Effects of oocytes exposure to bovine diarrhea viruses BVDV-1, BVDV-2 and Hobi-like virus on in vitro-produced bovine embryo development and viral infection.

As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 × 102 - 2.50 × 103 TCID50/100 µL), BVDV-2 (6.25 × 101 - 5.20 × 102 TCID50/100 µL) or Hobi-like virus (1.90 × 102 - 1.58 × 103 TCID50/100 µL) for 22-24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50-100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2-64.1% vs 49.8%; P = 0.003-0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20-50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3-38.0% vs 49.8% for negative controls; P ≤ 0.001-0.014). Blastocyst rates were only reduced in the 7.9 × 102 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P = 0.009), when calculated by oocyte number. 50-80% of degenerated embryos tested positive for Hobi-like virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and -2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.

Changes in acetylation of lysine 5 on histone H4 in canine oocytes following in vitro maturation.

Post-translational modifications of histones, such as acetylation, are involved in regulating chromatin remodelling and gene expression. Proper in vitro maturation (IVM) of canine oocytes, for many reasons, is up to now inefficient. This study aimed to evaluate the post-translational histone H4 acetylation at lysine 5 (H4K5) in immature and post-IVM canine oocytes. Oocyte nuclear stage was assessed using Hoechst 33342 staining. Acetylation patterns were determined by indirect immunofluorescence staining of immature and post-IVM oocytes, using an antibody against the acetylated lysine 5 residue on histone 4 (H4K5ac). The experiment was repeated four times, with a total of 7-17 oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIHimagej software. Data were expressed as a percentage of the average fluorescence intensity of the specific antibody over the intensity of DNA, as determined by Hoescht staining. H4K5ac displayed a significantly higher acetylated pattern in immature oocytes (0.97 ± 0.08) when compared to post-IVM oocytes at different nuclear stages. There was a decrease in the fluorescence level of the matured oocytes with the progression of meiosis (GVBD: 0.47 ± 0.06 and MI/MII: 0.35 ± 0.04). Similarly to other domestic species, we hypothesized that post-translational modification of histone acetylation takes place during meiosis of in vitro matured canine oocytes. However, it remains to be investigated whether these changes occur during in vivo maturation.

Aberrant expression of E-cadherin and ß-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer.

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and ß-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or ß-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total ß-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-ß-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/ß-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and ß-catenin proteins, along with defective ß-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Placental insufficiencies in cloned animals - a workshop report.

This workshop focused on describing clinical problems identified in the placentae of cloned animals and some of the potential biological mechanisms by which these anomalies arise. It was shown that placental anomalies related to somatic cell nuclear transfer (SCNT) in cattle often can be detected by ultrasonography early in gestation, enabling preventive clinical intervention. On the mechanistic front, the vascular defects in the placenta appear to be associated with anomalies in the expression of VEGF system, which could lead to the aberrant placentomes and generalized oedema seen in some gestations. Moreover, an upstream transcription factor (Mash2) controlling the differentiation of trophoblast into binucleate cells may be involved in the poor implantation rates of SCNT embryos. Finally, epigenetic patterns in placenta can be disrupted by fairly simple in vitro manipulations, which could explain the extreme anomalies observed in the placenta of SCNT pregnancies.

Characterization of the placenta specific bovine mammalian achaete scute-like homologue 2 (Mash2) gene.

Mash2, a basic helix-loop-helix transcription factor, stimulates mononucleate trophoblast cell proliferation and inhibits giant/binucleate cell formation. In mice, Mash2 is a maternally expressed imprinted gene. Regulation of bovine Mash2 is unclear due to limited genetic knowledge. Our objectives were to clone and characterize bovine Mash2 and evaluate its imprinting status by utilizing Bos taurus taurus and Bos taurus indicus interspecies crossing. Bovine Mash2 mRNA shares 78% and 70% homology with human and mouse Mash2, with the DNA binding domain (88%) and bHLH region (95%) being highly conserved. Expression of Mash2 mRNA was seen exclusively in cotyledonary areas of the placenta. The greatest abundance of Mash2 mRNA was in day 17 filamentous embryos, during the time of rapid trophoblast proliferation. Reduction in Mash2 mRNA abundance was detected in day 8 parthenogenetic blastocysts suggesting paternal regulation of gene expression. Prior to implantation (days 8 and 17), Mash2 mRNA appears to have biallelic expression, but is paternally silenced after implantation (days 40 and 60). In conclusion, the Mash2 is highly conserved across species and is specifically expressed in the bovine placenta. Bovine Mash2 appears to be maternally expressed after implantation, but the paternal genome plays a role in regulating expression.

