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PubMed1996

Identification of differentially expressed mRNA species during HIV infection by RNA arbitrarily primed PCR.

BACKGROUND: A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression. OBJECTIVES: To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR. STUDY DESIGN: RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets. RESULTS: Three genes, gamma-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of gamma-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that gamma-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation. CONCLUSIONS: These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.

Actins↗

PubMed1997

A process-oriented reasoner about physiology.

This paper presents the RAP system: a reasoner about physiology. RAP performs two tasks: (1) it infers the behaviour of a complex physiological process using the behaviours of its subprocesses and the relationships between them; (2) it reasons about the effect of introducing a fault into the model. In order to reason about the behaviour of a complex process, RAP uses a mechanism which: (i) represents how subprocesses behave; (ii) establishes how these subprocesses affect each others behaviors; (iii) 'aggregates' these behaviors together to obtain the behavior of the top level process; (iv) gives that process a temporal context in which to act. RAP uses limited common sense knowledge about faults to reason about their effect in terms of the generation of new processes and the misbehavior of existing ones. The effects are then propagated throughout the model to obtain the overall effect of the fault.

Artificial Intelligence↗

PubMed1995

Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins. The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding. Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M*. The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

NMR structural studies of domain 1 of receptor-associated protein.

The 39 kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds tightly to the low-density lipoprotein receptor-related protein (LRP) as well as to other members of the low-density lipoprotein receptor superfamily. The association of RAP with LRP prevents this receptor from interacting with ligands. RAP is a three-domain protein that contains two independent LRP binding sites; one located within domains 1 and 2, and one located within domain 3. As the first step toward defining the structure of the full-length protein and understanding the interaction between RAP and this family of receptors, we have determined the 3D structure of domain 1 using constraints derived from heteronuclear multi-dimensional NMR spectra, including NOEs, dihedral angles, J-couplings and chemical shifts, as well as two sets of non-correlated residual dipolar couplings measured from the protein solutions in anisotropic media of Pf1 and 6% polyacrylamide gel. The backbone C(alpha) rmsd between the current structure and a homo-nuclear NOE-based structure is about 2 A. The large rmsd mainly reflects the significant differences in helical orientation and in the structural details of the long helix (helix 2) between the two structures.

Acrylic Resins↗

PubMed2001

Immunolocalisation of cartilage-derived retinoic acid-sensitive protein in pleomorphic adenoma of the parotid salivary gland.

Pleomorphic adenoma of the parotid salivary glands often contains chondroid elements and may exhibit cartilaginous and osseous differentiation, although the latter is extremely rare. Twenty-nine pleomorphic adenomas (PAs) of the parotid gland were examined immunohistochemically for the distribution of cartilage-derived retinoic acid-sensitive protein (CD-RAP), a recently described marker of chondrocytes, which may be important in the morphogenesis and development of the salivary gland. In the normal parotid gland, the ductal cells expressed CD-RAP, but not the myoepithelial cells. In the pleomorphic salivary adenomas, the duct-like cells, but not the myoepithelial cells, expressed CD-RAP. Since many authorities consider myoepithelial cells to be the source of the chondroid matrix, it is surprising that these cells do not express the chondrocytic marker, CD-RAP. Putative neoplastic myoepithelium in the pleomorphic adenoma and some cells in the myxochondroid areas expressed S-100 and calponin.

Adenoma, Pleomorphic↗

PubMed1993

Psychosocial correlates of recurrent childhood pain: a comparison of pediatric patients with recurrent abdominal pain, organic illness, and psychiatric disorders.

Pediatric patients with recurrent abdominal pain (RAP) were compared with patients with peptic disease, patients with emotional disorders, and well children with regard to (a) emotional and somatic symptoms and (b) theoretically derived variables, including negative life events, competence, family functioning, and the modeling and encouragement of illness behavior. RAP patients had levels of emotional distress and somatic complaints higher than those of well children and lower than those of psychiatric patients, but not different from those of patients with peptic disease. RAP patients had fewer negative life events, better family functioning, and higher competence than children with emotional disorders. In comparison with well children and psychiatric patients, both RAP and peptic disease patients had a higher incidence of illness in other family members and perceived greater parental encouragement of illness behavior for abdominal symptoms.

