Estrogen replacement therapy and cardioprotection: mechanisms and controversies

Epidemiological and case-controlled studies suggest that estrogen replacement therapy might be beneficial in terms of primary prevention of coronary heart disease (CHD). This beneficial effect of estrogens was initially considered to be due to the reduction of low density lipoproteins (LDL) and to increases in high density lipoproteins (HDL). Recent studies have shown that estrogens protect against oxidative stress and decrease LDL oxidation. Estrogens have direct effects on the arterial tissue and modulate vascular reactivity through nitric oxide and prostaglandin synthesis. While many of the effects of estrogen on vascular tissue are believed to be mediated by estrogen receptors alpha and ß, there is evidence for `immediate non-genomic' effects. The role of HDL in interacting with 17ß-estradiol including its esterification and transfer of esterified estrogens to LDL is beginning to be elucidated. Despite the suggested positive effects of estrogens, two recent placebo-controlled clinical trials in women with CHD did not detect any beneficial effects on overall coronary events with estrogen therapy. In fact, there was an increase in CHD events in some women. Mutations in thrombogenic genes (factor V Leiden, prothrombin mutation, etc.) in a subset of women may play a role in this unexpected finding. Thus, the cardioprotective effect of estrogens appears to be more complicated than originally thought and requires more research.

Nitric oxide, cholesterol oxides and endothelium-dependent vasodilation in plasma of patients with essential hypertension

The objective of the present study was to identify disturbances of nitric oxide radical (·NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of·NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 µM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 µM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ·NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ·NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

A leucine-supplemented diet improved protein content of skeletal muscle in young tumor-bearing rats

Cancer cachexia induces host protein wastage but the mechanisms are poorly understood. Branched-chain amino acids play a regulatory role in the modulation of both protein synthesis and degradation in host tissues. Leucine, an important amino acid in skeletal muscle, is higher oxidized in tumor-bearing animals. A leucine-supplemented diet was used to analyze the effects of Walker 256 tumor growth on body composition in young weanling Wistar rats divided into two main dietary groups: normal diet (N, 18% protein) and leucine-rich diet (L, 15% protein plus 3% leucine), which were further subdivided into control (N or L) or tumor-bearing (W or LW) subgroups. After 12 days, the animals were sacrificed and their carcass analyzed. The tumor-bearing groups showed a decrease in body weight and fat content. Lean carcass mass was lower in the W and LW groups (W = 19.9 ± 0.6, LW = 23.1 ± 1.0 g vs N = 29.4 ± 1.3, L = 28.1 ± 1.9 g, P < 0.05). Tumor weight was similar in both tumor-bearing groups fed either diet. Western blot analysis showed that myosin protein content in gastrocnemius muscle was reduced in tumor-bearing animals (W = 0.234 ± 0.033 vs LW = 0.598 ± 0.036, N = 0.623 ± 0.062, L = 0.697 ± 0.065 arbitrary intensity, P < 0.05). Despite accelerated tumor growth, LW animals exhibited a smaller reduction in lean carcass mass and muscle myosin maintenance, suggesting that excess leucine in the diet could counteract, at least in part, the high host protein wasting in weanling tumor-bearing rats.

Cardiovascular complications and increased levels of circulating modified low density lipoprotein in HIV patients and patients with lipodystrophy

The introduction of highly active antiretroviral therapy (HAART) for patients infected with HIV has significantly prolonged the life expectancy and to some extent has restored a functional immune response. However, the premature introduction of HAART has led to a significant and alarming increase in cardiovascular complications, including myocardial infarction and the appearance of abnormal distribution of body fat seen as lipodystrophy. One key element in the development of ischemic coronary artery disease is the presence of circulating and tissue-fixed modified low density lipoprotein (mLDL) that contributes to the initiation and progression of arterial lesions and to the formation of foam cells. Even though not completely elucidated, the most likely mechanism involves mLDL in the inflammatory response and the induction of a specific immune response against mLDL. Circulating antibodies against mLDL can serve as an indirect marker of the presence of circulating and vessel-fixed mLDL. In the present study, we measured antibodies to mLDL and correlated them with immune status (i.e., number of CD4+ T cells) in 59 HIV patients and with the clinical manifestation of lipodystrophy in 10 patients. We observed a significant reduction in anti-mLDL antibody levels related both to lipodystrophy and to an immunocompromised state in HIV patients. We speculate that these antibodies may explain in part the rapid development of ischemic coronary artery disease in some patients.

