Biomedical subjects

P Ye

Publications and source records attributed to P Ye.

At least 37 records · Page 2Linked to original sources

PubMed2001

Carotid intima-media thickness and the association with cardiovascular risk factors in the elderly.

OBJECTIVE: To investigate the association of carotid arterial intima-media thickness (IMT) with principal cardiovascular risk factors in the elderly. METHODS: Carotid arterial IMT was measured by high resolution B mode ultrasound in 94 elderly subjects (old-aged group), and compared with subjects aged < 60 (middle-aged group). RESULTS: In comparison with the middle-aged group, the prevalence of coronary heart disease, cerebral vascular disease, hypertension and diabetes mellitus was significantly higher, and serum cholesterol and systolic blood pressure were also significantly higher in old-aged group. Although there was no obvious difference in IMT between the two groups, carotid plaque and carotid wall thickening were more frequently found in old-aged group. Age, systolic blood pressure and serum cholesterol were shown as the independent determinants for carotid IMT in the total participants, whereas no such independent relation was found in old-aged group. CONCLUSION: Age is the major risk factor for carotid atherosclerosis in the elderly. In other words, the occurrence of carotid atherosclerosis is the result of advancing age combined with the effect of multiple cardiovascular risk factors.

Aged↗

PubMed2000

Insulin-like growth factor-I (IGF-I) protects myelination from undernutritional insult: studies of transgenic mice overexpressing IGF-I in brain.

Using insulin-like growth factor-I (IGF-I)-overexpressing transgenic (Tg) mice as a model, we have shown that IGF-I promotes myelination by increasing the number of oligodendrocytes and stimulating the expression of myelin-specific protein genes. In the present study, we investigated whether IGF-I protects myelination from undernutritional insult in Tg mice. Mice were undernourished beginning from postnatal (P) day 1, a time coincident with the onset of transgene expression, and sacrificed at P20. Consistently with our previous studies, brain weights of undernourished non-Tg control mice were decreased by approximately 18%. Brain weights of undernourished IGF-I Tg mice, however, were the same as those of well-fed control mice and much greater than those of undernourished control mice. The expression of two major myelin proteins [myelin basic protein (MBP) and proteolipid protein (PLP)] in cerebral cortex (CTX) and hippocampus (HIP) was decreased by 73-92% in undernourished control mice, as judged by Northern and Western blot hybridization. The abundances of MBP and PLP mRNAs and proteins, however, were decreased by only 40-70% in undernourished IGF-I Tg mice. To assess the number of oligodendrocytes and their precursors, antibodies specific for carbonic anhydrase II (CAII; an oligodendrocyte marker) and NG2 (a precursor marker) were used. Compared to their well-fed counterparts, undernourished control mice exhibited 17-30% decreases in the number of oligodendrocytes and their precursors in CTX and corpus callosum (CC), whereas well-fed IGF-I Tg mice had 21-35% increases in CTX and CC. Undernourished IGF-I Tg mice exhibited cell numbers similar to those of well-fed control mice. These data indicate that IGF-I protects myelination from undernutrition damage during development.

Animals↗

PubMed2000

Insulin-like growth factor-I promotes neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development.

The in vivo actions of insulin-like growth factor-I (IGF-I) on the growth and development of the hippocampal dentate gyrus were investigated in transgenic mice that overexpress IGF-I postnatally in the brain and in normal nontransgenic littermate controls. Stereological analyses of the dentate gyrus were performed by light and electron microscopy on days 7, 14, 21, 28, 35, and 130 to determine postnatal changes in the numerical density and total number of neurons and synapses. The volumes of both the granule cell layer and the molecular layer of the dentate gyrus were significantly increased by 27-69% in transgenic mice after day 7, with the greatest relative increases occurring by day 35. Although the numerical density of neurons in the granule cell layer did not differ significantly between transgenic and control mice at any age studied, the total number of neurons was significantly greater in transgenic mice by 29-61% beginning on day 14. The total number of synapses in the molecular layer was significantly increased by 42-105% in transgenic mice from day 14 to day 130. A transient increase in the synapse-to-neuron ratio was found in transgenic mice at postnatal days 28 and 35 but not at day 130. This finding indicates a disproportionate increase in synaptogenesis, exceeding that expected for the observed increase in neuron number. Our results demonstrate that IGF-I overexpression produces persistent increases in the total number of neurons and synapses in the dentate gyrus, indicating that IGF-I promotes both neurogenesis and synaptogenesis in the developing hippocampus in vivo.

