The impact of a rural emergency department rotation on applicant ranking of a US emergency medicine residency program.

INTRODUCTION: Few US emergency medicine (EM) residency programs have been located in rural states due to program requirements for emergency department (ED) patient volume. Recent revision to the program requirements now permits 'educationally justifiable exceptions' to the patient population requirement, 'such as clinical sites in a rural setting', and some EM residency programs now plan to offer rural ED clinical experiences as a required curricular component. The impact of a required rural EM rotation on the ranking decisions of applicants is important to residency programs seeking to attract the most desirable applicants. OBJECTIVE: To assess the impact of a required rural ED rotation on applicant ranking of an EM residency program in the US National Resident Matching Program (NMRP). METHODS: All applicants to the study's EM residency program completing the interview portion of the application process received a mailed and emailed survey following the release of the 2004 NMRP results. The survey included questions addressing the rural/non-rural classification of the location of the applicants' childhood home, medical school, and anticipated future practice. RESULTS: Of 46 eligible subjects, 32 (69.6%) completed the survey. Of subjects with a rural childhood, 73.3% reported a positive impact on rank order (95% CI 50.9-95.7%) and 26.7% reported no impact (CI 4.3-49.1%); 81.3% of subjects with non-rural backgrounds reported no impact (CI 62.2-100%), 12.5% higher rank (CI 0-28.7%), and 6.3% lower (CI 0-18.2%). If planning a future practice in a rural community, 83.3% reported positive impact (CI 62.2-100%) and 16.7% no impact (CI 0-37.8%); 78.9% of subjects anticipating future practice in non-rural communities reported no impact (CI 60.6-97.3%), 15.8% higher rank (CI 0-32.2%), and 5.3% lower (CI 0-15.4). Of the subjects attending medical school in rural states, 52.2% reported a positive impact (CI 31.8-72.6%) and 47.8% no impact (CI 27.4-68.2%), while 75% of graduates of medical schools in non-rural states reported no impact (CI 32.6-100%) and 25% (CI 0-67.4%) a negative impact. CONCLUSION: The presence of a rural ED rotation did not adversely impact EM residency applicants' ranking of the program.

Respiratory syncytial virus pneumonia in the hospital setting: length of stay, charges, and mortality.

BACKGROUND: Respiratory syncytial virus (RSV) is the most important viral cause of lower respiratory tract infection in infants and young children worldwide. No vaccine against RSV is available, but prophylactic interventions have been shown to be safe and effective in clinical trials. OBJECTIVES: This retrospective analysis was conducted to examine the health and economic burden of hospitalization for RSV pneumonia. METHODS: Nationally weighted hospital discharges for RSV pneumonia among children 4 years old and younger were analyzed by using the Healthcare Cost and Utilization Project National Inpatient Sample. RESULTS: In 1993, there were estimated to be 16,500 hospital discharges with RSV pneumonia, which increased to 19,700 and 20,800 in 1994 and 1995, respectively. Children less than 1 year of age accounted for over 70% of these discharges. Hospital charges (in 1998 dollars) for RSV pneumonia-associated episodes were $295,100,000 in 1993; $392,300,000 in 1994; and $295,800,000 in 1995. CONCLUSIONS: With inpatient charges of $300 to $400 million per year in the United States, the disease burden of RSV pneumonia is very high in terms of both morbidity and economic costs. Emerging prophylactic interventions should have an impact on the high burden of RSV pneumonia.

CDB-2914: anti-progestational/anti-glucocorticoid profile and post-coital anti-fertility activity in rats and rabbits.

