Cell compensatory responses of fungi to damage of the cell wall induced by Calcofluor White and Congo Red with emphasis on Sporothrix schenckii and Sporothrix globosa. A review.

The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, ß-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White.

(1,3)-beta-D-glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa.

The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-beta-D-glucan synthase activity. fz, sg, os-1 segregants from slime x wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-beta-glucan, chitin, and other polysaccharides and possess (1,3)-beta-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate x wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-beta-glucan synthase activity and cell wall.

Presence and chemical composition of glycoproteic layer on human spermatozoa.

Studies from electronic microscopy disclosed the presence of an electrodense stranded and branch-like electrodense layer that extends toward extracellular space. Chemical composition of this glycoproteic layer showed that protein and total sugar content is similar (0.98 microgram/microgram protein). As for the total sugar content of this glycoproteic constituent, sialic acid accounts for 40%, hexosamines 27%, and fucose 30%. Electrophoresis characterization of this constituent showed the presence of 6 different motility bands. Risen levels of sialic acid indicate the contribution of sialic residue in the net charge of the sperm membrane, its role during capacitation, and its possible participation in the formation of binding bridges between sperm membrane and ovum.

Perspective: measurement of circulating glycated proteins to monitor intermediate-term changes in glycaemic control.

The effective clinical management of diabetes requires accurate means to measure and monitor prevailing blood glucose concentrations. This can be accomplished with regular home glucose monitoring and periodic measurement of indicators that reflect ambient glycaemia during defined temporal intervals. The amount of glycated haemoglobin provides an index of integrated long-term glycaemia, whereas the amount of glycated albumin provides an index of short to intermediate term integrated glycaemic control. However, there is confusion regarding the various methods for estimation of glycated serum or plasma proteins, in part because the different methods use different reaction principles, measure different substances, and express results in different units. This article reviews the principles underlying different assays for measurement of glycated albumin and glycated serum proteins, compares their ranges of values, and discusses their clinical relevance assessed by studies published in the recent medical literature. The methods evaluated include commercially available assays for measuring glycated albumin with monoclonal antibodies and with affinity chromatography (including HPLC), and the fructosamine assay for glycated serum proteins.

Dosagem de frutosamina na avaliaçäo de pacientes diabéticos: comparaçäo com o método de dosagem das proteínas glicosilados por cromatografia de afinidade / Dosage of fructosamine in the avaliation of diabetic patients: comparison with the dosage method of glycosylated proteins by affinity chromatography

Este trabalho compara o método colorimétrico automatizado (frutosamina) com o método de cromatografia de afinidade para a dosagem das proteínas séricas glicosiladas. Näo houve diferença estatística entre os valores encontrados pelos dois métodos foi em indivíduos normais. Em pacientes diabéticos do tipo I os níveis de proteína glicosilada por cromatografia de afinidade (6,1 ñ 2,2%) foram mais altos do que os observados pelo método da frutosamina (4,3 ñ 0,9 mmol/l). A correlaçäo entre os dois métodos foi 0,88.(p 0,001> O método da frutosamina apresenta as vantagens de ser mais simples e barato, e poder ser utilizado em analisadores automatizados. Já o método de cromatografia de afinidade é um melhor indicador do controle metabólico do paciente diabético

Carbohydrate utilization by the avian oviduct during the laying cycle.

In the present work the carbohydrate utilization by the avian oviduct during the egg formation was studied. The pattern obtained for total hexose, fucose, hexosamines, sialic acid, fructose and sulphate demonstrated that in the domestic fowl's oviduct the isthmus and the shell gland secrete the precursors for the protein carbohydrate substance of the mammillae and shell matrix and may contribute during the shell formation with the substrates necessary for the metabolic energy of the organ. The infundibulum only participates as a donor for the metabolic energy.

Histo-biochemical correlation in the oviduct of Bufo arenarum (Hensel).

A histo-biochemical correlation was carried out in oviducts of Bufo arenarum (Hensel), in trophic animals in the preovulatory period. Histochemical techniques were used to detect acidic mucoproteins, whereas complex mucopolysaccharides were biochemically assayed, thus simplifying the histologic methodology. The existence of acetylhexosamines, hexoses, phosphate and sialic acid in the oviduct was put in evidence through histochemical and biochemical methods. Results show a high content in sialic aicd, total phosphate and acetylhexosamines in the middle and terminal portions of the preconvoluted zone. Sialomucins, acetylhexosamines and hexosamines show a maximum in the last loops of the convoluted zone, coincident with clearly determined histologic areas. All components detected exhibit minimal values in the proximal and distal intermediate zones. Results found for phosphate show it is combined, and determines the reactivity to the stains used. This type of mucin has not been found in other species.