Publications by authors named "Kenneth P Roberts"

28 Publications

  • Page 1 of 1

Enhanced hot electron lifetimes in quantum wells with inhibited phonon coupling.

Sci Rep 2018 Aug 20;8(1):12473. Epub 2018 Aug 20.

Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, 73019, USA.

Hot electrons established by the absorption of high-energy photons typically thermalize on a picosecond time scale in a semiconductor, dissipating energy via various phonon-mediated relaxation pathways. Here it is shown that a strong hot carrier distribution can be produced using a type-II quantum well structure. In such systems it is shown that the dominant hot carrier thermalization process is limited by the radiative recombination lifetime of electrons with reduced wavefunction overlap with holes. It is proposed that the subsequent reabsorption of acoustic and optical phonons is facilitated by a mismatch in phonon dispersions at the InAs-AlAsSb interface and serves to further stabilize hot electrons in this system. This lengthens the time scale for thermalization to nanoseconds and results in a hot electron distribution with a temperature of 490 K for a quantum well structure under steady-state illumination at room temperature.
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http://dx.doi.org/10.1038/s41598-018-30894-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102289PMC
August 2018

Hospital-level Variation in the Quality of Benign Inpatient Urologic Surgery.

Urology 2016 Jan 27;87:82-7. Epub 2015 Oct 27.

Department of Urology, University of Washington School of Medicine, Seattle, WA. Electronic address:

Objective: To examine hospital-level variation in outcomes following benign urologic surgeries given that hospital-level variation in surgical outcomes can portend quality and appropriateness of care concerns and identify quality improvement opportunities in perioperative care.

Materials And Methods: Using the Washington State Comprehensive Hospital Abstract Reporting System, we identified patients who underwent transurethral resection of the prostate (TURP), percutaneous nephrostolithotomy (PCNL), and pyeloplasty from 2003 to 2008. We classified prolonged postoperative length of stay (LOS) as that exceeding the 75th percentile, and we measured the rate of Agency for Healthcare Quality Patient Safety Indicators, readmissions, and death. We calculated hospital-specific observed-to-expected event rates using random effects multilevel multivariable models adjusted for age and comorbidity.

Results: We identified 6699 TURP patients at 54 hospitals, 2541 PCNL patients at 45 hospitals, and 584 pyeloplasty patients at 36 hospitals. Complication rates were highest after PCNL (22.9% prolonged LOS vs 17.3% for TURP and 13.9% for pyeloplasty, P < .001; 3.4% 90-day mortality vs 0.6% for TURP and 0% for pyeloplasty). Hospital-level variation was most substantial for LOS after TURP and pyeloplasty (8.1% and 14.3% of variance in prolonged LOS, respectively).

Conclusion: Hospital-level variation is common after benign inpatient urologic surgeries and may relate to difference in perioperative provider practice patterns. The morbidity of PCNL in this study was higher than expected and merits further investigation.
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http://dx.doi.org/10.1016/j.urology.2015.07.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861261PMC
January 2016

Comparative study on coating CdSe nanocrystals with surfactants.

Mikrochim Acta 2013 8;180:1341-1350. Epub 2013 Aug 8.

Faculty of Chemistry, Department of Analytical Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warsaw, Poland.

