Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 µmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast.

An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma

A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasmaproteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol develope in our experiments involves coating well with 10 µg/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 µg/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levelsin small samples of mouse plasma.

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways. Indeed, if alternative (compensative) pathway(s) can operate after blockage of the de novo PC biosynthesis pathway this would indicate a potential mechanism for resistance acquisition. Up until now, there is no evidence of such a compensative process occurring in Plasmodium-infected erythrocytes under physiological conditions. Besides, the discovery of a highly parasite-specific pathway (serine decarboxylation and the presence of PS synthase) constitutes a very attractive and promising target, which could be attacked if resistances are built up against choline analogs. Indeed, potential inhibitions of the serine decarboxylase pathway could be very useful in acting instead of, or in surgery with, choline analogs.

Trypanosoma cruzi: inhibition of alpha-hydroxyacid dehydrogenase isozyme II by N-allyl and N-propyl oxamates and their effects on intact epimastigotes

N-allyl (NAOx) and N-propyl (NPOx) oxamates were designed as inhibitors of alpha-hydroxyacid dehydrogenase (HADH) isozyme II from Trypanosoma cruzi. The kinetic studies showed that NAOx and NPOx were competitive inhibitors of HADH-isozyme II (Ki = 72 µM, IC50 = 0.33 mM and 70 µM, IC50 = 0.32 mM, respectively). The attachment of the allylic and propylic chains to nitrogen of the competitive inhibitor oxamate (Ki = 0.91 mM, IC50 = 4.25 mM), increased 12.6 and 13-folds respectively, the affinity for T. cruzi HADH-isozyme II. NAOx and NPOx were selective inhibitors of HADH-isozyme II, because other T. cruzi dehydrogenases were not inhibited by these substances. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with these inhibitors. However, we were not able to detect any trypanocidal activity with these oxamates. When the corresponding ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested as a possible trypanocidal prodrugs, in comparison with nifurtimox and benznidazole, the expected trypanocidal effects were obtained.

Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

DNA topoisomerase inhibitors: biflavonoids from Ouratea species

Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats

The frequent use of nonsteroidal anti-inflammatory drugs (NSAID) in combination with gentamicin poses the additional risk of nephrotoxic renal failure. Cyclooxygenase-1 (COX-1) is the main enzyme responsible for the synthesis of renal vasodilator prostaglandins, while COX-2 participates predominantly in the inflammatory process. Both are inhibited by non-selective NSAID such as indomethacin. Selective COX-2 inhibitors such as rofecoxib seem to have fewer renal side effects than non-selective inhibitors. The objective of the present study was to determine whether the combined use of rofecoxib and gentamicin can prevent the increased renal injury caused by gentamicin and indomethacin. Male Wistar rats (250-300 g) were treated with gentamicin (100 mg/kg body weight, ip, N = 7), indomethacin (5 mg/kg, orally, N = 7), rofecoxib (1.4 mg/kg, orally, N = 7), gentamicin + rofecoxib (100 and 1.4 mg/kg, respectively) or gentamicin + indomethacin (100 and 5 mg/kg, respectively, N = 8) for 5 days. Creatinine clearance and alpha-glutathione-S-transferase concentrations were used as markers of renal injury. Animals were anesthetized with ether and sacrificed for blood collection. The use of gentamicin plus indomethacin led to worsened renal function (0.199 ± 0.019 ml/min), as opposed to the absence of a nephrotoxic effect of rofecoxib when gentamicin plus rofexicob was used (0.242 ± 0.011 ml/min). These results indicate that COX-2-selective inhibitors can be used as an alternative treatment to conventional NSAID, especially in situations in which risk factors for nephrotoxicity are present.

Action of auxin inhibitors on growth and grain yield of soybean

Soybean genotypes grown in sub-tropical climate may exhibit lodging. The plant lodging is influenced by soil type and fertility level, sowing date, latitude and altitude of the location, plant population and conditions of crop development. Plant regulators and herbicides are able to avoid or reduce plant lodging. This study aimed to verify the effects of the growth regulators TIBA and daminozide on vegetative growth and yield of soybean cultivar CD 214 RR. The experiment was carried out at a field in randomized block design with four replications in a factorial scheme. The A factor was represented by the combination of regulators TIBA and daminozide and its concentrations, and the Factor B was seven times of evaluation of injury and plant height or eight times of evaluation of lodging. In the range of doses used, the application of daminozide resulted in greater injury to soybean plants than TIBA. The smaller plant height was achieved by the application of 6 g ha-1 of TIBA and 1200 g ha-¹ of daminozide. Treatments with daminozide (100 g ha-¹) and TIBA (10 g ha-1) stood out due to the reduced lodging of soybean plants. Grain weight increased linearly when the levels of TIBA increased. There was a negative correlation between lodging and grain yield and a positive correlation between plant height and lodging. There was also a negative correlation between injury caused by the application of plant regulators and lodging.