Abdominal Pain↗

PubMed2004

Immobility--a major risk factor for development of pressure ulcers among adult hospitalized patients: a prospective study.

OBJECTIVE: To identify risk factors associated with pressure ulcer development among adult hospitalized medical and surgical patients. DESIGN: A prospective comparative study including 530 adult patients from medical and surgical wards. Registered Nurses made the data collection on admission and once a week for up to 12 weeks. The risk assessment scale used was the Risk Assessment Pressure Sore (RAPS) scale, including the following variables; general physical condition, activity, mobility, moisture, food intake, fluid intake, sensory perception, friction and shear, body temperature and serum albumin. RESULTS: Sixty-two (11.7%) patients developed 85 pressure ulcers. The most common pressure ulcer was that of nonblanchable erythema. Patients who developed pressure ulcers were significantly older, hospitalized for a longer time, had lower scores on the total RAPS scale, had lower weight and lower diastolic blood pressure than nonpressure ulcer patients did. In the multiple logistic regression analyses using variables included in the RAPS scale immobility emerged as a strong risk factor. When adding remaining significant variables in the analyses, mobility, time of hospitalization, age, surgical treatment and weight were found to be risk factors for pressure ulcer development. CONCLUSION: It is confirmed that immobility is a risk factor of major importance for pressure ulcer development among adult hospitalized patients. The results also indicate that the RAPS scale may be useful for prediction of pressure ulcer development in clinical practice.

Activities of Daily Living↗

PubMed2001

Nitric oxide and the role of blood pressure variability to the kidney.

Blood pressure variability is buffered by at least two mechanisms: the arterial baroreceptor reflex and nitric oxide (NO). Only recently is the importance of blood pressure variations on cardiovascular control being investigated. Here we report of a study performed in conscious dogs, in which renovascular hypertension was induced. Reduction of renal arterial pressure (RAP) to 85 mmHg for 24 h elicited profound hypertension by 60 mmHg (vs. control: 110 +/- 3 mmHg; P < 0.01). This was accompanied by reduced volume and sodium excretion (-48% of control, P < 0.01 and -80% of control, P < 0.01, respectively) and augmented renin release by more than two-fold (P < 0.01). This intervention was compared with a protocol in which RAP was reduced to the same mean value, however, RAP oscillated by +/-10 mmHg at 0.1 Hz. This manoeuvre led to a transient increase in NO3 excretion in urine (P < 0.01), blunted antidiuresis (-14% of control) as well as antinatriuresis (-40% of control) and attenuated the increased renin release by 30% (P < 0.05). In consequence, the magnitude of blood pressure increase was only half as high as that observed during static reduction of RAP (P < 0.01). It is concluded that blood pressure oscillations to the kidney have a profound influence on water and electrolyte balance and on renin release, which alleviates the onset of Goldblatt hypertension.

Animals↗

PubMed2000

T cell lines specific for a synthetic Heymann nephritis peptide derived from the receptor-associated protein.

Pathogenic antigens involved in the induction of Heymann nephritis (HN), an experimental rat model of human membranous nephritis, have been identified in megalin (gp330) and the receptor-associated protein (RAP) [1,2]. A pathogenic epitope has been identified in RAP (amino acid 1-86) that plays a significant role in the formation of immune deposits in glomeruli in HN. A synthetic peptide (P31-53) derived from RAP1-86 contains a pathogenic epitope recognized by antibodies eluted from glomerular immune deposits and includes two putative RT-1B1 MHC class II-binding motifs. We have investigated whether RAP P31-53 can be recognized by T cells. Five peptide-specific T cell lines were generated from regional lymph node (LN) T cells from Lewis rats immunized with P31-53. The T cell lines were characterized by using a T cell proliferation assay for their specificity, FACS and MHC restriction assay for the phenotype, reverse transcription-polymerase chain reaction for TCR Vbeta repertoire and cytokine expression, and cloning and sequencing for the analysis of the CDR3 sequence of TCR. The helper function of the T cell line was confirmed by autoantibody production in vitro. In this study, we clearly identify that the synthetic pathogenic peptide P31-53 contains a T cell epitope recognized by CD4+ Th2 cells in Lewis rats. This recognition was restricted by MHC class II RT1.B1. These CD4+ Th2 cells were able to promote B cells to produce specific antibodies and used a restricted set of TCR Vbeta genes with preferential usage of Vbeta18. A charged amino acid motif at the CDR3 region of predominant TCR Vbeta subfamilies may contribute to the specific ability of these cells to recognize the immunogenic T cell epitope within RAP peptide P31-53.

