Immunohistochemical studies on electron transport proteins associated with cytochromes P-450 in steroidogenic tissues. II. Microsomal NADPH-cytochrome c reductase in the rat adrenal.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum produced against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase technique and an indirect fluorescent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found to differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Mutants of FtsZ targeting the protofilament interface: effects on cell division and GTPase activity

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 microM, in contrast to the high GTPase activity of wild-type FtsZ above 1 muM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 microM K(d) for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.     

Immunohistochemical localization of epoxide hydratase in rat liver

An antibody produced against epoxide hydratase (EC 4.2.1.63) which had been purified to apparent homogeneity from hepatic microsomes of phenobarbital pretreated rats was employed in an unlabeled antibody peroxidase-antiperoxidase technique to localize the enzyme at the light microscopic level in the livers of untreated rats. Immunohistochemical staining for epoxide hydratase was detected in parenchymal cells throughout the liver lobule. Cells within the centrilobular regions, however, were observed to be stained more intensely than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that epoxide hydratase does not exhibit a uniform pattern of distribution within the liver lobule in untreated rats.     

The Anabolic Androgenic Steroid Nandrolone Decanoate Disrupts Redox Homeostasis in Liver,Heart and Kidney of Male Wistar Rats

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g⁻¹ body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.