Prior sexual behavior of teenagers attending rap sessions for the first time.
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Adolescent↗
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Retrograde autologous priming of the cardiopulmonary bypass circuit: safety and impact on postoperative outcomes.
OBJECTIVES: Retrograde autologous priming (RAP) is a blood conservation technique used to limit the severity of hemodilution during cardiopulmonary bypass and reduce perioperative transfusions. The aim of this investigation was to examine the safety of RAP and to determine the effect of RAP on adverse outcomes after cardiac surgery. DESIGN: Retrospective cohort study. SETTING: University hospital. PARTICIPANTS: Five hundred fifty-nine undergoing cardiopulmonary bypass. INTERVENTIONS: Data were retrospectively collected on 2 cohorts of adult cardiac surgical patients operated on by a single surgeon. In the RAP group (n = 256), outcome data were analyzed on all subjects over a 2-year period during which RAP was used routinely. This group was compared with a similar cohort of patients undergoing cardiopulmonary bypass over a 2-year period immediately before the introduction of RAP into the clinical practice (no-RAP group, n = 287). MEASUREMENTS AND MAIN RESULTS: In-hospital mortality was not significantly different between the RAP group (2.7%) and the no-RAP group (3.8%, p = 0.636). The incidence of postoperative cardiac arrest was significantly less in the RAP group (1 patient) compared to the no-RAP group (9 patients, p = 0.040). There were no differences between the 2 groups in the incidence of several other postoperative complications, including postoperative delirium (1.6% RAP v 3.1% no RAP), heart block (1.6% RAP v 4.2% no RAP), atrial fibrillation (19.1% RAP v 22.7% no RAP), and requiring postoperative ventilation >24 hours (2.7% RAP v 5.2% no RAP). CONCLUSIONS: The authors observed no evidence of any increase in adverse events in the RAP group of this retrospective cohort study, but they did observe a decrease in the incidence of postoperative cardiac arrest in the RAP group. These findings suggest that RAP is a safe technique and may have a beneficial effect on postoperative outcomes.
Adult↗
Development of a multidimensional measure for recurrent abdominal pain in children: population-based studies in three settings.
OBJECTIVE: Recurrent abdominal pain (RAP) is a common problem in children and adolescents. Evaluation and treatment of children with RAP continue to challenge physicians because of the lack of a psychometrically sound measure for RAP. A major obstacle to progress in research on RAP has been the lack of a biological marker for RAP and the lack of a reliable and valid clinical measure for RAP. The objectives of this study were (1) to develop and test a multidimensional measure for RAP (MM-RAP) in children to serve as a primary outcome measure for clinical trials, (2) to evaluate the reliability of the measure and compare its responses across different populations, and (3) to examine the reliabilities of the measure scales in relation to the demographic variables of the studied population. METHODS: We conducted 3 cross-sectional studies. Two studies were clinic-based studies that enrolled children with RAP from 1 pediatric gastroenterology clinic and 6 primary care clinics. The third study was a community-based study in which children from 1 elementary and 2 middle schools were screened for frequent episodes of abdominal pain. The 3 studies were conducted in Houston, Texas. Inclusion criteria for the clinic-based studies were (1) age of 4 to 18 years; (2) abdominal pain that had persisted for 3 or more months; (3) abdominal pain that was moderate to severe and interfered with some or all regular activities; (4) abdominal pain that may or may not be accompanied by upper-gastrointestinal symptoms; and (5) children were accompanied by a parent or guardian who was capable of giving informed consent, and children over the age of 10 years were capable of giving informed assent. The community-based study used standardized questionnaires that were offered to 1080 children/parents from the 3 participating schools; 700 completed and returned the questionnaires (65% response rate). The questionnaire was designed to elicit data concerning the history of abdominal pain or discomfort. A total of 160 children met Apley's criteria and were classified as having RAP. Inclusion criteria were identical to those criteria for the clinic-based studies. Participating children in