Long term follow-up and patterns of response of ALT in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha

The response to interferon treatment in chronic hepatitis NANB/C has usually been classified as complete, partial or absent, according to the behavior of serum alanine aminotransferase (ALT). However, a more detailed observation of the enzymatic activity has shown that the patterns may be more complex. The aim of this study was to describe the long term follow-up and patterns of ALT response in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha. A follow-up of 6 months or more after interferon-a was achieved in 44 patients. We have classified the serum ALT responses into six patterns and the observed frequencies were as follows: I. Long term response = 9 (20.5%); II. Normalization followed by persistent relapse after IFN = 7 (15.9%); III. Normalization with transient relapse = 5 (11.9%); IV. Temporary normalization and relapse during IFN = 4 (9.1%); V. Partial response (more than 50% of ALT decrease) = 7 (15.9%); VI. No response = 12 (27.3%). In conclusion, ALT patterns vary widely during and after IFN treatment and can be classified in at least 6 types.

Autoantibodies before, during and after administration of recombinant interferon-alpha for chronic viral hepatitis

This study was undertaken to investigate the presence of autoantibodies in patients with chronic viral hepatitis B and C, before, during and after interferon-alpha (IFN-alpha) therapy and to study their relation to dose and type of IFN-alpha and response to treatment. Fifty patients with chronic hepatitis were divided in two groups, a control-group of 21 patients (10 type B and 11 type C) who were followed for 6 months without treatment and an IFN-group consisting of 29 patients (8 type B and 21 type C) who received IFN therapy for 6 months. Serum samples were tested for a range of antibodies at the start of the study, during therapy and at the end of the 6 month period. Antibodies tested for included: antinuclear, smooth muscle, antimitochondrial, parietal cell and thyroid microsomal. Four (8%) of the total patient group had autoantibodies at the beginning of the study (two in each group). During the follow-up period no patient in the control group developed antibodies compared with 3 (11%) patients in the treatment group. Autoantibodies developed in patients treated with higher doses of IFN and were found in those patients who tended to show a poor response to IFN-therapy. Further studies are needed to establish the relationship between poor response to IFN-alpha and development of autoantibodies.

Plasma levels of tumor necrosis factor-alpha in patients with visceral leishmaniasis (Kala-Azar). Association with activity of the disease and clinical remisson following antimonial therapy

Evaluation of TNF-alpha in patients with Kala-azar has drawn increasing interest due to its regulatory role on the immune system, in addition to its cachetizing activity. The objective of this study was to examine the association between plasma levels of TNF-alpha, measured by immunore-activity (ELISA) and bioactivity (cytotoxicity assay with L-929 cells), and clinical manifestations of visceral leishmaniasis. Plasma samples from 19 patients with Kala-azar were obtained before, during and at the end of antimonial therapy. TNF-alpha determinations was done by using the cytotoxicity assay (all patients) and the enzyme-linked immunoassay (ELISA - 14 patients). A discrepancy between results obtained by ELISA and cytotoxicity assay was observed. Levels of circulating TNF-alpha, assessed by ELISA, were higher in patients than in healthy controls, and declined significantly with improvement in clinical and laboratory parameters. Plasma levels before treatment were 124.7 ± 93.3 pg/ml (mean ± SD) and were higher than at the end of therapy 13.9 ± 25.1 pg/ml (mean ± SD) (p = 0.001). In contrast, plasma levels of TNF-alpha evaluated by cytotoxicity assay did not follow a predicted course during follow-up. Lysis, in this case, might be not totally attributed to TNF-alpha. The discrepancy might be attributed to the presence of factor(s) known to influence the release and activity of TNF-alpha.

Chronic hepatitis C virus infections in Brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-a) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-a, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0.005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-a therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

The pattern of immune cell infiltration in chromoblastomycosis: involvement of macrophage inflammatory protein-1 alpha/CCL3 and fungi persistence

Chromoblastomycosis (CR) is a subcutaneous chronic mycosis characterized by a granulomatous inflammatory response. However, little is known regarding the pattern of leukocyte subsets in CR and the pathways involved in their recruitment. The objective of this study was to assess the cellular subsets, chemokine, chemokine receptors and enzymes in CR. The inflammatory infiltrate was characterized by immunohistochemistry using antibodies against macrophages (CD68), Langerhans'cells (S100), lymphocytes (CD3, CD4, CD8, CD45RO, CD20 and CD56) and neutrophils (CD15). The expression of MIP-1alpha (Macrophage inflammatory protein-1alpha), chemokine receptors (CXCR3 and CCR1) and enzymes (superoxide dismutase-SOD and nitric oxide synthase-iNOS) was also evaluated by the same method. We observed an increase in all populations evaluated when compared with the controls. Numbers of CD15+ and CD56+ were significantly lower than CD3+, CD4+, CD20+ and CD68+ cells. Statistical analysis revealed an association of fungi numbers with CD3, CD45RO and iNOS-positive cells. Furthermore, MIP-1alpha expression was associated with CD45RO, CD68, iNOS and CXCR3. Our results suggest a possible role of MIP-1alpha and fungi persistence in the cell infiltration in CR sites.