Plasma and acrosomal membrane lipid content of saltwater crocodile spermatozoa.

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is documented. The mean (±s.d.) ratio of unsaturated to saturated membrane fatty acids within the plasma membrane was 2.57±0.50, and was determined to be higher than a similar analysis of the lipids found in the acrosomal membrane (0.70±0.10). The saltwater crocodile sperm plasma membrane also contained remarkably high levels of cholesterol (mean (±s.d.) 40.7±4.5 nmol per 106 sperm cells) compared with the spermatozoa of other amniote species that have so far been documented. We suggest that this high cholesterol content could be conferring stability to the crocodile sperm membrane, allowing it to tolerate extreme osmotic fluxes and rapid changes in temperature. Our descriptive analysis now provides those interested in reptile and comparative sperm physiology an improved baseline database for interpreting biochemical changes associated with preservation pathology (e.g. cold shock and cryoinjury), epididymal sperm maturation and capacitation/acrosome reaction.

Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.

Investigation of the expression and functional significance of the novel mouse sperm protein, a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10 (ADAMTS10).

Fertilization represents the culmination of a series of complex interactions between male and female gametes. Despite advances in our understanding, the precise molecular mechanisms underlying these fundamental interactions remain largely uncharacterized. There is however growing recognition that this process requires the concerted action of multiple sperm receptors that possess affinity for complementary zona pellucida ligands and those that reside on the surface of the oolemma. Among the candidate sperm proteins that have been implicated in fertilization, those belonging to the ADAM (a disintegrin and metalloprotease) family of proteases have received considerable attention. The focus of the studies described herein has been the characterization of a closely related member of this protease family, ADAMTS10 (a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10). We have demonstrated that ADAMTS10 is expressed during the later stages of mouse spermatogenesis and incorporated into the acrosomal domain of developing spermatids. During sperm maturation, the protein appears to be processed before being expressed on the surface of the peri-acrosomal region of the head. Our collective data suggest that, from this position, ADAMTS10 participates in sperm adhesion to the zona pellucida. Indeed, pre-incubation of capacitated spermatozoa with either galardin, a broad spectrum inhibitor of metalloprotease activity, or anti-ADAMTS10 antisera elicited a significant reduction in their ability to engage in zona adhesion. Overall, these studies support the notion that sperm-oocyte interactions involve considerable functional redundancy and identify ADAMTS10 as a novel candidate in the mediation of these fundamentally important events.

Dietary patterns, insulin sensitivity and inflammation in older adults.

BACKGROUND/OBJECTIVES: Several studies have linked dietary patterns to insulin sensitivity and systemic inflammation, which affect risk of multiple chronic diseases. The purpose of this study was to investigate the dietary patterns of a cohort of older adults, and to examine relationships of dietary patterns with markers of insulin sensitivity and systemic inflammation. SUBJECTS/METHODS: The Health, Aging and Body Composition (Health ABC) Study is a prospective cohort study of 3075 older adults. In Health ABC, multiple indicators of glucose metabolism and systemic inflammation were assessed. Food intake was estimated with a modified Block food frequency questionnaire. In this study, dietary patterns of 1751 participants with complete data were derived by cluster analysis. RESULTS: Six clusters were identified, including a 'healthy foods' cluster, characterized by higher intake of low-fat dairy products, fruit, whole grains, poultry, fish and vegetables. In the main analysis, the 'healthy foods' cluster had significantly lower fasting insulin and homeostasis model assessment of insulin resistance values than the 'breakfast cereal' and 'high-fat dairy products' clusters, and lower fasting glucose than the 'high-fat dairy products' cluster (P≤0.05). No differences were found in 2-h glucose. With respect to inflammation, the 'healthy foods' cluster had lower interleukin-6 than the 'sweets and desserts' and 'high-fat dairy products' clusters, and no differences were seen in C-reactive protein or tumor necrosis factor-α. CONCLUSIONS: A dietary pattern high in low-fat dairy products, fruit, whole grains, poultry, fish and vegetables may be associated with greater insulin sensitivity and lower systemic inflammation in older adults.

Successful management of recurrent coccygeal hernia with the de-epithelialised rectus abdominis musculocutaneous flap.