Effect of dietary restriction, pregnancy, and fetal type on intestinal cellularity and vascularity in Columbia and Romanov ewes.

The objectives of this study were to evaluate intestinal cellularity and vascularity in mature ewes in response to dietary restriction and pregnancy status and to quantify the response of these variables to increased nutrient demand of fetal growth. In Exp. 1, 28 mature Dorset x crossbred white-faced ewes (61.6+/-1.8 kg initial BW) were fed a pelleted, forage-based diet. Treatments were arranged in a 2 x 3 factorial, with dietary restriction (60% restriction vs. 100% maintenance for respective states of pregnancy) and pregnancy status (nonpregnant, NP; d 90 and 130) as main effects. Dietary treatments were initiated on d 50 of gestation and remained at 60 or 100% maintenance throughout the experiment. Nonpregnant ewes were fed dietary treatments for 40 d. In Exp. 2, four Romanov ewes were naturally serviced (Romanov fetus and Romanov dam; R/R); two Romanov embryos per recipient were transferred to four Columbia recipients (Romanov fetus and Columbia recipient; R/C), and three Columbia ewes were naturally serviced (Columbia fetus and Columbia dam; C/C). In Exp. 1, dietary restriction and pregnancy status interacted with regard to maternal jejunal DNA concentration (P < 0.01), with restricted ewes having a greater DNA concentration (mg/g; fresh basis) at d 130. Vascularity (percentage of total tissue area) in the jejunum was increased (P < 0.06) as a result of dietary restriction and pregnancy status. Total microvascular volume ofjejunal tissue was not altered by dietary restriction and increased (P < 0.01) at d 130 of pregnancy. In Exp. 2, R/R ewes had less (P < 0.09) DNA (g) in the jejunum compared with R/C and C/C ewes. Jejunal vascularity (%) was increased (P < 0.05) in R/R ewes compared with R/C or C/C ewes, whereas total jejunal microvascular volume remained unchanged. These data demonstrate intestinal vascular density responds to changes in diet and physiological state. In addition, pregnancy increased total jejunal microvascular volume.

Verbal instruction for correcting errors versus such instructions plus videotape replay on learning the overhand throw.

The influence of two methods of providing information to women, ages 19 to 22 years who were novices and learning the overhand throw with the nondominant arm, was examined. One group received verbal information on correcting errors, and a second group received the same information immediately prior to viewing a videotaped replay of a just completed throw. Performance was assessed quantitatively with respect to outcome (distance thrown) and qualitatively with respect to throwing form as measured on a 7-point rating scale by judges with a working knowledge of the overhand throw and with respect to throwing mechanics rated by a panel of experts in biomechanics using a scale of Leme and Shambes. Although the treatments led to better learning and performance, there was no significant difference between groups for distance thrown on the Leme and Shambes scale in Sessions 1-6 of 10 trials each on Session 7. The mean rating also indicated increased scores for both groups and better retention at posttest by the group receiving only verbal corrections. These results suggest that information provided by adding videotaped replay may be redundant and unnecessary for those in Sessions 1-6.

Photosensitized (electron-transfer) deconjugation of 1-arylcyclohexenes.

[figure: see text] A series of 1-arylcyclohexenes have been deconjugated to the corresponding 3-arylcyclohexenes via a photosensitized electron-transfer reaction. The introduction of substituents on the aryl group has provided insight into the underlying mechanism and has defined the scope and limitations of the reaction.

Notch signaling induces cell cycle arrest in small cell lung cancer cells.

