Identification of an alpha2-macroglobulin receptor in human mammary epithelial cells

Several cases of zinc (Zn) deficiency in human infants caused by abnormally low concentrations of Zn in breast milk were recently reported, the underlying mechanism of which is not known. Alpha2-macroglobulin (alpha2-M), a major Zn-binding ligand in serum, presents a potential vehicle for mammary Zn uptake. This study was conducted to determine if an alpha2-M receptor is present in human mammary epithelial cells, where it may be involved in the endocytosis of alpha2-M into the mammary gland. Normal human mammary epithelial cells were grown to confluency in serum-free medium. For all binding and uptake studies, alpha2-M, preactivated with methylamine and labeled with 125I, was added to cells for varied lengths of time to determine saturation over time and at varied concentrations to determine saturation over increasing concentration of ligand. Nonspecific and competitive binding were measured by addition of a 100-fold molar excess of unlabeled alpha2-M and serum albumin or lactoferrin, respectively. Binding at 4 degreesC was specific for alpha2-M and approached saturation kinetics at 56 nmol/L. Scatchard plot analysis of the binding data demonstrated more than one binding site: a high affinity, saturable binding site and a low affinity, nonsaturable binding site. Uptake of alpha2-M at 37 degreesC was rapid and continuous over increasing concentrations of alpha2-M, and internalized alpha2-M was rapidly degraded. Results from this study present evidence for receptor-mediated uptake of alpha2-M in human mammary epithelial cells, which in turn, provides a potential mechanism for Zn acquisition by the cell.     

Association of bone mineral density with apolipoprotein E phenotype

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47-82 years (64.0 +/- 1.0 years, mean +/- SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4-/- (E3/2 and E3/3, n = 217, Apo E4 +/- (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4-/- (0.91 +/- 0.01 g/cm2), E4 +/- (0.85 +/- 0.02 g/cm2), and E4+/+ (0.83 +/- 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 +/- 5.7 ng/ml) was higher than in E4-/- (7.7 +/- 0.3 ng/ml) or E4 +/- (7.7 +/- 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (-0.493 +/- 0.152) was not significantly different from that in the AAB of VDR (-0.616 +/- 0.225) or PPxx of ER (-0.785 +/- 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.     

Association of apolipoprotein E epsilon2 and vasculopathy in cerebral amyloid angiopathy

Endogenous opioid peptides appear to be involved in acute behavioral effects induced by single doses of ethanol. However, its role in repeated ethanol exposure has not been studied. In the present study ethanol was given to rats at the doses of 2 and 4 g/kg by a stomach gauge for 15 days, and its effects on spontaneous motility, open-field activity, and active avoidance behavior recorded on the 3rd, the 6th and the 15th days. Then the effect of naloxone (0.5 and 2 mg/kg by intraperitoneal route) was tested against a challenge ethanol dose, administrated by oral route, on the 16th day. Control animals received tap water and saline instead of ethanol or naloxone, respectively. Both doses of ethanol induced a decrease in spontaneous motility that was antagonized by naloxone. Open-field ambulations were increased by the high dose of ethanol, low-dose lacking effect; naloxone did not modify these ethanol effects. The low dose of ethanol shortened latency time in shuttlebox, the high dose causing escape and freezing responses; none of these effects were modified by naloxone. Therefore, endogenous opioid peptides appear to play a limited role in the chronic effects of ethanol in rats; particularly its effects in tests inducing an increase in the level of anxiety were resistant to naloxone.     

Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases

The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.     

Influence of macrophage-derived apolipoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apolipoprotein E-deficient mice

Two-dimensional multiple-quantum magic angle spinning (MQMAS) NMR and MAS NMR of 11B at various magnetic fields, were applied to elucidate the structure of vitreous (glassy) boron trioxide (v-B2O3), vitreous boron trisulfide (v-B2S3) and crystalline boron trisulfide (c-B2S3). These techniques, when combined with computer simulations of the resulting spectra, provide the isotropic chemical shifts and the quadrupole parameters, as well as a quantitative measure of the intensities of various boron resonances. The MAS NMR of v-B2O3 produced overlapping anisotropic lineshapes corresponding to the -1/2<-->1/2 transition in two distinct types of BO3 units with 3(+/-0.08):] intensity ratio. A combination of MAS and the multiple-quantum method resulted in a better resolved, isotropic 11B spectrum of v-B2O3. A remarkable enhancement of resolution of the MQMAS NMR proved instrumental in finding and identifying various impurities present in v-B2S3 and c-B2S3. In addition to the resonances from boron in two types of BS3 groups, four other structural units, BOS2, BO2S, BO3 and BS4, were elucidated from the spectra of vitreous and crystalline samples. The effects of various experimental parameters, such as the magnitude of the B0 and B1 fields, on the resolution of the MAS and MQMAS techniques are also shown.     