OBJECTIVE: Little data exist about the optimal management of the rare coccygeal hernia. A novel method of repair is reported. METHODS: A 46-year-old woman presented with a symptomatic coccygeal hernia after resection of the coccyx for a tumour. She had previously been reconstructed with an on-lay polytetrafluorethylene (PTFE) mesh but subsequently developed a hernia. A de-epithelialised vertical rectus abdominis musculocutaneous flap was elevated and passed through the hernia defect. The de-epithelialised dermis was secured to the levator ani and to the periosteum of the sacrum via access through a posterior approach. The gluteal skin was closed primarily over the inset flap. RESULTS: The de-epithelialised rectus abdominis musculocutaneous flap is a viable option for the treatment of coccygeal hernia. RELEVANCE: The de-epithelialised rectus abdominis flap has several advantages over other techniques including mesh repair and anterior or posterior flap repairs of the coccygeal hernia. The transposed muscle blocks herniation through the pelvic floor and does not create the dead space that is associated with posterior flap repairs such as the bilateral gluteal advancements. It also has the advantages of the posterior approach mesh repair, as the de-epithelialised dermis provides significant strength when secured like mesh to healthy local tissue.

Vulval intraepithelial neoplasia and periungual Bowen's disease concordant for mucosal (HPV-34) and epidermodysplasia verruciformis (HPV-21) human papillomavirus types.

Human papillomavirus (HPV) infection is associated with genital malignancy and specific cutaneous malignancies. We report a case of an HPV-associated concurrent vulval intraepithelial neoplasia and periungual Bowen's disease in a young immunocompetent Afro-Caribbean woman with no known risk factors for either disease. HPV genotyping studies detected multiple alpha and beta papillomaviruses with concordance for HPV-34 [a high-risk (HR) mucosal type], and HPV-21 [an epidermodyslasia verruciformis (EV) type] in both vulval and finger tissue. Although the HR-mucosal viruses detected are likely to have a pathogenic role in vulval intraepithelial neoplasia, this is the first report of concordance for EV HPV types in both genital and nongenital skin premalignancies. This case, in the context of accumulating epidemiological and experimental data in cutaneous SCC, raises the question of whether EV HPV may contribute to vulval malignancy, and further study is merited.

Trials and tribulations in deploying digital imaging network and picture archiving and communication system--film to filmless in 80 days.

The Naval Medical Center Portsmouth (NMCP) was constructed with the intention of deploying a picture archiving and communications system (PACS). All necessary infrastructures were installed and considerable planning was done during construction with the belief that this would make the deployment of the PACS much simpler. This was true during the early deployment; however, as time passed and the system was more heavily used, significant problems arose. User/operator-related and hardware/software-related problems were encountered. Although, most have been corrected, some have persisted and will require considerable manpower and/or fiscal resources to correct. The lesson learned is that no matter how much preparation is done for deploying PACS, many significant problems will surface as the system is used that will require the continued attention of the deployment project officer.

Quality-control issues on high-resolution diagnostic monitors.

Previous literature indicates a need for more data collection in the area of quality control of high-resolution diagnostic monitors. Throughout acceptance testing, which began in June 2000, stability of monitor calibration was analyzed. Although image quality on all monitors was found to be acceptable upon initial acceptance testing using VeriLUM software by Image Smiths, Inc (Germantown, MD), it was determined to be unacceptable during the clinical phase of acceptance testing. High-resolution monitors were evaluated for quality assurance on a weekly basis from installation through acceptance testing and beyond. During clinical utilization determination (CUD), monitor calibration was identified as a problem and the manufacturer returned and recalibrated all workstations. From that time through final acceptance testing, high-resolution monitor calibration and monitor failure rate remained a problem. The monitor vendor then returned to the site to address these areas. Monitor defocus was still noticeable and calibration checks were increased to three times per week. White and black level drift on medium-resolution monitors had been attributed to raster size settings. Measurements of white and black level at several different size settings were taken to determine the effect of size on white and black level settings. Black level remained steady with size change. White level appeared to increase by 2.0 cd/m2 for every 0.1 inches decrease in horizontal raster size. This was determined not to be the cause of the observed brightness drift. Frequency of calibration/testing is an issue in a clinical environment. The increased frequency required at our site cannot be sustained. The medical physics division cannot provide dedicated personnel to conduct the quality-assurance testing on all monitors at this interval due to other physics commitments throughout the hospital. Monitor access is also an issue due to radiologists' need to read images. Some workstations are in use 7 AM to 11 PM daily. An appropriate monitor calibration frequency must be established during acceptance testing to ensure unacceptable drift is not masked by excessive calibration frequency. Standards for acceptable black level and white level drift also need to be determined. The monitor vendor and hospital staff agree that currently, very small printed text is an acceptable method of determining monitor blur, however, a better method of determining monitor blur is being pursued. Although monitors may show acceptable quality during initial acceptance testing, they need to show sustained quality during the clinical acceptance-testing phase. Defocus, black level, and white level are image quality concerns, which need to be evaluated during the clinical phase of acceptance testing. Image quality deficiencies can have a negative impact on patient care and raise serious medical-legal concerns. The attention to quality control required of the hospital staff needs to be realistic and not have a significant impact on radiology workflow.