Analysis of Variance↗

PubMed2000

Insulin-like growth factor-1 inhibits mature oligodendrocyte apoptosis during primary demyelination.

Metabolic insult results in apoptosis and depletion of mature oligodendrocytes during demyelination. To examine the role of insulin-like growth factor-1 (IGF-1) during acute demyelination and remyelination in the adult CNS, we exposed transgenic mice that continuously express IGF-1 (IGF-1 tg) to cuprizone intoxication. Demyelination was observed within the corpus callosum in both wild-type and IGF-1 tg mice 3 weeks after exposure to cuprizone. Wild-type mice showed significant apoptotic mature oligodendrocytes and a dramatic loss of these cells within the lesion that resulted in near complete depletion and demyelination by week 5. In contrast, the demyelinated corpus callosum of the IGF-1 tg mice was near full recovery by week 5. This rapid recovery was apparently caused by survival of the mature oligodendrocyte population because apoptosis was negligible, and by week 4, the mature oligodendrocyte population was completely restored. Furthermore, despite demyelination in both wild-type and IGF-1 tg mice, oligodendrocyte progenitors accumulated only in the absence of mature oligodendrocytes and failed to accumulate if the mature oligodendrocytes remained as demonstrated in the IGF-1 tg mice. These results suggest that IGF-1 may be important in preventing the depletion of mature oligodendrocytes in vivo and thus facilitates an early recovery from demyelination.

Acute Disease↗

PubMed2000

Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis.

IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.

3T3 Cells↗

PubMed2000

Expression patterns of E-cadherin, involucrin, and connexin gap junction proteins in the lining epithelia of inflamed gingiva.

The structural integrity and functional differentiation of the lining epithelium were studied in relation to inflammatory changes associated with destructive periodontitis. In the different regions of lining epithelia from clinically healthy gingiva and periodontitis, comparisons were made of the expression patterns of E-cadherin, which is critical in intercellular adhesion; of proteins associated with gap junction communication channels; and of involucrin, which is a key marker of differentiation in stratified epithelia. Filamentous actin (F-actin), which is important in cell structural integrity, attachment, and migration, was also examined. Semiquantitative immunohistochemical analysis revealed that in both clinically healthy gingiva and lesions of advanced periodontitis, expression patterns of E-cadherin, involucrin, and connexins 26 and 43 were similar, with a statistically significant reduction in staining intensity from the external oral epithelium, through the gingival sulcus, to the junctional epithelium or pocket epithelium, respectively. Furthermore, there was a striking reduction in staining for E-cadherin, involucrin, and both connexins in the pathological lining epithelium of the periodontal pocket. These changes were associated with marked alterations of filamentous actin expression, collectively indicating profound perturbation of the epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised.

Actins↗

PubMed2000

Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.

Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.

ADAM Proteins↗

PubMed2000

Interferon-gamma protects against cuprizone-induced demyelination.

Evidence suggests that interferon-gamma (IFN-gamma), a proinflammatory cytokine secreted by activated T lymphocytes, contributes a deleterious effect to immune-mediated demyelinating disorders such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Nevertheless, mouse strains that are normally resistant to EAE induction become susceptible when the gene encoding either IFN-gamma or its receptor is mutated, demonstrating that the role that this cytokine plays in demyelinating disorders is complex. We have examined the effect of IFN-gamma in a chemically induced model of CNS demyelination. Mice that receive through their diet the copper chelator cuprizone display extensive demyelination of the corpus callosum. Remarkably, transgenic mice that ectopically express low levels of IFN-gamma in the CNS did not display evidence of demyelination when treated with cuprizone, nor did they shows signs of oligodendroglial death, astrogliosis, or microgliosis, which are typically seen in treated animals. Myelin protein gene expression was, however, dramatically reduced in both the treated control and the transgenic animals, indicating that demyelination is not an obligatory consequence of a large diminution of myelin protein synthesis. Interestingly, the CNS of the IFN-gamma-expressing mice contained elevated levels of insulin-like growth factor I, which has been demonstrated to have a protective effect against the demyelinating action of cuprizone.