the 3 studies received a standardized questionnaire that asked about socioeconomic variables, abdominal pain (intensity; frequency; duration; nature of abdominal pain, if present, and possible relationships with school activities; and other upper gastrointestinal symptoms). We used 4 scales for the MM-RAP: pain intensity scale (3 items), nonpain symptoms scale (12 items), disability scale (3 items), and satisfaction scale (2 items). Age 7 was used as a cutoff point for the analysis as the 7-year-olds have been shown to exhibit more sophisticated knowledge of illness than younger children. RESULTS: A total of 295 children who were aged 4 to 18 years participated in the study: 155 children from the pediatric gastroenterology clinics, 82 from the primary care clinics, and 58 from the schools. The interitem consistency (Cronbach's coefficient alpha) for the pain intensity items, nonpain symptoms items, disability items, and satisfaction items were 0.75, 0.81, 0.80, and 0.78, respectively, demonstrating good reliability of the measure. The internal consistencies of the 4 scales did not significantly differ between younger (< or =7 years) and older (>7 years) children. There was also no significant variation in the coefficient alpha of each of the 4 scales in relation to gender or the level of the parent's education. Reliability was identical for the pain-intensity items (0.74) among children who sought medical attention from primary care or pediatric gastroenterology clinics. The intercorrelations of factor scores among the 4 scales showed a strong relationship among the factors but not high enough that correlations would be expected to be measuring the same items. The results of the factor analysis identified 5 components instead of 4 components representing the 4 scales. The 12 items of the nonpain symptoms scale were classified into 2 components; 1 component included heartburn, burping, passing gas, bloating, problem with ingestion of milk, bad breath, and sour taste (nonpain symptoms I), and the other included nausea/vomiting, diarrhea, and constipation (nonpain symptoms II). The program ordered the 5 components on the basis of the percentage of the total variance explained by each component and consequently by the strength of each components in the following order: nonpain symptoms I, pain intensity, pain disability, satisfaction, and nonpain symptoms II. Of the 20 items that composed the MM-RAP, 17 met the inclusion criteria of having a correlation of > or =0.40 on the primary factor analyses. The 3 items that assessed pain intensity met the inclusion criteria as well as the 2 items that assessed satisfaction. Two of the 3 items that assessed disability met the inclusion criteria; however, the missed school item did not. The sleep problem and the loss of appetite items in the nonpain items also did not meet the inclusion criteria in both components of the nonpain symptoms scale. However, the loss of appetite item met the inclusion criteria in the disability scale with a correlation of 0.6. The 2 items that did not meet the inclusion criteria (missed school days and sour taste) will be eliminated in the revised measure for RAP. CONCLUSION: The MM-RAP demonstrated good reliability evidence in population samples. Children who have RAP and are seen at pediatric gastroenterology or primary care pediatric clinics have similar responses, showing that the measure performed well across several populations. Age did not affect the reliability of responses. The MM-RAP included 4 dimensions, each with several items that may identify disease-specific dimensions. In addition, dividing the nonpain symptoms scale into 2 components instead of 1 component could assist in creating a disease-specific measure. The present study focused exclusively on developing the multidimensional measure for RAP in children that could assist physicians in evaluating the efficacy of RAP treatment independent of psychological evaluations. In addition, the measure was designed for use in clinical trials that evaluate the efficacy of RAP treatment and to allow comparison between intervention studies. In conclusion, we were able to identify 4 dimensions of RAP in children (pain intensity, nonpain symptoms, pain disability, and satisfaction with health). We demonstrated that these dimensions can be measured in a reliable manner that is applicable to children who experience RAP in various settings.
Abdominal Pain↗
Oral administration of rapamycin and cyclosporine differentially alter intestinal function in rabbits.
