throughout the mesophyll cells or aligned with the respective vascular bundles. Immature cells demonstrated the capacity to synthesize all these reserves, but gradually the pre-determined cells produced mainly lipid-protein reserves. Besides the unique characteristics of the T. cacao products, the lipid-protein synthesis capacities of T. grandiflorum, T. subincanum and T. bicolor suggest various possibilities for new industrialized food, pharmaceutical and cosmetic products.

Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)

(Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)). Colleters are secretory structures formed by a parenchymatic axis with vascular bundles, bound by a layer of secretory palisade-like epidermis. Some studies regarding the structure of colleters have focused on secretory cells structure, but not distinguished the secretory and senescent phases. Generally, in mucilage-secreting cells such as colleters, the endoplasmic reticulum and Golgi apparatus are involved in secretion production and transport. In these study, colleters structure of Bathysa gymnocarpa K. Schum. and B. stipulata (Vell.) C. Presl. (Rubiaceae) were determined in two phases: a secretory phase and a senescence one. Samples were collected and processed by usual light and electron microscopy techniques. Studied colleters are constituted by an epidermal palisade layer and a central axis formed by parenchymatic cells with rare vascular traces. During the secretory phase, epidermal cells presented a dense cytoplasm, small vacuoles, enhanced rough and smooth endoplasmic reticulum, and a Golgi apparatus close to large vesicles. During the senescence phase epidermal cells presented a disorganized membrane system. No intact organelles or vesicles were observed. The outer cell wall exhibited similar layers to that observed during the secretory phase. The senescent phase is easily defined by the morphology of the colleters, but not well defined at subcellular level. Our research suggests that programmed cell death starts on secretory phase. However, more evidences are needed to evaluate the phenomena.

Hypoglycemic activity of polysaccharide fractions containing ß-glucans from extracts of Rhynchelytrum repens (Willd.) C.E. Hubb., Poaceae

ß-Glucans are soluble fibers with physiological functions, such as interference with absorption of sugars and reduction of serum lipid levels. The objective of the present study was to analyze the distribution of ß-glucans in different tissues of the African grass species Rhynchelytrum repens and also to evaluate their hypoglycemic activity. Leaf blades, sheaths, stems, and young leaves of R. repens were submitted to extraction with 4 M KOH. Analysis of the fractions revealed the presence of arabinose, glucose, xylose, and traces of rhamnose and galactose. The presence of ß-glucan in these fractions was confirmed by hydrolyzing the polymers with endo-ß-glucanase from Bacillus subtilis, followed by HPLC analysis of the characteristic oligosaccharides produced. The 4 M KOH fractions from different tissues were subjected to gel permeation chromatography on Sepharose 4B, with separation of polysaccharides with different degrees of polymerization, the highest molecular mass (above 2000 kDa) being found in young leaves. The molecular mass of the leaf blade polymers was similar (250 kDa) to that of maize coleoptile ß-glucan used for comparison. The 4 M KOH fraction injected into rats with streptozotocin-induced diabetes showed hypoglycemic activity, reducing blood sugar to normal levels for approximately 24 h. This performance was better than that obtained with pure ß-glucan from barley, which decreased blood sugar levels for about 4 h. These results suggest that the activity of ß-glucans from R. repens is responsible for the use of this plant extract as a hypoglycemic drug in folk medicine.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Mechanical and water vapor permeability properties of biodegradables films based on methylcellulose, glucomannan, pectin and gelatin

Mimic biological structures such as the cell wall of plant tissues may be an alternative to obtain biodegradable films with improved mechanical and water vapor barrier properties. This study aims to evaluate the mechanical properties and water vapor permeability (WVP) of films produced by using the solvent-casting technique from blended methylcellulose, glucomannan, pectin and gelatin. First, films from polysaccharides at pH 4 were produced. The film with the best mechanical performance (tensile strength = 72.63 MPa; elongation = 9.85%) was obtained from methylcellulose-glucomannan-pectin at ratio 1:4:1, respectively. Then, gelatin was added to this polysaccharide blend and the pH was adjusted to 4, 5 and 6. Results showed significant improvement in WVP when films were made at pH 5 and at polysaccharides/gelatin ratio of 90/10 and 10/90, reaching 0.094 and 0.118 g.mm/h.m².kPa as values, respectively. Films with the best mechanical properties were obtained from the blend of polysaccharides, whereas WVP was improved from the blend of polysaccharides and gelatin at pH 5.