We have synthesized CdSe nanocrystals (NCs) in sizes from 2.2 to 5.1 nm passivated with hydrophobic trioctylphosphine oxide (TOPO) in combination trioctylphosphine (TOP) or tributylphosphine (TBP) to obtain particles of the type CdSe/TOPO/TOP or CdSe/TOPO/TBP. These NCs were then dispersed in aqueous solution of ionic or non-ionic surfactants (such as stearate, oleic acid, Tween) using a biphase (water and chloroform or hexane) transfer method. It is found that both the structure of the surfactant and the native surface of the ligand govern the coating of the NCs with surfactants. More specifically, the hydrophobicity-hydrophilicity balance of the surfactant regulates the coating efficacy, thereby transferring the NC from the organic to the aqueous phase. The type of ligand on the NCs and the kind of coating surfactant also affect photoluminescence (PL). The ratio of PL and absorbance unit (defined as PL per 0.1 AU) was implemented as a tool to monitor changes in PL intensity and wavelength as a function of size, coatings and surface defects. Finally, the distribution of CdSe nanocrystals between pseudophases in cloud point extraction was discussed based on experimental results. It was concluded that the size of CdSe nanocrystal present in an appropriate pseudophase is correlated with the way in which the non-ionic surfactant coats CdSe nanocrystals. FigureCoating of CdSe semiconductor nanocrystals with surfactants impacts nanocrystals' spectral features. Absorbance of first exciton absorption band was used to estimate ability of surfactant to disperse CdSe nanocrystals. Photoluminescence (PL) intensity and position of PL band were analysed in terms of nanocrystal's surface phenomena via surfactants applied for coating.
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http://dx.doi.org/10.1007/s00604-013-1062-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779019PMC
August 2013

Capillary zone electrophoresis of quantum dots dispersed in mixed micelles: new evidence of the concentration effect.

J Chromatogr A 2013 Aug 16;1305:320-7. Epub 2013 Jul 16.

Warsaw University of Technology, Faculty of Chemistry, Chair of Analytical Chemistry, Noakowskiego 3, 00664 Warsaw, Poland.

CZE was applied to characterizing a nanoparticle/micellar system, in which CdSe nanoparticles dispersed in a mixture of non-ionic and ionic surfactants are introduced into a capillary to form the sample zone that migrates being sandwiched by the background electrolyte (BGE). Parameters that affect the migration of the surface-modified CdSe nanoparticles and conditions under which they are focused within the micellar zone (or released from it into bulk BGE) were explored, including the sample composition, sample plug length, and applied voltage. The observed migration behavior was analyzed within the framework of a concept of formation of a mixed pseudomicellar system, according to which a nanoparticle coated with an ionic surfactant is treated as a micelle-like entity. It was found out that the nanoparticles are subject to transformation that results in building-up a mixed pseudomicellar system (with regular micelles), with a consequence of exhibiting distinctive migration phenomena. Particularly observed and brought into focus is the event of focusing of the nanoparticles.
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http://dx.doi.org/10.1016/j.chroma.2013.07.047DOI Listing
August 2013

A systematic analysis of a deep mouse epididymal sperm proteome.

Biol Reprod 2012 Jun 21;87(6):141. Epub 2012 Dec 21.

School of Molecular Biosciences, Washington State University, Spokane, Washington 99201, USA.

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It therefore is theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end, we performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and confidently identified 2850 proteins, which to our knowledge is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, beta-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.
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http://dx.doi.org/10.1095/biolreprod.112.104208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435428PMC
June 2012

Characterization of CdSe nanocrystals coated with amphiphiles. A capillary electrophoresis study.

Mikrochim Acta 2012 Feb 4;176(3-4):345-358. Epub 2011 Nov 4.

We have synthesized CdSe nanocrystals (NCs) possessing a trioctylphosphine surface passivation layer and modified with amphiphilic molecules to form a surface bilayer. The NCs covered with single amphiphiles are not stable in aqueous solution, but a mixed amphiphilic system is shown to provide stability in solution over several months. The solutions of the modified NCs were characterized by UV-Vis absorbance, photoluminescence, and transmission electron microscopy. An electrophoretic study revealed two operational modes. The first relies on the enrichment of NCs using a micellar plug as a tool. The accumulation of NCs at the plug-electrolyte buffer interface results in a sharp peak. By controlling the electrophoretic conditions, nanocrystals were forced to exit a micellar plug into an electrolyte buffer. We conclude that a system consisting of modified nanocrystals and a micellar plug can act as a mixed pseudomicellar system, where modified nanocrystals play the role of pseudomicelles.FigureElectrophoretic focusing of amphiphile coated CdSe nanocrystals using a micellar plug. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00604-011-0727-8) contains supplementary material, which is available to authorized users.
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http://dx.doi.org/10.1007/s00604-011-0727-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267930PMC
February 2012

Hospital-level variation in the quality of urologic cancer surgery.