The immunosuppressive drugs rapamycin (Rap) and cyclosporine A (CsA) are used clinically to modify or abolish immune-mediated functions. This study examined the effect of orally administered regimens of Rap, CsA, and a combination of Rap/CsA on intestinal function in male New Zealand white rabbits. Animals received oral doses of CsA (15 mg/kg/body weight/day), low-dose (LD) and high-dose (HD) Rap (0.25 or 1 mg/kg/body wt/day, respectively), or Rap/CsA (0.25 and 5 mg/kg/body wt/day, or 0.5 and 5 mg/kg/body wt/day, respectively) for 20 days. We measured in vitro uptake of nutrients and permeability, and morphometric measurements in the jejunum and ileum were made. Animals receiving HD-Rap or HD-Rap/CsA had decreased food intake, body weight, and intestinal weight, when compared with LD-Rap, LD-Rap/CsA, CsA, or controls. The maximal transport rate (Vmax) for the active jejunal uptake of D-glucose was increased in HD-Rap and CsA, but not in the HD-Rap/CsA-treated animals. The jejunal Vmax of D-glucose in the LD-Rap- or -Rap/CsA-treated animals was no different from controls. In the HD-Rap- and HD-Rap/ CsA-treated animals, jejunal rates of uptake of stearic, linoleic, and linolenic acids were reduced when compared with controls. Jejunal and ileal permeability (as assessed by the passive uptake of L-glucose, tissue conductance, and mucosal-to-serosal flux of [3H]inulin) was increased in animals treated with HD-Rap or HD-Rap/CsA, when compared with CsA or controls. These parameters of permeability were no different at lower doses of Rap or Rap/CsA. The jejunal and ileal villous surface area was increased in CsA, but decreased in HD-Rap or HD-Rap/CsA animals. Thus, HD-Rap given alone or in combination with CsA reduced body weight gain, in part due to reduced food intake and malabsorption of lipids, which was due at least in part to reduced intestinal surface area. The relevance of these findings to patients undergoing chronic immunosuppressive drug therapy needs to be established.
Administration, Oral↗
Effect of nifekalant on acute electrical remodelling in rapid atrial pacing canine model.
BACKGROUND: Nifekalant may prevent atrial fibrillation (AF) and possibly be useful in treatment of atrial tachyarrhythmia in patients with severe heart failure. This study investigated the electophysiologic effect of nifekalant on the acute atrial remodeling in rapid atrial pacing (RAP) model of canine. METHODS: Twelve mongrel dogs subjected to rapid stimulation (400 beats/min) at left atrial appendage (LAA) for 24 hours, were randomized into the control group (rapid pacing only, n = 6) and the nifekalant group (intravenous nifekalant therapy immediately after RAP, n = 6). Atrial electrophysiological parameters were measured in right atrium, coronary sinus, LAA, posterior wall of left atrium (PWLA) and left superior pulmonary vein (LSPV), before and after the RAP. RESULTS: In the control group, the effective refractory periods (ERP) were shortened greatly at all sites, paced dogs had substantially shorter ERPs in the high right atrium, LAA, and LSPV, but fewer changes in the PWLA, the coefficient variation of ERP (COV ERP) was increased significantly. After rapid atrial stimulation, the inducibility of AF increased significantly [induction number: pre-RAP vs post-RAP, 1.00 +/- 0.89 vs 8.17 +/- 2.79, P < 0.01; duration of AF: pre-RAP vs post-RAP, (450.34 +/- 362.59) ms vs (9975.77 +/- 4376.99) ms, P < 0.01]. In the nifekalant group, although the ERPs were prolonged at all sites compared with those in pre-RAP state, only the value at LSPV differed significantly from that in pre-RAP state [pre-RAP vs post-RAP, (102.50 +/- 5.24) ms vs (132.51 +/- 5.20) ms, P < 0.01]; the COV ERP did not change statistically in this group. The inducibility of AF slightly increased but insignificantly after pacing [induction number: pre-RAP vs post-RAP, 0.83 +/- 0.75 vs 1.67 +/- 0.82, P = 0.19; duration of AF: pre-RAP vs post-RAP, (378.67 +/- 317.88) ms vs (1124.08 +/- 1109.77) ms, P = 0.06]. Conduction time values did not alter significantly in either of the two groups after RAP. CONCLUSIONS: In canine RAP model, nifekalant inhibited ERP shortening and ERP heterogeneity increasing, decreased AF induction. Nifekalant can reverse acute electrical remodeling effect in this model.
Animals↗
Role of receptor-associated 39/45 kD protein in active Heymann nephritis.
