Search PubMed⌕ Search

Biomedical subjects

R Hyman

Publications and source records attributed to R Hyman.

At least 19 recordsLinked to original sources

Adding value to clinical data by linkage to a public death registry.

We describe the methodology and impact of merging detailed statewide mortality data into the master patient index tables of the clinical data repository (CDR) of the University of Virginia Health System (UVAHS). We employ three broadly inclusive linkage passes (designed to result in large numbers of false positives) to match the patients in the CDR to those in the statewide files using the following criteria: a) Social Security Number; b) Patient Last Name and Birth Date; c) Patient Last Name and Patient First Name. The results from these initial matches are refined by calculation and assignment of a total score comprised of partial scores depending on the quality of matching between the various identifiers. In order to validate our scoring algorithm, we used those patients known to have died at UVAHS over the eight year period as an internal control. We conclude that we are able to update our CDR with 97% of the deaths from the state source using this scheme. We illustrate the potential of the resulting system to assist caregivers in identification of at-risk patient groups by description of those patients in the CDR who were found to have committed suicide. We suggest that our approach represents an efficient and inexpensive way to enrich hospital data with important outcomes information.

Death Certificates↗

Hyaluronan binding by cell surface CD44.

CD44 is the primary cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. Here we determined the relative avidities of unlabeled hyaluronan preparations for cell surface CD44 by their ability to block the binding of fluorescein-conjugated hyaluronan to a variety of cells. We show that hyaluronan binding at the cell surface is a complex interplay of multivalent binding events affected by the size of the multivalent hyaluronan ligand, the quantity and density of cell surface CD44, and the activation state of CD44 as determined by cell-specific factors and/or treatment with CD44-specific monoclonal antibody (mAb). Using low M(r) hyaluronan oligomers of defined sizes, we observed monovalent binding between 6 and 18 sugars. At approximately 20 to approximately 38 sugars, there was an increase in avidity (approximately 3x), suggesting that divalent binding was occurring. In the presence of the inducing mAb IRAWB14, monovalent binding avidity was similar to that of noninduced CD44, but beginning at approximately 20 residues, there was a dramatic and progressive increase in avidity with increasing oligomer size ( approximately 22 < 26 < 30 < 34 < 38 sugars). Kinetic studies of binding and dissociation of fluorescein-conjugated hyaluronan indicated that inducing mAb treatment had little effect on the binding kinetics, but dissociation from the cell surface was greatly delayed by inducing mAb.

Animals↗

Adhesion of lymphoid cells to CD44-specific substrata: the consequences of attachment depend on the ligand.

Interactions between cell-surface adhesion receptors and immobilized specific substrata can exert profound effects on cell morphology. Using phase-contrast microscopy, we show that CD44-expressing mouse lymphoid cells display a spread morphology when adhering to CD44-specific monoclonal antibody (mAb) immobilized on plastic. This spread morphology is different from that of these same cells when adhering to immobilized hyaluronan, the natural ligand of CD44. Morphometric measurements, in combination with intracellular actin staining and fluorescence microscopy, revealed that the adhesion of lymphoid cells to hyaluronan required essentially no cytoskeletal reorganization and resulted in no fundamental change in morphology. On the other hand, cells adhering to immobilized CD44-specific mAb rearranged their actin structure and established multiple membrane contact sites (spread). Cell spreading on antibody, but not attachment to hyaluronan, was inhibited by cytoskeleton-disrupting agents. Transfection of CD44-negative lymphoid cells with full-length and tailless CD44 enabled these cells to bind to both immobilized hyaluronan and mAb. However, the transfectant lacking the cytoplasmic tail of CD44 spread only transiently on the antibody-coated surface. Our results suggest that CD44 may mediate lymphocyte attachment to its carbohydrate ligand hyaluronan by mechanisms broadly similar to those used by selectins. When immobilized CD44-specific antibody is the ligand, however, CD44 may regulate the activity of the cytoskeleton by mechanisms broadly similar to those used by integrins. In the latter case, the cytoplasmic domain of CD44 contributes to cell spreading.

Actins↗

The role of the CD44 cytoplasmic and transmembrane domains in constitutive and inducible hyaluronan binding.