Animals↗

PubMed2000

Inhibitory effect of fluvastatin on aortic intimal thickening in normocholesterolemic rabbits.

OBJECTIVE: The anti-atherosclerotic effect of fluvastatin at does insufficient to lower serum cholesterol on the catheter-induced intimal thickering and possible mechanism were investigated in abdominal aorta of rabbits. METHODS: Fifty-six rabbits were randomly divided into eight groups (n = 7, each). Fluvastatin was given mixed with food at daily dose of 8 mg/kg starting 5 days before catheterization. Light microscope, immunohistochemistry, transmission electron microscope and RT-PCR assay were applied to assess vascular smooth muscle cell (VSMC) proliferation and apoptosis, as well as oncogene expression in vascular wall. RESULTS: At day 10 and day 15 after catheter induced denudation intima/media (I/M) thickness ratio was obviously higher, and also the percentage of PCNA-positive cells and TUNEL-positive cells in media was significantly higher compared with controls. The intimal hyperplasia was mostly composed of alpha-SM-actin-positive cells. In rabbits given fluvastatin I/M ratio and the percentage of these positive cells significantly decreased compared with those without fluvastatin. The overexpression of proto-oncogene H-ras mRNA and decreased expression of anti-oncogene p53 mRNA were found after vascular injury, whereas fluvastatin significantly reduced H-ras mRNA and increased p53 mRNA expression. CONCLUSION: Proliferation of VSMC in the media and the migration to the intima can be inhibited, and apoptosis of VSMC be induced by short-term use of fluvastatin after balloon catheter denudation, independent of serum lipid change. The underlying mechanism is presumably associated with the influence of fluvastatin on oncogene expression in the injured vascular wall.

Animals↗

PubMed1999

Increased insulin-like growth factor-I (IGF-I) expression during early postnatal development differentially increases neuron number and growth in medullary nuclei of the mouse.

Morphometric analyses of the medulla were performed in transgenic mice that overexpress insulin-like growth factor-I (IGF-I) postnatally and in non-transgenic littermates. The total volume of the medulla was increased in transgenic mice at all postnatal ages studied: 14 days (18%), 21 days (23%), 28 days (23%), and 35 days (27%). By 35 days of age, the volumes of individual medullary nuclei were also increased: nucleus of the tractus solitarius (NTS, 59%), dorsal motor nucleus of the vagus (DMV, 84%), hypoglossal nucleus (HN, 29%) and facial nucleus (FN, 21%). Neuron number in transgenic mice was significantly greater in NTS (50%) and DMV (53%), but not in the HN or the FN. Motor neurons in DMV, HN and FN of transgenic mice exhibited increases in mean profile areas of the soma and decreased numerical densities, suggesting increases in neuritic outgrowth. These results point to IGF-I actions in promoting neuron survival and growth, and suggest that IGF-I has differential effects on distinct neuron populations, possibly depending upon its time of expression.

Animals↗

PubMed1999

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells.

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.

Adenoviridae↗

PubMed1999

Insulin-like growth factor-I (IGF-I) ameliorates and IGF binding protein-1 (IGFBP-1) exacerbates the effects of undernutrition on brain growth during early postnatal life: studies in IGF-I and IGFBP-1 transgenic mice.

