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Vitamin A palmitate and aciclovir biodegradable microspheres for intraocular sustained release.

The aim of this study was to obtain a prolonged release of Vitamin A palmitate (RAP) and aciclovir from biodegradable microspheres for intraocular administration with an antiviral action and to be capable of preventing the inherent risks of intravitreal administration. The RAP effect on the microsphere characteristics was also studied. Poly(D,L-lactic-co-glycolic) acid microspheres were prepared by the solvent evaporation method. Different quantities of aciclovir (40-80 mg) and RAP (10-80 mg) were added to the internal phase of the emulsion. Microspheres were analysed by scanning electron microscopy, which revealed a spherical surface and a porous structure, and granulometric analysis that showed an adequate particle size for intraocular administration. The aciclovir loading efficiency increased when Vitamin A palmitate was added. Differential scanning calorimetry detected no differences in the polymer glass transition temperature and the aciclovir melting endotherm in all formulations. The release of aciclovir during the first days of the in vitro assay was improved with respect to microspheres without RAP. The microspheres showed a constant release of aciclovir and RAP for 49 days. Best results were obtained for microspheres prepared with 40 mg aciclovir, 80 mg RAP and 400mg polymer. A dose of 4.74 mg of microspheres would be therapeutic for the herpes simplex and Epstein-Barr viruses' treatment in an animal model and would reduce the intravitreal adverse effects. The injectability of a suspension of microspheres in isotonic saline solution resulted appropriate for its injection through a 27 G needle.

Acyclovir↗

Alpha2-macroglobulin, lipoprotein receptor-related protein and lipoprotein receptor-associated protein and the genetic risk for developing Alzheimer's disease.

Alpha2-macroglobulin (alpha2M) as well as its receptor, the low-density lipoprotein receptor-related (LRP) and the receptor-associated protein (RAP) are involved in the clearance of cerebral A beta. Current evidence suggests that polymorphisms in the genes of alpha2M, LRP and RAP may have functional effects on the proteins. Two independent association samples of 271 AD patients and 280 representative controls were investigated whether the risk for developing AD is altered in carriers of polymorphisms in the alpha2M-gene (Va1000Ile), in the LRP-gene (Ala216Val) and in the RAP-gene (Val311Met). Genotypes were determined by standard PCR and restriction fragment length polymorphism. The results were adjusted for age, gender and apolipoprotein E-epsilon4 (APOE) polymorphism. Inheritance of alpha2M conferred a small increased risk for sporadic AD with an estimated Mantel-Haenszel odds ratio of 1.47. There was no age- or gender-dependent increase in alpha2M Val1000Ile allele frequencies in AD patients compared to controls. There was no significant difference in the allele frequencies among control and AD subjects for the LRP and RAP polymorphisms. We found no evidence of an interaction between the alpha2M and RAP or LRP with regard to conferred risk. Our data suggest that the alpha2M Val1000Ile polymorphism is weakly associated with AD. Although LRP as well as RAP seem to play an essential role in the metabolism of alpha2M and APOE, there is no increase in the genetic risk for AD in patients carrying the investigated polymorphisms.

Aged↗

The Rater Applied Performance Scale: development and reliability.

Previous studies of rater performance and interrater reliability have used passive scoring tasks such as rating patients from a videotaped interview. Little is known, however, about how well raters conduct assessments on real patients or how reliably they apply scoring criteria during actual assessment sessions. With growing recognition of the importance of monitoring and review of actual evaluation sessions, there is need for a systematic approach to quantify raters' applied performance. The Rater Applied Performance Scale (RAPS) measures six dimensions of rater performance (adherence, follow-up, clarification, neutrality, rapport, and accuracy) based on reviews of audiotaped or videotaped assessment sessions or on live monitoring of assessment sessions. We tested this new scale by having two reviewers rate 20 Hamilton Depression Scale rating sessions ascertained from a multi-site depression trial. We found good internal consistency for the RAPS. Interrater (i.e. inter-reviewer) reliability was satisfactory for RAPS total score ratings. In addition, RAPS ratings correlated with quantitative measures of scoring accuracy based on independent expert ratings. Preliminary psychometric data suggest that the RAPS may be a valuable tool for quantifying the performance of clinical raters. Potential applications of the RAPS are considered.

Major Depressive Disorder↗

Correlations among critical closing pressure, pulsatility index and cerebrovascular resistance.

