Characterization of progesterone 11 alpha-hydroxylase of Aspergillus ochraceus TS: a cytochrome P-450 linked monooxygenase

The monooxygenase of Aspergillus ochraceus TS capable of 11 alpha-hydroxylation of progesterone has been resolved into three components and characterized as (i) cytochrome P450, (ii) NADPH-cytochrome P450-reductase and (iii) phosphatidyl choline. The 11 alpha-hydroxylase was observed to be NADPH dependent, and hydroxylation was enhanced by a NADPH regenerating system. This fungal monooxygenase has many features in common with that of mammalian liver microsomes. The role of mammalian cytochrome P450 inducers were tested for induction of 11 alpha-hydroxylase in Aspergillus ochraceus TS. The reductase has been partially purified.     

m-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na⁺/Ca²⁺ exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 μM) in presence of CaCl₂ (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca²⁺ chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca²⁺ overload, which could arise due to inhibition of Ca²⁺ efflux by the forward-mode NCX and that could lead to sustained Ca²⁺ overload in the smooth muscle leading to pulmonary hypertension.     

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.     

Fish kairomones, its benefits and detriments: A model based study both from releaser and acceptor perspective

Kairomones have been documented as an infochemicals to convey information between individuals in aquatic system. However, whether the effect of fish kairomones on acceptor (zooplankton) is beneficial or detrimental is a debatable and unanswered issue. This may be due to lack of feasibility of experimentation. In this study, we theoretically explore how fish kairomones affect the aquatic food chain and provide possible explanation of such different behaviors of kairomones. To do this, we propose two hypotheses and formulate two simple mathematical models which resemble more realistic scenario synergetic with natural complex system. Our study suggests that vertical migration helps zooplankton species for proper conservation of its abundance by avoiding unnecessary over predation.     

Uses for humanised mouse models in precision medicine for neurodegenerative disease

Mammalian Genome - Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are...     

Comparative proteomic network signatures in seminal plasma of infertile men as a function of reactive oxygen species

Background

Reactive oxygen species (ROS) plays a major role in the pathology of male infertility. It is an independent biomarker of sperm function. Seminal plasma is a natural reservoir of antioxidants responsible for the nourishment, protection, capacitation, and motility of sperm within the female reproductive tract resulting in successful fertilization and implantation of the embryo. A comparative proteomic analysis of seminal plasma proteins from fertile men and infertile men with varying levels of ROS was carried out to identify signature proteins involved in ROS-mediated reproductive dysfunction.

Methods

A total of 42 infertile men presenting with infertility and 17 proven fertile donors were enrolled in the study. ROS levels were measured in the seminal ejaculates by chemiluminescence assay. Infertile men were subdivided into Low ROS (0–<93 RLU/s/10⁶ sperm; n = 11), Medium ROS (>93–500 RLU/s/10⁶ sperm; n = 17) and High ROS (>500 RLU/s/10⁶ sperm; n = 14) groups and compared with fertile men (4–50 RLU/s/10⁶ sperm). 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. 1D gel electrophoresis followed by in-gel digestion and LC/MS–MS in a LTQ-Orbitrap Elite hybrid mass spectrometer system was used for proteome analysis. Identification of differentially expressed proteins (DEPs), their cellular localization and involvement in different pathways were examined utilizing bioinformatics tools.

Results

The results indicate that proteins involved in biomolecule metabolism, protein folding and protein degradation are differentially modulated in all three infertile patient groups in comparison to fertile controls. Membrane metallo-endopeptidase (MME) was uniformly overexpressed (>2 fold) in all infertile groups. Pathway involving 35 focus proteins in post-translational modification of proteins, protein folding (heat shock proteins, molecular chaperones) and developmental disorder was overexpressed in the High ROS group compared with fertile control group. MME was one of the key proteins in the pathway. FAM3D was uniquely expressed in fertile group.

Conclusion

We have for the first time demonstrated the presence of 35 DEPs of a single pathway that may lead to impairment of sperm function in men with Low, Medium or High ROS levels by altering protein turn over. MME and FAM3D along with ROS levels in the seminal plasma may serve as good markers for diagnosis of male infertility.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9094-5) contains supplementary material, which is available to authorized users.