An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA.

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.

Metabolism and renal clearance of 111In-labeled DOTA-conjugated antibody fragments.

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that 111In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-epsilon-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s-5720s]. To determine the metabolic profile of another widely used metal-chelate, [111In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [111In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [111In]DOTA-epsilon-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [111In]DOTA-Fab. Since injection of [111In]DOTA-epsilon-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [111In]DOTA-Fab, followed by lysosomal degradation to [111In]DOTA-epsilon-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [111In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [111In]DOTA-epsilon-amino-lysine.

Hepatitis B virus transcription and replication.

Hepatitis B virus (HBV) replicates by the reverse transcription of the 3.5-kb viral pregenomic RNA. Therefore, the regulation of the transcription of the pregenomic RNA is a critical step in the viral life cycle. Various ubiquitous and liver-enriched transcription factors have been shown to modulate the level of RNA synthesis from the core promoter. The nuclear hormone receptors HNF4 and RXRalpha plus PPARalpha appear to have a critical role in governing pregenomic RNA synthesis from the core promoter in cell culture and probably represent a major determinant governing the hepatotropism of this virus. The level of 3.5-kb HBV RNA synthesis is approximately proportional to the level of viral replication in cell culture; however, this is not the case in the liver of HBV transgenic mice. Directly modulating the levels or activities of specific transcription factors known to regulate HBV transcription in cell culture can increase viral replication in HBV transgenic mice without greatly changing the levels of HBV transcripts. Various immune stimuli that alter transcription factor activities involved in regulating viral RNA synthesis can negatively affect viral replication without affecting HBV transcription. These observations suggest that in vivo very subtle changes in HBV transcription may contribute to large alterations, either negative or positive, in viral replication. Investigation of transcription factor-null HBV transgenic mice under various physiological conditions will be required to establish the putative role of specific transcription factors in regulating viral replication in vivo.

High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment.

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

An apparatus for membrane-confined analytical electrophoresis.

A membrane-confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377-382). Presented here is a design for this apparatus, which permits the on-line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady-state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused-silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 microL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.

Nurse practitioners, certified nurse midwives, and physician assistants in California.

California's health care industry includes workers prepared in many ways to perform many jobs. One significant group of health care workers prepared to provide care that often overlaps with physician-generated services is known as "nonphysician providers." Commonly, this label refers to nurse practitioners (NPs), certified nurse midwives (CNMs), and physician assistants (PAs). In this article, we will describe this group in five main areas: (1) the characteristics of the current and projected workforce and programs preparing these professionals; (2) the current skill expectations and knowledge bases of each; (3) trends in the education of these health professionals; (4) innovative models of education of these health professionals; and (5) the inclusion of NPs, PAs, and CNMs in workforce planning in a changing health care system. We conclude that, particularly in light of the overlapping functions of this provider group with many physician functions, the NP, CNM, and NP workforces must be recognized and considered when planning for the future of the physician workforce.

Practice environment for nurse practitioners in California. Identifying barriers.

