Reproducibility of a commercial in vitro allergen-specific assay for immunoglobulin E in dogs.

Samples of serum taken from 42 dogs with clinical signs and histories indicating pruritic skin disease and/or diseases of the ear were tested in a commercial allergen-specific assay for immunoglobulin E. Dogs which had been treated with glucocorticoids and/or antihistamines were not excluded. The samples were separated into two equal aliquots, given different randomised numbers, and analysed in two batches on two separate days. The laboratory was blinded to the identification numbers and the history of each dog, but knew the purpose of the study. The results for 48 allergens were expressed in modified absorbance units (MAU). The overall median MAU was 29. For each allergen the mean difference between the MAU values of the paired duplicates was determined and the difference was compared to zero by a paired t test. The number of means that were not 0 (P<0.05) in each allergen group were: seven of 10 grasses, seven of nine weeds, two of 13 trees, six of 10 fungi, and three of six environmental allergens. A single 2 x 2 table for the 48 allergens was created with MAU > or = 60 defined as 'positive' and < 60 as 'negative'. There were 116 of 188 (62 per cent) pairs that were reproducibly 'positive' and 1756 of 1828 (96 per cent) pairs that were reproducibly 'negative'.

Phosphorylation is a regulatory mechanism in apolipoprotein B mRNA editing.

The editing of apolipoprotein B (apoB) mRNA is under tissue-specific, developmental and metabolic regulation. We found that multiple protein kinase inhibitors or activators increased apoB mRNA editing up to 2.5-fold in Caco-2 cells and 3-8-fold in McA7777 and FAO rat cells respectively. The phosphorylation-agent-induced modulation is independent of the apolipoprotein B editing catalytic subunit 1 (APOBEC-1) and of apoB mRNA expression levels, indicating the involvement of a protein modification, such as phosphorylation, regulating the cellular editing of apoB mRNA. Transient expression of protein kinase C-θ more than doubled apoB mRNA editing in FAO cells. Chronic exposure to ethanol, a treatment known to increase the expression of protein kinases and to change protein phosphorylation status, increased apoB mRNA editing in FAO cells up to 2.5-fold without increasing the mRNA abundance of APOBEC-1. The elimination of potential phosphorylation sites 47 and 72 of human APOBEC-1 decreased its activity to approx. one-eighth of control levels by a Ser(47)-->Ala mutation, but more than doubled the activity by a Ser(72)-->Ala mutation. The activity modulation was reversed by a Ser-->Asp mutation at sites 47 and 72, which introduced a phosphorylation-like carbonic acid group. Both human APOBEC-1 dephosphorylated by alkaline phosphase and the Ser(47,72)-to-alanine double mutant protein demonstrated a shifted isoelectric focusing pattern compared with the wild type, indicating phosphorylation at these sites. Taken together, these results suggest that phosphorylation might be an important mechanism in the regulation of apoB mRNA editing.

Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein.

A new human 95 kDa high density lipoprotein (HDL)-binding protein (HBP) corresponding to a high affinity HDL-binding site with K(d) = 1.67 microg/mL and a capacity of 13.4 ng/mg was identified in human fetal hepatocytes. The HDL binding with the 95 kDa HBP plateaus at 2.5-5 microg/mL under reducing and nonreducing conditions. The association of HDL(3) with the 95 kDa HBP plateaued in 15-30 min while dissociation was complete in 30 min. HDL(3), apoA-I, and apoA-II were recognized by the 95 kDa HBP while low density lipoproteins (LDL) and tetranitromethane-modified HDL were not. The 95 kDa HBP predominantly resides on the surface of cells since trypsin treatment of HepG2 cells eliminated nearly 70% of HDL binding. All studied human cells and cell lines (HepG2, Caco-2, HeLa, fibroblasts, SKOV-3, PA-I) demonstrated the presence of the 95 kDa protein. Both RT-PCR and Western blotting for HB-2/ALCAM were negative in human fetal hepatocytes while Gp96/GRP94 was clearly differentiated from the 95 kDa HBP by two-dimensional electrophoretic mobility. Moreover, deglycosylation of HepG2 membrane preparations did not affect either HDL binding to the 95 kDa HBP or its size, while in contrast it affected the molecular weights of HB-2/ALCAM and SR-BI/CLA-1. We conclude that the 95 kDa HBP is a new HDL receptor candidate widely expressed in human cells and cell lines.

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter.

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.

Calcium increases apolipoprotein B mRNA editing.