The role of the CD44 cytoplasmic domain in cells displaying constitutive or inducible hyalu ronan (HA) binding mediated by wild-type CD44 was investigated using mutant CD44 constructs with the cytoplasmic domain truncated or replaced by foreign sequences. In cell lines in which wild-type CD44 bound HA constitutively, chimeric constructs consisting of the CD44 external domain and the transmembrane plus cytoplasmic domains of beta5 integrin bound as well as wild-type CD44, arguing that the specific sequence of the cytoplasmic and transmembrane domains was not critical for HA binding by the external domain. The cytoplasmic domain sequence did not contribute to the 'inducible' phenotype in cell lines which did not bind HA constitutively, but which could be induced to bind by CD44-specific mAb. Tailless CD44 was inducible in these cells, as was chimeric CD44 with the integrin beta5 cytoplasmic domain. Dimer- or oligomerization of CD44 by addition of AP1510 to cells containing CD44 / FKBP chimeric constructs caused a modest enhancement of HA binding in cells that bound constitutively, but did not alter the inducible phenotype. This result suggests that clustering of CD44 from inside the cell is not a sufficient 'inside-out' signal to activate HA binding in inducible cells.

Amino Acid Sequence↗

Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3'-kinase is essential for male fertility.

The c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R) is required for normal haematopoiesis, melanogenesis and gametogenesis. However, the roles of individual Kit/SCF-R-induced signalling pathways in the control of developmental processes in the intact animal are completely unknown. To examine the function of SCF-induced phosphatidylinositol (PI) 3'-kinase activation in vivo, we employed the Cre-loxP system to mutate the codon for Tyr719, the PI 3'-kinase binding site in Kit/SCF-R, to Phe in the genome of mice by homologous recombination. Homozygous (Y719F/Y719F) mutant mice are viable. The mutation completely disrupted PI 3'-kinase binding to Kit/SCF-R and reduced SCF-induced PI 3'-kinase-dependent activation of Akt by 90%. The mutation induced a gender- and tissue-specific defect. Although there are no haematopoietic or pigmentation defects in homozygous mutant mice, males are sterile due to a block in spermatogenesis, with initially decreased proliferation and subsequent extensive apoptosis occurring at the spermatogonial stem-cell level. In contrast, female homozygotes are fully fertile. This is the first report so far demonstrating the role of an individual signalling pathway downstream of Kit/SCF-R in the intact animal. It provides the first in vivo model for male sterility caused by a discrete signalling pathway defect affecting early germ cells.

Amino Acid Substitution↗

Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding.

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.

Antibodies, Monoclonal↗

CD44 structure and function.

In this review we discuss the structural elements of CD44 that have been shown to be involved in specific functions. To this end, we focus primarily on experiments in which CD44 constructs are transfected into cells whose function is then assayed. The hyaluronan binding function of CD44 has been assayed in cell lines and in fusion proteins, termed CD44-Igs, consisting of the external domain of CD44 coupled to the hinge, CH2 and CH3 regions of human IgG1. These studies have shown that hyaluronan binding by CD44 is regulated by the cells in which it is expressed, and that at least part of this regulation is determined by cell specific posttranslational modifications, especially N-glycosylation, of CD44 itself. Variant isoforms of CD44 determined by alternative splicing of 11 optional exons in the middle of the gene determine additional functions of CD44, as well as contributing to the regulation of hyaluronan binding. Soluble CD44 may modulate the function of cell surface CD44. The cytoplasmic domain of CD44 contributes to ligand binding in a way that remains obscure. It also determines membrane localization in polarized epithelial cells, and is probably involved in CD44 interactions with the cytoskeleton and in mediating post-ligand binding events.

Animals↗

CD44 in inflammation and metastasis.

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered.

Animals↗

Cognitive deficits and psychopathology in elderly schizophrenic patients.

The authors investigated the syndromal and cognitive profiles of 25 DSM-III-R older schizophrenic inpatients with continuous acute psychotic symptoms and compared them with 20 younger schizophrenic patients by means of a multidimensional assessment battery. Subjects were medically well and without neurological comorbidity and were comparable in length of current hospitalization and medication regimens. There were no significant differences between the two groups on various symptom rating scores or on neurological variables. The older group's mean scores for various cognitive measures did not reach the value for senile dementia. They also scored significantly better on a memory test and on formal cognitive functions. These findings support the notion of a stable encephalopathy, rather than a dementia-like process, underlying the course of the illness. Authors discussed limitations and implications of these findings.

Adult↗

Tiazofurin enhances the anabolism and toxicity of 5-fluorouracil.