Barriers exist that prevent nurse practitioners from using their primary health care knowledge and skills. We present the incidence of and specific barriers experienced by nurse practitioner respondents in California, the state with the largest number of nurse practitioners in the nation. A January 1995 survey was sent to all nurse practitioners certified in California to elicit their experiences regarding legal or social barriers in their practice, with space for an open-ended response. Of an estimated 3,895 nurse practitioners in California, 2,741 (70%) returned surveys. Most nurse practitioner (65%) respondents in California are providing primary care. Perceived barriers to practice are lack of prescriptive authority, lack of support from physicians, reimbursement difficulties, and lack of public awareness. Current barriers to nurse practitioner practice in California are similar to national barriers discovered in 1992 data. The development of interprofessional dialogue and the recognition of the contributions of all primary care professionals are some of the steps that can be taken to reduce these barriers and increase the use and effectiveness of nurse practitioners in primary care.

Runner with gout and an aortic valve nodule.

A 33-yr-old male ran 10 miles, drank some beer, and developed pain in his left knee and ankle. He took some leftover antibiotics but was no better after 6 d, when a heart murmur and an aortic valve nodule were discovered. He was presumed to have endocarditis with septic arthritis and was started on intravenous antibiotics. On the second hospital day, synovial fluid analysis revealed acute gout, and the patient improved very rapidly on anti-gout therapy. The valvular nodule remained unexplained, but one very rare cause of valvular heart nodules is visceral gout. An unsuccessful attempt to resorb the nodule was made by using allopurinol. This patient demonstrates several points about gout in endurance athletes: 1) acute gout can mimic infectious endocarditis, 2) misdiagnosed or undertreated gout often leads to multiple joint involvement and sometimes to visceral tophi, and 3) athletes who exercise in warm weather and quench their thirst with cold beer are at risk for acute gout.

Calibration of a 241Am wound-monitoring system using Monte Carlo techniques.

Monte Carlo techniques have been used to establish calibration factors and to predict gamma spectra for well-defined measurements. These techniques are routinely used to predict shielding requirements and critical specifications. The Lawrence Livermore National Laboratory is researching the feasibility of using Monte Carlo techniques to establish calibration factors for in vivo measurement systems. A pilot study was conducted to demonstrate the use of the Monte Carlo technique to calibrate in vivo measurement systems, to predict the efficiency of a wound measurement system and compare the predicted efficiency with the measured efficiency, and to investigate the effects of the source geometry and the detector size on the measured efficiency. Results of this study demonstrate good agreement between the Monte-Carlo-predicted efficiency and the measured efficiency for a wound calibration phantom. The effects of the source geometry and the detector size tend to conform to the physical processes that govern the measurement process. These results demonstrate that the Monte Carlo technique accurately predicts the in vivo measurement efficiency if the characteristics of the attenuating material and the Monte Carlo source geometry are properly established.

Radioimmunodetection of Hodgkin's disease and non-Hodgkin's lymphomas with monoclonal antibody to eosinophil peroxidase.

The purpose of this study was to determine if a radiolabeled murine monoclonal antibody (EOS) directed against eosinophil peroxidase would localize specifically to tumor sites in patients with lymphomas infiltrated by eosinophils. Ten patients with Hodgkin's disease and eosinophilia, three patients with non-Hodgkin's lymphomas and eosinophilia and five control patients received an intravenous injection of 3-10 mg of EOS antibody radiolabeled with 74-155 MBq (2.0-4.2 mCi) of 111In. At intervals of 24, 48 and 72 hr after injection, gamma camera images were obtained along with blood and urine specimens and the imaging results were correlated with the results of other staging modalities. As early as 24 hr after antibody injection, there was clear visualization of identifiable sites of lymphoma with eosinophilia greater than 1 cm in size, including the spleen, bone marrow and lymph nodes. Although EOS also localized nonspecifically to the liver and, in some patients, to the nasopharynx, there was no appreciable uptake in normal bone marrow, spleen, uninvolved lymph nodes, lymphomas without eosinophilia or various other pathologic conditions without eosinophilia. Except for transient pain at tumor sites in three patients, no adverse reactions were noted. We conclude that a radiolabeled monoclonal antibody directed against eosinophil peroxidase localizes to lymphoma sites infiltrated by eosinophils.