Genome features of Leptospira interrogans serovar Copenhageni

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4ºC, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(³H-methyl)-methionine was used. The O-methylated derivatives were oxidized to ³H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.

Effect of a cholesterol-rich diet on the metabolism of the free and esterified cholesterol components of a nanoemulsion that resembles LDL in rabbits

We have shown that the free cholesterol (FC) and the cholesteryl ester (CE) moieties of a nanoemulsion with lipidic structure resembling low-density lipoproteins show distinct metabolic fate in subjects and that this may be related to the presence of dyslipidemia and atherosclerosis. The question was raised whether induction of hyperlipidemia and atherosclerosis in rabbits would affect the metabolic behavior of the two cholesterol forms. Male New Zealand rabbits aged 4-5 months were allocated to a control group (N = 17) fed regular chow and to a 1% cholesterol-fed group (N = 13) during a 2-month period. Subsequently, the nanoemulsion labeled with ³H-FC and 14C-CE was injected intravenously for the determination of plasma kinetics and tissue uptake of the radioactive labels. In controls, FC and CE had similar plasma kinetics (fractional clearance rate, FCR = 0.234 ± 0.056 and 0.170 ± 0.038 h-1, respectively; P = 0.065). In cholesterol-fed rabbits, the clearance of both labels was delayed and, as a remarkable feature, FC-FCR (0.089 ± 0.033 h-1) was considerably greater than CE-FCR (0.046 ± 0.010 h-1; P = 0.026). In the liver, the major nanoemulsion uptake site, uptake of the labels was similar in control animals (FC = 0.2256 ± 0.1475 and CE = 0.2135 ± 0.1580%/g) but in cholesterol-fed animals FC uptake (0.0890 ± 0.0319%/g) was greater than CE uptake (0.0595 ± 0.0207%/g; P < 0.05). Therefore, whereas in controls, FC and CE have similar metabolism, the induction of dyslipidemia and atherosclerosis resulted in dissociation of the two forms of cholesterol.

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.

Association of HDL cholesterol and triglycerides with mortality in patients with heart failure

It has been demonstrated that there is an association between serum lipoproteins and survival rate in patients with ischemic cardiomyopathy, as well as in patients with non-ischemic causes of heart failure. We tested the hypothesis of an association between serum lipoprotein levels and prognosis in a cohort of outpatients with heart failure, including Chagas' heart disease. The lipid profile of 833 outpatients with heart failure in functional classes III and IV of the New York Heart Association, with a mean age of 46.9 ± 10.6 years, 655 (78.6%) men and 178 (21.4%) women, was studied from April 1991 to June 2003. The survival rate was estimated by the Kaplan-Meyer's method and the Cox proportional hazards models. Etiology of heart failure was ischemic cardiomyopathy in 171 (21%) patients, Chagas' heart disease in 144 (17%), hypertensive cardiomyopathy in 136 (16%), and other etiologies in 83 (10%). In 299 (36%) patients, heart failure was ascribed to idiopathic dilated cardiomyopathy. Variables significantly associated with mortality were age (hazard ratio, HR = 1.02; 95%CI = 1.01-1.03; P = 0.0074), male gender (HR = 1.77; 95%CI = 1.2-2.62; P = 0.004), idiopathic dilated cardiomyopathy (HR = 1.81; 95%CI = 1.16-2.82; P = 0.0085), serum triglycerides (HR = 0.97; 95%CI = 0.96-0.98; P < 0.0001), and HDL cholesterol (HR = 0.99; 95%CI = 0.99-1.0; P = 0.0280). Therefore, higher serum HDL cholesterol and higher serum triglycerides were associated with lower mortality in this cohort of outpatients with heart failure.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

Effect of (-)-∆9-tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered ∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: ∆9-THC (N = 10), treated with 10 mg/kg body weight ∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Δ9-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Δ9-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Δ9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.