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PubMed1990

Mapping of low abundance latency-associated RNA in the trigeminal ganglia of mice latently infected with herpes simplex virus type 1.

During herpes simplex virus type 1 (HSV-1) latent infection of the mouse trigeminal ganglion there is limited viral gene expression. The latency-associated transcripts (LAT) map approximately to the PstI-MluI fragment within the BamHI B and BamHI E fragments (long repeat regions) of the viral genome. Additional weak hybridization signals have been detected by in situ hybridization that correspond to transcription from HSV-1 DNA fragments adjacent tot he PstI-MluI fragment. We mapped the region encoding this additional transcription. This minor latency-associated RNA (m-LAT) was shown to map to a group of contiguous fragments (approximately 8.3 kb of DNA), which are adjacent to the 3' end of LAT and to a (2.0 kb) fragment adjacent to the 5' end of the LAT. Using single-stranded probes in in situ hybridization experiments, we showed that the KpnI-BamHI and BamHI-SacI regions of m-LAT are transcribed in a rightward direction within the long internal repeat region. This low abundance RNA may be related to the previously described LAT.

Animals↗
PubMed1994

Purification and characterization of ensconsin, a novel microtubule stabilizing protein.

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.

Cell Extracts↗
PubMed2006

Design and analysis of genetic association studies to finely map a locus identified by linkage analysis: sample size and power calculations.

Association (e.g. case-control) studies are often used to finely map loci identified by linkage analysis. We investigated the influence of various parameters on power and sample size requirements for such a study. Calculations were performed for various values of a high-risk functional allele (fA), frequency of a marker allele associated with the high risk allele (f1), degree of linkage disquilibrium between functional and marker alleles (D') and trait heritability attributable to the functional locus (h2). The calculations show that if cases and controls are selected from equal but opposite extreme quantiles of a quantitative trait, the primary determinants of power are h2 and the specific quantiles selected. For a dichotomous trait, power also depends on population prevalence. Power is optimal if functional alleles are studied (fA= f1 and D'= 1.0) and can decrease substantially as D' diverges from 1.0 or as f(1) diverges from fA. These analyses suggest that association studies to finely map loci are most powerful if potential functional polymorphisms are identified a priori or if markers are typed to maximize haplotypic diversity. In the absence of such information, expected minimum power at a given location for a given sample size can be calculated by specifying a range of potential frequencies for fA (e.g. 0.1-0.9) and determining power for all markers within the region with specification of the expected D' between the markers and the functional locus. This method is illustrated for a fine-mapping project with 662 single nucleotide polymorphisms in 24 Mb. Regions differed by marker density and allele frequencies. Thus, in some, power was near its theoretical maximum and little additional information is expected from additional markers, while in others, additional markers appear to be necessary. These methods may be useful in the analysis and interpretation of fine-mapping studies.

Alleles↗
PubMed2005

Noninvasive hemodynamic assessment of the effect of mean arterial pressure on the amplitude of pulse pressure.

BACKGROUND: The present study compares the relationships between mean arterial pressure (MAP) and pulse pressure (PP) in young and older men and whether MAP determines age-related changes in PP. METHODS: Impedance cardiography was used to evaluate systemic hemodynamics noninvasively in 189 unmedicated men referred to our hypertension unit. Patients were divided by age according to the median value (< and >/=40 years). RESULTS: In younger patients, increasing supine MAP was associated with a transition in the blood pressure pattern from systolic to diastolic, whereas in older patients, increasing MAP was associated with change from diastolic to systolic hypertension. In young patients elevation in MAP was associated with a parallel decrease in PP (P < .001) and stroke volume index (SVI) (P < .001), whereas in older patients higher MAP was associated with higher PP (P < .001) and PP/SVI ratio (P < .001), a measure of arterial stiffness. When the sample was divided according to the median value of MAP (< and >/=101 mm Hg), differences in the age-related change in PP and SVI became apparent. With a lower MAP, changes in PP and SVI remained parallel until the sixth decade, after which they began a progressive dissociation. In contrast, at higher MAP, the dissociation between PP and SVI began two decades earlier, indicating an acceleration of the age-related increase in arterial stiffness. Therefore, the relationship between MAP and PP was negative in younger men at the expense of a decrease in SVI, and positive in older men due to increasing arterial stiffness. CONCLUSIONS: Noninvasive hemodynamic parameters from impedance cardiography provide a useful method to characterize the mechanism of increased blood pressure.

