Thermosensitive ion channel TRPV1 is endogenously expressed in the sperm of a fresh water teleost fish (Labeo rohita) and regulates sperm motility.

Sperm cells exhibit extremely high sensitivity in response to slight changes in temperature, osmotic pressure and/or presence of various chemical stimuli. In most cases throughout the evolution, these physico-chemical stimuli trigger Ca (2+)-signaling and subsequently alter structure, cellular function, motility and survival of the sperm cells. Few reports have recently demonstrated the presence of Transient Receptor Potential (TRP) channels in the sperm cells from higher eukaryotes, mainly from higher mammals. In this work, we have explored if the sperm cells from lower vertebrates can also have thermo-sensitive TRP channels. In this paper, we demonstrate the endogenous presence of one specific thermo-sensitive ion channel, namely Transient Receptor Potential Vanilloid family member sub type 1 (TRPV1) in the sperm cells collected from fresh water teleost fish, Labeo rohita. By using western blot analysis, fluorescence assisted cell sorting (FACS) and confocal microscopy; we confirm the presence of this non-selective cation channel. Activation of TRPV1 by an endogenous activator NADA significantly increases the quality as well as the duration of fish sperm movement. The sperm cell specific expression of TRPV1 matches well with our in silico sequence analysis. The results demonstrate that TRPV1 gene is conserved in various fishes, ranging from 1-3 in copy number, and it originated by fish-specific duplication events within the last 320 million years (MY). To the best of our knowledge, this is the first report demonstrating the presence of any thermo-sensitive TRP channels in the sperm cells of early vertebrates as well as of aquatic animals, which undergo external fertilization in fresh water. This observation may have implications in the aquaculture, breeding of several fresh water and marine fish species and cryopreservation of fish sperms.

Protocol for a mammalian cell-based assay for monitoring the HIV-1 protease activity.

Proteases are essential at different stages of the viral life cycle and for the establishment of a successful infection. Monitoring the catalytic activity of proteases in an easy and straightforward manner can thus drastically facilitate the discovery of novel antivirals, as well as help elucidate the activity and mechanism of action of the viral protease under study. In our laboratory, we have developed an assay in T-cells with a robust read-out to monitor the proteolytic activity of HIV-1 Protease (PR). The assay utilizes the prototypic transcription factor Gal4, which consists of the N-terminal DNA-binding domain and the C-terminal trans-activation domain. The assay is based upon (1) introduction of PR in between the two Gal4 domains to obtain a PR/Gal4 fusion protein and (2) utilization of the enhanced Green Fluorescent Protein as reporter of PR activity.In order to overcome the possible cellular cytotoxicity of PR, the fusion protein in our assay is under the control of a tetracycline-inducible promoter. This ensures that it will be expressed only when needed, upon the addition of tetracycline or doxycycline. When active, PR has autocatalytic activity and cleaves itself from the Gal4 domains, resulting in the inability to induce eGFP expression. However, if PR activity is blocked or it is inactive, the two domains remain intact, resulting in eGFP expression. The assay can therefore be utilized to analyze the inhibitory effects of factors, peptides or compounds, designed on a rational- or nonrational-based approach, in the natural milieu of infection, where eGFP serves as a biosensor for PR activity.