Angiotensin (1–7) and its receptor Mas are expressed in the human testis: implications for male infertility

The presence of classical components of the renin-angiotensin system has been demonstrated in the male reproductive tract, mainly in the testes and epididymis. The objective of this study was to verify the localization of angiotensin (Ang)-(1–7) and its receptor Mas in human testis. The study included 12 men with previously proven fertility submitted to orchiectomy for prostate cancer and 20 infertile men submitted to testicular biopsy for infertility work-up, comprising a subgroup with obstructive azoospermia/normal spermatogenesis (n = 8) and another with non-obstructive azoospermia and severely impaired spermatogenesis (n = 12). Testicular tissue samples were processed by immunohistochemistry and real time polymerase chain reaction. Ang-(1–7) was strongly expressed in the interstitial compartment, mainly in Leydig cells, with similar intensity in all groups evaluated. The peptide was also detected in the seminiferous tubules, but with much less intensity compared to interstitial cells. The receptor Mas was equally distributed between interstitial and tubular compartments and was found in all layers of the normal seminiferous epithelium. However, neither Ang-(1–7) nor Mas were detected in the seminiferous tubules of samples with impaired spermatogenesis. The testicular samples of infertile men with impaired spermatogenesis (non-obstructive azoospermia) expressed Mas and ACE2 mRNA at lower concentrations (fold change = 0.06 and 0.04, respectively, P < 0.05) than samples with full spermatogenesis (obstructive azoospermia). This shows, for the first time, the immunolocalization of Ang-(1–7) and its receptor Mas in testes of fertile and infertile men, and suggests that this system may be altered when spermatogenesis is severely impaired.     

Isolation and characterization of microsatellite loci in Santalum lanceolatum and Santalum leptocladum (Santalaceae)

? Premise of the study: Microsatellite primers were developed for the first time in the native Australian sandalwood species Santalum lanceolatum. ? Methods and Results: Using an enrichment cloning protocol, five novel polymorphic codominant loci were developed and characterized in S. lanceolatum and S. leptocladum. In addition to these, three existing microsatellite loci from other sandalwood species were successfully amplified and characterized for S. lanceolatum and S. leptocladum. Among the eight loci, allelic diversity ranged from 4 to 29. ? Conclusions: Primers will be useful for studies of clonality, genetic diversity and spatial genetic structure in wild populations. When coupled with other molecular techniques will help investigate the relationship between S. lanceolatum and S. leptocladum, species of commercial and conservation interest.     

Fungal communities associated with degradation of polyester polyurethane in soil

Soil fungal communities involved in the biodegradation of polyester polyurethane (PU) were investigated. PU coupons were buried in two sandy loam soils with different levels of organic carbon: one was acidic (pH 5.5), and the other was more neutral (pH 6.7). After 5 months of burial, the fungal communities on the surface of the PU were compared with the native soil communities using culture-based and molecular techniques. Putative PU-degrading fungi were common in both soils, as <45% of the fungal colonies cleared the colloidal PU dispersion Impranil on solid medium. Denaturing gradient gel electrophoresis showed that fungal communities on the PU were less diverse than in the soil, and only a few species in the PU communities were detectable in the soil, indicating that only a small subset of the soil fungal communities colonized the PU. Soil type influenced the composition of the PU fungal communities. Geomyces pannorum and a Phoma sp. were the dominant species recovered by culturing from the PU buried in the acidic and neutral soils, respectively. Both fungi degraded Impranil and represented >80% of cultivable colonies from each plastic. However, PU was highly susceptible to degradation in both soils, losing up to 95% of its tensile strength. Therefore, different fungi are associated with PU degradation in different soils but the physical process is independent of soil type.     

Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.     

Isoproterenol-induced impairment of heart function and remodeling are attenuated by the nonpeptide angiotensin-(1-7) analogue AVE 0991

The aim of this study was to evaluate the effects of AVE 0991 (AVE), a nonpeptide compound that mimics Ang-(1-7) actions, on cardiac remodeling. Heart hypertrophy and heart dysfunction were induced by isoproterenol (ISO) (2 mg/kg i.p./day for 7 days) in male Wistar rats. At the end of the 7-day period, the hearts were perfused according to the Langendorff method to evaluate cardiac function. The hearts, atria, and right and left ventricles wet weights were recorded, normalized for body weight and then expressed as muscle mass index (mg/g). In addition, serial sections from left ventricle were stained with hematoxylin-eosin for cell morphometry and with collagen-specific Masson's trichrome for detection of fibrosis. Immunofluorescence-labeling and confocal microscopy were used to investigate the distribution and deposition of collagen types I, III, VI, and fibronectin. AVE reduced the ISO-induced hypertrophy as quantified by myocyte diameter measurements (Control: 10.60+/-0.08 microm; ISO: 14.60+/-0.11 mum; ISO+AVE: 11.22+/-0.08 microm, n = 5). In addition, AVE markedly attenuated the increase of extracellular matrix proteins induced by ISO. AVE treatment also attenuated the decrease in systolic tension and +/-dT/dt and exacerbated the vasodilatation induced by ISO. These results show that AVE has a cardioprotective effect on ISO-induced cardiac remodeling.     

