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1.
J Immigr Minor Health ; 25(3): 674-679, 2023 Jun.
Article in English | MEDLINE | ID: mdl-36251203

ABSTRACT

Over 25 million individuals living in America are limited English proficient, many of whom live in rural communities. Adequate language accommodations are critical to providing effective healthcare for these populations. An online questionnaire was delivered to 42 rural facilities in Washington State. It included questions about their demand for language services, modalities of interpretation, translated documentation and barriers to providing accommodations. Fifteen of 42 (35.7%) responded. Spanish, Russian, Vietnamese, Japanese, Ukrainian and Mam were encountered daily. Telephonic and virtual remote interpreter services were the most widely available. Not all facilities had vital documents translated to frequently encountered languages. Challenges to providing language access were reported by nearly all participants. The rural facilities surveyed all encountered LEP patient populations and offered oral interpretation. Overall, these facilities were meeting requirements for providing language accommodation services. Even so, many facilities reported experiencing barriers to providing these services.


Subject(s)
Limited English Proficiency , Humans , Rural Health , Communication Barriers , Language , Translating
2.
RSC Adv ; 12(30): 19431-19444, 2022 Jun 29.
Article in English | MEDLINE | ID: mdl-35865562

ABSTRACT

This paper describes the synthesis of enamino carbonyl compounds by the copper(i)-catalyzed coupling of acceptor-substituted diazo compounds and tertiary thioamides. We plan to use this method to synthesize indolizidine (-)-237D analogs to find α6-selective antismoking agents. Therefore, we also performed in silico α6-nAchRs binding studies of selected products. Compounds with low root-mean-square deviation values showed more favorable binding free energies. We also report preliminary pharmacokinetic data on indolizidine (-)-237D and found it to have weak activity at CYP3A4. In addition, as enamino carbonyl compounds are also known for antimicrobial properties, we screened previously reported and new enamino carbonyl compounds for antibacterial, antimicrobial, and antifungal properties. Eleven compounds showed significant antimicrobial activities.

3.
Sci Rep ; 8(1): 12473, 2018 Aug 20.
Article in English | MEDLINE | ID: mdl-30127507

ABSTRACT

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.

4.
Urology ; 87: 82-7, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26519000

ABSTRACT

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.


Subject(s)
Hospitals , Inpatients , Prostatic Hyperplasia/surgery , Quality Indicators, Health Care , Transurethral Resection of Prostate/standards , Aged , Humans , Length of Stay/trends , Male , Middle Aged , Retrospective Studies , Treatment Outcome , Washington
5.
J Health Econ Outcomes Res ; 4(1): 47-54, 2016.
Article in English | MEDLINE | ID: mdl-34414247

ABSTRACT

Background: Increased surgical volume is associated with better patient outcomes and shorter lengths of hospitalization. As a consequence, traveling to receive care from a high volume provider may be associated with better outcomes. However, travel may also be associated with a decision by the healthcare provider to increase the length of stay due to a decreased ability to return to the primary hospital should complications arise. Thus, research is needed to understand the relationship between the distance a patient must travel and their outcomes following urologic surgery. Objective: The purpose of this study was to determine whether the distance a patient travels to receive urologic surgery is associated with their length of hospital stay and direct medical hospitalization costs. Methods: This was a retrospective observational cohort study of 12 106 patients over 50 years of age undergoing transurethral resection of the prostate (TURP), radical prostatectomy (RP) or radical cystectomy (RC) in Washington State hospitals between 2009 and 2013. Distance traveled was determined by calculating the linear distance between zip code centroids of patient residence and the hospital performing their procedure. Patients were sorted into four groups classified by distance traveled (≤5 miles, 6-20 miles, 21-50 miles and ≥51 miles) and cost calculated using a charges-to-reimbursement ratio for each hospital. Statistical significance was determined using a Kruskal-Wallis test. Results: Patients traveling greater distances had significantly lower median medical costs compared with patients who lived closer to the hospitals where they underwent TURP and RP (TURP: ≤5 miles, $6243 and ≥51 miles, $5105, p≤0.001; RP: ≤5 miles, $12 407 and ≥51 miles, $11 882, p≤0.001), whereas there was no significant difference for patients undergoing RC (≤5 miles, $27 554 and ≥51 miles, $26 761, p=0.17). Likewise, patients traveling greater distances had significantly lower median lengths of hospitalization for TURP and RP (TURP: p≤0.001, RP: p≤0.001), while there was no difference for RC (p=0.50). Conclusions: Patient travel burden does appear to play a role in cost and length of hospital stay for select urologic procedures with variable levels of morbidity and recovery time. Although these findings are statistically significant, the magnitude of the effect is small.

6.
Mikrochim Acta ; 180: 1341-1350, 2013.
Article in English | MEDLINE | ID: mdl-24078747

ABSTRACT

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.

7.
J Chromatogr A ; 1305: 320-7, 2013 Aug 30.
Article in English | MEDLINE | ID: mdl-23891371

ABSTRACT

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.


Subject(s)
Electrophoresis, Capillary/methods , Micelles , Quantum Dots , Surface-Active Agents
8.
Biol Reprod ; 87(6): 141, 2012 Jun.
Article in English | MEDLINE | ID: mdl-23115268

ABSTRACT

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.