Cancer 2012 Feb 26;118(4):987-96. Epub 2011 Jul 26.

Department of Urology, University of Washington School of Medicine, Seattle, Washington 98195, USA.

Background: Unexplained variation in outcomes after common surgeries raises concerns about the quality and appropriateness of surgical care. Understanding variation in surgical outcomes may identify processes that could affect the quality of surgical and postoperative care. The authors of this report examined hospital-level variation in outcomes after inpatient urologic oncology procedures.

Methods: Patients who underwent radical cystectomy, radical nephrectomy, and radical prostatectomy were identified from the Washington State Comprehensive Hospital Abstract Reporting System for the years 2003 through 2007. The postoperative length of stay (LOS) was measured, and LOS that exceeded the 75th percentile was classified as prolonged. The occurrence of Agency for Healthcare Quality patient safety indicators (PSIs), readmissions, and deaths also were measured. Analyses were adjusted for patient age and comorbidity in random effects, multilevel, multivariable models that assessed hospital-level outcomes.

Results: The authors identified 853 patients from 37 hospitals who underwent cystectomy, 3018 patients who underwent nephrectomy from 51 hospitals, and 8228 patients who underwent prostatectomy from 51 hospitals. Complications captured by PSIs were rare. Hospital-level variation was most profound for LOS outcomes after nephrectomy and prostatectomy (variance in prolonged LOS, 8.1% and 26.7%, respectively), thromboembolic events after nephrectomy (8% of variance), and mortality after cystectomy (7.1% of variance).

Conclusions: Hospital-level variation confounds the care of urologic cancer patients in the state of Washington. The authors concluded that transparent reporting of surgical outcomes and local quality-improvement initiatives should be considered to ameliorate the observed variation and improve the quality of cystectomy, nephrectomy, and prostatectomy care.
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http://dx.doi.org/10.1002/cncr.26373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273633PMC
February 2012

Capillary electrophoretic separation of nanoparticles.

Anal Bioanal Chem 2011 Mar 26;399(8):2831-42. Epub 2011 Jan 26.

Faculty of Chemistry, Department of Analytical Chemistry, Warsaw University of Technology, Poland.

In the present work, CdSe nanocrystals (NCs) synthesized with a trioctylphosphine surface passivation layer were modified using amphiphilic molecules to form a surface bilayer capable of providing stable NCs aqueous solutions. Such modified nanocrystals were used as a test solute in order to analyze new electrophoretic phenomena, by applying a micellar plug as a separation tool for discriminating nanocrystals between micellar and micelle-free zones during electrophoresis. The distribution of NCs between both zones depended on the affinity of nanocrystals towards the micellar zone, and this relies on the kind of surface ligands attached to the NCs, as well as electrophoretic conditions applied. In this case, the NCs that migrated within a micellar zone can be focused using a preconcentration mechanism. By modifying electrophoretic conditions, NCs were forced to migrate outside the micellar zone in the form of a typical CZE peak. In this situation, a two-order difference in separation efficiencies, in terms of theoretical plates, was observed between focused NCs (N ~ 10(7)) and a typical CZE peak for NCs (N ~ 10(5)). By applying the amino-functionalized NCs the preconcentration of NCs, using a micellar plug, was examined, with the conclusion that preconcentration efficiency, in terms of the enhancement factor for peak height (SEF(height)) can be, at least 20. The distribution effect was applied to separate CdSe/ZnS NCs encapsulated in silica, as well as surface-modified with DNA, which allows the estimation of the yield of conjugation of biologically active molecules to a particle surface.
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http://dx.doi.org/10.1007/s00216-011-4650-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3043243PMC
March 2011

Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa.