Adult↗
PubMed2003

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins.

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

Animals↗
PubMed1995

Three distinct axonal transport rates for tau, tubulin, and other microtubule-associated proteins: evidence for dynamic interactions of tau with microtubules in vivo.

Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50-60 kDa tau isoforms and a second 90-95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S-methionine intravitreously and, after 6-30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2-0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1-0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature.

Animals↗
PubMed1993

Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes.

The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.

Animals↗
PubMed1992

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

Adenosine Triphosphate↗
PubMed1998

The distribution of murine 115-kDa epithelial microtubule-associated protein (E-MAP-115) during embryogenesis and in adult organs suggests a role in epithelial polarization and differentiation.

In interphase cells microtubules play fundamental roles in the intracellular distribution and movement of organelles and vesicles and thereby contribute to cellular polarization and differentiation. The organization of microtubules varies with the cell type and is presumably controlled by tissue-specific microtubule-associated proteins (MAPs). The 115-kDa epithelial MAP (E-MAP-115) has been identified as a microtubule-stabilizing protein predominantly expressed in cell lines of epithelial origin. To assess a putative function of E-MAP-115 in epithelial morphogenesis in vivo, we have cloned the cDNA encoding the murine protein and studied the cellular distribution of E-MAP-115 mRNA and protein during murine embryogenesis and in adult organs. Analysis of the predicted amino acid sequence of murine E-MAP-115 revealed 81% sequence identity with its human homolog, the best-conserved part of the protein being the microtubule-binding site. Our data indicate that E-MAP-115 is expressed in several epithelia from 9.5 days of embryogenesis onwards and that its expression levels increase during development. From 14.5 days onwards, E-MAP-115 mRNA is found in some neuronal cells as well. In adult organs, E-MAP-115 is most abundant in epithelial cells of kidney tubules, in absorptive cells of the intestine and is widely distributed in the testis. E-MAP-115 expression correlates with the differentiation of certain epithelial cell types: in the adult intestine, for example, E-MAP-115 mRNA and protein are more abundant in the differentiating than in the proliferative cell compartment. Moreover, E-MAP-115 expression clearly correlates with the degree of cellular apicobasal polarity. In the developing kidney, E-MAP-115 mRNA is detected in the cuboidal cells of S-shaped bodies, of primitive tubules and glomerula, whereas, E-MAP-115 mRNA and protein are absent from mature podocytes which have lost their initial apico-basal polarity. The pattern of distribution of E-MAP-115 in vivo is so far unique for a MAP. Taken together, our results provide support for a role of E-MAP-115 in reorganizing the microtubule cytoskeleton during epithelial cell polarization and differentiation.

Aging↗
PubMed1991

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.

Animals↗
PubMed2006

Fine-mapping of coccidia-resistant quantitative trait loci in chickens.

Two commercial, pure broiler lines with different susceptibility to coccidiosis were used to fine-map QTL associated with the previously identified marker LEI0101, located at 259 cM on chromosome 1 and shown to be significantly associated with disease resistance. Eight additional microsatellite markers linked to LEI0101 were used for genotyping of F(1) parents and F(2) offspring (n = 314), and their associations with oocyst shedding, as a marker of disease resistance, were determined in birds experimentally infected with Eimeria maxima. Single-point analysis of 4 families showed that logarithm of odds (LOD) scores at all marker loci were > 0.5, with the exception of marker LEI0071, located at 242 cM (LOD score = 2.45). Multipoint analysis showed a maximum LOD score between LEI0071 and LEI0101 at 254 cM (LOD score = 3.74). Although the LEI0071 marker was mapped near LEI0101 by linkage analysis, the physical location of LEI0071 was not identified. Further studies to determine the physical locations of these and other markers will allow additional application association mapping techniques using single nucleotide polymorphism markers.