Tiazofurin, a clinically active anticancer agent, is undergoing additional clinical testing in combination with other agents. We found that tiazofurin is an effective biochemical modulator of 5-fluorouracil anabolism. Pretreatment of cultured L1210 cells with tiazofurin at concentrations of 1-100 microM results in an increase in the rate of conversion of 5-fluorouracil to phosphorylated metabolites. Concentrations of tiazofurin effective in increasing 5-fluorouracil anabolism cause a corresponding increase in the 5-phosphoribosyl-1-pyrophosphate pool. Studies in mice show that tiazofurin increases the lethal toxicity of 5-fluorouracil and increases the antitumor effectiveness of low doses of 5-fluorouracil: however, the combination is not more effective than an optimal dose of 5-fluorouracil given alone. These results indicate that caution should be exercised in the concurrent use of tiazofurin with other drugs, particularly 5-fluorouracil, that require 5-phosphoribosyl-1-pyrophosphate for activation or that are affected by a decrease in pyrimidine nucleotide synthesis.

Animals↗

Central brain atrophy in childhood AIDS encephalopathy.

OBJECTIVE: To determine differential patterns of brain atrophy in pediatric AIDS encephalopathy. DESIGN: We measured the bicaudate, bifrontal, and ventricle-brain ratio in brain magnetic resonance imaging scans of 42 control children, nine children with progressive AIDS encephalopathy, 25 AIDS children without progressive encephalopathy, and 23 children with cerebral atrophy of other causes. RESULTS: When compared with controls, encephalopathy patients showed significantly increased bicaudate and ventricle-brain ratios, but no significant increase in bifrontal ratio, whereas children with brain atrophy from causes other than AIDS showed increases in all three ratios. CONCLUSION: Children with AIDS encephalopathy demonstrate a specific pattern of brain atrophy distinct from other etiologies: a central atrophy, primarily affecting the subcortical white matter or the basal ganglia regions.

AIDS Dementia Complex↗

Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation.

CD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence-activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N-linked glycosylation. Furthermore, culture in tunicamycin for 2-3 d converted parental and inducible cell lines into cells showing constitutive CD44-mediated HA binding. Also, removal of cell surface glycosaminoglycan chains by culture of cells in p-nitrophenyl beta-D-xylopyranoside or treatment with chondroitinase ABC resulted in conversion of cells with an inactive CD44 receptor to an inducible state. These results indicate that carbohydrate side chains of CD44 and/or other molecules on the cell surface that interact with CD44 are potentially involved in regulating the HA-binding function of CD44 on the cell surface.

Amino Acid Sequence↗

Role of CD44 cytoplasmic domain in hyaluronan binding.

The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T-lymphoma cells was evaluated by flow cytometry using fluorescein-conjugated HA (Fl-HA). Previous studies showed that wild-type hematopoietic CD44 bound Fl-HA when expressed in AKR1, but that truncated "tailless" CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide-bonded dimer of CD44, formed by substituting the transmembrane region of CD3 zeta chain for that of CD44, binds Fl-HA, even when the cytoplasmic domain of the CD44 dimer is absent. We conclude that dimerization of CD44 abrogates the requirement for the cytoplasmic domain, suggesting that the cytoplasmic domain of CD44 may contribute to HA binding by promoting CD44 clustering. These results suggest that changes in the distribution of CD44 on the cell surface, induced by molecular interactions either from within the cell or from outside, may regulate its role as a receptor. Further studies sought to localize the region of the CD44 cytoplasmic domain contributing to HA binding by the construction of a series of cytoplasmic domain truncation mutants and internal deletion mutants. All of the mutant CD44 molecules bound Fl-HA similarly to wild-type CD44. Thus, it was not possible to assign the function mediating HA binding to a specific region of the cytoplasmic domain, suggesting either that multiple regions of the cytoplasmic domain can promote enhancement of HA binding, or that the role of the cytoplasmic domain in mediating this function does not require a specific amino acid sequence.

Amino Acid Sequence↗

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

Amino Acid Sequence↗

Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts.

The hyaluronan receptor CD44 is an abundant glycoprotein expressed on a variety of different cell types. In fibroblasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extraction. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton. In this study we examined the structural features of CD44 required for its Triton X-100 insolubility in murine fibroblasts. We expressed in L cells the wild-type hematopoietic form of CD44, a mutant CD44 lacking the cytoplasmic domain, and two mutant CD44 molecules with substituted transmembrane domains. Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined. No difference in detergent solubility was observed between wild-type and tailless mutant-transfected molecules. However, both CD44 mutants with a heterologous transmembrane domain, derived from either the CD3 zeta chain or CD45, were completely soluble in Triton X-100. These results demonstrate that the transmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells. No obvious colocalization of CD44 and actin stress fibers was observed before or after treatment with cytochalasin D, and no change in the detergent extraction profile of wild-type and mutant CD44 molecules was effected by cytochalasin D. In equilibrium density sucrose gradients the Triton-insoluble CD44 component was found in the low density fractions, indicating an association with Triton X-100-insoluble lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response.