Reprod Fertil Dev 2010 ;22(6):893-900

Departments of Integrative Biology and Physiology and Urologic Surgery, University of Minnesota, Minneapolis, MN 55455, USA.

Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 microg mL(-1). No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 microg mL(-1). The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa.
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http://dx.doi.org/10.1071/RD09274DOI Listing
September 2010

Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility.

Biol Reprod 2009 Oct 17;81(4):700-6. Epub 2009 Jun 17.

Departments of Mechanical Engineering, Biomedical Engineering, Urologic Surgery, and Integrative Biology & Physiology, University of Minnesota, Minneapolis, Minnesota, USA.

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.
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http://dx.doi.org/10.1095/biolreprod.109.076075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754885PMC
October 2009

Feasibility of using a computer modeling approach to study SUI Induced by landing a jump.

Ann Biomed Eng 2009 Jul 5;37(7):1425-33. Epub 2009 May 5.

Department of Urologic Surgery, University of Minnesota, 725 Mayo Memorial Building, 420 Delaware Street S.E., Minneapolis, MN 55455, USA.

Stress urinary incontinence (SUI) occurs due to anatomic and/or neurologic factors involving connective tissues, muscles and nerves. Although SUI is more common in post-menopausal and multiparous women, studies have also shown a high prevalence of SUI in young, physically fit female athletes. With a goal toward dynamic subject-specific mechanical characterization of the interaction between anatomical structures during physical activities that elicit SUI in females during physical or daily activities, a computer aided design (CAD)-based computer model of the female pelvis has been developed to test the feasibility of the computer modeling approach in understanding the measurable differences between stress-continent and stress-incontinent women. In the present study, a fluid-structure interaction analysis was conducted by using the finite element (FE) analysis technique based on the CAD-based computer model of the female pelvis to investigate the urine leakage in females during jumping. To the best of our knowledge, this is the first application of a fluid-structure interaction FE analysis approach in understanding the mechanisms of SUI in females. Through a series of computer simulations, the effects of varying impact forces determined by jumping height and bladder volume were investigated. The dynamic computer simulation results revealed that jumping heights have a significant influence on the volume of urine leakage caused by the landing impact of jumping. Bladder volume did not have a significant influence on leakage when the jumping heights were smaller than 1 ft, which indicates that normal walking (corresponds to a jumping height smaller than 0.1 ft) is not the primary cause of urine leakage for healthy females. The computer simulation results also showed that the deformation difference between the anterior and posterior portion of the female pelvis causes opening of the urethra and resultant urine leakage. The present study demonstrates the feasibility of using a computer modeling approach to study female SUI during physical and daily activities.
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http://dx.doi.org/10.1007/s10439-009-9705-2DOI Listing
July 2009

Lead placement and associated nerve distribution of an implantable periurethral electrostimulator.

Int Urogynecol J Pelvic Floor Dysfunct 2009 Mar 4;20(3):325-9. Epub 2008 Dec 4.

Department of Obstetrics and Gynecology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA.

The aim of this study was to determine gross and neuroanatomic features of a novel periurethral neuromuscular electrostimulator. Periurethral leads were placed in eight female cadavers. In two cases, leads were imaged after placement to enhance anatomic understanding. Pelvic viscera were removed en bloc for analysis of lead placement in the six remaining cadavers. Excised tissue was sectioned and immunostained to identify general, afferent, sympathetic, and nitric oxide synthase efferent nerve fibers. The electrodes were found within/lateral (n = 4), within/posterolateral (n = 9), and anterolateral (n = 1) to the external urethral sphincter (distance 0.25 +/- 0.5, 2.9 +/- 3.3, and 1.0 +/- 0.0 mm, respectively). The electrode to the urethra and vagina distance averaged 7.6 +/- 3.4 and 8.8 +/- 4.3 mm, respectively. Variable density staining for all nerve types was found around the electrode. A periurethral electrode interfaces the external urethral sphincter, and the adjacent distribution of nerve fibers supports proposed neuromuscular therapeutic mechanisms.
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http://dx.doi.org/10.1007/s00192-008-0776-7DOI Listing
March 2009

Molecular cloning and expression of the CRISP family of proteins in the boar.