Animals↗
PubMed1991

Activation-associated marker proteins: peptide mapping and their expression in macrophage cell lines.

The activation-associated markers, p47b and p71/73, have been recognized as minor proteins in peritoneal and bone marrow culture-derived macrophages activated for tumor cell killing. Proteins with identical characteristics of migration on 2-dimensional gels and comparable Cleveland peptide maps are described here in macrophage cell lines that could be activated for tumor cell killing (J774A.1, RAW 264, UNC-2). Macrophage cell lines that could not be activated (P388D1 and PU5-1.8) did not express the markers. The expression of these markers in activatable macrophage cell lines strengthens their association with the activation process and provides a bulk source of the proteins for purification studies.

Animals↗
PubMed1992

Changes in microtubules, microtubule-associated proteins, and intermediate filaments during the differentiation of HL-60 leukemia cells.

The cytoskeleton is composed mainly of microtubules (MT), microfilaments, and intermediate filaments (IF) that form a structural network which connects cellular membranes, cytoplasmic organelles, and the nucleus. Since the cytoskeleton may be involved in modulating signal transduction and in the morphological and structural changes that occur during cellular proliferation and differentiation, cytoskeletal changes were measured by immunofluorescence microscopy and fluorescence-activated cell sorter analysis during the differentiation of HL-60 leukemia cells induced by retinoic acid (RA). Differentiated HL-60 cells exhibited increased staining intensity and altered organization of MT and IF, as visualized by immunofluorescence microscopy with anti-tubulin monoclonal antibody and anti-vimentin antibody, respectively. A new procedure was developed and used to measure the content of the cytoskeletal components of HL-60 cells during the process of maturation. HL-60 cells were fixed with formaldehyde in an MT-stabilizing buffer, permeabilized using L-lysophosphatidylcholine, stained for immunofluorescent measurement with antibodies specific for particular cytoskeletal components, and analyzed by flow cytometry. Terminally differentiated cells produced by exposure to RA contained larger amounts of MT and the IF vimentin. During the course of the maturation process, a transient increase in the amounts of the microtubule-associated proteins, (MAPs) MAP2 and tau, occurred. An RA-supersensitive clone, designated HL-60/S4, and an RA-resistant clone, designated HL-60/R3, were developed by mutagenization and selection. Use of these clones supported the concept that the observed changes in MT, MAPs, and vimentin were associated with the differentiation process rather than being due to other effects produced by the retinoid. Thus, the findings suggest that changes in MT, MAPs, and IF are important to the terminal maturation of leukemia cells.

Cell Differentiation↗
PubMed2005

Fine mapping by linkage and association in nuclear family and case-control designs.

This report summarizes the Genetic Analysis Workshop 14 contributions related to fine-mapping strategies, in which examining smaller regions by association with single-nucleotide polymorphisms (SNPs) can yield savings in genotyping and multiple-testing penalties. The aim of the analyses conducted in Group 7 contributions was to localize disease susceptibility loci from either the simulated or the Collaborative Study on the Genetics of Alcoholism (COGA) data within identified regions of linkage. Among the 10 contributions, most groups analyzed the simulated data, one group analyzed the COGA data only, and one group analyzed both data sets. The research questions included evaluation of new methods of analysis, as well as comparisons among alternative methods, analytic strategies, and study designs. Methods of interest included an algorithm for SNP marker ordering, a locally weighted transmission disequilibrium test statistic, a likelihood-ratio test statistic for family-based association in nuclear families, a robust test statistic for case-control association studies, and Bayesian spatial modeling methods for haplotype clustering and association. Evaluations included comparisons among confidence intervals for loci detected via linkage, effects of multiple testing adjustments and trade-offs between type I error and power, comparisons among haplotype-based (multilocus) and genotype-based (multilocus and single-locus) association analyses, and design of fine-mapping and replication studies. While several promising new approaches were identified, further development and evaluation of methods for multiple testing, regression modeling of association with multiple markers and haplotypes, and combined treatment of linkage and association data are necessary if we are to identify many of the genes that contribute to complex traits.