A radioimmunometric assay for urinary alpha 2-macroglobulin

To measure urinary alpha 2-macroglobulin levels, a sensitive radioimmunometric assay was established. The least detectable level of this assay was 225 pg/ml. A linear correlation between alpha 2-macroglobulin levels and serial dilution of urine samples was found. Western blot analysis and study on column chromatography revealed that the molecular weight of alpha 2-macroglobulin in urine was identical to that of serum alpha 2-macroglobulin. The findings suggested that urinary substance detected by the present assay was truly alpha 2-macroglobulin. Timed overnight urine samples from 49 diabetic patients with retinopathy and 20 healthy controls were measured by the present assay. Patients were classified as Albustix-negative and Albustix-positive patients. The highest urinary alpha 2-macroglobulin excretion rates (alpha 2-MER) was found in Albustix-positive patients followed by Albustix-negative patients and the healthy controls. In view of the fact that the stroke radius of alpha 2-macroglobulin (88A) is larger than that of the restrictive pore (56A), the present finding suggests that leakage of alpha 2-macroglobulin in urine may be induced by defect of non-discriminatory pores in the glomerular basement membrane proposed by Deen and colleagues.     

Similar architectures of native and transformed human alpha2-macroglobulin suggest the transformation mechanism

The refined three-dimensional structure of native human alpha2-macroglobulin (alpha2M) has been determined by cryoelectron microscopy and three-dimensional reconstruction. New features corresponding to "sigmoid arches," "basal bodies," and "apical connections" were observed in the molecule. Since similar elements are found in the architecture of transformed alpha2M, the 2 volumes were aligned in three dimensions. In their common orientations, they show many similarities except near the openings of the central chamber. In the native conformation, these apertures are fully opened, allowing the proteases to access the central chamber of the molecule, while in the transformed structure, they are partially closed. These structures suggest that alpha2M conformational change involves a strong lateral compression and a vertical stretching of the native particle seen in its four-petaled flower view to produce the H view of the transformed form. A model of structural transformation, in which all the parts of the alpha2M molecule seem involved in the entrapment of the proteinases is proposed.     

Association of apolipoprotein E epsilon 4 allele with bulbar-onset motor neuron disease

BACKGROUND: Variants of the apolipoprotein E (APOE) gene influence the age of onset of Alzheimer's disease. APOE may influence the presentation of other neurological diseases. We investigated the relationship between the allelic variants of apolipoprotein E and clinical presentation in motor neuron disease. METHODS: 123 patients with motor neuron disease and 121 controls were studied. Diagnosis, location of onset and date of onset were recorded prospectively. Genotyping was performed blind to clinical information. FINDINGS: Possession of at least one epsilon 4 allele was significantly more common in patients with bulbar onset motor neuron disease (14/33, 42%) than in limb onset patients (20/90, 22%) and controls (26/121, 21%) (chi 2 = 4.93, p = 0.026 and chi 2 = 5.91, p = 0.015, respectively). INTERPRETATION: These results suggest that the apolipoprotein E epsilon 4 allele may influence the pattern of motor neuron loss in motor neuron disease and that it may affect neuronal function in ways unrelated to the deposition of beta-amyloid or accumulation of neurofibrillary tangles.     

Transcription factor AP-2 regulates human apolipoprotein E gene expression in astrocytoma cells

Apolipoprotein E (apoE), one of the major plasma lipoproteins, also is expressed in a variety of cell types, including the glial cells of the nervous system. apoE is involved in processes of degeneration and regeneration after nerve lesions as well as in the pathogenesis of Alzheimer's disease (AD). Glial synthesis of apoE is activated in response to injury both in the peripheral and central nervous system. We now report that the activity of the proximal apoE promoter in astrocytes is upregulated by cAMP and retinoic acid, which act synergistically. Sequence analysis of the apoE promoter indicated the presence of several AP-2 consensus sequences that could mediate the stimulatory effect of cAMP and retinoic acid. The possible functional role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with an apoE promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated apoE promoter activity. DNase I footprinting technique revealed the existence of two binding sites for recombinant AP-2 in regions from -48 to -74 and from -107 to -135 of the apoE promoter. Mutations in these regions markedly impaired the trans-stimulatory effect of AP-2. These results indicate the existence of functional AP-2 sites in the promoter region of apoE that could contribute to the complex regulation of this gene in developmental, degenerative, and regenerative processess of the nervous system.     