Biol Reprod 2008 Dec 20;79(6):1129-34. Epub 2008 Aug 20.

Departments of Integrative Biology & Physiology and Urologic Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.
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http://dx.doi.org/10.1095/biolreprod.108.070177DOI Listing
December 2008

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm.

Biol Reprod 2008 Dec 13;79(6):1046-53. Epub 2008 Aug 13.

Departments of Integrative Biology & Physiology, Cell Biology, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.
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http://dx.doi.org/10.1095/biolreprod.108.070664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844498PMC
December 2008

Structure and function of epididymal protein cysteine-rich secretory protein-1.

Asian J Androl 2007 Jul;9(4):508-14

Department of Urologic Surgery, University of Minnesota, MMC 394, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

Cysteine-rich secretory protein-1 (CRISP-1) is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP-1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract.
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http://dx.doi.org/10.1111/j.1745-7262.2007.00318.xDOI Listing
July 2007

Quantification of penicillin G during labor and delivery by capillary electrophoresis.

J Biochem Biophys Methods 2008 Apr 21;70(6):992-8. Epub 2007 May 21.

Department of Chemistry and Biochemistry, The University of Tulsa, 600 South College Avenue, Tulsa, OK 74104, United States.

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.
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http://dx.doi.org/10.1016/j.jbbm.2007.05.002DOI Listing
April 2008

Effects of seminal plasma on cooling-induced capacitative changes in boar sperm.

J Androl 2007 May-Jun;28(3):416-22. Epub 2006 Dec 27.

Department of Urologic Surgery, University of Minnesota Medical School, MMC 394, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

Porcine seminal plasma (SP) has been shown to contain factors that have a decapacitative or capacitation-inhibiting effect on sperm. The objectives of the present study were to compare the capacitative changes observed in cooled sperm with those seen in sperm after in vitro capacitation and to determine whether SP could prevent these changes. Sperm were subjected to incubation or to slow cooling under noncapacitating or capacitating conditions. The effect of SP on protein tyrosine phosphorylation and the ability of the sperm to undergo an acrosome reaction (AR) were determined. Cooled sperm displayed an increased level of tyrosine phosphorylation and a higher percentage of induced AR sperm compared to incubated sperm. The addition of SP inhibited the number of ARs that occurred during incubation and cooling. These results suggest that cooling of sperm augments the capacitative changes in sperm, and that SP contains a factor(s) that effectively prevents these changes.
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http://dx.doi.org/10.2164/jandrol.106.001826DOI Listing
November 2007

Acute effect of vasectomy on the function of the rat epididymal epithelium and vas deferens.

J Androl 2006 Nov-Dec;27(6):826-36. Epub 2006 Jul 12.

Department of Urologic Surgery, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