Our goal was to determine the endocrine and post-coital anti-fertility activity of CDB-2914. Concurrent administration of progesterone to rats on day 4 post-mating blocked the anti-fertility activity of a single oral 2 mg dose of CDB-2914. CDB-2914 did not exhibit progestational activity in the oestradiol-primed immature female rabbit at doses that exhibited anti-progestational activity. CDB-2914 antagonized exogenous and endogenous progesterone-stimulated uterine haptoglobin synthesis and secretion in immature and adult mated rabbits respectively. Neither CDB-2914 nor mifepristone exhibited glucocorticoid activity as determined by thymus involution in rats; mifepristone was twice as potent as CDB-2914 in antagonizing glucocorticoid action. Post-coital CDB-2914 treatment resulted in a dose-dependent reduction in implantation sites and pregnancy rates in rabbits. CDB-2914-induced inhibition of uterine weight increase, endometrial glandular arborization and uterine haptoglobin synthesis/secretion correlated with inhibition of pregnancy in mated rabbits. A single oral dose of 64 mg CDB-2914/rabbit was effective at blocking pregnancy when administered on day 4, 5, or 6 post-mating, whereas 32 mg/rabbit was only partially effective in this regard. These data demonstrate that CDB-2914 is a potent, orally active anti-progestin with weak anti-glucocorticoid activity. CDB-2914 inhibited implantation in adult rats and rabbits demonstrating its potential as a post-coital contraceptive drug.

Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina.

PURPOSE: Transvascular leakage occurs in diabetic retinopathy. The tight junction proteins occludin and zonula occludens-1 (ZO-1) and adherens junction protein cadherin-5 are critical to the maintenance of endothelial barrier. We report a comparison of junction protein expression in the normal and diabetic retina. METHOD: Case report. Postmortem retinal cryosections were prepared from the left eye of a 73-year-old woman with diabetic retinopathy. Cryosections were immunostained for cadherin-5, occludin, and ZO-1 and compared with retinal cryosections from the right eye of a 72-year-old man with no progression of retinal disease. RESULTS: Immunofluorescence showed positive retinal vessel staining for occludin and ZO-1 in both eyes and cadherin-5 in the normal eye but reduced cadherin-5 staining in the retinal vessels of the diabetic eye. CONCLUSION: Increases in transvascular leakage observed in diabetic retinal vasculature may be associated with reduction in the expression of the critical adherens junction protein, cadherin-5.

Psychological risk factors and early complications after bone marrow transplantation in adults.

Complications of bone marrow transplantation can compromise its effectiveness, and often it is not possible to predict who is at greatest risk. In a previous study we reported that certain psychological factors correlated with a high incidence of post-transplant mortality, and here we analyze the associated complications and causes of death. Prior to receiving high-dose chemotherapy and bone marrow transplantation, 112 patients underwent a psychodynamically oriented psychiatric assessment (the 'FIT' assessment). Mortality and associated complications were ascertained by a retrospective chart review. The results of the 'FIT' assessment correlated with the incidence of complications and death, whether or not the transplant was performed for hematologic or solid organ cancers, or was from an allogeneic or autologous source. Most individuals with a high risk profile died of progressive major organ dysfunction or recurrent/refractory neoplastic disease in the first year after transplant. We propose that such a psychiatric assessment might identify a subgroup of individuals in whom pre-emptive therapeutic interventions could be most effective.

Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma.

Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.

Red blood cell transfusions for elective hip and knee arthroplasty: opportunity to improve quality of care and documentation.

OBJECTIVES: To assess current practice for red blood cell transfusion relative to the American College of Physicians guideline for red blood cell transfusion; to determine comparative rates and relative appropriateness of autologous versus allogeneic blood use; and, to assess cost implications of current transfusion practices. DESIGN: Computerized quality-of-care algorithm applied retrospectively to medical-record and blood-bank data. SETTING: Twenty-six hospitals in Colorado, Connecticut, Georgia, Oklahoma, and Virginia. PATIENTS: Medicare beneficiaries (2,137) who were hospitalized in 1993 for two elective surgical procedures: total hip arthroplasty and total knee arthroplasty. Of the 1,195 patients who received a preoperative or postoperative transfusion, 728 were excluded from the analysis because the hospital medical record did not contain the clinical documentation necessary to apply the American College of Physicians guideline to each unit transfused. The remaining 467 patients comprised the sample. RESULTS: For 467 patients who underwent these two procedures and received a total of 651 units of preoperative or postoperative blood, there were 256 excess units transfused. Two hundred four of these units were autologous, and 52 were allogeneic. These excess units accounted for $48,200 of the total $121,000 direct cost of transfused units. CONCLUSIONS: These findings demonstrate that current medical records lack the documentation necessary to evaluate transfusion practice for the majority of Medicare beneficiaries undergoing elective hip and knee arthroplasty. The direct costs of preoperative and postoperative blood transfusion for these two procedures could be reduced by nearly 40% through adherence to the American College of Physicians guideline. The majority of this cost saving would be realized through reduction in unnecessary collection and use of autologous blood.