UNLABELLED: Active Heymann nephritis (HN) of rat, an autoimmune glomerular disease, is an experimental model of human membranous glomerulonephropathy (MGN), a common human glomerular disease. The putative autoantigen of HN is believed to be a large glycoprotein (gp330) present in kidneys of rat and human. Gp330 has sequence homology to low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and co-purifies with another 45 kD protein called receptor-associated protein (RAP) which binds to both gp330 and LRP/alpha 2-macroglobulin receptor. Since RAP co-purifies with gp330 and is thus present in the immunizing material used to induce active HN, the present study was undertaken to determine if gp330 or RAP individually could produce active HN and whether RAP had a role in the pathogenesis of active HN. Rats immunized with the traditional crude antigen (Fx1A) containing both gp330 and RAP developed typical HN. Although these rats developed low titers of autoantibody to RAP in their sera, they had no deposition of antibody to RAP in their kidneys. Rats immunized with gp330 also developed typical HN but had no autoantibody to RAP in their sera or kidneys. Rats immunized with RAP developed high titers of autoantibody to RAP in their sera but had no antibody in their kidneys and did not develop HN. Three rats were injected intravenously with polyclonal antibody to RAP and assessed seven days later for the development of passive HN. All three developed mild passive HN characterized by granular staining of polyclonal antibody along the capillary loops. CONCLUSIONS: (1) This is the first report to show that gp330 alone without the accompanying RAP can induce active HN; (2) RAP by itself does not induce active HN; (3) Autoantibodies to RAP do not appear to be involved in the pathogenesis of active HN induced with the traditional crude antigen, Fx1A; and (4) We confirm that polyclonal antibody to RAP can induce passive HN.
Animals↗
Production of synergistic but nonidentical mechanisms of immunosuppression by rapamycin and cyclosporine.
We report that the mechanism of rapamycin (RAP) inhibition is synergistic, but nonidentical, to the mechanism of CsA inhibition. Like CsA, RAP inhibits T cell proliferation following mitogen (PHA) and/or alloantigen (MLR) stimulation. RAP levels of 100, 33, 11, 3.6, 1.2, and less than 1 ng/ml reduced PHA stimulation by 81%, 84%, 81%, 83%, 62%, and 33%, respectively, without cytotoxicity. The RAP concentration required to achieve 50% proliferative inhibition of either mitogen (PHA) or MLR assays revealed an interindividual variability of 5 pg/ml RAP (2 individuals), 1 ng/ml (3 individuals), and 100 ng/ml (2 individuals). Unlike CsA, RAP proliferative inhibition was not restricted to the G0 phase of the cell cycle. Addition of 100, 10, or 1 ng/ml RAP at the onset (G0), or 24 hr following cultivation (G1) similarly inhibited DNA synthesis by 42%, 42%, and 41% compared with 44%, 48%, and 47%, respectively. PWM-stimulated B cell proliferation was primarily RAP-sensitive during the G0 phase of the cell cycle. RAP at 100, 10, and 1 ng/ml inhibited B cell proliferation 46%, 51%, and 50% when added during G0 but only 15%, 20%, and 20% when added during G1. Generation of a cyclosporine-sensitive cytoplasmic activation signal, activator of DNA replication (ADR), was reduced by RAP. RAP reduction did not correlate directly with T cell proliferative inhibition (as does CsA). RAP-induced proliferative inhibition of 40% and 80% resulted in ADR inhibition of 16% and 33%. Proliferative inhibition was synergistically increased when CsA and RAP were used in combination, whereas ADR inhibition was only additively enhanced. Mechanistic disparity between RAP and CsA may potentiate clinical immunosuppression when RAP and CsA are used together.
B-Lymphocytes↗
Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma.
Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.
Animals↗
Robotic computer-assisted pyeloplasty versus conventional laparoscopic pyeloplasty.