ApoB-100 and apoB-48 are major components of chylomicrons, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). The two proteins are generated from a single apoB mRNA by apoB mRNA editing which induces an in-frame stop codon in apoB mRNA. Apolipoprotein B (apoB) mRNA editing is an important determinant of the proportion of full-length (apoB-100) and truncated (apoB-48) proteins in total apoB metabolism. Calcium is involved in the regulation of secretion and synthesis of VLDL and apoB. In this paper, we demonstrate for the first time that the amount of edited apoB mRNA in the cultured cells Caco-2 and McA7777 is markedly increased by calcium. Increasing extracellular calcium concentration, calcium ionophore (A23187 and ionomycin) treatment, and depleting calcium stores and raising cytoplasmic calcium concentration by thapsigargin increase apoB mRNA editing up to threefold in a dose dependent manner. Calcium has no direct stimulative effect on apoB mRNA editing in an in vitro editing system. The editing increase by extracellular calcium is not related to alterations of APOBEC-1 mRNA expression. These data suggest that calcium is not only involved in the regulation of apolipoprotein metabolism but also apoB mRNA editing.

Heat shock protein 60 is a high-affinity high-density lipoprotein binding protein.

A new 55-kDa HDL/apolipoprotein binding protein was demonstrated in plasma membrane preparations of the human cell lines and primary cultured hepatocytes. Analysis of specific binding by ligand immunoblots of HDL, apoA-I, and apoA-II to a partially purified 55-kDa PA-I plasma membrane preparation demonstrated a K(d,HDL) = 50 nM (10 microg/ml), K(d,apoA-II) = 20 nM (0.4 microg/ml), and K(d, apoA-I) = 330 nM (10 microg/ml). Following preparative SDS-PAGE electrophoresis of a plasma membrane preparation isolated from human PA-I cells, fractions with apoA-II binding activity were collected, concentrated, and subjected to two-dimensional electrophoresis. Internal microprotein sequencing of the 55-kDa protein band revealed the binding protein as being heat shock protein 60 (hsp60). The hsp60 monoclonal antibody LK-1 blocked apoA-II binding to the 55-kDa HBP preparation. In summary, these results provide a potential mechanism to explain the known association between immunity developed against hsp60 and the development of atherosclerosis.

Xenotransplantation: the potential and the challenges.

Clinical use of xenotransplants is a potential way to provide care for a population of seriously ill patients and alleviate the demand for human organs. However, xenotransplantation also presents a spectrum of concerns, not only for individual patients but also for the public health, that must be discussed and dealt with in a science-based and public manner. Such discussions should take place on a national level and should include scientists, physicians, and policy makers from all countries in which the clinical use of xenografts is being considered.

Development of databases and registries. International issues.

This paper describes the U.S. Public Health Service (PHS) efforts to develop a National Xenotransplantation Registry (NXR). The function of the registry is discussed, paying particular attention to data collection and harmonization issues. The need for standardization and coordination on an international scale is presented and the benefits of such efforts are discussed. Finally, a recommendation for an international effort aimed at harmonization is made.

Therapeutic response to medium-chain triglycerides and omega-3 fatty acids in a patient with the familial chylomicronemia syndrome.

We have studied the underlying molecular defect in a patient presenting with recurrent pancreatitis, hypertriglyceridemia, and virtually undetectable postheparin plasma lipoprotein lipase (LPL) mass and activity, who normalized her triglycerides 3 to 6 months after initiation of either medium-chain triglyceride (MCT) oil or omega-3 fatty acid (omega-3-FA) therapy. After treatment, postheparin plasma LPL activity and mass ranged from 24% to 39% of normal and LPL specific activity was normal (1.0 nmol.ng-1.min-1). On discontinuation of MCT oil or omega-3-FA, plasma triglyceride increased to > 2000 mg/dL. Northern blotting revealed both normal size and abundance of LPL mRNA isolated from adipocytes as well as macrophages. Sequence analysis of the LPL gene, which included all 10 exons, intron-exon splice junctions, and 1.7 kb of the 5'-flanking region, and of LPL cDNA failed to identify any mutations. ApoC-II activity and mass assays revealed the presence of normal levels of a fully functional cofactor as well as the absence of circulating plasma inhibitors of lipase function. In summary, we describe a unique patient presenting with classical features of the familial chylomicronemia syndrome who manifests an unusually beneficial therapeutic response to MCT oil and omega-3-FA therapy. Unlike that in most patients with LPL deficiency, the chylomicronemia in this patient is not caused by a mutation in the structural LPL gene but possibly by a posttranscriptional defect. Thus, a subset of LPL-deficient patients with unique genetic defects respond to therapy by normalizing fasting plasma triglycerides; a therapeutic trial with MCT oil should be considered in all patients presenting with the familial chylomicronemia syndrome.

Ontogenetic regulation of apolipoprotein B mRNA editing during human and rat development in vivo.

