Sulfated bromophenols from Osmundaria obtusiloba (C. Agardh) R. E. Norris (Rhodophyta, Ceramiales)

Two new sulfated oligobromophenols from the marine red algae Osmundaria obtusiloba, 4-(1'-potassium sulfate, 2,3-dibromo, 1',4,5-trihydroxybenzyl) - 4''-(1'''-potassium sulfate, 2'',3''-dibromo, 1''',4'',5''-trihydroxybenzyl) sulfate and 1'-(2, 3-dibromo, 4-potassium sulfate, 1', 4, 5-trihydroxybenzyl) - 4''-(1''' potassium sulfate, 2'', 3''-dibromo, 1''', 4'', 5'' trihydroxybenzyl) sulfate, are herein reported. Besides them it was obtained 2, 2', 3, 3'-tetrabromo-4, 4', 5, 5'-tetrahydroxydiphenylmethane, 2, 3-dibromo-p-hydroxybenzyl methyl ether (methyl lanosol), dipotassium 2,3-dibromo-5-hydroxybenzyl 1',4-disulfate, 24-methylenecolest-5-en-3-beta-ol and alpha-D-mannopyranosyl-(1->2')-glycerate (digeneaside). The structures were determined by analysis of the spectroscopic data (IR, NMR and MS) and comparison with the literature. The 13CNMR data for dipotassium 2,3-dibromo-5hydroxybenzyl-1',4-disulfate are described here for the first time. The present study also suggests a mild method to isolate and to purify sulfated compounds.

Copper accumulation in Xanthomonas campestris pv. vesicatoria

A strain of Xanthomonas campestris pv. vesicatoria showing resistance to 1.2 mM cupric sulfate was analyzed by atomic absorption spectroscopy and ESI (electron spectrophotometry imaging). Accumulation of copper was detected in the periphery of the cell membrane region, suggesting that the mechanism of copper resistance is similar to that previously described for Pseudomonas species. The ESI technique was used to detect copper in the membrane region. Copper-resistance in X. campestris pv. vesicatoria 484 is inducible and occurs by accumulation of the metal and not by efflux mechanism as has been suggested. The growth curve also showed that this system is inducible.

Correlation between high molecular weight gluten subunits composition and bread-making quality in Brazilian wheat

Bread-making quality is one of the most important targets in the genetic improvement of wheat. Although extensive analyses of quality traits such as farinography, sodium dodecyl sulfate (SDS) sedimentation, alveography, and baking are made in breeding programs, these analyses require high amounts of seeds which are obtained only in late generations. In this experiment the statistical correlations between the high molecular weight subunit of glutenin and bread-making quality measured by alveograph, farinograph and SDS sedimentation were evaluated. Seventeen wheat genotypes were grown under the same conditions, each producing about 1 kg of seeds for the evaluations. The high molecular weight (HMW) glutenin subunits were analyzed by SDS-PAGE. Statistical correlations were highly significant between HMW glutenin subunits and alveograph and SDS sedimentation. These results indicate the possibility of manipulating major genes for wheat seed quality by coupling traditional breeding with non-destructive single seed analysis. Only half seed is necessary to perform the SDS-PAGE analysis. Therefore, the other half seed can be planted to generate the progeny. Seed yield and SDS sedimentation were statistically correlated, indicating the possibility of simultaneous selection for both traits

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Radioactive fucose as a tool for studying glycoprotein secretion

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest levels of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming

Structure and function of the selectin ligand PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4[Fuca1®3]GlcNAcß1®R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