Among the various forms of human lung cancer, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype. Neural and NE differentiation in SCLC depend, in part, on the action of the basic-helix-loop-helix (bHLH) transcription factor human achaete-scute homologue-1 (hASH1). In nervous system development, the Notch signaling pathway is a critical negative regulator of bHLH factors, including hASH1, controlling cell fate commitment and differentiation. To characterize Notch pathway function in SCLC, we explored the consequences of constitutively active Notch signaling in cultured SCLC cells. Recombinant adenoviruses were used to overexpress active forms of Notch1, Notch2, or the Notch effector protein human hairy enhancer of split-1 (HES1) in DMS53 and NCI-H209 SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G1 cell cycle block. We found up-regulation of p21(waf1/cip1) and p27kip1 in concert with the cell cycle changes. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2, findings that have been shown to be associated with cell cycle arrest in SCLC cells. These data suggest that the previously described function of Notch proteins as proto-oncogenes is highly context-dependent. Notch activation, in the setting of a highly proliferative hASH1-dependent NE neoplasm, can be associated with growth arrest and apparent reduction in neoplastic potential.

Interferon-tau modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells.

Antiluteolytic actions of bovine interferon-tau (bIFN-tau) require suppression of prostaglandin F(2 alpha) (PGF(2 alpha)) production. Our objective was to test whether bIFN-tau could block PGF(2 alpha) production and synthesis of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-tau. Medium samples were analyzed for concentrations of PGF(2 alpha), whole-cell extracts were analyzed for abundance of PLA(2) and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF(2 alpha) between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA(2) by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-tau suppressed PGF(2 alpha) production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA(2) proteins. Added after a 3-h stimulation with PDBu alone, bIFN-tau suppressed PGF(2 alpha) production after 1 h. Bovine IFN-tau inhibited intracellular mechanisms responsible for PGF(2 alpha) production in BEND cells, and this could be through both cytosolic and nuclear actions.

Intracellular regulation of endometrial PGF(2a) and PGE(2) production in dairy cows during early pregnancy and following treatment with recombinant interferon-tau.

Objectives were to examine how the conceptus and recombinant bovine interferon-tau (rbIFN-tau) regulate intracellular components of the PGF(2a) synthetic pathway and to determine if arachidonic acid (AA) is limiting in endometrial tissue of pregnant cows. In Experiment 1, uteri were collected from either cyclic or pregnant dairy cows on Day 17 post-estrus. Intercaruncular explants were dissected and incubated for 60 min to quantify PGF(2a) production in response to oxytocin (10(-6) M), A23187 (10(-5) M), melittin (10(-5) M), and phorbol 12, 13 dibutyrate (PDBu, 10(-6) M). Additional explants from the same cows were incubated for 24 h with and without AA. Oxytocin and A23187 did not stimulate PGF(2a) in explants from either cyclic or pregnant cows. Both PDBu, melittin, and A23187 + melittin stimulated PGF(2a) production in explants of cyclic cows, but not in explants of pregnant cows. The addition of AA to explant cultures for 24 hr did not increase PGF(2a) production during a subsequent 60-min incubation. In Experiment 2, explants were collected from cows that received intrauterine infusions of either BSA (1.9 mg/1.2 ml) or rbIFN-tau (0.2 mg rbIFN-tau + 1.7 mg BSA/1.2 ml) twice a day from Days 14 to 17 of the estrous cycle. Treatments of rbIFN-tau attenuated PGF(2a) secretion induced by in vitro PDBu and A23187 treatments. However, rbIFN-tau treatment in vivo had no effect on the in vitro induction of PGF(2a) secretion by melittin. IFN-tau may regulate the PGF(2a) synthetic pathway by reducing activity of PKC or PKC mediated events.

Maxillary sinus augmentation using different grafting materials and dental implants in monkeys. Part IV. Evaluation of hydroxyapatite-coated implants.