BACKGROUND AND PURPOSE: Laparoscopic pyeloplasty (LP) for the repair of ureteropelvic junction (UPJ) obstruction provides results similar to those of open pyeloplasty with less morbidity, but its use has been limited, as it requires advanced laparoscopic skills. Robotic computer-assisted pyeloplasty (RAP) has the potential to reduce the technical challenges of the reconstructive portion of the operation. We compare our RAP experience with our recent LP cases. PATIENTS AND METHODS: Fourteen patients underwent LP, and 31 underwent RAP. The demographics of the two groups were similar. Three patients in the RAP group had been treated previously for UPJ obstruction. All procedures were performed transperitoneally. For RAP, conventional laparoscopic dissection and exposure preceded robot-assisted reconstruction. A Double-J stent was placed cystoscopically in all patients. Patient demographics and operative, postoperative, and follow-up data were compared. Success was defined strictly as the unequivocal absence of both obstruction and postoperative pain. Also technical success was defined as no evidence of persistent high-grade obstruction, no loss of function, no symptomatic obstruction, and no necessity for further treatment. RESULTS: The diagnosis of UPJ obstruction was confirmed intraoperatively in all cases. No difference was found in operative and postoperative outcomes of the two procedures. Operative time, including cystoscopy, was 299 minutes in the LP group and 271 minutes in the RAP group. The median estimated blood loss was <100 mL in both groups. The median console time for RAP was 76 minutes (range 54-124 minutes) and consisted of preparation and completion of the anastomosis. The median robotic docking and undocking time was 16 minutes (range 5-30 minutes). The anastomotic times for LP were not recorded. There were no conversions to open surgery and no intraoperative complications. The mean length of stay (LOS) was 2 days in both groups. There were two postoperative complications in each group: In the LP group, one large retroperitoneal hematoma and one umbilical hernia; in the RAP group, one nonfebrile urinary-tract infection and one urine leak. The mean follow-up was 10 months (range 1-31 months) for LP and 6 months (range 1-21 months) for RAP. Strict success was seen in 64% of the LP patients and 66% of the RAP patients. There was one technical failure in the RAP group, resulting in a technical success rate of 100% for LP and 97% for RAP. Technical success was seen in two LP patients and five RAP patients with partial obstruction on early postoperative renography and three LP patients and four RAP patients with occasional postoperative pain. CONCLUSION: Robotic computer-assisted pyeloplasty provides short-term results similar to those of conventional laparoscopic pyeloplasty at our institution.
Adolescent↗
Evaluation of mean right atrial pressure by two-dimensional and Doppler echocardiography in patients with cardiac disease.
BACKGROUND: Several approaches have been used for noninvasive estimation of right atrial pressure (RAP), but, no currently available method has gained any definite validation. The purpose of this study was to evaluate the accuracy of two-dimensional and Doppler echocardiography in estimating mean RAP in patients with cardiac disease. METHODS: We examined the relation of mean RAP to right atrial size and function, size and respiratory changes of inferior vena cava and Doppler parameters of tricuspid and hepatic vein flow in 114 consecutive patients (77 men, 37 women; mean age 57 +/- 12 years) with various cardiac diseases undergoing cardiac catheterization. Echocardiographic studies were performed within 24 hours before catheterization (mean interval 6 +/- 3 hours). Patients were assigned to 3 groups according to the values of mean RAP (group 1, < or = 8 mmHg; group 2, between 9 and 12 mmHg; group 3, > 12 mmHg). RESULTS: Mean RAP ranged from 3 to 20 mmHg (mean 9.1 +/- 4.3 mmHg). It correlated most strongly with the collapsibility index of inferior vena cava (IVCCI) (r = -0.76), minimal inspiratory diameter of inferior vena cava (r = 0.72) and deceleration time of early tricuspid flow (DT) (r = -0.61). Discriminant analysis demonstrated that IVCCI and DT were major determinants of mean RAP with 81.6% of cases correctly assigned to study groups: 96% of patients of group 1 and 87% of patients of group 3 were identified, whereas the accuracy in identifying the patients of group 2 was lower (46%). An IVCCI > 45% was the best cutoff point in predicting a mean RAP < or = 8 mmHg; an IVCCI < 35% and a DT < 150 msec were the best cutoff points in predicting a mean RAP > or = 15 mmHg. The best multivariate equation predicting mean RAP was: mean RAP = 23.3 - 0.2 IVCCI -0.026 DT (r = 0.80, R2 = 0.64). This equation was 81% sensitive and 84% specific in detecting a mean RAP < or = 8 mmHg and 74% sensitive and 97% specific in detecting a mean RAP > 12 mmHg. CONCLUSIONS: Mean RAP can be estimated noninvasively by two-dimensional and Doppler echocardiography. The combined analysis of IVCCI and DT provides an accurate prediction on mean RAP < or = 8 mmHg and > 12 mmHg, whereas the prediction of intermediate values is less accurate.