We attempted to explore the relationships among critical closing pressure (CrCP), resistance-area product (RAP) and traditional resistance indices of cerebral hemodynamics. Twenty healthy volunteers were studied. Blood pressure was obtained with servo-controlled plethysmography. Cerebral blood flow velocity (CBFV) was monitored by transcranial Doppler. Hemodynamic changes were induced by hyperventilation and by 5% CO(2) inhalation. Beat-to-beat CrCP and RAP values were extracted by linear regression analysis of instantaneous arterial blood pressure (ABP) and CBFV tracings. Gosling's pulsatility index (PI) and cerebrovascular resistance (CVR) were calculated. RAP correlated well with CVR at rest and during provocative tests (p = 0.006 approximately <0.001). There was no correlation among CrCP, CVR and PI. The changes in CVR correlated with those in RAP (p = 0.008 for the 5% CO(2) test and p = 0.014 for the hyperventilation test). The changes in PI and CrCP showed significant correlation (p = 0.004 for the 5% CO(2) test and p = 0.003 for the hyperventilation test). RAP reliably reflected cerebrovascular resistance. The changes in CrCP were valuable in assessing cerebrovascular regulation. Estimating changes in CrCP and RAP provided better understanding of the nature of cerebrovascular regulation.

Adolescent↗

Use of a stringent dimerizer-regulated gene expression system for controlled BMP2 delivery.

Gene therapy using constitutively active viral promoters to drive expression of bone morphogenetic proteins (BMPs) has been extensively evaluated as a strategy for inducing bone regeneration. However, this approach offers little control over the concentration, timing, or duration of BMP synthesis. To gain greater control over BMP kinetics, we developed a new inducible system for the controlled expression of BMP2 using a two-component transcription factor that is dimerized with rapamycin (Rap). This approach provided stringent control over BMP2 synthesis with no BMP expression detected in the uninduced state. Rapamycin or the less immunosuppressive analogue, AP21967, rapidly and reversibly induced BMP2 in a dose-dependent manner (range 0.1-10 nM). Subcutaneous implants of fibroblasts containing the Rap-inducible system in syngeneic C57BL/6 mice were highly responsive to ip Rap injection (0.1-1 mg/kg). Peak BMP2 levels were detected within 24 h of a single Rap injection and declined to undetectable levels after 8-10 days. Alternate-day Rap injections (1 mg/kg) for 6 weeks induced subcutaneous ectopic bone formation. Rap-dependent healing of a critical-sized cranial defect was also achieved using this system. This regulated BMP2 expression system will be extremely useful for examining the role of timing and sequence of BMP delivery on bone regeneration.

Animals↗

Expression of cartilage-derived retinoic acid-sensitive protein during healing of the rat tooth-extraction socket.

Cartilage-derived retinoic acid sensitive protein (CD-RAP) is a recently described, cartilage-specific protein. During early healing of the tooth-extraction socket, cells express both chondrogenic and osteogenic cell markers, but no cartilage is formed. Cartilaginous collagen type II protein, a major constituent of hyaline cartilage, has not been detected in the healing socket, although type IX collagen, which coats these fibres, has been detected transiently in early socket healing. This study investigated the spatial and temporal expression of CD-RAP and various osteoblast cell markers, i.e., alkaline phosphatase, osteopontin, osteonectin and osteocalcin, during healing. Immunolocalization of these proteins was determined in the rat tooth socket at 3, 4, 5, 6, 7, 8, 10 and 14 days after extraction. CD-RAP was expressed by preosteoblast cells maximally at 6, 7, and 8 days after extraction. Fully differentiated osteoblasts expressed osteocalcin, a specific osteoblast marker. Preosteoblasts and fibroblasts did not express osteocalcin. On double immunofluorescent staining, some preosteoblasts coexpressed CD-RAP (indicative of chondrogenic differentiation), and either alkaline phosphatase or osteopontin (markers of osteogenic stem-cell maturation). There was no colocalization between osteopontin and osteonectin. CD-RAP was unique amongst the cell markers used in that it was expressed by preosteoblasts, but not by osteoblasts lining the newly formed trabeculae. CD-RAP may have an important role in osteoblast cell differentiation during bone healing.

Alkaline Phosphatase↗

Recurrent abdominal pain in children: evidence from a population-based study that social and familial factors play a major role but not Helicobacter pylori infection.

OBJECTIVE: To analyze the relationship between social and familial factors, Helicobacter pylori infection and recurrent abdominal pain (RAP) in children in a population-based cross-sectional study among 1221 preschool children aged 5-8 years. METHODS: H. pylori infection status was determined by 13C-urea breath test (13C-UBT) and information on medical history of the child and on RAP as well as on family demographics was obtained by a standardized questionnaire. RESULTS: Overall, 129 children (11.3%) were infected with H. pylori and 29 children were identified as having RAP within the past 3 months (2.5%). Analysis by multiple logistic regression demonstrated a clear relationship of RAP with living in a single parent household [odds ratio (OR) 2.9, 95% confidence interval (95% CI) 1.2-6.7], with parental history of peptic ulcer (OR 3.7, 95% CI 1.3-10.4) and with parental history of nonulcer gastrointestinal disorders (OR 5.3, 95% CI 2.1-13.2). By contrast, there was a nonsignificant relation between H. pylori infection and occurrence of RAP (OR 1.6, 95% CI 0.5-5.5). CONCLUSION: Social and familial factors play a major role but not H. pylori infection in RAP.

