The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-α1,3-[man-α1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates. Published in 2004.     

Enhancement on the Europium emission band of Europium chlortetracycline complex in the presence of LDL

Low-density lipoprotein (LDL) particles are the major cholesterol-carrying lipoprotein in the human circulation from the liver to peripheral tissues. High levels of LDL-Cholesterol (LDL-C) are known risk factor for the development of coronary artery disease (CAD). The most common approach to determine the LDL-C in the clinical laboratory involves the Friedewald formula. However, in certain situations, this approach is inadequate. In this paper we report on the enhancement on the Europium emission band of Europium chlortetracycline complex (CTEu) in the presence of LDL. The emission intensity at 615 nm of the CTEu increases with increasing amounts of LDL. This phenomenon allowed us to propose a method to determine the LDL concentration in a sample composed by an aqueous solution of LDL. With this result we obtained LDL calibration curve, LOD (limit of detection) of 0.49 mg/mL and SD (standard deviation) of 0.003. We observed that CTEu complex provides a wider dynamic concentration-range for LDL determination than that from Eu-tetracycline previously. The averaged emission lifetimes of the CTEu and CTEu with LDL (1.5 mg/mL) complexes were measured as 15 and 46 μs, respectively. Study with some metallic interferents is presented.     

Structural features of sulfated glycans from the tunic of Styela plicata (Chordata-Tunicata). A unique occurrence of L-galactose in sulfated polysaccharides

The sulfated polysaccharides in the tunic of Styela plicata occur as three fractions that differ markedly in molecular mass and chemical composition. The high-molecular-mass fraction has a high galactose content and a strong negative optical rotation while the low-molecular-mass fractions have a higher proportion of amino sugars and glucose. The galactose occurs in these polysaccharides entirely in the L-enantiomeric form. Although L-galactose is a constituent of several polysaccharides, this is the first report of sulfated polysaccharides that contain high amounts of L-galactose, and that lack the D enantiomorph of this sugar. Furthermore, the structure of the high-molecular-mass fraction, which is composed mainly of a core of alpha-L-galactopyranose residues, sulfated at position 3, linked glycosidically though position 1----4, and with non-sulfated L-galactopyranose non-reducing end-units, is unique among other previously described sulfated glycans. These data are of considerable interest as they show an unusual example of possible variants of polyanionic glycans with structure function in living tissues.     

Prevalence and Serological Diagnosis of Relapse in Paracoccidioidomycosis Patients

A review of 400 clinical records of paracoccidioidomycosis (PCM) patients, 93 with the acute/subacute (AF) and 307 with the chronic form (CF), attended from 1977 to 2011, selected as to the schedule of release for study by the Office of Medical Records at the University Hospital of the Faculdade de Medicina de Botucatu – São Paulo State University – UNESP, was performed to detect cases in relapse. The control of cure was performed by clinical and serological evaluation using the double agar gel immunodiffusion test (DID). In the diagnosis of relapse, DID, enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay (IBgp70 and IBgp43) were evaluated. Out of 400 patients, 21 (5.2%) went through relapse, 18 of them were male and 3 were female, 6∶1 male/female ratio. Out of the 21 patients in relapse, 15 (4.8%) showed the CF, and 6 (6.4%) the AF (p>0.05). The sensitivity of DID and ELISA before treatment was the same (76.1%). DID presented higher sensitivity in pre-treatment (80%) than at relapse (45%; p = 0.017), while ELISA showed the same sensitivity (80% vs 65%; p = 0.125). The serological methods for identifying PCM patients in relapse showed low rates of sensitivity, from 12.5% in IBgp70 to 65.0% in IBgp43 identification and 68.8% in ELISA. The sensitivity of ELISA in diagnosing PCM relapse showed a strong tendency to be higher than DID (p = 0.06) and is equal to IBgp43 (p = 0.11). In sum, prevalence of relapse was not high in PCM patients whose treatment duration was based on immunological parameters. However, the used methods for serological diagnosis present low sensitivity. While more accurate serological methods are not available, we pay special attention to the mycological and histopathological diagnosis of PCM relapse. Hence, direct mycological, cytopathological, and histopathological examinations and isolation in culture for P. brasiliensis must be appropriately and routinely performed when the hypothesis of relapse is considered.     

Paracoccidioimycosis and white individuals: Susceptibility and biogeographic aspects in an important endemic area in Brazil

Paracoccidioidomycosis (PCM) is a neglected mycosis most commonly occurring in Latin America. The etiologic agents are thermo dimorphic fungi of the genus Paracoccidioides, and cause an important granulomatous response in affected tissues. The Botucatu Medical School, from São Paulo State University (UNESP), is a PCM study pole, located in São Paulo State Midwest region, which is classified as a hyperendemic area in the Southeast region in Brazil. This study aimed to perform a retrospective epidemiological, geographical, and clinical analysis by the information available in medical records. It was listed as socio-demographic data along with clinical characteristics from patients diagnosed and treated during a 10-year period in Botucatu, totaling 177 patients with Paracoccidioidomycosis confirmed by the histopathological test. It was observed that the main clinical presentation was the chronic type (76,3%), most commonly identified in white male individuals over the age of 29 years old, smokers, and alcoholics, providing evidences for the first time that white individuals were more affected by the disease, in comparison to non-white individuals that may be more resistant to infection. This data opens new avenues for study within ancestry, resistance and susceptibility in paracoccidioidomycosis.     

Histochemical and morphological characterization of reticular fibers

Summary The results presented in this paper show that collagen fibers can be clearly distinguished from reticular fibers using the picrosirius-polarization method. A morphologic and morphometric study of these two types of fibers with electron microscopy shows that reticular fibers are characterized by the smaller diameter of their fibrillar components and the higher content of interfibrillar material, resulting in a loose arrangement of the fibrils. The evidences presented suggest that the amorphous matrix in which fibrils are embedded is responsible for the silver impregnation of reticular fibers. Our results show that the matrix of reticular fibers is characteristically rich in heparitin sulfate, and that the glycosaminoglycans present show a high interaction with the fibrillar component of these fibers.