Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity

Adhesion of Albumin to FDA Group I Contact Lenses

Tear protein adhesion can contaminate contact lenses and reduce their effectiveness for the treatment of vision abnormalities.. Protein adhesion depends upon the type of contact lens material, and fluctuates with tear secretion rate and pathology. We examined the adhesion of albumin, a major tear protein, to FDA Group I contact lenses over a four day period. Never worn Optima FW contact lenses (Bausch and Lomb, Inc., Rochester, NY) were incubated in albumin dissolved in optical saline (0.2 mg/ml) contained in borosilicate glass vials. The concentration of albumin in the vials, and adhering to the lenses, was monitored with bicinchoninic acid (BCA). Albumin concentration in the vials decreased on day three, and then regained initial levels. After one day of incubation, albumin adhesion to lenses reached a plateau that was stable through the remainder of the incubation period. This pattern is in contrast to that of lysozyme, which adheres to lenses in an up-down-up-down pattern regardless of lens material. Both albumin and lysozyme adhered to a lesser degree to these lenses than to FDA group IV lenses. These results clearly indicate the need for further studies of this complex interaction