Adrenal incidentaloma

Incidentally discovered adrenal masses, or adrenal incidentalomas, have become a common clinical problem owing to wide application of radiologic imaging techniques. This definition encompasses a heterogeneous spectrum of pathologic entities, including primary adrenocortical and medullary tumors, benign or malignant lesions, hormonally active or inactive lesions, metastases, and infections. Once an adrenal mass is detected, the clinician needs to address two crucial questions: is the mass malignant, and is it hormonally active? This article provides an overview of the diagnostic clinical approach and management of the adrenal incidentaloma. Mass size is the most reliable variable to distinguish benign and malignant adrenal masses. Adrenalectomy should be recommended for masses greater than 4.0 cm because of the increased risk of malignancy. Adrenal scintigraphy has proved useful in discriminating between benign and malignant lesions. Finally, fine-needle aspiration biopsy is an important tool in the evaluation of oncological patients and it may be useful in establishing the presence of metastatic disease. The majority of adrenal incidentalomas are non-hypersecretory cortical adenomas but an endocrine evaluation can lead to the identification of a significant number of cases with subclinical Cushing's syndrome (5-15%), pheochromocytoma (1.5-13%) and aldosteronoma (0-7%). The first step of hormonal screening should include an overnight low dose dexamethasone suppression test, the measure of urinary catecholamines or metanephrines, serum potassium and, in hypertensive patients, upright plasma aldosterone/plasma renin activity ratio. Dehydroepiandrosterone sulfate measurement may show evidence of adrenal androgen excess.

Adrenocortical tumors in children

Childhood adrenocortical tumors (ACT) are rare. In the USA, only about 25 new cases occur each year. In Southern Brazil, however, approximately 10 times that many cases are diagnosed each year. Most cases occur in the contiguous states of São Paulo and Paraná. The cause of this higher rate has not been identified. Familial genetic predisposition to cancer (p53 mutations) and selected genetic syndromes (Beckwith-Wiedemann syndrome) have been associated with childhood ACT in general but not with the Brazilian counterpart. Most of the affected children are young girls with classic endocrine syndromes (virilizing and/or Cushing). Levels of urinary 17-ketosteroids and plasma dehydroepiandrosterone sulfate (DHEA-S), which are abnormal in approximately 90% of the cases, provide the pivotal clue to a diagnosis of ACT. Typical imaging findings of pediatric ACT consist of a large, well-defined suprarenal tumor containing calcifications with a thin capsule and central necrosis or hemorrhage. The pathologic classification of pediatric ACT is troublesome. Even an experienced pathologist can find it difficult to differentiate carcinoma from adenoma. Surgery is the single most important procedure in the successful treatment of ACT. The role of chemotherapy in the management of childhood ACT has not been established although occasional tumors are responsive to mitotane or cisplatin-containing regimens. Because of the heterogeneity and rarity of the disease, prognostic factors have been difficult to establish in pediatric ACT. Patients with incomplete tumor resection or with metastatic disease at diagnosis have a dismal prognosis. In patients with localized and completely resected tumors, the size of the tumor has predictive value. Patients with large tumors have a much higher relapse rate than those with small tumors.

The extracellular matrix in multiple sclerosis: an update

Extracellular matrix (ECM) molecules play important roles in the pathobiology of the major human central nervous system (CNS) inflammatory/demyelinating disease multiple sclerosis (MS). This mini-review highlights some recent work on CNS endothelial cell interactions with vascular basement membrane ECM as part of the cellular immune response, and roles for white matter ECM molecules in demyelination and remyelination in MS lesions. Recent basic and clinical investigations of MS emphasize axonal injury, not only in chronic MS plaques, but also in acute lesions; progressive axonal degeneration in normal-appearing white matter also may contribute to brain and spinal cord atrophy in MS patients. Remodeling of the interstitial white matter ECM molecules that affect axon regeneration, however, is incompletely characterized. Our ongoing immunohistochemical studies demonstrate enhanced ECM versican, a neurite and axon growth-inhibiting white matter ECM proteoglycan, and dermatan sulfate proteoglycans at the edges of inflammatory MS lesions. This suggests that enhanced proteoglycan deposition in the ECM and axonal growth inhibition may occur early and are involved in expansion of active lesions. Decreased ECM proteoglycans and their phagocytosis by macrophages along with myelin in plaque centers imply that there is "injury" to the ECM itself. These results indicate that white matter ECM proteoglycan alterations are integral to MS pathology at all disease stages and that they contribute to a CNS ECM that is inhospitable to axon regrowth/regeneration.