Human alpha 2-macroglobulin is an osteogenic growth peptide-binding protein

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations < 50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.     

Further characterization of alpha 2-macroglobulin binding properties of Actinomyces pyogenes

OBJECTIVE: To investigate and compare agreement within two groups of dental practitioners, family dentists and oral surgeons, in their decisions regarding removal of asymptomatic mandibular third molars. SUBJECTS: Ten oral surgeons and 18 family dentists from South Wales with experience ranging from 5 to 28 years. METHODOLOGY: Participants were presented with periapical radiographs of 36 asymptomatic, mandibular third molars and were informed of the age and gender of the patients and the degree of eruption of the third molars. Participants were asked to indicate whether they thought that the third molar should be removed or not. The degree of agreement between participants was measured by kappa indices for multiple raters. RESULTS: The kappa indices were 0.14 for the oral surgeons and 0.09 for the family dentists, indicating poor agreement beyond chance. Although in most cases the participants decided not to remove the third molar, they did so inconsistently, that is, they did not make this decision on the same cases. There were also differences in the inclination of the participants to suggest removal of the 36 third molars. CONCLUSION: Poor inter-observer agreement suggested that treatment decisions regarding asymptomatic third molars are based more on subjective beliefs and habitual practices than on rational decision making.     

Different expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein in human keratinocytes and fibroblasts

BACKGROUND AND OBJECTIVES: Although partner notification has been a long-standing intervention and prevention strategy for sexually transmitted diseases (STD), variations in partner notification practices across sites have never been documented. GOALS OF THE STUDY: To describe provider-assisted partner notification practices in four STD programs in the United States. STUDY DESIGN: Eleven disease intervention specialists (DIS) in each of three urban sites and seven DIS in one rural site documented their activities and clients for 14 working days using a personal digital assistant. RESULTS: Of 2,506 recorded activity hours across sites, 37.4% of the recorded time was spent on partner notification (PN) activities with 1148 clients. Field visits to locate contacts accounted for the largest proportion of time spent on PN. Overall, PN clients were cases of or were contacts to nonprimary and secondary (P&S) syphilis (39.6%), gonorrhea (25.5%), chlamydia (18.0%), HIV/AIDS (10.4%), and P&S syphilis (6.5%). CONCLUSION: The activities which constitute PN, the diseases for which PN is used, and the time spent on each PN client vary across sites. More research is needed on the determinants of these variations and their association with the ultimate goal of disease prevention.     

Radioimmunoassay of prostaglandins E1, E2, and F2alpha in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE1, PGE2, and PGF2alpha is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE1, PGE2, and PGF2alpha. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE1 and PGE2, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Levuglandin E2-protein adducts in human plasma and vasculature

The prostaglandin endoperoxide PGH2 rearranges nonenzymatically to generate prostaglandins and secoprostanoic acid levulinaldehyde derivatives such as PGE2 and levuglandin (LG) E2, respectively. Direct detection of LGE2 in biological samples is complicated because it is rapidly sequestered by covalent adduction to endogenous nucleophiles including proteins, which produces LGE2-derived protein-bound pyrroles. Therefore, to detect LGE2-protein adducts in vivo, antibodies were raised against a covalent adduct of LGE2 with keyhole limpet hemocyanin (KLH). This antigen enabled the production of high-titer antibodies that exhibit minimal cross-specificity and are sensitive for detecting LGE2-derived pyrroles. Although pyrrole yields are low at LG/protein ratios found in vivo, an enzyme-linked immunosorbent assay with the LGE2-KLH antibodies detects LGE2-derived protein-bound pyrrole immunoreactivity in human plasma from specific patient populations. Furthermore, prominent immunocytochemical staining of human brain thin sections revealed the presence of LGE2-derived pyrrole immunoreactivity, especially in the meningeal vessels of some patients. This demonstration of LG-protein adducts in human plasma and vasculature provides the first evidence for the biological occurrence of levuglandins in vivo and further suggests that these antibodies might prove useful in diagnostic and mechanistic studies of various disease conditions.     

