A novel approach for screening immunogenic proteins in Penicillium marneffei using the DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus

Using serum from guinea-pigs immunized with a DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus to screen a cDNA library of A. fumigatus, we cloned a novel immunogenic 57-kDa protein in A. fumigatus. We also cloned its 55-kDa homologue in Penicillium marneffei, which was possibly related to amino acid biosynthesis and metabolism, with homologues present only in the subphylum Pezizomycotina of Ascomycota. The recombinant 55-kDa protein of P. marneffei reacted strongly with guinea-pig serum immunized with P. marneffei and with the sera of patients with P. marneffei infection. A similar approach could be applied to immunogenic protein screening in other microorganisms for serological diagnosis, epidemiological studies and the study of vaccines.     

Induction of mitochondrial nitrative damage and cardiac dysfunction by chronic provision of dietary omega-6 polyunsaturated fatty acids

Increased awareness of obesity has led to a dietary shift toward "heart-friendly" vegetable oils containing omega-6 polyunsaturated fatty acid (omega-6 PUFA). In addition to its beneficial effects, omega-6 PUFA also exhibits proinflammatory and prooxidative properties. We hypothesized that chronic dietary omega-6 PUFA can induce free radical generation, predisposing the cardiac mitochondria to oxidative damage. Male Wistar rats were fed a diet supplemented with 20% w/w sunflower oil, rich in omega-6 PUFA (HP) or normal laboratory chow (LP) for 4 weeks. HP feeding augmented phospholipase A(2) activity and breakdown of cardiolipin, a mitochondrial phospholipid. HP hearts also demonstrated elevated inducible nitric oxide synthase expression, loss of Mn superoxide dismutase, and increased mitochondrial nitrotyrosine levels. In these hearts, oxidative damage to mitochondrial DNA (mDNA) was demonstrated by 8-hydroxyguanosine immunopositivity, overexpression of DNA repair enzymes, and a decrease in the mRNA expression of specific respiratory subunits encoded by the mDNA. Functionally, at higher workloads, HP hearts also demonstrated a greater decline in cardiac work than LP, suggesting a compromised mitochondrial reserve. Our study, for the first time, demonstrates that consumption of a high fat diet rich in omega-6 PUFA for only 4 weeks instigates mitochondrial nitrosative damage and causes cardiac dysfunction at high afterloads.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.     

Insulin resistance is an intrinsic defect independent of fat mass in women with Turner's syndrome

BACKGROUND/AIMS: Turner's syndrome (TS) is associated with increased insulin resistance and adiposity, which might be associated with type 2 diabetes in later life. We aimed to determine whether the defect in insulin sensitivity is a primary intrinsic defect in TS or dependent on variation in body composition. METHODS: Sixteen women with TS not on growth hormone replacement but receiving oestrogen replacement therapy [age (mean +/- SD): 30.2 +/- 8.5 years; height-corrected fat-free mass: 26.1 +/- 3.1 kg/height] and a control group of 16 normal healthy women (age: 30.1 +/- 8.2 years; height-corrected fat-free mass: 25.9 +/- 2.4 kg/height) were studied. Fasting blood samples were obtained for measurement of glucose, insulin, IGF-I, IGFBP-1, IGFBP-3 and lipid levels. The hyperinsulinaemic euglycaemic clamp was performed to assess peripheral insulin sensitivity (M value), and the Homeostasis Model Assessment (HOMA-S) was used to estimate fasting insulin sensitivity. Body composition was assessed using a dual-energy X-ray absorptiometry scan. RESULTS: Fasting insulin sensitivity (HOMA-S 103.2 +/- 78.6 vs. 193.9 +/- 93.5, p = 0.006) was lower in TS subjects compared to controls as was whole-body insulin sensitivity (M value 2.9 +/- 1.9 vs. 5.5 +/- 2.6 mg/kg/min, p = 0.003). In a multiple regression analysis the Turner karyotype was significantly related to insulin sensitivity (p = 0.008) independent of any differences in fat-free mass and percent whole-body fat mass. CONCLUSION: The increased insulin resistance in women with TS is independent of measures of body composition and may represent an intrinsic defect related to their chromosomal abnormality.     

Chronic estrogen treatment reduces vaso-constrictor responses in insulin resistant rats

Previous experiments have shown that chronic estrogen treatment via subcutaneous implants prevented insulin-induced blood pressure elevation and increased insulin sensitivity in ovariectomized female rats. In vitro vascular studies were performed using isolated mesenteric arteries to determine the effect of chronic estrogen and insulin treatments on vascular responses to vasoconstrictor agents. Female Wistar rats were assigned to the following groups: sham-operated, sham-operated plus insulin, sham-operated plus insulin plus estrogen, ovariectomized, ovariectomized plus insulin, and ovariectomized plus insulin plus estrogen. Chronic insulin and estrogen treatments were initiated with subcutaneous placement of insulin implants (2 U/d) and 17beta-estradiol implants (0.5 mg/pellet, 60 day release) at the back of the neck. After 8 weeks of treatment, mesenteric arteries were isolated for assessment of constrictor responses to norepinephrine and the thromboxane A2 analogue U46619 in the presence or absence of the endothelium. The results show that chronic estrogen treatment attenuated the vascular constrictor responses to norepinephrine and U46619 only in endothelium intact vessels. Incubation with insulin did not significantly affect norepinephrine-induced vascular smooth muscle contraction. The study provides evidence that the mechanism by which estrogen prevents insulin-induced blood pressure elevation in insulin-treated ovariectomized rats is by influencing endothelium-derived vasoactive factors such as thromboxane A2.     

Effects of endothelin receptor blockade on hypervasoreactivity in streptozotocin-diabetic rats: vessel-specific involvement of thromboxane A2

Increased vasoconstrictor response to norepinephrine (NE) and endothelin (ET)-1 in arteries from diabetic animals is ameliorated by chronic endothelin receptor blockade with bosentan and was absent in endothelium-denuded arteries, suggesting the involvement of ET-1 and an endothelium-derived contracting factor such as thromboxane A2 (TxA2). To examine this possibility, we determined the effects of acute blockade of ET receptors or inhibition of TxA2 synthesis on the vascular function of superior mesenteric arteries (SMA) and renal arteries (RA) isolated from nondiabetic and 11-week streptozotocin (STZ) diabetic rats chronically treated with either bosentan or vehicle. Both in vitro incubation with bosentan and a selective ETA receptor blocker, BQ123, eradicated the increase in NE contractile responses in diabetic SMA. Additionally, in vitro incubation with the thromboxane synthase inhibitor, dazmegrel, abrogated the exaggerated NE and ET-1 contractile responses in diabetic SMA. Conversely, in RA, no significant acute effect of bosentan, BQ123, nor dazmegrel on vascular responses to NE was observed. Dazmegrel incubation attenuated the maximum contractile responses to ET-1 in diabetic RA; however, these responses in diabetic RA remained significantly greater than those of other groups. Diabetic RA but not SMA exhibited an enhanced contractile response to the TxA2 analogue U46619, which was corrected by chronic bosentan treatment. Immunohistochemical analyses in diabetic SMA revealed an increase in ETA receptor level that was normalized by chronic bosentan treatment. These data indicate that an interaction between ET-1 and TxA2 may be involved in mediating the exaggerated vasoconstrictor responses in diabetic arteries. Furthermore, the underlying mechanisms appear to be vessel specific.     

Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.