Persistent infertility after apparently successful vasectomy reversal is common. One possible etiology is epididymal epithelial dysfunction resulting in improper sperm maturation after vasectomy reversal. The epididymal epithelium secretes a number of proteins that are thought to be required for the maturation of sperm. Ligation of the vas deferens during vasectomy may affect the synthesis of some of these proteins. In the present study, the function of the epididymal epithelium was assessed at early times after vasectomy (1, 4, and 7 days) by measuring the level of mRNA of 4 secreted proteins: Crisp-1, clusterin, osteopontin, and transferrin. In addition, the site of synthesis of these proteins was determined by immunocytochemistry. The results demonstrated that the expression of Crisp-1 and clusterin, representative epididymal secretory proteins, was largely unaffected by vasectomy. However, osteopontin mRNA increased in the vas deferens in response to vasectomy. Immunocytochemical localization of osteopontin suggested that both infiltrating immune cells and deferential luminal epithelium were responsible for this up-regulation. Transferrin expression was viewed as a marker for immune cells at the site of injury. However, both the caput epididymis and deferential epithelia were found to express transferrin, in addition to immune cells. In conclusion, there appear to be only minor changes in expression of genes encoding epididymal secretory proteins acutely after vasectomy, but, not surprisingly, there was evidence of an inflammatory response after vasectomy.
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http://dx.doi.org/10.2164/jandrol.106.000745DOI Listing
December 2006

Determination of apurinic/apyrimidinic lesions in DNA with high-performance liquid chromatography and tandem mass spectrometry.

Chem Res Toxicol 2006 Feb;19(2):300-9

Department of Chemistry and Biochemistry, The University of Tulsa, Tulsa, Oklahoma 74104, USA.

A new method has been developed to accurately measure apurinic and apyrimidinic (AP) DNA damage sites, which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts. If AP sites are left unrepaired (or if improperly repaired), these sites can lead to DNA mutations that may ultimately result in the formation of cancer. Hence, detection of AP sites may provide a useful indicator of exposure and susceptibility to chemical carcinogens and oxidative stress. AP detection is currently accomplished by immunodetection methods using an aldehyde reactive probe [Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S.-Y., Kow, Y. W., and Swenberg, J. A. (1998) Cancer Res. 58, 222-225; Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 686-691]; however, these approaches lack the specificity required for unequivocal identification of the AP site. Therefore, we have developed an accurate method based on mass spectrometry detection of AP sites from AP DNA that have been prelabeled with O-4-nitrobenzylhydroxylamine (NBHA). Once labeled and once the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, followed by isolation and detection with high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Optimization and validation of the experimental procedures and detection limits have been established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates well-defined AP sites in both single- and double-stranded DNA. The addition of NBHA labels the AP site in the oligomer, creating a labeled 11-mer. HPLC-ESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer. The NBHA-tagged oligomer underwent endo- and exonuclease digestion to the 5'-deoxyribose monophosphate (5'-dRp) level, thereby releasing free 5'-dRp-NBHA. The 5'-dRp-NBHA product was partially purified by solid phase extraction and quantified by LC-MS/MS using several transitions of the deprotonated molecule ([M - H]- at m/z 363) and isotopically labeled 5'-dRp-NBHA as an internal standard. Further experiments with 5',3'-bisphosphate-deoxyribose and heat/acid-treated calf thymus DNA showed similar labeling, digestion, and detection results. Initial results show a quantification limit with 100 mug of DNA to be 100 fmol (three abasic sites per 10(7) bases).
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http://dx.doi.org/10.1021/tx0502589DOI Listing
February 2006

Identification of rat cysteine-rich secretory protein 4 (Crisp4) as the ortholog to human CRISP1 and mouse Crisp4.

Biol Reprod 2006 May 8;74(5):984-91. Epub 2006 Feb 8.

Contraception, Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426, USA.

Cysteine-rich secretory proteins (CRISPs) are present in a diverse population of organisms and are defined by 16 conserved cysteine residues spanning a plant pathogenesis related-1 and a C-terminal cysteine-rich domain. To date, the diversification of mammalian CRISPs is evidenced by the existence of two, three, and four paralogous genes in the rat, human, and mouse, respectively. The current study identifies a third rat Crisp paralog we term Crisp4. The gene for Crisp4 is on rat chromosome 9 within 1 Mb of both the Crisp1 and Crisp2 genes. The full-length transcript for this gene was cloned from rat epididymal RNA and encodes a protein that shares 69% and 91% similarity with human CRISP1 and mouse CRISP4, respectively. Expression of rat Crisp4 is most abundant in the epididymis, with the highest levels of transcription observed in the caput and corpus epididymis. In contrast, rat CRISP4 protein is most abundant in the corpus and cauda regions of the epididymis. Rat CRISP4 protein is also present in caudal sperm extracts, appearing as a detergent-soluble form at the predicted MWR (26 kDa). Our data identify rat Crisp4 as the true ortholog to human CRISP1 and mouse Crisp4, and demonstrate its interaction with spermatozoa in the epididymis.
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http://dx.doi.org/10.1095/biolreprod.105.048298DOI Listing
May 2006

