Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Relationship between the actions of atrial natriuretic peptide (ANP), guanylin and uroguanylin on the isolated kidney

Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 µg/ml, N = 12) or uroguanylin (0.5 µg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 µg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 ± 0.86 to 64.62 ± 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 ± 0.01 to 0.15 ± 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 ± 0.86 to 85.19 ± 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins.

Interaction between substance P and gastrin-releasing peptide on thyrotropin secretion by rat pituitary in vitro

The effect of substance P (SP) on thyrotropin (TSH) secretion is controversial. In this study we evaluated the effect of SP on TSH secretion by hemipituitaries of 3-month-old Wistar rats in vitro and its interaction with gastrin-releasing peptide (GRP) at equimolar concentrations (1 µM and 10 µM). TSH release was measured under basal conditions and 30 min after incubation in the absence or presence of SP, GRP or both peptides. Pituitary TSH content was also measured in the pituitary homogenate after incubation. SP at both concentrations caused a significant (P<0.05) increase in TSH secretion compared with all other groups, which was approximately 60% (1 µM) and 85% (10 µM) higher than that of the control group (23.3 ± 3.0 ng/ml). GRP at the lower concentration did not produce a statistically significant change in TSH secretion, whereas at the concentration of 10 µM it produced a 50% reduction in TSH. GRP co-incubated with substance P completely blocked the stimulatory effect of SP at both concentrations. Pituitary TSH content decreased in the SP-treated group compared to controls (0.75 ± 0.03 µg/hemipituitary) at the same proportion as the increase in TSH secretion, and this effect was also blocked when GRP and SP were co-incubated. In conclusion, in an in vitro system, SP increased TSH secretion acting directly at the pituitary level and this effect was blocked by GRP, suggesting that GRP is more potent than SP on TSH secretion, and that this inhibitory effect could be the predominant effect in vivo.

The expression of HLA-B27 modulates intracellular signaling in human monocytic macrophages

Reactive arthritis (ReA) is an inflammatory joint disease triggered by certain bacterial infections e.g. gastroenteritis caused by Salmonella. ReA is strongly associated to HLA-B27. However, the mechanism behind this association is unknown but it is suggested that the bacteria or bacterial compartments persist in the body. In this study, it was investigated whether the intracellular signaling is altered in HLA-B27- transfected U937 monocytic macrophages. Moreover, the contribution of HLA–B27 heavy chain (HC) misfolding was of interest. The study revealed that p38 activity plays a crucial role in controlling intracellular Salmonella Enteritidis in U937 cells. The replication of intracellular bacteria was dependent on p38 kinase and the activity of p38 was dysregulated in HLA-B27- transfected cells expressing misfolding heavy chains (HCs). Also the double-stranded RNA -dependent kinase (PKR) that modifies p38 signaling was overexpressed and hypophosphorylated upon infection and lipopolysaccharide stimulation. The expression of CCAAT enhancer binding protein beta (C/EBPβ) was found to be increased after infection and stimulation. Increased amount of full length human antigen R (HuR), disturbed HuR cleavage and reduced dependence on PKR after infection were observed. All the findings were linked to HLA-B27 HCs containing misfoldingassociated glutamic acid 45 (Glu45) at the peptide binding groove. The results indicate that the expression of HLA-B27 modulates the intracellular environment of U937 monocytic macrophages by altering signaling. This phenomenon is at least partially associated to the HLA-B27 misfolding. These observations offer a novel explanation how HLA-B27 may modulate inflammatory response induced by ReA-triggering bacteria.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Interactions of laminin with the amyloid ß peptide: Implications for Alzheimer's disease

Extensive neuronal cell loss is observed in Alzheimer's disease. Laminin immunoreactivity colocalizes with senile plaques, the characteristic extracellular histopathological lesions of Alzheimer brain, which consist of the amyloid ß (Aß) peptide polymerized into amyloid fibrils. These lesions have neurotoxic effects and have been proposed to be a main cause of neurodegeneration. In order to understand the pathological significance of the interaction between laminin and amyloid, we investigated the effect of laminin on amyloid structure and toxicity. We found that laminin interacts with the Aß1-40 peptide, blocking fibril formation and even inducing depolymerization of preformed fibrils. Protofilaments known to be intermediate species of Aß fibril formation were also detected as intermediate species of laminin-induced Aß fibril depolymerization. Moreover, laminin-amyloid interactions inhibited the toxic effects on rat primary hippocampal neurons. As a whole, our results indicate a putative anti-amyloidogenic role of laminin which may be of biological and therapeutic interest for controlling amyloidosis, such as those observed in cerebral angiopathy and Alzheimer's disease.

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

The brain decade in debate: VIII. Peptide hormones and behavior: cholecystokinin and prolactin

This article is a transcription of an electronic symposium held on November 28, 2000 in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the peptide field with particular focus on central actions of prolactin and cholecystokinin. The comments in this symposium reflect the diversity of prolactin and cholecystokinin research and demonstrate how the field has matured. Since both peptides play a role in reproductive behaviors, particularly mother-infant interactions, this was the starting point of the discussion. Recent findings on the role of the receptor subtypes as well as interaction with other peptides in this context were also discussed. Another issue discussed was the possible role of these peptides in dopamine-mediated rewarding systems. Both prolactin and cholecystokinin are involved in mechanisms controlling food intake and somatic pain thresholds. The role of peripheral inputs through vagal afferents modulating behavior was stressed. The advent of knockout animals as potential generators of new knowledge in this field was also addressed. Finally, interactions with other neuropeptides and investigation of the role of these peptides in other fields such as immunology were mentioned. Knowledge about the central functions of prolactin and cholecystokinin has shown important advances. The role of these peptides in neurological and psychiatric syndromes such as anorexia, drug abuse and physiological disturbances that lead to a compromised maternal behavior seems relevant.