The solubilization and delivery of lipids in plasma rely on both forms of apolipoprotein B (apo B): apo B-100 and apo B-48. Apo B-48 is the translational product of apo B-100 mRNA that undergoes peritranscriptional conversion of C----U, replacing codon CAA (glutamine 2,153) with the inframe stop codon (UAA). We examined mRNA editing activity in the human and the rat by reverse transcription-polymerase chain reaction primer-extension analysis of intestine and liver total RNA. In rat intestine the percentage of apo B transcripts that undergo editing increases dramatically the day before birth (from approximately 1% to 80%), whereas the rat liver acquires an adult level of editing activity during the third postnatal week (rising from approximately 8% to 30%), when weaning is completed, bile acid composition matures, and plasma thyroid hormone levels peak. In contrast to the rat, the human intestine acquires adult levels of apo B mRNA editing relatively early in fetal development, rising from 10% at 10 weeks to approximately 80% by the end of the second trimester. Our results establish that apo B mRNA editing is 1) developmentally regulated in a tissue- and species-specific manner; 2) fully developed prenatally in both human and rat intestine, suggesting a crucial role of apo B-48 in mammalian fetal adaptation to extrauterine life; and 3) acquired early in human fetal intestine, implying a potential role for apo B-48 in prenatal lipid metabolism.

The amino acid sequence of the insulin receptor is normal in an insulin-resistant Pima Indian.

Insulin resistance is an early predictor of development of noninsulin-dependent diabetes mellitus (NIDDM) in Pima Indians, a population with the highest reported prevalence of NIDDM. The insulin receptor plays a central role in mediating insulin action, and previous studies have demonstrated that mutations in the insulin receptor gene may cause insulin resistance. Therefore, we have cloned the insulin receptor cDNA from an insulin-resistant Pima Indian to determine if there is a mutation in the patient's insulin receptor gene. We obtained nine cDNA clones spanning exons 4-10 and 12-22 of the patient's insulin receptor gene. Polymorphisms in the nucleotide sequences for codons 523 (Ala), 1058 (His), and 1062 (Leu) provided useful markers to differentiate the patient's two alleles of the insulin receptor gene. These substitutions were silent, in that they did not alter the predicted amino acid sequence. The sequence of exons 1-3 and 11 was determined directly from genomic DNA that had been amplified using the polymerase chain reaction catalyzed by Taq DNA polymerase. Other investigators have reported defects in insulin binding and insulin receptor tyrosine kinase activity in diabetic Pima Indians. However, we did not detect any mutations in this patient's insulin receptor gene. Thus, these observations are consistent with the interpretation that the defects in insulin receptor function are acquired rather than derived from defects in the primary structure of the receptor.

Increased plasma and pituitary prolactin concentrations in adult male rats with selective elevation of FSH levels may be explained by reduced testosterone and increased estradiol production.

The roles of testosterone and estradiol in regulating prolactin concentrations were studied in acutely castrated adult male rats receiving subcutaneous Silastic implants of the sex steroids. Testosterone was administered in increasing doses, from subphysiologic to intact levels, both alone and in combination with a small, single dose of estradiol. The study was designed to assess whether a change in the relative rates of sex steroid production could account for an increase in PRL release in the absence of other testicular factors. At very low levels of plasma testosterone, FSH and LH levels were indistinguishable from castrate controls. As plasma testosterone concentration increased, both plasma FSH and LH levels were suppressed progressively to intact levels. When a subphysiologic dose of testosterone was coadministered with a small dose of estradiol, the combined effects produced a midcastrate level of FSH but maintained a normal level of LH similar to the selective increase in FSH concentration observed in men with germinal aplasia. Although PRL levels were indistinguishable in intact and castrate controls, testosterone replacement by capsule increased prolactin in a dose-related manner so that, at the physiologic level of testosterone, prolactin was elevated two-fold (P less than 0.01), similar to the level achieved with estradiol replacement alone. Pituitary prolactin levels also increased with increasing doses of testosterone but values remained within the range measured in intact controls. When estradiol was coadministered with testosterone, the combination produced different effects depending on the testosterone dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration in the plasma testosterone: estradiol ratio: an alternative to the inhibin hypothesis.

The data suggest that in the absence of the testis: (1) testosterone can maintain both FSH and LH concentrations chronically within the physiological range; (2) that estradiol preferentially suppresses plasma LH concentration, indicating that the androgenic component of testosterone modulates FSH secretion; and (3) that subphysiological testosterone concentrations accompanied by physiological estradiol levels permit FSH to escape to midcastrate levels while maintaining LH concentration at intact levels. An alteration in the testosterone: estradiol ratio can account for a selective FSH elevation when testosterone production is low. The data provide an alternative explanation for the inhibin phenomenon.