Amino Acid Sequence↗

PubMed1999

Evaluation of cerebral perfusion pressure changes in laboring women: effects of epidural anesthesia.

OBJECTIVE: To compare the effect of epidural anesthesia on cerebral perfusion pressure in laboring women. STUDY DESIGN: Maternal cerebral blood flow velocity was assessed in seven laboring patients with continuous epidural anesthesia and 15 without, using transcranial Doppler. Maternal cerebral blood flow velocity was assessed during the first stage at the trough of a contraction, at the peak of a contraction and at the second stage during pushing over the course of four contractions. Calculated estimated cerebral perfusion pressure: eCPP = Vmean/(Vmean - Vdiastolic) x (mean BP - diastolic BP), where V is velocity and BP is blood pressure; modified from Aaslid and colleagues. An index of cerebrovascular resistance, the resistance area product, was calculated: RAP = mean BP/mean velocity. We calculated an index of cerebral blood flow (cerebral blood flow index): CBF index = eCPP/RAP. RESULTS: In non-epidural patients, the eCPP fell significantly at the peak of a contraction and during pushing. Cerebrovascular resistance, RAP, rose significantly during the peak of a contraction, although cerebral blood flow did not change. In patients undergoing epidural anesthesia, the stages of labor had no significant effect on eCPP or RAP; however, these values were lower than those in patients without epidural anesthesia. CONCLUSIONS: The epidural group had a lower eCPP and RAP and cerebral blood flow index compared to the non-epidural group. In the non-epidural group, the mean arterial pressure was higher in all stages of labor with a trend towards an increase in eCPP and cerebral blood flow index.

Adult↗

PubMed1995

Estimation of critical closing pressure in the cerebral circulation of newborns.

We have developed a method to estimate the critical closing pressure (CrCP) of the cerebral circulation based on the intrinsic variability of arterial blood pressure (BP) around stable values of mean arterial pressure (MAP). A consecutive cohort of 33 premature newborns was studied at 6, 12, 24, 48 and 72 hours of age. Cerebral blood flow velocity (CBFV) was measured with Doppler ultrasound in the middle cerebral artery and BP was recorded in the abdominal aorta or in a peripheral artery. Continuous measurements lasting five minutes were recorded on digital magnetic tape and signals were digitized at a rate of 200 samples/seconds for processing on a digital computer. Mean values of BP (mBP) and CBFV (mBV) were computed for each cardiac cycle and CrCP was determined as the pressure axis intercept of the regression line of mBV as a function of mBP using 100 sequential heart beats. The resistance-area product (RAP) was obtained from the slope of the regression line. For 57 records (30 patients) the mean +/- SD values of CrCP and RAP were 23.9 +/- 11.6 mmHg and 4.07 +/- 1.83 x 10(4) kg.m-2.s-1, respectively. CrCP has a highly significant correlation with mean arterial pressure (p < 0.001) but RAP has not. Neither CrCP nor RAP are significantly correlated (p > 0.05) with PO2, PCO2, pH, haematocrit, gestational age, birthweight, postnatal age, heart rate on Pourcelot's resistance index. Our results suggest that cerebral perfusion pressure should be defined as MAP-CrCP for normal values of intracranial pressure.

Blood Pressure↗

PubMed1984

Angina pectoris and blood lactate concentration during graded exercise.