Insulin-like growth factor-I (IGF-I) plays an important role in the stimulation of postnatal brain growth. In transgenic (Tg) mice, IGF-I overexpression stimulates postnatal brain growth, whereas decreased IGF-I availability caused by ectopic brain expression of IGF binding protein-1 [(IGFBP-1), an inhibitor of IGF-I action] retards postnatal brain growth. Because undernutrition during early postnatal development profoundly retards growth and maturation of the brain in rodents, we sought to determine the influence of IGF-I on undernutrition-induced brain growth retardation. Caloric restriction was imposed on IGF-I Tg mice, IGFBP-1 Tg mice, and their non-Tg littermates by separating half of each litter from their dams during the suckling period, postnatal d 1 to 21. Undernutrition reduced the brain growth of each group of mice, but the growth of undernourished IGF-I Tg mice was comparable to that of well-fed control mice (increased 4.13- and 4.22-fold, respectively) and greater than that of undernourished control mice (increased 3.45-fold), whereas undernourished IGFBP-1 Tg mice exhibited less growth (increased 3.15-fold) than undernourished control mice. When the effects of undernutrition were examined in specific brain regions of each group, the same pattern was observed, and IGF-I was found to be more effective in preserving the growth of the regions with the highest transgene expression (cerebral cortex, hippocampus, and diencephalon). Despite undernutrition, IGF-I transgene expression stimulated overgrowth of these regions as well as that of the posterior medial barrel subfield, a somatosensory area of the cerebral cortex in which IGF-I may be especially important in development. These data indicate that IGF-I can ameliorate the brain growth retardation caused by undernutrition imposed during development, although it is unclear whether IGF-I directly opposes the impact of undernutrition or acts independently of nutritional status. Nonetheless, these findings raise the possibility that the relatively high IGF-I expression during early postnatal life may be responsible for sparing the brain from the full impact of undernutrition during this time in development.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important modulators of IGF action in many tissues including human prostate. IGFBPs and the androgen receptor (AR) are expressed in CWR22, an androgen-dependent epithelial cell human CaP xenograft that retains biological characteristics of human CaPs, including regression following androgen withdrawal and recurrent growth of AR-containing cells in the absence of testicular androgens beginning several months after castration. Northern blot and in situ hybridization analyses demonstrated that IGFBP-5 is androgen-regulated in CWR22. IGFBP-5 messenger RNA (mRNA) decreased by 90% following castration of tumor-bearing mice compared with noncastrate androgen-stimulated mice. Testosterone treatment of CWR22 tumor-bearing mice 6 or 12 days after castration increased IGFBP-5 mRNA 10- to 12-fold. Levels of other IGFBP mRNAs did not change following androgen withdrawal and replacement. IGFBP-5 protein in tumor extracts bound 125I-labeled IGF-I in ligand blot assays and the amounts of IGFBP-5 measured by immunoblotting paralleled the levels of IGFBP-5 mRNA. Androgen-induced expression of IGFBP-5 was at a maximum level within 24 h after testosterone replacement, whereas the major increase in cell proliferation as measured by Ki-67 immunostaining occurred between 24-48 h. This time course suggested IGFBP-5 may be a mediator of androgen-induced growth of CWR22. In tumors that recurred several months following castration, IGFBP-5 mRNA and protein increased to levels that approached those in androgen-stimulated CWR22 tumors from noncastrate mice. IGFBP-5 immunohistochemical staining of prostate tissue specimens from patients was stronger in androgen-dependent and androgen-independent CaP than in areas of intraepithelial neoplasia (PIN) or benign prostatic hyperplasia (BPH). IGFBP-5 mRNA in these specimens was localized predominantly to stromal cells and IGFBP-5 protein to epithelial cell membranes.

Animals↗

PubMed1999

Insulin-like growth factor I protects oligodendrocytes from tumor necrosis factor-alpha-induced injury.

Tumor necrosis factor-alpha (TNF-alpha) has been causally implicated in several demyelinating disorders, including multiple sclerosis. Because insulin-like growth factor I (IGF-I) is a potent stimulator of myelination, we investigated whether it can protect oligodendrocytes and myelination from TNF-alpha-induced damage using mouse glial cultures as a model. Compared with controls, TNF-alpha decreased oligodendrocyte number by approximately 40% and doubled the number of apoptotic oligodendrocytes and their precursors. Addition of Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), an inhibitor of interleukin-1beta converting enzyme (ICE)/caspase proteases, blocked TNF-alpha-induced reductions in oligodendrocytes, indicating that the TNF-alpha-induced reduction in oligodendrocytes is, at least in part, due to apoptosis, and that ICE/caspases are one of TNF-alpha action mediators. Simultaneous addition of IGF-I to TNF-alpha-treated cultures negated these TNF-alpha effects nearly completely. Furthermore, IGF-I promoted oligodendrocyte precursor proliferation and/or differentiation in TNF-alpha-treated cultures. To analyze TNF-alpha and IGF-I actions on oligodendrocyte function, we measured the abundance of messenger RNAs (mRNAs) for two major myelin-specific proteins, myelin basic protein (MBP) and proteolipid protein (PLP). While TNF-alpha decreased MBP and PLP mRNA abundance by 5- to 6-fold, IGF-I abrogated TNF-alpha-induced reductions in a dose- and time-dependent manner. The changes in MBP and PLP mRNA abundance could not be completely explained by the changes in oligodendrocyte number, indicating that myelin protein gene expression is regulated by both TNF-alpha and IGF-I. These data support the hypothesis that TNF-alpha can mediate oligodendrocyte and myelin damage, and indicate that IGF-I protects oligodendrocytes from TNF-alpha insults by blocking TNF-alpha-induced apoptosis, and by promoting oligodendrocyte and precursor proliferation/differentiation and myelin protein gene expression.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Structure and function of alleles in the 3' end region of human apoB gene.