Genetic factors precipitating type III hyperlipoproteinemia in hypolipidemic transgenic mice expressing human apolipoprotein E2

Several factors are hypothesized to precipitate or exacerbate type III hyperlipoproteinemia (HLP) in humans. Among such factors are those that directly overload remnant lipoprotein production or disrupt removal pathways, including an increased ratio of apolipoprotein (apo) E2 to normal apoE, overproduction of apoB-containing lipoproteins, and decreased LDL receptor activity. Hypolipidemic apoE2-transgenic mice bred onto an apoE-null background had dramatically higher plasma total cholesterol (192 +/- 26 mg/dL for males, 203 +/- 40 mg/dL for females) and triglyceride (295 +/- 51 mg/dL for males, 277 +/- 58 mg/dL for females) levels than apoE2 mice with endogenous mouse apoE. Thus, eliminating normal apoE in the presence of apoE2 (thereby increasing the relative abundance of the defective ligand) can convert a hypolipidemic to a hyperlipidemic phenotype. Hypolipidemic apoE2 transgenic mice overexpressing human apoB had moderate remnant accumulation compared with apoE2-only or apoB-only transgenic mice, indicating that overproduction of apoB-containing lipoproteins in the presence of apoE2 can augment remnant production. Hypolipidemic apoE2 transgenic mice bred-onto an LDL receptor-null background had markedly higher plasma total cholesterol (288 +/- 51 mg/dL for males, 298 +/- 73 mg/dL for females) and triglyceride (356 +/- 72 mg/dL for males, 317 +/- 88 mg/dL for females) levels than apoE2-only mice, and remnant accumulation increased even in apoE2 mice with a heterozygous LDL receptor-knockout background (compared with apoE2-only mice), suggesting that reducing or eliminating a major receptor-mediated remnant-removal pathway in the presence of apoE2 can also precipitate a hyperlipidemic phenotype. In all cases where either lipoprotein remnant production or removal pathways were severely stressed, increased remnant accumulation was apparent. As judged by the chemical characteristics of the remnant lipoproteins, the lipoprotein phenotype was quite similar to that of human type III HLP, especially in the apoE2-expressing mice with no endogenous apoE or LDL receptors, and thus these mice represent improved models of the disorder.     

Presence and formation of 'free apolipoprotein A-I-like' particles in human plasma

The influence of dilution on apolipoprotein (apo) A-I-containing subpopulations was studied in human plasma. Agarose electrophoresis and two-dimensional agarose nondenaturing gradient polyacrylamide gel electrophoresis were used. Both in one- and two-dimensional electrophoresis, an increase of charge was observed that resulted in an increase of subpopulations with pre-alpha mobility. Dilution of plasma also resulted in a decrease in the size of apo A-I-containing pre-beta 1 subpopulations. The existence of smaller pre-beta 1 particles was confirmed by subjecting undiluted and 8x diluted plasma to 3% to 16% nondenaturing gradient gel electrophoresis for 4 hours. In addition to the generally observed pre-beta 1 subpopulations, smaller particles similar in size to the free apo A-I were detected even in the undiluted plasma. During dilution, the proportion of larger pre-beta 1 particles decreased while the smaller ones increased, and in 8x diluted plasma, almost all the pre-beta 1 was present in smaller sizes. Using 3% to 35% nondenaturing polyacrylamide gels run for 24 hours, no pre-beta 1 particles could be detected in 8x diluted plasma because the small pre-beta 1 electrophoresed out. These studies show that pre-beta 1 particles can be converted to smaller ones during dilution. It also was demonstrated that "free apo A-I-like" pre-beta 1 particles are present in undiluted plasma. The presence of these particles may have important physiological and pathophysiological functions.     

Regulation of interleukin-8 binding and function by heparin and alpha2-macroglobulin

The effect of radiation on speech and swallowing function was assessed for 18 patients surgically treated for oral and oropharyngeal cancer. Nine patients received surgical intervention and postoperative radiation therapy, and nine received surgery only. Patients were matched regarding percentage of oral tongue resected, percentage of tongue base resected, locus of resection, and method of reconstruction. Speech and swallowing function was assessed before and at 1, 3, 6, and 12 months after surgery following a standardized protocol. Speech tasks included an audio recording of a brief conversation and of a standard articulation test; swallowing function was examined with videofluoroscopy. Statistical testing indicated that overall speech function did not differ between the irradiated and nonirradiated patients. Irradiated patients had significantly reduced oral and pharyngeal swallowing performance, specifically, longer oral transit times on paste boluses, lower oropharyngeal swallow efficiency, increased pharyngeal residue, and reduced cricopharyngeal opening duration. Impaired function may be the result of radiation effects such as edema, fibrosis, and reduced salivary flow. Increased use of tongue range-of-motion exercises during and after radiation treatment may reduce the formation of fibrotic tissue in the oral cavity and may improve pharyngeal clearance by maintaining adequate tongue base-to-pharyngeal wall contact.     

On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule

The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.