Antioxidant properties of Krebs cycle intermediates against malonate pro-oxidant activity in vitro: a comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates

A variety of Krebs cycle intermediaries has been shown to possess antioxidant properties in different in vivo and in vitro systems. Here we examined whether citrate, succinate, malate, oxaloacetate, fumarate and alpha-ketoglutarate could modulate malonate-induced thiobarbituric acid-reactive species (TBARS) production in rat brain homogenate. The mechanisms involved in their antioxidant activity were also determined using two analytical methods: 1) a popular spectrophotometric method (Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351-358.) and a high performance liquid chromatographic (HPLC) procedure (Grotto, D., Santa Maria, L. D., Boeira, S., Valentini, J., Char?o, M. F., Moro, A. M., Nascimento, P. C., Pomblum, V. J., Garcia, S. C., 2006. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. Journal of Pharmaceutical and Biomedical Analysis 43, 619-624.). Citrate, malate, and oxaloacetate reduced both basal and malonate-induced TBARS production. Their effects were not changed by pre-treatment of rat brain homogenates at 100 degrees C for 10 min. alpha-Ketoglutarate increased basal TBARS without changing malonate-induced TBARS production in fresh and heat-treated homogenates. Succinate reduced basal--without altering malonate-induced TBARS production. Its antioxidant activity was abolished by KCN or heat treatment. Fumarate reduced malonate-induced TBARS production in fresh homogenates; however, its effect was completely abolished by heat treatment. There were minimal differences among the studied methods. Citrate, oxaloacetate, malate, alpha-ketoglutarate and malonate showed iron-chelating activity. We suggest that antioxidant properties of citrate, malate and oxaloacetate were due to their ability to cancel iron redox activity by forming inactive complexes, whereas alpha-ketoglutarate and malonate pro-oxidant activity can be due to formation of active complexes with iron. In contrast, succinate and fumarate antioxidant activity was probably due to some enzymatic system.     

Study of angiotensin-(1-7) vasoactive peptide and its beta-cyclodextrin inclusion complexes: complete sequence-specific NMR assignments and structural studies

We report the complete sequence-specific hydrogen NMR assignments of vasoactive peptide angiotensin-(1-7) (Ang-(1-7)). Assignments of the majority of the resonances were accomplished by COSY, TOCSY, and ROESY peak coordinates at 400MHz and 600MHz. Long-side-chain amino acid spin system identification was facilitated by long-range coherence transfer experiments (TOCSY). Problems with overlapped resonance signals were solved by analysis of heteronuclear 2D experiments (HSQC and HMBC). Nuclear Overhauser effects (NOE) results were used to probe peptide conformation. We show that the inclusion of the angiotensin-(1-7) tyrosine residue is favored in inclusion complexes with beta-cyclodextrin. QM/MM simulations at the DFTB/UFF level confirm the experimental NMR findings and provide detailed structural information on these compounds in aqueous solution.     

Fungal Communities Associated with Degradation of Polyester Polyurethane in Soil

Soil fungal communities involved in the biodegradation of polyester polyurethane (PU) were investigated. PU coupons were buried in two sandy loam soils with different levels of organic carbon: one was acidic (pH 5.5), and the other was more neutral (pH 6.7). After 5 months of burial, the fungal communities on the surface of the PU were compared with the native soil communities using culture-based and molecular techniques. Putative PU-degrading fungi were common in both soils, as <45% of the fungal colonies cleared the colloidal PU dispersion Impranil on solid medium. Denaturing gradient gel electrophoresis showed that fungal communities on the PU were less diverse than in the soil, and only a few species in the PU communities were detectable in the soil, indicating that only a small subset of the soil fungal communities colonized the PU. Soil type influenced the composition of the PU fungal communities. Geomyces pannorum and a Phoma sp. were the dominant species recovered by culturing from the PU buried in the acidic and neutral soils, respectively. Both fungi degraded Impranil and represented >80% of cultivable colonies from each plastic. However, PU was highly susceptible to degradation in both soils, losing up to 95% of its tensile strength. Therefore, different fungi are associated with PU degradation in different soils but the physical process is independent of soil type.