Psychiatric assessment of candidates for bone marrow transplantation: a psychodynamically-oriented approach.

OBJECTIVE: To seek possible relationships between psychological factors and survival after an intensive medical therapy, using bone marrow transplantation as a model. METHOD: Candidates for bone marrow transplantation underwent two to three psychodynamically-oriented psychiatric interviews that explored family functioning ("F"), individual psychological maturity ("I"), and the capacity to form and communicate a mature psychological construct of the transplant ("T") process. The results were recorded in a semiquantitative manner, assigning a possible score of 1 to 3 for each parameter, for a possible total of 3 to 9 (the "F.I.T." assessment). Survival after the transplant was analyzed retrospectively in relation to the F.I.T. assessment. RESULTS: In a series of 112 candidates interviewed prior to transplant, those with the lowest F.I.T. assessment tended not to survive as long. By one year, 95 percent of individuals assigned the lowest score (F.I.T. = 3) had died, whereas 96 percent of those assigned the highest scores (F.I.T. = 7-9) remained alive. The strongest predictors were the "I" and "T" parameters. CONCLUSION: This approach to assessment of candidates for bone marrow transplantation may identify individuals who require added, supportive measures, both medical and psychological. Furthermore, the results suggest possible leads in the search for how psychological factors might influence the physiologic response to a toxic stress.

Partial characterization of the human retinal endothelial cell tight and adherens junction complexes.

PURPOSE: In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized. METHODS: Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina. RESULTS: Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin. CONCLUSIONS: This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.

A novel transcription initiation factor (TIF), TIF-IE, is required for homogeneous Acanthamoeba castellanii TIF-IB (SL1) to form a committed complex.

The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460, 000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 +/- 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

Glucose-induced increase in paracellular permeability and disruption of beta-receptor signaling in retinal endothelium.

PURPOSE: To examine the effects of high glucose concentrations on retinal endothelial permeability in an in vitro model of the retinal microvasculature. METHODS: The permeability of the endothelial barrier to small solutes was measured in a chromatographic cell column consisting of bovine retinal endothelial cells cultured on porous fibronectin-coated microcarriers. In each cell column, permeability changes were evaluated by comparing the treatment permeability response over time with the initial baseline permeability. Short-term (2-hour) barrier effects of glucose were examined by measuring permeability at 15-minute intervals after an increase in perfusate concentration from baseline (5.5 mM) to high (25 mM) glucose. Long-term (to 57 days) effects were tested by addition of 25 mM glucose to microcarrier cultures. The effect of glucose on beta-receptor signaling was tested by measuring its effect on the permeability decrease produced by 1 microM isoproterenol. RESULTS: An increase from 5.5 mM to 25 mM glucose concentration did not change retinal endothelial cell monolayer permeability (n=6) during 2 hours. However, an increase in monolayer permeability was observed after 19 days (n=8) in the 25-mM glucose culture. Paralleling this time course, short-term exposure to 25 mM glucose did not prevent a decrease in permeability triggered by the beta-receptor agonist isoproterenol. However, the permeability effect of the agonist was blocked by long-term culture in 25 mM glucose. Permeability of retinal endothelial monolayers cultured in 5.5 mM glucose and treated with 1 microM isoproterenol decreased significantly to 0.71+/-0.06 of baseline (n=4; mean+/-SEM). However, permeability did not change in parallel cell columns made from microcarriers cultured in 25 mM glucose (0.97+/-0.2 of baseline permeability; n=4; mean+/-SEM). CONCLUSIONS: High-glucose culture decreases the retinal endothelial barrier and blocks the response to beta-adrenergic receptors. This model may prove valuable in exploring other hypotheses of increased permeability associated with diabetic retinopathy or other retinal diseases that break down the retinal vascular barrier.

