María Laura Martínez

Purification and biological characterization of N‐acetyl β‐D glucosaminidase from Bufo arenarum spermatozoa

Fertilization in Bufo arenarum requires the sperm to penetrate the egg envelopes. The incubation of isolated vitelline envelopes with sperm induces the acrosome reaction, releasing proteases and glycosidases to the media. In the present work N‐acetyl‐β‐D‐glucosaminidase, β‐D‐galactosidase, β‐D‐glucosidase, α‐D‐mannosidase, α‐L‐fucosidase, and α‐D‐glucosidase activities are measured in spermatozoa. N‐acetyl‐β‐D‐glucosaminidase is the major sperm glycosidase activity assayed. However, N‐acetyl‐β‐D‐galactosamine show competitive inhibitory effect. The glycosidase pH optimum is 3.5 being inhibited at pHs higher than 7.5. In our study, N‐acetyl‐β‐D‐glucosaminidase is the only glycosidase that in vitro binds to vitelline envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertilization. The enzyme has been purified in a two‐step procedure. After native gel electrophoresis, the activity‐stained band was cut out and the eluted enzyme was finally subjected to ConA–sepharose chromatography. In SDS‐PAGE, the denatured enzyme migrates as a single band with a molecular mass of 45 kDa. Furthermore, analysis by size‐exclusion on HPLC showed a peak of activity at around 45 kDa. Preliminary localization studies showed higher relative activity in the acrosomal content. In addition, 10% of the N‐acetyl‐β‐D‐glucosaminidase activity was associated with the reacted sperm. By in vitro fertilization assay, it was observed that the inhibition of the enzyme results in the inhibition of fertilization. This last study shows that N‐acetyl‐β‐D‐glucosaminidase plays an important role in toad fertilization. Mol. Reprod. Dev. 57:194–203, 2000.

Assessment of the acrosome reastion in Bufo arenarum Spermatozoa by immunostaining: comparison with other methods

The acrosome reaction in Bufo arenarum spermatozoa has been visualised only by electron microscopy. Different staining procedures for spermatozoa are described in the present study. By light microscopy the acorsomal status cannot be determined, In some reacted sperm, stained with Coomassie blue, a filament that could be the ‘perforatorium’ was observed, as seen by electron microscopy. Several fluorescent lectins were used, but only FITC-PNA binds to acrosomal proteins. However, the fluorescence observed was weak. Indirect immunofluorescence, with antibodies raised against acrosomal matrix, showed different staining patterns between acrosome-reacted and intact spermatozoa. The technique is specific for evaluating acrosomal status in a large population of spermatozoa. Moreover, immunostaining, in contrast with lectin staining, can be carried out in the presence of glycoconjugates such as oocyte extracellular matrix without interference.

Novel micromorphological features of wood and bark of Argentinean Simaroubaceae

ABSTRACT Simaroubaceae is a family of about 22 genera and 100 species of mainly tropical and subtropical trees and shrubs and it is generally considered to be a heterogeneous group. In Argentina, Simaroubaceae is represented by three genera and four species, one of these is exotic: Ailanthus altissima (Mill.) Swingle while the other three are native: Castela coccinea Griseb., C. tweedii Planch. and Picrasma crenata (Vell.) Engl. Wood and bark anatomies of the Argentinean species were studied in an effort to find distinctive features for the genera and species within this family. Transverse, tangential and radial sections of the specimens were prepared and macerated according to conventional techniques; microscopic examination was performed by light microscopy and scanning electron microscopy. It was possible to make a key for the species and genera using a combination of qualitative (presence/absence of wood growth rings, porosity, abundance of parenchyma, occurrence of crystals, etc.), and quantitative features (vessels per square millimetre, tangential diameter of vessel lumina, etc.). The wood and bark characteristics obtained provide new evidence of the heterogeneous nature of the family. From an ecological point of view, according to vessel size, vessel number, mesomorphy and vulnerability index, the wood of the studied species belonging to Castela genus were characterised as xeromorphic, while the other genera were characterised as mesomorphic.

Quality Control of Herbal Medicines with Spectrophotometry and Chemometric Techniques - Application to Baccharis L. Species Belonging to Sect - Caulopterae DC. (Asteraceae)

Medicinal plants constitute a rich cultural and biological heritage in many countries, which could be very useful in meeting the therapeutic needs of the population (Rodriguez, 2010a). Traditional herbal medicines have been widely used for many years in many eastern countries (Liang et al., 2004). However, little work has been done to validate and standardize these products properly in order to match phytotherapy to chemotherapy which currently receives almost unconditional support from formal systems of health care. For several years now activities have been undertaken to systematize the identification, validation, production and use of medicinal plants, for both primary health care as well as a semi-industrial or industrial process, which implies their transformation into safe, reliable and stable phytopharmaceutical products. Therefore it is suggested that medicinal plants and their derived products would be a viable option for national development as an agricultural and therapeutic alternative, but standardization and industrialization, involving sustained yields, a quality control system and honest and reliable marketing would be needed for widespread implementation and official support. Consequently, on account of the above, education and research should be in agreement if any advance is to be made in this area (Rodriguez, 2010a). However, the necessary criteria for data quality, safety and efficacy of traditional medicine that would support its use in the world do not exist. Appropriate, accepted research methodology for evaluating traditional medicine is also lacking (Liang et al., 2004).