Adult↗
Rapamycin inhibits IL-1-mediated interferon-gamma production in the YAC-1 T cell lymphoma.
Rapamycin (RAP) exerts potent immunosuppressive effects that are thought to reflect primarily an inhibition of T cell proliferation driven by growth-promoting cytokines. In the present study, we examined whether RAP would be able to alter other T cell responses mediated by cytokines, such as lymphokine production. As a model system, we used the murine T cell lymphoma, YAC-1, that can be induced to produce IFN-gamma when activated with either ionomycin + PMA or IL-1 alpha + PMA. As previously found in other systems, induction of this lymphokine by ionomycin + PMA was not inhibited by RAP, although it was highly sensitive to inhibition by FK-506, an immunosuppressive macrolide structurally related to RAP. In contrast, the induction of the same lymphokine by IL-1 alpha + PMA was highly sensitive to RAP but resistant to FK-506. This inhibition of IFN-gamma production by RAP was detectable at the mRNA level. Such an inhibition was specific as it did not occur in a mutant clone selected for resistance to the antiproliferative effects of RAP. Moreover, kinetics analysis revealed that RAP still inhibited IFN-gamma mRNA accumulation in YAC-1 cells, when added as late as 12-18 h after initial stimulation of the cells with IL-1 alpha + PMA. While the inhibitory action of RAP could not be removed by extensive washing of the cells, it was readily reversed by a hundred-fold excess of FK-506 added to the cultures simultaneously with RAP or up to 16-18 h later. Altogether, these data indicate that the action of RAP in T cells is not limited to the inhibition of proliferation but also affects the regulation of lymphokine gene expression mediated by IL-1 alpha. The inhibition of lymphokine gene expression by RAP takes place at a late stage of the inductive response, and through a mechanism that involves interaction with the same cellular binding proteins as FK-506. Such an inhibitory effect of RAP may contribute significantly to its ability to suppress immune responses.
Animals↗
Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates.
Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.
Binding, Competitive↗
Responses of regional kidney perfusion to vasoconstrictors in anaesthetized rabbits: dependence on agent and renal artery pressure.
1. We tested the effects of intravenous infusions of angiotensin II (AngII; 300 ng/kg per min) and the vasopressin V1 receptor agonist [Phe2,Ile3,Orn8]-vasopressin (30 ng/kg per min) on regional kidney perfusion in an extracorporeal circuit model in anaesthetized rabbits in which renal artery pressure (RAP) can be set independently of systemic mean arterial pressure. To test whether the level of RAP can influence the renal vascular response to [Phe2,Ile3,Orn8]-vasopressin, we compared its effects when RAP was initially set at approximately 65 mmHg with those when RAP was set at approximately 130 mmHg. 2. When RAP was initially set at approximately 65 mmHg, a 20min infusion of AngII increased RAP (13%) and reduced renal blood flow (RBF; 50%) and cortical perfusion (CBF; 43%). Medullary perfusion (MBF) transiently increased during the first 10 min of infusion, but was not significantly different from control levels during the final 5 min of infusion. 3. When RAP was initially set at approximately 65 mmHg, a 20 min infusion of [Phe2,Ile3,Orn8]-vasopressin increased RAP (9%) and reduced RBF (21%); MBF was reduced by 57%, but CBF was reduced by only 15%. In contrast, when RAP was initially set at approximately 130 mmHg, infusion of [Phe2,Ile3,Orn8]-vasopressin reduced RAP (7%) and increased RBF (13%). In these experiments, MBF was reduced by 38%, but CBF increased by 6%. 4. Our experiments show that AngII preferentially reduces CBF, while [Phe2,Ile3,Orn8]-vasopressin preferentially reduces MBF. The renal vascular responses to [Phe2,Ile3,Orn8]-vasopressin appear to be profoundly affected by the level of RAP, because increasing RAP from approximately 65 to approximately 130 mmHg transforms its cortical vasoconstrictor effect into cortical vasodilatation while leaving the response of the medullary microvasculature relatively unchanged. Whether renal vascular responses to other vasoactive agents (e.g. AngII) are similarly affected by the level of RAP remains to be determined.