Blastocyst-dependent upregulation of metalloproteinase/disintegrin MDC9 expression in rabbit endometrium.

Blastocyst attachment to mammalian uteri at implantation involves the adhesion of the trophoblast to the uterine epithelial surface. In the rabbit, fusion between adjacent epithelial cells precedes the initial attachment phase and is followed by fusion between the trophoblast and the epithelium. The reverse transcription/polymerase chain reaction method has been used to prepare a partial cDNA (rbMDC9) from periimplantation-stage endometrium; this represents the rabbit ortholog of MDC9, a member of the cellular metalloproteinase/disintegrin (ADAM) gene family. We demonstrate here the reproductive stage-specific expression of rbMDC9 mRNA in uterine epithelium during the periimplantation period. Furthermore, this expression is upregulated at implantation sites, and in situ hybridization analysis has revealed that the epithelial cells with the most prominent signal are those apposed to blastocysts. Immunostaining with E-cadherin has been used to trace lateral membranes of epithelial cells and, together with nuclear staining, has enabled the identification of cells fusing to become multinucleated cells, and later, to become an epithelial syncytium (symplasma). These fusing cells express the highest level of rbMDC9 mRNA. The results suggest that MDC9, a transmembrane modular protein with domains having potential integrin-binding, metalloproteinase, and fusogenic functions, is probably critical for the cellular interactions accompanying blastocyst implantation.

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Progesterone and implanting blastocysts regulate Muc1 expression in rabbit uterine epithelium.

Mammalian uteri are unreceptive to blastocyst implantation except during a relatively brief period. The transmembrane, cell surface mucin, Muc1, is present on epithelial cells of nonreceptive uteri in various species and has been demonstrated to have antiadhesive properties. These activities of Muc1 may prevent interaction of the embryonic trophoblast cells with the uterine epithelium. A previous study indicated that Muc1 expression in the rabbit, as in primates, is up-regulated by progesterone. This response would be expected to create a nonadhesive uterine surface during the progesterone-dominated receptive phase. In the current study, Northern blot analysis was used to evaluate Muc1 messenger RNA expression in the endometrium of estrous and progesterone-treated estrous rabbits and in endometrium from different stages of pregnancy or pseudopregnancy. Steady state levels of Muc1 messenger RNA were increased 10-fold when estrous animals were treated with progesterone for 5 days. Muc1 message was elevated 2- to 6-fold over estrous levels in endometrium of pseudopregnant females and 30-fold in preimplantation stage (6.75 days postcoitum) uteri. During implantation (7.25 day postcoitum), the high level of Muc1 expression continued in nonimplantation regions, but was dramatically reduced in endometrium from implantation sites. Using immunofluorescence localization, Muc1 protein was present on the apical surface of epithelial cells of estrous, pseudopregnant (4 and 6.75 days), preimplantation (6.75 days), and implantation (7.25 day) stage uteri. At the latter stage, luminal epithelium apposed to blastocysts had a marked reduction or absence of Muc1 immunostaining. Muc1-immunoreactive cells included luminal and cryptal epithelium in pregnant/pseudopregnant uteri, whereas the glandular cells stained weakly. Short term coculture of uterine epithelial cells with trophoblastic vesicles derived from 6.75-day blastocysts also resulted in a local reduction in apical epithelial Muc1 staining. These findings demonstrate that Muc1 expression is up-regulated by progesterone in the rabbit uterine epithelium and increases incrementally during pre- and periimplantation stages. Removal of Muc1 from the epithelial surface at implantation sites is accomplished locally via signals apparently produced by the blastocyst.

Impact of managed care on quality of healthcare: theory and evidence.