Alcoholism↗
PubMed1992

Age-dependent changes in the ultrastructure and in the molecular composition of rat brain microtubules.

An age-dependent increase of a cathepsin D-like protease activity that preferentially degrades high molecular weight microtubule-associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age-dependent changes in the relative amounts of MAPs and MT-associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of tau molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP-dependent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age-dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2-bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm-like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.

Aging↗
PubMed2006

Increased renal alpha-ENaC and NCC abundance and elevated blood pressure are independent of hyperaldosteronism in vasopressin escape.

Previously, we demonstrated that rats undergoing vasopressin escape had increased mean arterial blood pressure (MAP), plasma and urine aldosterone, and increased renal protein abundance of the alpha-subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter (NCC), and the 70-kDa band of gamma-ENaC (Song J, Hu X, Khan O, Tian Y, Verbalis JG, and Ecelbarger CA. Am J Physiol Renal Physiol 287: F1076-F1083, 2004; Ecelbarger CA, Knepper MA, and Verbalis JG. J Am Soc Nephrol 12: 207-217, 2001). Here, we determine whether changes in these renal proteins and MAP require elevated aldosterone levels. We performed adrenalectomies (ADX) or sham surgeries on male Sprague-Dawley rats. Corticosterone and aldosterone were replaced to clamp these hormone levels. MAP was monitored by radiotelemetry. Rats were infused with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) via osmotic minipumps (5 ng/h). At day 3 of dDAVP infusion, seven rats in each group were offered a liquid diet [water load (WL)] or continued on a solid diet (SD). Plasma aldosterone and corticosterone and urine aldosterone were increased by WL in sham rats. ADX-WL rats escaped, as assessed by early natriuresis followed by diuresis; however, urine volume and natriuresis were somewhat blunted. WL did not reduce the abundance or activity of 11-beta-hydroxsteroid dehydrogenase type 2. Furthermore, the previously observed increase in renal aldosterone-sensitive proteins and escape-associated increased MAP persisted in clamped rats. The densitometry of immunoblots for NCC, alpha- and gamma-70 kDa ENaC, respectively, were (% sham-SD): sham-WL, 159, 278, 233; ADX-SD, 69, 212, 171; ADX-WL, 116, 302, 161. However, clamping corticosteroids blunted the rise at least for NCC and gamma-ENaC (70 kDa). Overall, the increase in aldosterone observed in vasopressin escape is not necessary for the increased expression of NCC, alpha- or gamma-ENaC or increased MAP associated with "escape."

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed1984

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

Animals↗
PubMed2000

Evaluation of the latency-associated nuclear antigen (ORF73) of Kaposi's sarcoma-associated herpesvirus by peptide mapping and bacterially expressed recombinant western blot assay.

Kaposi's sarcoma (KS)-associated herpesvirus open-reading frame (ORF) 73 encodes a latency-associated nuclear antigen (LANA) that is the basis for several serologic assays. Immunoreactive epitopes were searched for by peptide mapping, and 171 cleavable, biotinylated 17-mer peptides offset by 5 residues were synthesized and screened with human serum samples by ELISA. The initial screen, which used highly reactive serum diluted 1:500, identified 38 immunoreactive peptides. These were subsequently tested on additional serum samples diluted 1:40. Thirteen peptides were more reactive with serum samples from patients with KS than with control serum samples. No single epitope was recognized by most KS patient serum samples. Combined use of these peptides did not increase test sensitivity to that of current indirect immunofluorescence assays for LANA (80%-90%). For comparison, full-length ORF73 was expressed in bacteria and analyzed by Western blot. The overall sensitivity was 67% (range, 100% among US patients with classic KS to 52% among Italian patients with classic KS). These studies suggest that LANA immunoreactivity may be due to variations in patient response or conformational epitopes.

Antigens, Viral↗