Anesthesia↗
Inhibitory effect on the lipolysis-stimulated receptor of the 39-kDa receptor-associated protein.
Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.
Animals↗
Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin.
Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.
ADP Ribose Transferases↗
Differential functions of triplicated repeats suggest two independent roles for the receptor-associated protein as a molecular chaperone.
The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that binds multiple ligands. The primary function of RAP has been defined as promotion of the correct folding of LRP, and prevention of premature interaction of ligands with LRP within the early secretory pathway. Previous examination of the RAP sequence revealed an internal triplication. However, the functional implication of the triplicated repeats was unknown. In the current study using various RAP and LRP domain constructs, we found that the carboxyl-terminal repeat of RAP possesses high affinities to each of the three ligand-binding domains on LRP, whereas the amino-terminal and central repeats of RAP exhibit only low affinity to the second and the fourth ligand-binding domains of LRP, respectively. Using truncated soluble minireceptors of LRP, we identified five independent RAP-binding sites, two on each of the second and fourth, and one on the third ligand-binding domain of LRP. By coexpressing soluble LRP minireceptors and RAP repeat constructs, we found that only the carboxyl-terminal repeat of RAP was able to promote the folding and subsequent secretion of the soluble LRP minireceptors. However, when the ability of each RAP repeat to inhibit ligand interactions with LRP was examined, differential effects were observed for individual LRP ligands. Most striking, both the amino-terminal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of alpha2-macroglobulin with LRP. These differential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.
Amino Acid Sequence↗
Inhalation and dermal exposure among asphalt paving workers.
The primary objective of this study was to identify determinants of inhalation and dermal exposure to polycyclic aromatic compounds (PACs) among asphalt paving workers. The study population included three groups of highway construction workers: 20 asphalt paving workers, as well as 12 millers and 6 roadside construction workers who did not work with hot-mix asphalt. During multiple consecutive work shifts, personal air samples were collected from each worker's breathing zone using a Teflon filter and cassette holder connected in series with an XAD-2 sorbent tube, while dermal patch samples were collected from the underside of each worker's wrist. All exposure samples were analyzed for PACs, pyrene and benzo[a]pyrene. Inhalation and dermal PAC exposures were highest among asphalt paving workers. Among paving workers, inhalation and dermal PAC exposures varied significantly by task, crew, recycled asphalt product (RAP) and work rate (inhalation only). Asphalt mix containing high RAP was associated with a 5-fold increase in inhalation PAC exposures and a 2-fold increase in dermal PAC exposure, compared with low RAP mix. The inhalation PAC exposures were consistent with the workers' proximity to the primary source of asphalt fume (paver operators > screedmen > rakers > roller operators), such that the adjusted mean exposures among paver operators (5.0 microg/m3, low RAP; 24 microg/m3, high RAP) were 12 times higher than among roller operators (0.4 microg/m3, low RAP; 2.0 microg/m3, high RAP). The dermal PAC exposures were consistent with the degree to which the workers have actual contact with asphalt-contaminated surfaces (rakers > screedmen > paver operators > roller operators), such that the adjusted mean exposures among rakers (175 ng/cm2, low RAP; 417 ng/cm2, high RAP) were approximately 6 times higher than among roller operators (27 ng/cm2, low RAP; 65 ng/cm2, high RAP). Paving task, RAP content and crew were also found to be significant determinants of inhalation and dermal exposure to pyrene. The effect of RAP content, as well as the fact that exposures were higher among paving workers than among millers and roadside construction workers, suggests that the PAC and pyrene exposures experienced by these paving workers were asphalt-related.
Construction Materials↗
CD4+ T-helper lymphocyte responses against Babesia bigemina rhoptry-associated protein I.
A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.
Animals↗