The aim of this study was to evaluate clinically, histologically and histometrically the use of hydroxyapatite-coated dental implants in conjunction with maxillary sinus augmentation procedures. In 4 adult male Rhesus monkeys (Macaca mulatta) the 3 maxillary molars on 1 side of the jaws were extracted and the remaining bone between the alveolar crest and the floor of the sinus was reduced to 3-4 mm. After 3 months, maxillary sinus augmentation procedures were performed in each monkey and the sinuses were grafted with a porous hydroxyapatite bone graft (Interpore-200). At the same time, 2 hydroxyapatite-coated cylinder implants (IMZ) were immediately placed into the augmented sinuses (i.e. simultaneous-implants-loaded group). Four months later, 2 additional similar implants were placed into the previously augmented sinuses (i.e. delayed-implants-loaded group). After 4 months, the abutment connection was performed and all 4 implants were loaded with a gold-alloy bridge for 6 months (i.e. until sacrifice of the animals). The contralateral side of each monkey received the same treatment with the exception that removal of the maxillary molars was performed 7 months after those in the opposite side, and that the implants in this side were not loaded. Thus, 2 additional study groups (i.e. simultaneous-implants-unloaded group and delayed-implants-unloaded group) were obtained. Clinically, all loaded and unloaded implants were stable the day of sacrifice. Histologically, the grafted sinuses exhibited a significant amount of new bone formation with integration of the porous hydroxyapatite graft particles and hydroxyapatite-coat of the dental implants to the new bone. Histometric analysis indicated that on the loaded side the implants placed simultaneously with the sinus lift procedure exhibited greater direct mineralized bone-to-implant contact than the delayed placed implants. In addition, the percentage of direct mineralized bone-to-implant contact was significantly greater in the residual bone in comparison to the augmented area in all groups. Loading of the implants exhibited a positive effect on the percentage of direct mineralized bone-to-implant contact in the augmented area. It could be concluded that hydroxyapatite-coated implants may be of benefit when used in conjunction with sinus augmentation procedures.

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Vitreous and aqueous humors contain a latent proteinase activity that abolishes IGF binding to specific IGF binding proteins.

Several distinct insulin-like growth factor binding proteins (IGFBPs) are present in tissues and fluids of the developing and adult eye. However, the mechanism(s) involved in the regulation of ocular IGFBP levels is unknown. We have now identified an endogenous factor in vitreous and aqueous humors that, when activated by sodium dodecyl sulfate (SDS), abolishes the capacity of specific low molecular weight IGFBPs (i.e. 24-30 kDa) to bind IGF as assessed by western ligand blotting. In contrast, IGF binding to the 46 and 32 kDa IGFBPs (IGFBP-3 and IGFBP-2 respectively) is not affected by the SDS-activated inhibitory factor (IF). Maximal activation of the IF occurs at an SDS concentration of approximately 0.015%. Incubations in the presence of the serine-proteinase inhibitor aprotinin result in marked inhibition of IF activity. Preliminary characterization by ultrafiltration suggests that the IF is large (< 100 kDa) and/or that it is present in a complex. The finding of a factor, most likely a serine proteinase, that specifically abolishes IGF binding to low molecular weight IGFBPs suggests a mechanism for regulating the levels of these IGFBPs and thus the functional activities of IGFs in ocular fluids under normal and/or pathological conditions.

Distribution of IGF-I and -II, IGF binding proteins (IGFBPs) and IGFBP mRNA in ocular fluids and tissues: potential sites of synthesis of IGFBPs in aqueous and vitreous.

Levels of insulin-like growth factor-I and II (IGF-I and IGF-II) in bovine aqueous humor are twice those found in the vitreous (aqueal IGF-I = 0.62 nM, vitreal IGF-I = 0.30 nM; aqueal IGF-II = 0.028 nM, vitreal IGF-II = 0.017 nM). IGF-I and II binding assays and IGF-II Western ligand blots indicate that aqueous and vitreous humor have equal overall levels of binding (binding assays, mean +/- S.E.M. bound/free per 50 microliters of fluid: vitreal IGF-II = 7.28 +/- 1.6, IGF-I = 0.3 +/- 0.078; aqueal IGF-II = 7.21 +/- 0.072; IGF-I = 0.3 +/- 0.078). In addition, the ligand blots reveal that aqueous and vitreous have markedly different complements of specific IGF binding proteins (IGFBPs). Aqueal levels of a 34 kDa IGFBP, immunologically identified as IGFBP-2, exceed those in the vitreous by two-fold. In contrast, the vitreous exhibits a two- to three-fold higher level of smaller (28-24 kDa), yet unidentified, IGFBPs. Aqueal and vitreal IGFBP patterns are also different from those found in serum. IGFBP-2 found in the aqueous and vitreous may be synthesized by ciliary body and/or cornea since these structures contain high levels of IGFBP-2 mRNA. Lens epithelial cells may also contribute IGFBP-2 to the aqueous since they also contain IGFBP-2 mRNA, albeit at substantially lower levels than the cornea and ciliary body. The retina has the lowest level of IGFBP-2 mRNA. IGF-II binding assays of cornea, ciliary body, retina and retinal pigment epithelium (RPE) indicate that the cornea has the highest level of binding (mean +/- S.E.M. IGF-II B/F per 50 micrograms protein: cornea = 84.52 +/- 28.8; iris/ciliary body complex = 0.61 +/- 0.078; retina = 0.47 +/- 0.096; RPE = 0.069 +/- 0.019). IGF-II ligand blots confirm these tissue-specific differences in binding and show that each ocular tissue contains IGFBP-2. In addition, ligand blots indicate that each ocular tissue contains a complex and distinctive population of IGFBPs. For example, the cornea and retina (but not the ciliary body, aqueous or vitreous) contain a 46 kDa IGFBP that may be IGFBP-3. The finding that cornea and retina also contain IGFBP-3 mRNA suggests that these structures may synthesize IGFBP-3 for local use within the eye.

Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: a characterization of binding and structural differences.

We have characterized insulin and insulin-like growth factor I (IGF-I) binding sites in developing chick retina and pigment epithelium (10- and 14-day embryonic, and 2-week post-hatched). For comparison, binding sites in brain and liver were also examined. Both the retina and pigment epithelium (PE) contain separate, specific, high affinity binding sites for insulin and IGF-I. In both tissues, IGF-I binding exceeds insulin binding by two to threefold. Insulin and IGF-I binding in the retina is four to six times greater than in PE. Insulin and IGF-I binding in the retina and PE exhibit independent developmental regulation. In the retina, the number of binding sites decreases by approximately 50% between embryonic day 10 and 2 weeks post-hatching. In the PE, binding decreases slightly between embryonic day 10 and 14 and then, in the 2-week post-hatched chick, increases threefold. Insulin receptor binding subunits in the retina and brain are similar in that both are neuraminidase insensitive and have apparent molecular weights of 116 kD. In contrast, binding subunits in the PE and liver have higher molecular weights (about 126 kD), and are sensitive to neuraminidase. At the embryonic stages examined, the levels of retinal insulin and IGF-I binding exceed those of the brain by five to 13-fold. Taken together, these data suggest that the retina is a major target of insulin and IGF-I and that the binding of these growth factors is developmentally regulated.

Insulin and IGF-1 binding in chick sclera.

The sclera of embryonic (days 10 and 14) and young adult (2-week posthatching chicks) contains distinct binding sites for insulin and for insulin-like growth factor-1 (IGF-1). Since there is a nearly 50% decrease in insulin and IGF-1 binding between embryonic day 10 and the 2nd week posthatching, it is clear that these sites are developmentally regulated. The affinity of each binding site for its ligand is stable across development. This suggests that the developmental decrease in binding is the result of a decrease in the number of binding sites. The insulin binding site in the sclera is specific for insulin since it has a high affinity for insulin and a lower affinity for IGF-1 (IC50 for unlabeled insulin = 0.4 nM; unlabeled IGF-1 = 5.0 nM). The embryonic chick sclera also contains two high-affinity IGF-1 binding sites. One of these sites exhibits poor binding specificity since it has an equal affinity for insulin and IGF-1. However, the specificity of this site increases in the young adult. The second IGF-1 binding site exhibits a more conventional specificity in that it has a higher affinity for IGF-1 than for insulin. The specificity of this binding site also improves in the young adult. The presence of insulin and IGF-1 receptor binding site subtypes is not correlated with structurally different receptor binding subunits since only a single population of binding subunits is observed (apparent molecular weight of 125 +/- 2.7 kD) in embryonic and adult sclera.(ABSTRACT TRUNCATED AT 250 WORDS)

Hospital challenge: using change theory and processes to adopt and implement the marketing concept.

Getting the marketing concept understood and accepted is one of the biggest challenges faced by a hospital. The authors discuss a variety of issues related to the process of changing a hospital's internal cultural values and managing those changes needed to embrace the marketing concept as part of the overall hospital culture.

Organization culture and the marketing concept: diagnostic keys for hospitals.

For successful adaptation to changing environmental conditions, hospital organizational cultures must incorporate the marketing concept to enhance flexibility and orientation toward the external environment. The authors propose procedures for diagnosing a hospital's culture and determining how well it has adopted and implemented the marketing concept.