Abdominal Pain↗

Pentapeptide regulation of aspartyl-phosphate phosphatases.

Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively. The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor. Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases. Phr peptide's recognition of the Rap phosphatase targets is remarkably specific. Specificity is dictated by the amino acid sequence of the pentapeptide. The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.

Amino Acid Sequence↗

Solution structure of the DNA-binding domain of human telomeric protein, hTRF1.

BACKGROUND: Mammalian telomeres consist of long tandem arrays of the double-stranded TTAGGG sequence motif packaged by a telomere repeat binding factor, TRF1. The DNA-binding domain of TRF1 shows sequence homology to each of three tandem repeats of the DNA-binding domain of the transcriptional activator c-Myb. The isolated c-Myb-like domain of human TRF1 (hTRF1) binds specifically to telomeric DNA as a monomer, in a similar manner to that of homeodomains. So far, the only three-dimensional structure of a telomeric protein to be determined is that of a yeast telomeric protein, Rap 1p. The DNA-binding domain of Rap 1p contains two subdomains that are structurally closely related to c-Myb repeats. We set out to determine the solution structure of the DNA-binding domain of hTRF1 in order to establish its mode of DNA binding. RESULTS: The solution structure of the DNA-binding domain of hTRF1 has been determined and shown to comprise three helices. The architecture of the three helices is very similar to that of each Rap 1p subdomain and also to that of each c-Myb repeat. The second and third helix form a helix-turn-helix (HTH) variant. The length of the third helix of hTRF1 is similar to that of the second subdomain of Rap 1p. CONCLUSIONS: The hTRF1 DNA-binding domain is likely to bind to DNA in a similar manner to that of the second subdomain of Rap 1p. On the basis of the Rap 1p-DNA complex, a model of the hTRF1 DNA-binding domain in complex with human telomeric DNA was constructed. In addition to DNA recognition by the HTH variant, a flexible N-terminal arm of hTRF1 is likely to interact with DNA.

Amino Acid Sequence↗

Interlobular arterial resistance: influence of renal arterial pressure and angiotensin II.

Pressure in the distal third of the interlobular arteries (Pila) was measured in anesthetized rats by micropuncture through corticotomy. Control Pila was 83.9 (SD 11.9) mm Hg at a renal arterial pressure (RAP) of 113.1 (SD 12.8) mm Hg. Reduction of RAP by 20 mm Hg caused no consistent change of total renal blood flow (RBF) or Pila. Relative interlobular arterial resistance, Rila = (RAP - Pila)/RBF, fell by 40 to 50%, and then remained practically unchanged at further reduction of RAP. Blood flow measured by radiolabeled microspheres (10.7 micron) showed similar values in intact cortex and in the tissue beneath the corticotomy, both varying in proportion to RBF. Intravenous infusion of angiotensin II (AII) 40 to 90 ng/min reduced RBF by 29% and increased RAP by 19 mm Hg. Pila rose by only 8 mm Hg and Rila increased to 209% of control. Reduction of RAP to control level during continued AII infusion did not change RBF, while Rila fell to 131% of control. We conclude that: dilatation and constriction of the interlobular arteries contribute importantly to autoregulation of outer cortical blood flow, probably through a myogenic mechanism (Bayliss); the constriction of interlobular arteries elicited by i.v. AII reflects mainly an autoregulatory response to increased arterial pressure, and to a smaller extent, a direct constrictor effect of AII.

Angiotensin II↗

Role of renal arterial pressure in the regulation of extracellular volume in conscious dogs.

