ABSTRACT
OBJECTIVE: To compare buoyant density and sedimentation kinetics of human immunodeficiency virus 1 (HIV-1) in two sperm-washing media, Percoll and Pureception. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): None. INTERVENTION(S): Buoyant density and sedimentation kinetics of HIV-1 particles (MOLT-4/LAI strain) were measured in Percoll and Pureception using isopyknic ultracentrifugation and continuous-density-gradient centrifugation. MAIN OUTCOME MEASURE(S): The HIV-1 particles were detected and semiquantified using a reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 RNA. RESULT(S): Calculated buoyant density of HIV-1 was approximately 1.042 in both media in isopyknic centrifugation. However, most HIV-1 particles were found in fractions with specific gravity less than 1.04 in both media, even after 40 minutes of density-gradient centrifugation at 1,600 g. Small viral accumulations were observed at the bottom of the tube in Pureception density gradients. CONCLUSION(S): Although we found very high efficiency of HIV-1 removal using density-gradient centrifugation, a minute quantity of virus was found at the bottom of the gradient tube when Pureception was used as the medium.
Subject(s)
Centrifugation, Density Gradient , Centrifugation, Isopycnic , HIV-1/isolation & purification , Semen/virology , Specimen Handling/methods , HIV-1/genetics , Humans , Kinetics , Male , Povidone , RNA, Viral/isolation & purification , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Silicon Dioxide , Viral LoadABSTRACT
Human sperm nucleoproteins consist of protamines and histones. Changes in composition of these proteins are thought to correlate with spermatogenesis and may be involved in some instances of male infertility. We sought to separate sperm nucleoproteins including variants of protamine using an improved two-dimensional electrophoretic method, with the aim of comprehensively analysing all sperm nucleoprotein constituents. After extracting nuclear basic proteins from the sperm of normal volunteers, we analysed these proteins on a gel sheet by a radical free, highly reducing method based on Kaltschmidt and Whittmann's two-dimensional electrophoresis. Basic proteins from sperm nuclei were separated clearly into 12 spots. By amino acid sequence analysis, these spots corresponded to protamine 1 (P1)- (five spots), protamine 2 (P2)-related proteins (six spots) and testis-specific histone H2B (one spot). The N-terminal amino acid sequences of the six P2-related proteins were compatible with those of HPI1, HPI2, HPS1, HPS2, HP2 and HP3, and quantitative comparison could be performed. In conclusion, human sperm nucleoproteins including all P2-related variants could be analysed quantitatively with high resolution on a single electrophoretic gel.