Each strategy for managing healthcare risk has important and unique implications for the patient-provider relationship and for quality of care. Not only are different incentive structures created by different risk-sharing arrangements, but these incentives differ from those in a fee-for-service environment. With fee-for-service and traditional indemnity insurance, physicians have incentives to provide healthcare services of marginal value to the patient; under managed care, physicians have fewer incentives to provide marginally beneficial services. However, the impact of financial arrangements on quality of care remains ambiguous, because it depends on the strategic behavior of physicians with regard to their informational advantage over their patients. Using the framework of an agency theory model, we surveyed the current scientific literature to assess the impact of managed care on quality of care. We considered three different dimensions of quality of care: patient satisfaction, clinical process and outcomes of care measures, and resource utilization. Although we found no systematic differences in patient satisfaction and clinical process and outcomes between managed care and fee-for-service plans, resource utilization appears to be decreased under managed care arrangements. Given the strengths and weaknesses of fee-for-service and managed care, it is unlikely that either will displace the other as the exclusive mechanism for arranging health insurance contracts. Policy makers may be able to take advantage of the strengths of both fee-for-service and managed care financial arrangements.

Expression of vascular endothelial growth factor (VEGF) and VEGF-receptor messenger ribonucleic acids in the peri-implantation rabbit uterus.

The endometrial vasculature undergoes expansion during preimplantation stages and, even more prominently, after implantation. In addition to angiogenesis, vascular hyperpermeability accompanies the attachment and invasion of blastocysts into the uterine lining. Vascular endothelial growth factor (VEGF) is an angiogenic factor expressed in mammalian uteri that also has potent activity in inducing vascular permeability. Rabbit uteri were examined using Northern and in situ hybridization to assess the temporal and spatial expression of VEGF and its receptor (Flk-1, Flt-1) mRNAs during the pre- and peri-implantation periods (Days 0-8). Steady-state levels of VEGF mRNA were highest in endometrium at estrous and peri-implantation stages (Days 6-8). In situ hybridization revealed a shift from uniform expression of VEGF transcripts throughout the uterus at estrus and Day 4, to an endometrial epithelial localization just before and during implantation. At implantation sites, a pronounced signal was present in the trophoblastic knobs, the syncytial aggregates that attach to and invade the endometrium. VEGF protein was detected by immunoblot analysis in peri-implantation-stage uteri but was below the limit of detection in estrous endometrium. VEGF receptor mRNAs were expressed in the uterus at all stages examined, with high levels of Flk-1 and Flt-1 at estrus and again just before implantation, 6-3/4 days pregnant. The high level just before implantation correlates with in situ hybridization results showing a prominent, but transient, signal for Flk-1 mRNA in the endometrial epithelium. During implantation, Flk-1 mRNA was associated with blood vessels of the endometrial stroma. We conclude that VEGF is a candidate factor for the induction of vascular hyperpermeability at implantation in the rabbit and in the angiogenic process that follows.

Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium.

A glycoprotein, termed GP42, was previously identified in uterine fluid obtained from peri-implantation-stage rabbits. N-terminus amino acid sequencing of purified GP42 demonstrated identity through the first 13 amino acids with the beta subunit of liver haptoglobin. The present study was undertaken to determine if GP42 is indeed identical to haptoglobin and, if so, to determine whether it is expressed in the uterus as opposed to being present as a transudate from plasma. Reverse transcription-PCR amplification of poly(A)+ RNA prepared from implantation-stage rabbit endometrium with GP42- and haptoglobin-specific primers yielded a predicted 667 bp cDNA product. Sequence analysis of the cloned cDNA confirmed the identity of GP42 with beta-haptoglobin. Northern blot analysis demonstrated the specific expression of haptoglobin mRNA in the peri-implantation-stage endometrium and the absence of its expression in the estrous or day 4 pseudopregnant endometrium. Non-isotopic in situ hybridization revealed that the haptoglobin mRNA was restricted to the epithelium lining the luminal surface and mucosal folds of day 6(3/4) pregnant or pseudo-pregnant uteri and that no haptoglobin mRNA was detectable in the epithelium of the deep glands or cells of the stroma or myometrium. Similarly, in situ hybridization revealed no expression of haptoglobin mRNA in any cell types of the estrous uterus. These data establish the identity of GP42 with beta-haptoglobin and demonstrate that it is expressed in a stage-specific manner just prior to implantation, correlating with uterine receptivity to blastocyst implantation. Endometrial GP42 mRNA expression is not dependent on the presence of blastocysts.