OBJECTIVE: To study the structure of alleles in the 3' end of the apoB gene in Han, Mongolian and Tibetan populations in China as well as the roles in the regulation of gene expression. METHODS: DNA were obtained from human leukocytes by phenol-chloroform extraction and ethanol precipitation. PCR were carried out in a 50 microliters volume containing 50 ng genomic DNA as template. The Ssp1-digested products were loaded on a gradient acrylamide gel and run for 3 hours. The constructs containing alleles were tested in cultured HepG2 and HeLa cells using transient assays. RESULTS: Sixteen alleles with different repeat number were characterized. All of the alleles varying from HVE22 to HVE52, allele HVE34 was the most common (58.4%), followed by allele HVE36 (13.8%) and HVE32 (10.5%). 258 PCR products were digested with Ssp1 and run in 4-12% PAGE. We detected the fragments of 266bp, 91 bp, 61 bp and 39 bp in almost all samples. The small alleles (including HVE22, HVE24, HVE26 and HVE36) decreased the expressive activity of the luciferase reporter, in contrary, the large alleles (including HVE44, HVE46 and HVE48) elevated obviously the expressive activity of the luciferase reporter. CONCLUSIONS: More alleles with different number of tandem repeats in 3' end of apoB gene exist in the Chinese populations. The alleles in 3' end minisatellite of human apoB gene could control the expression of the gene itself.

Alleles↗

PubMed1999

[Analyses on structural and physicochemical properties of collagen hemostatic sponge].

The ultraviolet spectrum and aminoacid content of collagen hemostatic sponge prepared from bovine tendon were analyzed, and the struture and composition of collagen spoge were confirmed. Physicochemical properties measurements showed that collagen hemostatic sponge had excellently stable quality. It was concluded that the technology for preparing collagen hemostatic sponge was reasonable and reliable.

Collagen↗

PubMed1998

IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines.

IL-17 is a novel cytokine secreted principally by CD4+ T cells. It has been shown to support the growth of hemopoietic progenitors in vitro; however, its in vivo effects are presently unknown. Adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to the liver (5 x 10(9) plaque-forming units (PFU) intravenous) resulted in a transiently transgenic phenotype, with dramatic effects on in vivo granulopoiesis. Initially, there was a significant increase (fivefold) in the peripheral white blood count (WBC), including a 10-fold rise in the absolute neutrophil count. This was associated with a doubling in the spleen size over 7-14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated. There was a profound stimulation of splenic hemopoiesis as demonstrated by an increase in total cellularity by 50% 7 days after gene transfer and an increase in hemopoietic colony formation. A maximal increase in frequency of high proliferative potential colonies (HPPC) (11-fold) and CFU-granulocyte-macrophage (GM) and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM) (CFU) (6-fold) was seen on day 3 after IL-17 gene transfer. Both CFU and HPPC remained significantly elevated in the spleen throughout day 21, but at reduced levels compared with day 3. Bone marrow CFU and HPPC were elevated on day 3 only by 75% and 25%, respectively, without changes in total cellularity. Thus, murine IL-17 is a cytokine that can stimulate granulopoiesis in vivo. Since IL-17 is principally produced by CD4+ T cells, this cytokine could have therapeutic implications in AIDS-related bone marrow failure and opportunistic infections.

3T3 Cells↗

PubMed1998

The domain structure of human receptor-associated protein. Protease sensitivity and guanidine HCl denaturation.

The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.

Amino Acid Sequence↗