Analysis of the human sperm proteome.

Ann N Y Acad Sci 2005 Dec;1061:190-202

Women's Health Research Institute, Wyeth Research, 500 Arcola Rd., N3169, Collegeville, PA 19426, USA.

As part of our effort to identify putative protein targets for the development of male contraceptives, we performed an in-depth proteomic analysis of human sperm by liquid chromatography and tandem mass spectrometry. Motile sperm were collected from a single fertile individual and fractionated into detergent-soluble and detergent-insoluble fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of these fractions, followed by manual cutting of the gel, yielded 35 gel sections for each fraction to include proteins across the full range of electrophoretic mobility. Proteomic analysis of these gel sections identified more than 1,760 proteins with high confidence, with 1,350 proteins identified in the soluble fraction, 719 identified in the insoluble fraction, and 309 identified in both fractions. This characterization of the human sperm proteome provides a high-resolution, physiologically relevant index of the proteins that comprise human sperm.
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http://dx.doi.org/10.1196/annals.1336.021DOI Listing
December 2005

Epididymal secreted protein Crisp-1 and sperm function.

Mol Cell Endocrinol 2006 May 18;250(1-2):122-7. Epub 2006 Jan 18.

Department of Urologic Surgery, University of Minnesota, 420 Delaware St. SE, MMC 394, Minneapolis, MN, USA.

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal-specific expression of Crisp-1, combined with its role in regulation of sperm capacitation and oocyte interaction, make it an attractive target for post-testicular contraceptive development.
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http://dx.doi.org/10.1016/j.mce.2005.12.034DOI Listing
May 2006

Effect of pH on radiation-induced p53 expression.

Int J Radiat Oncol Biol Phys 2004 Nov;60(4):1264-71

Department of Therapeutic Radiology, College of Medicine, University of Ulsan, Seoul, Korea.

Purpose: In most tumors, the intratumor environment is acidic. The purpose of this study was to elucidate the effect of acidic extracellular environment on the radiation-induced expression of p53 and related molecular signals.

Methods And Materials: Cultured RKO.C human colorectal cancer cells carrying wild-type p53 were used. Cells grown in pH 7.5 medium or pH 6.6 medium were irradiated with gamma-rays, and the expression of p53 and p53 mRNA, as well as the degradation rate of the molecules, was determined. The transcriptional activity for p53 was investigated using cells transfected with a p53 reporter construct. The expression of Mdm2 and the phosphorylation of p53, essential factors for p53 degradation, were also investigated.

Results: The pH 6.6 environment prolonged the radiation-induced expression of p53 and p53 mRNA. The radiation-induced increase in transcriptional activity of p53 lasted longer in pH 6.6 medium than in pH 7.5 medium. The degradation of p53 was delayed at pH 6.6. The radiation-induced expression of Mdm2 was markedly suppressed, whereas the phosphorylation of p53 was markedly increased after irradiation in pH 6.6 medium.

Conclusion: Acidic environment significantly enhances the radiation-induced expression of p53, partly by increasing the formation of p53 and also partly by slowing down the degradation of p53 through inhibiting p53-Mdm2 complex formation. The potential implication of acidic intratumor microenvironment for the response of tumors to radiotherapy remains to be elucidated.
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http://dx.doi.org/10.1016/j.ijrobp.2004.04.043DOI Listing
November 2004

Mechanical property characterization of mouse zona pellucida.