The POU gene ceh-18 promotes gonadal sheath cell differentiation and function required for meiotic maturation and ovulation in Caenorhabditis elegans.

In Caenorhabditis elegans, specialized contractile myoepithelial cells of the somatic gonad, the gonadal sheath cells, are closely apposed to oocytes and are required for normal meiotic maturation and ovulation. Previously we found that mutations in the ceh-18 gene, which encodes a POU-class homeoprotein expressed in sheath cells, result in oocyte defects. To determine the basis for these oocyte defects, we have used time-lapse video Nomarski microscopy to observe meiotic maturation, ovulation, and early embryogenesis in ceh-18 mutants. In ceh-18 mutants sheath cell contractions are weaker, less frequent, and uncoordinated throughout the sequence of ovulation events, and ovulation is defective. Defective ovulation can result in the formation of endomitotic oocytes in the gonad, the formation of haploid embryos, and reversals in embryonic polarity. ceh-18 mutant oocytes exhibit defects prior to nuclear envelope breakdown, suggesting that they are physiologically different from the wild type. We observed delays in meiotic maturation, as well as maturation out of the normal spatial and temporal sequence, suggesting that proximal sheath cells directly or indirectly promote and spatially restrict meiotic maturation. Analysis of sheath cell differentiation in ceh-18 mutants using antibodies to proteins of the contractile apparatus reveals that although contractile proteins are expressed, the sheath cells appear disorganized. Transmission electron microscopy reveals that ceh-18 mutant sheath cells are morphologically irregular and only loosely cover oocytes. Taken together, these observations indicate that ceh-18 is a crucial determinant of sheath cell differentiation, a function required for normal meiotic maturation and ovulation.

Modulation of retinal endothelial barrier in an in vitro model of the retinal microvasculature.

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.

A haptoglobin-like glycoprotein is produced by implantation-stage rabbit endometrium.

A polypeptide of 42 kDa was previously identified in rabbit uterine epithelium during the peri-implantation period as a progesterone-dependent, stage-specific protein. Binding of the lectin RCA-I to the 42-kDa band on Western blots demonstrated that it was a glycoprotein, here designated GP42. With use of a polyclonal antiserum to this glycoprotein, strong immunostaining was present on the surface of epithelial cells in implantation-stage uteri (6-7 days pregnant). Uteri of 4-day pseudopregnant females had only trace reactivity, and estrous uteri were devoid of immunostaining. Comparison of GP42 staining on immunoblots of uterine luminal samples, obtained using buffer with or without the detergent Triton X-100, demonstrated that GP42 is either loosely associated with the epithelial surface or is a secretory product. The N-terminal sequence of GP42 was identical through 13 amino acids with the beta subunit of haptoglobin, an acute-phase protein secreted by the liver. Additional immunoblot analyses were carried out after one- or two-dimensional PAGE separation of polypeptides of rabbit uterine samples and human haptoglobin. These employed anti-GP42 as well as antibodies directed against haptoglobin, and results confirmed the similarity of GP42 with beta-haptoglobin. In nonreducing gels, reactivity with anti-GP42 was present in a band of 110 kDa. This is comparable to the molecular size of serum haptoglobin, which occurs as a tetramer of two alpha (15-kDa) and two beta (38-42-kDa) chains. Cultured epithelial cells, derived from 4-day pseudopregnant uteri, released GP42 into the medium, but stromal cells appeared not to produce the glycoprotein. We conclude that GP42 is a uterine glycoprotein, related or identical to haptoglobin, and produced by rabbit uterine epithelial cells during the peri-implantation period. Possible roles for GP42 in relation to ovo-implantation are discussed in light of known functions for haptoglobin and haptoglobin-related protein.