1. This study in conscious dogs examined the quantitative effects of a reduction in the renal arterial pressure on the renal homeostatic responses to an acute extracellular fluid volume expansion. 2. Seven female beagle dogs were chronically instrumented with two aortic catheters, one central venous catheter and a suprarenal aortic cuff, and were kept under standardized conditions on a constant high dietary sodium intake (14.5 mmol of Na+ day-1 kg-1 body weight). 3. After a 60 min control period, 0.9% (w/v) NaCl was infused at a rate of 1 ml min-1 kg-1 body weight for 60 min (infusion period). Two different protocols were applied during the infusion period: renal arterial pressure was maintained at 102 +/- 1 mmHg by means of a servo-feedback control circuit (RAP-sc, 14 experiments) or was left free (RAP-f, 14 experiments). 4. During the infusion period, in the RAP-sc protocol as well as in the RAP-f protocol, the mean arterial pressure increased by 10 mmHg, the heart rate increased by 20 beats/min, the central venous pressure increased by 4 cmH2O and the glomerular filtration rate (control 5.1 +/- 0.3 ml min-1 kg-1 body weight, mean +/- SEM) increased by 1 ml min-1 kg-1. 5. Plasma renin activity [control 0.85 +/- 0.15 (RAP-f) and 1.08 +/- 0.23 (RAP-sc) pmol of angiotensin I h-1 ml-1] decreased similarly in both protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination.

BACKGROUND: The ninR region of phage lambda contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage lambda. RESULTS: When lambda DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of lambda decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap- phenotype. CONCLUSION: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type lambda grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap- phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function. A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants.

Bacterial Proteins↗

Correlation between measured inferior vena cava diameter and right atrial pressure depends on the echocardiographic method used in patients who are mechanically ventilated.

In patients who are mechanically ventilated, the correlation between inferior vena cava diameter (IVCD) measurements and mean right atrial pressure (RAP) varies in the literature. The purpose of this study was to test if the correlation between IVCD and RAP measurement in patients who are critically ill depends on the transthoracic echocardiography (TTE) methodology used. Twenty patients who were critically ill, sedated, and required respiratory support were prospectively studied by TTE during mechanical ventilation in a controlled mode. The TTE measures of IVCD were made, using methods previously cited. First, IVCD was measured at end-expiration and end-diastole, with ECG synchronization, using the M-mode, on short-axis view 2 cm below the right atrium. Second, IVCD was assessed at end-expiration, without ECG synchronization, using the 2-dimensional long-axis view at the same location. RAP was measured simultaneously by using a central venous catheter positioned in the superior vena cava. All measurements were taken in the supine position. IVCD at end-expiration and end-diastole, with ECG synchronization, using the M-mode, and IVCD at end-expiration, without ECG synchronization, using the 2-dimensional long-axis view, correlate linearly with RAP (0.81, P <.0001 and 0.71, P =.0004). Mean bias between the 2 TTE methods (Bland-Altman analysis) was 1.6 mm (SD +/- 2.03 mm). In conclusion, this study confirms that variation of correlation between TTE IVCD measurement and RAP depends on the ultrasonographic methodology used and the timing of measurement during the cardiac cycle. IVCD at end-expiration and end-diastole, with ECG synchronization, using the M-mode (IVCD-MM) correlates more satisfactory with RAP than with IVCD at end-expiration, without ECG synchronization, using the 2-dimensional long-axis view, in patients during mechanical ventilation.

Adult↗

Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins.

The immunosuppressant rapamycin (RAP) has been demonstrated to specifically inhibit the activity of p70 S6 kinase (p70s6k) and subsequent phosphorylation of ribosomal S6 protein in mammalian cells. Addition of RAP to proliferating lymphoid cells resulted in inhibition of protein synthesis before any changes in the rate of cell proliferation. When the cellular composition of proteins was examined by gel electrophoresis, RAP dramatically inhibited synthesis of selective proteins, particularly elongation factor 2 (eEF-2). The inhibition of eEF-2 synthesis by RAP was at the translational level. Further, RAP inhibited the polysomal association of mRNAs encoding not only eEF-2 but also elongation factor 1-alpha and ribosomal proteins without affecting mRNA translation of any of a number of nonribosomal proteins. Since levels of activity of p70s6k are correlated with the rate of biosynthesis of eEF-2, p70s6k might be involved in coordinate translational regulation of ribosomal protein mRNAs in higher eukaryotes, which have a conserved sequence at their 5' end. Specific inhibition of ribosomal protein synthesis likely explains the differential antiproliferative effect of RAP on proliferating and mitogen-activated quiescent cells.

Base Sequence↗

Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.

The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo.

Animals↗

Ca2+ and receptor-associated protein are independently required for proper folding and disulfide bond formation of the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca2+ and its relationship to RAP during LRP folding. We found that depletion of Ca2+ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca2+ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca2+ depletion. The role of Ca2+ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca2+ depletion. Furthermore, Ca2+ depletion-induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca2+ and RAP independently participate in LRP folding.

Calcium↗

Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein.

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

Animals↗

Analysis of expression of genes involved in apolipoprotein E-based lipoprotein metabolism in pregnant mice deficient in the receptor-associated protein, the low density lipoprotein receptor, or apolipoprotein E.

Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP(-/-) mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP(-/-) pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.

Animals↗