Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.     

Possible link of different slow calcium signals generated by membrane potential and hormones to differential gene expression in cultured muscle cells

The study of fluorescent calcium signals from cultured rat myotubes has provided interesting results in the past few years. Both K+ depolarization and tetanic electrical stimulation were shown to produce slow Ca2+ signals, unrelated to contraction and associated to regulation of gene expression in cultured rat myotubes. We studied the effect of IGF-I, insulin and testosterone on intracellular Ca2+ in cultured muscle cells. Insulin produced a fast (< 1 s) and transient [Ca2+] increase lasting less than 10 s. IGF-I induced a transient [Ca2+] increase, reaching a fluorescence peak 6 s after stimulus, to return to basal values after 60 s. Testosterone induced delayed (35 s) and long lasting (100-200 s) signals, frequently associated with oscillations. IGF-I, testosterone and electrical stimulation-induced Ca2+ signals were shown to be dependent on IP3 production. All of these Ca2+ signals were blocked by inhibitors of the IP3 pathway. On the other hand, insulin-induced Ca2+ increase was dependent on ryanodine receptors and blocked by either nifedipine or ryanodine. The different intracellular Ca2+ patterns produced by electrical stimulation, testosterone, IGF-I and insulin, may help to understand the role of intracellular calcium kinetics in the regulation of gene expression by various stimuli in skeletal muscle cells.     

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.     

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood and nasal secretion of Brazilian household contacts

DNA samples from blood and nasal swabs of 125 healthy household contacts was submitted to amplification by polymerase chain reaction (PCR) using a Mycobacterium leprae-specific sequence as a target for the detection of subclinical infection with M. leprae. All samples were submitted to hybridization analysis in order to exclude any false positive or negative results. Two positive samples were confirmed from blood out of 119 (1.7%) and two positive samples from nasal secretion out of 120 (1.7%). The analysis of the families with positive individuals showed that 2.5% (n = 3) of the contacts were relatives of multibacilary patients while 0.8% of the cases (n = 1) had a paucibacilary as an index case. All positive contacts were followed up and after one year none of them presented clinical signs of the disease. In spite of the PCR sensitivity to detect the presence of the M. leprae in a subclinical stage, this molecular approach did not seem to be a valuable tool to screen household contacts, since we determined a spurious association of the PCR positivity and further development of leprosy.     

Effects of irrigation and air humidity preconditioning on water relations, growth and survival of Rosmarinus officinalis plants during and after transplanting

The effect of different irrigation and air humidity conditioning treatments on the morphological and physiological responses of Rosmarinus officinalis in nursery conditions was investigated in order to evaluate the degree of hardening resulting from these conditions. Rosmarinus officinalis seedlings were pot-grown during 4 months in two greenhouses (nursery period), in which two irrigation treatments were used (control and deficit). In one of these greenhouses, air humidity was controlled using a dehumidifying system (low humidity), in the other greenhouse the air conditions were not artificially modified (control humidity). After the nursery period, the plants of all treatments were transplanted and well watered (100% water holding capacity for 1 month, transplanting period). After this period, they received no water (establishment period). At the end of the nursery period it was seen that deficit irrigation had altered the morphology of the R. officinalis plants by reducing plant height, stem diameter, leaf area, total dry weight, and root length, while humidity influenced the parameters related with plant water relations. Low air humidity and deficit irrigation-induced tissue dehydration and lower stomatal conductance values (gs). The plants subjected to deficit irrigation developed leaf osmotic adjustment, which was maintained during the transplanting period. At that time, the plants that had been exposed to deficit irrigation and low humidity showed efficient stomatal regulation (lower gs values). After transplanting and during the establishment period, these plants showed a better water status (higher psil and gs values). Their post-planting survival rate improved as a result of acclimation processes.     

Phenotypic flexibility of digestive morphology and physiology of the South American omnivorous rodent Akodon azarae (Rodentia: Sigmodontinae)

We have determined the occurrence of responses at different levels (morphological, physiological and biochemical) in the omnivorous rodent Akodon azarae upon cold acclimation (15 degrees C). A short-term enhancement in food consumption appeared to account for the maintenance of both mass and body composition. At the morphological level, the main response was an increase in the dimensions of small intestine, which constitutes the section of the gut where absorption and secretion take place. An increase in sucrase specific activity was only found in small intestine. Sucrose independent maltase activity was very low since 99.8% of total maltase activity was due to sucrase-isomaltase (SI) complex. Protease specific activities were not affected. The fact that resting metabolic rates determined at 15 and 23 degrees C were similar in cold acclimated animals suggests a change in lower critical temperature. In conclusion, our results show that A. azarae exhibits different strategies to support cold environment that could lead to an enhancement in digestion and absorption efficiency. Furthermore, this work suggests that low temperature is an independent cue of other environmental factors to trigger the strategies allowing the maintenance of body condition in A. azarae.     

High molecular weight chitosan and sodium alginate effect on secretory acid proteinase of Candida albicans

The effect of high molecular weight chitosan (HMWCh) and sodium alginate (NaAL) on acid proteinase secretion of Candida albicans (one of culture collection and five isolates) was evaluated. The secretion of acid proteinase was induced in the presence and the absence of these polymers in different concentrations and their enzymatic activity was determined. HMWCh and NaAL significantly diminished the enzymatic activity (>76% for the collection strains and > 89% for the isolates, p < 0.05). HMWCh did not modify protein concentrations, but NaAL did. It can be concluded that both polymers can inhibit the proteinase activity of Candida albicans.     

Systemic host inflammatory and coagulation response in the Dengue virus primo-infection

BACKGROUND: Dengue virus infection has been rising in tropical countries. Clinical manifestations range from fever and general malaise to hemorrhagic manifestations and death. The role of endothelial damage and cytokines has not been well established for dengue infection. OBJECTIVE: Determine the profile of the pro-inflammatory cytokines and several markers of coagulopathy of dengue infection. METHODS: Patients admitted between September 2000 and April 2001, who met the WHO dengue diagnosis criteria, were enrolled. Blood samples were collected at 0, 6, 12, 24, 48, 72 h and 5 and 7 days after hospitalization. Profile of pro-inflammatory cytokines, markers of coagulopathy, protein C, protein S, d-dimer, prothrombin time, activated partial thromboplastin time, fibrinogen and activated protein C levels were determined. RESULTS: Thirty-three patients were enrolled. Median (range) age was 31 (13-70) years; 51.5% (17/33) were female. Ten of 33 (30%) presented with hemorrhagic manifestations. Patients were classified: Grade 1: 23/33 (70%), Grade II: 10/33 (30%). At study entry IL-6 was the most elevated, followed by IL-8 and TNF alpha. IL-10 was not elevated. No significant differences (P < 0.05) were demonstrated in the levels of any of the haemostatic or cytokine markers by disease severity (Grade I versus Grade II patients). CONCLUSION: The systemic host inflammatory and coagulation activation response occurs early in patients with dengue viral infection in the absence of severe hemorrhagic manifestations, and provides the basis for considering future clinical study in the use of recombinant human activated protein C to treat patients with severe sepsis from dengue infection.     

Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity

Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.