Nitrergic modulation of vasopressin, oxytocin and atrial natriuretic peptide secretion in response to sodium intake and hypertonic blood volume expansion

The central nervous system plays an important role in the control of renal sodium excretion. We present here a brief review of physiologic regulation of hydromineral balance and discuss recent results from our laboratory that focus on the participation of nitrergic, vasopressinergic, and oxytocinergic systems in the regulation of water and sodium excretion under different salt intake and hypertonic blood volume expansion (BVE) conditions. High sodium intake induced a significant increase in nitric oxide synthase (NOS) activity in the medial basal hypothalamus and neural lobe, while a low sodium diet decreased NOS activity in the neural lobe, suggesting that central NOS is involved in the control of sodium balance. An increase in plasma concentrations in vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP), and nitrate after hypertonic BVE was also demonstrated. The central inhibition of NOS by L-NAME caused a decrease in plasma AVP and no change in plasma OT or ANP levels after BVE. These data indicate that the increase in AVP release after hypertonic BVE depends on nitric oxide production. In contrast, the pattern of OT secretion was similar to that of ANP secretion, supporting the view that OT is a neuromodulator of ANP secretion during hypertonic BVE. Thus, neurohypophyseal hormones and ANP are secreted under hypertonic BVE in order to correct the changes induced in blood volume and osmolality, and the secretion of AVP in this particular situation depends on NOS activity.

Silicon-containing species in used lube oil re-refining

Nowadays, the re-refining of the used lube oils has gained worldwide a lot of attention due to the necessity for added environmental protection and increasingly stringent environmental legislation. One of the parameters determining the quality of the produced base oils is the composition of feedstock. Estimation of the chemical composition of the used oil collected from several European locations showed that the hydrocarbon structure of the motor oil is changed insignificantly during its operation and the major part of the changes is accounted for with depleted oil additives. In the lube oil re-refining industry silicon, coming mainly from antifoaming agents, is recognized to be a contaminant generating undesired solid deposits in various locations in the re-refining units. In this thesis, a particular attention was paid to the mechanism of solid product formation during the alkali treatment process of silicon-containing used lube oils. The transformations of a model siloxane, tetramethyldisiloxane (TMDS), were studied in a batch reactor at industrially relevant alkali treatment conditions (low temperature, short reaction time) using different alkali agents. The reaction mechanism involving solid alkali metal silanolates was proposed. The experimental data obtained demonstrated that the solids were dominant products at low temperature and short reaction time. The liquid products in the low temperature reactions were represented mainly by linear siloxanes. The prolongation of reaction time resulted in reduction of solids, whereas both temperature and time increase led to dominance of cyclic products in the reaction mixture. Experiments with the varied reaction time demonstrated that the concentration of cyclic trimer being the dominant in the beginning of the reaction diminished with time, whereas the cyclic tetramer tended to increase. Experiments with lower sodium hydroxide concentration showed the same effect. In addition, a decrease of alkali agent concentration in the initial reaction mixture accelerated TMDS transformation reactions resulting in solely liquid cyclic siloxanes yields. Comparison of sodium and potassium hydroxides applied as an alkali agent demonstrated that potassium hydroxide was more efficient, since the activation energy in KOH presence was almost 2-fold lower than that for sodium hydroxide containing reaction mixture. Application of potassium hydroxide for TMDS transformation at 100° C with 3 hours reaction time resulted in 20 % decrease of solid yields compared to NaOH-containing mixture. Moreover, TMDS transformations in the presence of sodium silanolate applied as an alkali agent led to formation of only liquid products without formation of the undesired solids. On the basis of experimental data and the proposed reaction mechanism, a kinetic model was developed, which provided a satisfactory description of the experimental results. Suitability of the selected siloxane as a relevant model of industrial silicon-containing compounds was verified by investigation of the commercially available antifoam agent in base-catalyzed conditions.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 µl, ip) and group Ci received a sublethal dose of microcystins (48.2 µg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

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.

Adsorption of arsenic from ammonia containing waste water by ferrous hydroxide waste

Arsenic is a toxic substance. The amount of arsenic in waste water is a raising problem because of increasing mining industry. Arsenic is connected to cancers in areas where arsenic concentration in drinking water is higher than recommendations. The main object in this master’s thesis was to research how ferrous hydroxide waste material is adsorbed arsenic from ammonia containing waste water. In this master’s thesis there is two parts: theoretical and experimental part. In theoretical part harmful effects of arsenic, theory of adsorption, isotherms modeling of adsorption and analysis methods of arsenic are described. In experimental part adsorption capacity of ferrous hydroxide waste material and adsorption time with different concentrations of arsenic were studied. Waste material was modified with two modification methods. Based on experimental results the adsorption capacity of waste material was high. The problem with waste material was that at same time with arsenic adsorption sulfur was dissolving in solution. Waste material was purified from sulfur but purification methods were not efficient enough. Purification methods require more research.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.