Nineteen patients with angina pectoris referred for preoperative evaluation were studied by psychophysical scaling of the chest pain according to Borg and blood lactate determination at rest and during a graded exercise test. The exercise tests consisted of stepwise increments of 10 W every min. Anginal pain, systolic blood pressure, heart rate (HR), and blood lactate concentration were determined at the end of each work load. The exercise test was interrupted at a rated anginal pain level (RAP) of 5-6/9 ("strong"), and this load was referred to as Wmax. The patients were divided into three groups: A with a Wmax less than or equal to 74 W, B 75-124 W, and C greater than or equal to 125 W. At submaximal loads HR was almost identical at the same absolute work load in all three groups. The heart rate at Wmax varied, however, and averaged 98 (A), 118 (B), and 136 (C), respectively. Corresponding peak values for RAP were 3.8, 4.5, and 4.8. Blood lactate at rest ranged from 1.0-1.6 mmol X 1-1 and increased curvilinearly with intensity, which was also the case for RAP. RAP was almost linearly related to blood lactate in all three groups suggesting a causal relationship between these two parameters.

Aged↗

PubMed2005

CTLA-4 up-regulation of lymphocyte function-associated antigen 1 adhesion and clustering as an alternate basis for coreceptor function.

Although cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T cell activation, the full range of functions mediated by this coreceptor has yet to be established. In this study, we report the surprising finding that CTLA-4 engagement by soluble antibody or CD80 potently up-regulates lymphocyte function-associated antigen 1 (LFA-1) adhesion to intercellular adhesion molecule-1 (ICAM-1) and receptor clustering concurrent with IL-2 inhibition. This effect was also observed with CTLA-4 ligation and not with other coreceptors. T cell antigen receptor (TcR)-induced lymphocyte function-associated antigen 1 function was also dependent on CTLA-4 expression as observed with reduced adhesion/clustering on CTLA-4(-/-) primary T cells. CTLA-4 up-regulated adhesion was mediated by regulator for cell adhesion and polarization type 1 (Rap-1) as shown by anti-CTLA-4-induced Rap-1 activation as well as Rap-1-N17 blockade and Rap-1-V12 mimicry of adhesion/clustering. Our findings identify a potent role for CTLA-4 in directing integrin adhesion and provide an alternate mechanism to account for aspects of CTLA-4 function in T cell immunity.

Animals↗

PubMed1984

Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas.

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions.

Adenocarcinoma↗

PubMed1991

The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex.

We found that transcription factor IIF mediates the association of RNA polymerase II with promoter sequences containing transcription factors IID, IIB, and IIA (DAB complex). The resulting DNA-protein complex contained RNA polymerase II and the two subunits of transcription factor IIF (RAP 30 and RAP 74). Cloned human RAP 30 was sufficient for the recruitment of RNA polymerase II to the DAB complex. This ability of RAP 30 to recruit RNA polymerase to a promoter is also a characteristic of sigma factors in prokaryotes.

Adenoviridae↗

PubMed1993

RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1.

RNA fingerprinting using arbitrarily primed PCR (RAP) samples an RNA population and allows the detection of differentially expressed genes between two or more populations. This method was applied to mink lung epithelial cells, which respond to treatment with transforming growth factor beta (TGF-beta) by undergoing cell cycle arrest at or near the G1/S-phase boundary. The steady-state abundances of approximately 200 RNAs were surveyed, a few of which displayed differential regulation in response to TGF-beta 1. Three products were isolated, cloned, and sequenced. One differentially regulated RNA corresponded to cyclin A, a gene known to be required for the progression of mammalian fibroblasts through S phase. Northern blot analysis confirmed that the cyclin A mRNA steady-state abundance decreased dramatically in response to a 24-hr TGF-beta 1 treatment and also in response to cell cycle arrest caused by contact inhibition. A second RAP product corresponded to a previously unknown 7.5-kb mRNA, the level of which decreased dramatically in response to TGF-beta 1 treatment. Unlike the cyclin A mRNA, the abundance of this transcript did not decrease in response to growth arrest induced by contact inhibition. A third RAP product corresponded to the mRNA for osteonectin, an extracellular matrix protein. The abundance of this mRNA increased at least 2-fold during TGF-beta 1 treatment. This observation is consistent with other reports of increases in extracellular matrix proteins during TGF-beta treatment. RAP should be able to identify many of the genes that change in steady-state expression during the cell cycle.

Animals↗

PubMed1994

Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionally expressed genes in prokaryotes. Differential gene expression in Salmonella typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP fingerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentially amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyR1 mutant that constitutively expressed a subset of hydrogen peroxide-inducible genes. Using pulsed-field gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionally expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.

Base Sequence↗

PubMed1995

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

Analysis of Variance↗