Subject(s)
Epididymis/cytology , Proteome/metabolism , Spermatozoa/metabolism , Animals , Chromatography, High Pressure Liquid , Cluster Analysis , Databases, Protein , Down-Regulation , Infertility, Male/etiology , Infertility, Male/metabolism , Male , Mice , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Proteome/chemistry , Proteomics/methods , Signal Transduction , Tandem Mass Spectrometry , Testis/cytology , Transcriptome , Up-Regulation
9.
Mikrochim Acta ; 176(3-4): 345-358, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22347727

ABSTRACT

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.

10.
Cancer ; 118(4): 987-96, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-21792864

ABSTRACT

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.


Subject(s)
Kidney Neoplasms/surgery , Prostatic Neoplasms/surgery , Quality of Health Care/standards , Surgery Department, Hospital/standards , Urinary Bladder Neoplasms/surgery , Adolescent , Adult , Aged , Aged, 80 and over , Cohort Studies , Cystectomy/standards , Female , Humans , Kidney Neoplasms/mortality , Length of Stay , Male , Middle Aged , Nephrectomy/standards , Outcome Assessment, Health Care , Prostatectomy/standards , Prostatic Neoplasms/mortality , Retrospective Studies , Urinary Bladder Neoplasms/mortality , Washington , Young Adult
11.
Anal Bioanal Chem ; 399(8): 2831-42, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21267714

ABSTRACT

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.


Subject(s)
Biological Assay/instrumentation , Electrophoresis, Capillary/methods , Nanoparticles/chemistry , Adsorption , DNA/chemistry
12.
Reprod Fertil Dev ; 22(6): 893-900, 2010.
Article in English | MEDLINE | ID: mdl-20591323

ABSTRACT

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.


Subject(s)
Seminal Plasma Proteins/pharmacology , Sperm Capacitation/drug effects , Spermatozoa/drug effects , Analysis of Variance , Animals , Cryopreservation , Flow Cytometry , Male , Mass Spectrometry , Semen/metabolism , Seminal Plasma Proteins/metabolism , Spermatozoa/metabolism , Swine
13.
Biol Reprod ; 81(4): 700-6, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19535788

ABSTRACT

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.


Subject(s)
Cryopreservation , Semen Preservation , Sperm Motility , Spermatozoa/metabolism , Water/metabolism , Animals , Calorimetry, Differential Scanning , Cell Membrane Permeability , Freezing , Male , Mice , Models, Biological , Rats
14.
Ann Biomed Eng ; 37(7): 1425-33, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19415493

ABSTRACT

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.


Subject(s)
Locomotion , Models, Biological , Pelvic Floor/physiopathology , Sports , Urinary Incontinence, Stress/physiopathology , Acceleration , Computer Simulation , Feasibility Studies , Female , Humans , Stress, Mechanical
15.
Article in English | MEDLINE | ID: mdl-19052686

ABSTRACT

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.


Subject(s)
Electric Stimulation Therapy/instrumentation , Urethra/anatomy & histology , Urethra/innervation , Aged , Cystitis, Interstitial/therapy , Electric Stimulation Therapy/methods , Electrodes , Electrodes, Implanted , Female , Humans , Urinary Bladder, Overactive/therapy , Urinary Incontinence, Stress/therapy
16.
Biol Reprod ; 79(6): 1046-53, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18703418

ABSTRACT

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.


Subject(s)
Epididymis/physiology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Spermatozoa/physiology , Acrosome/metabolism , Acrosome/ultrastructure , Animals , Blotting, Western , Coculture Techniques , Detergents/pharmacology , Epididymal Secretory Proteins , Epididymis/cytology , Glucosides/pharmacology , Immunohistochemistry , Indicators and Reagents , Male , Membrane Glycoproteins/biosynthesis , Protein Binding , Rats , Rats, Sprague-Dawley , Temperature
17.
Biol Reprod ; 79(6): 1129-34, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18716287

ABSTRACT

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.


Subject(s)
Gene Expression Regulation/physiology , Membrane Glycoproteins/genetics , Acrosome Reaction/physiology , Amino Acid Sequence , Animals , Cloning, Molecular , Computational Biology , Culture Media , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Epididymis/cytology , Epididymis/metabolism , Male , Membrane Glycoproteins/biosynthesis , Molecular Sequence Data , RNA/biosynthesis , RNA/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Semen/physiology , Sperm Capacitation/physiology , Spermatozoa/metabolism , Swine , Testis/metabolism
18.
J Biochem Biophys Methods ; 70(6): 992-8, 2008 Apr 24.
Article in English | MEDLINE | ID: mdl-17588670

ABSTRACT

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.


Subject(s)
Delivery, Obstetric , Electrophoresis, Capillary/methods , Fetus/metabolism , Labor, Obstetric , Penicillin G/analysis , Penicillin G/pharmacokinetics , Calibration , Female , Humans , Hydrophobic and Hydrophilic Interactions , Penicillin G/administration & dosage , Penicillin G/blood , Penicillin G/urine , Pregnancy , Streptococcus agalactiae/drug effects
19.
Asian J Androl ; 9(4): 508-14, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17589788

ABSTRACT

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.


Subject(s)
Membrane Glycoproteins/genetics , Spermatozoa/physiology , Amino Acid Sequence , Animals , Conserved Sequence , Humans , Male , Mammals , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Rats
20.
J Androl ; 28(3): 416-22, 2007.
Article in English | MEDLINE | ID: mdl-17192595

ABSTRACT

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.


Subject(s)
Cold Temperature , Phosphotyrosine/metabolism , Semen/physiology , Sperm Capacitation/physiology , Spermatozoa/metabolism , Acrosome Reaction/physiology , Animals , Male , Signal Transduction/physiology , Swine
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