IEEE Trans Nanobioscience 2003 Dec;2(4):279-86

Swiss Federal Institute of Technology (ETH) Zürich, Ch-8092 Zürich, Switzerland.

Previous intracytoplasmic sperm injection (ICSI) studies have indicated significant variation in ICSI success rates among different species. In mouse ICSI, the zona pellucida (ZP) undergoes a "hardening" process at fertilization in order to prevent subsequent sperm from penetrating. There have been few studies investigating changes in the mechanical properties of mouse ZP post fertilization. To characterize mouse ZP mechanical properties and quantitate the mechanical property differences of the ZP before and after fertilization, a microelectromechanical systems-based multiaxis cellular force sensor has been developed. A microrobotic cell manipulation system employing the multiaxis cellular force sensor is used to conduct mouse ZP force sensing, establishing a quantitative relationship between applied forces and biomembrane structural deformations on both mouse oocytes and embryos. An analytical biomembrane elastic model is constructed to describe biomembrane mechanical properties. The characterized elastic modulus of embryos is 2.3 times that of oocytes, and the measured forces for puncturing embryo ZP are 1.7 times those for oocyte ZP. The technique and model presented in this paper can be applied to investigations into the mechanical properties of other biomembranes, such as the plasma membrane of oocytes or other cell types.
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http://dx.doi.org/10.1109/tnb.2003.820273DOI Listing
December 2003

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

Technol Cancer Res Treat 2004 Jun;3(3):245-51

University of Minnesota, Department of Therapeutic Radiology, 420 Delaware St. SE, MMC 494, Minneapolis, MN 55455, USA.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.
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http://dx.doi.org/10.1177/153303460400300302DOI Listing
June 2004

Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1.

Biol Reprod 2003 Aug 16;69(2):572-81. Epub 2003 Apr 16.

Department of Urologic Surgery, University of Minnesota, Minneapolis 55455, USA.

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.
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http://dx.doi.org/10.1095/biolreprod.102.013771DOI Listing
August 2003

Sensitization of thermotolerant SCK cells to hyperthermia and freezing with reduction of intracellular pH: implications for cryosurgery.

J Surg Oncol 2003 Mar;82(3):160-9

Department of Urologic Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Background And Objectives: During cryosurgery, cells frozen slowly at the outer part of the ice ball undergo severe dehydration and are subject to solute effects injury, which may be caused in part by protein denaturation. This study was undertaken to determine whether heat shock proteins (HSPs), the molecular chaperones that stabilize proteins against denaturation, have a protective effect on cells during slow freezing. In addition, we aimed to determine whether acidic conditions, similar to those found in many solid tumors, would effect this protection.

Methods: SCK cells were frozen at 5 degrees C/min to -10 degrees C or -20 degrees C before or after induction of thermotolerance, and at neutral or low pH conditions. Lethal damage was determined by clonogenics.

Results: Clonogenic survival was decreased by 50% in thermotolerant cells frozen to -10 degrees C after culture in acidic conditions (pH 6.6) compared with non-thermotolerant cells cultured at neutral pH. Induction of thermotolerance alone or low pH alone did not significantly sensitize SCK cells to freezing. All treatment groups were equally susceptible to killing when frozen to -20 degrees C.

Conclusions: Our results show that induction of thermal tolerance does not protect SCK cells against subsequent freezing injury and that a low pH environment actually sensitizes these cells to freeze injury.
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http://dx.doi.org/10.1002/jso.10214DOI Listing
March 2003

A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D and E.

Biol Reprod 2002 Aug;67(2):525-33

Department of Genetics, Cell Biology, and Development, University of Minnesota, 321 Church Street SW, Minneapolis, MN 55455, USA.

The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.
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http://dx.doi.org/10.1095/biolreprod67.2.525DOI Listing
August 2002
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