Though CD44 functions as a cell surface receptor for hyaluronan (HA) in some cell lines, most normal hematopoietic cells expressing CD44 do not bind HA. Certain CD44-specific monoclonal antibodies (mAbs) can rapidly induce CD44-mediated HA binding in normal murine T cells. This observation suggests that in vivo mechanisms may exist for activating the HA receptor function of CD44 on normal T cells. Here, it is shown that up to one third of splenic T cells are capable of CD44-mediated binding of fluorescein-conjugated HA (Fl-HA) during an in vivo allogeneic response. HA binding activity peaks at 7-8 d postinjection and declines rapidly. These rapid kinetics could be the result of transient activation of CD44 function and/or differentiation or expansion of short-lived population(s) that have constitutive HA-binding function. Both CD4 and CD8 T cells are included in the HA binding population which is strongly CD44 positive. After separation of HA-binding cells from nonbinding cells by cell sorting, it is shown that almost all cytotoxic effector cells are found in the HA-binding population. However, there is no evidence that CD44-mediated HA recognition is directly involved in the killing of target cells, since cytotoxicity could not be inhibited by CD44-specific mAbs that inhibit HA binding or by soluble HA. PCR amplification of cDNA reverse transcribed from RNA of sorted HA-binding cells indicated no evidence for CD44 isoforms other than the standard (hematopoietic) form. Though CD44 expression is known to be elevated upon T cell activation, and, as shown here, HA-binding function is induced in a portion of CD44-expressing T cells including cytotoxic effector cells, the role of CD44 and HA-recognition in immune responses is not known.

Animals↗

Effect of high-protein diet on pyrimidine synthesis and response to PALA in mouse tissues.

BACKGROUND: High-protein diets have been found to protect mice from the lethal effects of cytotoxic pyrimidine analogues and to reduce the toxicity of the antipyrimidine fluorouracil (5-FU), but the biochemical explanation for these effects is not known. PALA potentiates the chemotherapeutic efficacy of 5-FU, and each of the two agents can produce dose-limiting intestinal toxic effects. We have shown that intraperitoneal infusion of ammonium chloride stimulates intestinal de novo pyrimidine synthesis. This stimulation with excess ammonia, which can also result from high-protein intake, is dependent on the presence of carbamoyl phosphate synthetase I, an enzyme in the liver and intestine but not in most tumors. These findings suggest that a high-protein diet can stimulate pyrimidine synthesis in the liver and intestine but leave it unchanged in tumor tissue. PURPOSE: The purpose of this study was to determine whether varying dietary protein causes pharmacologically relevant and preferential changes in de novo pyrimidine synthesis. METHODS: Mice were fed diets containing 18%, 35%, or 50% casein. Dietary effects on de novo pyrimidine synthesis were measured in the intestine, liver, and B16 mouse melanoma in mice treated with PALA and in untreated mice. De novo synthesis was measured by infusion of [15N]alanine into intact animals, determination of 15N incorporation into uracil by use of gas chromatography-mass spectrometry, and calculation of the fraction of the uracil nucleotide pool formed by de novo synthesis. RESULTS: In mice on a 50% casein diet (high protein), de novo pyrimidine synthesis increased substantially in the liver and intestine, compared with synthesis in mice receiving 18% casein. Increase in pyrimidine synthesis in B16 tumor tissue was negligible. The high-protein diet protected the intestine and liver from depletion of uracil nucleotide pools by PALA, and toxicity in tumor-free animals was reduced, as determined by mortality after PALA treatment. Sensitivity of the B16 tumor to the biochemical and cytotoxic effects of PALA was not diminished. CONCLUSIONS: We propose that the basis for these effects of a high-protein diet is the generation of excess carbamoyl phosphate in tissues containing carbamoyl phosphate synthetase I. This carbamoyl phosphate can stimulate de novo pyrimidine synthesis and compete with drugs that interact with enzymes of the de novo pathway, thereby selectively protecting the liver and intestine. IMPLICATIONS: These data provide a biochemical explanation for reported effects of high-protein diet on toxicity of antipyrimidines like 5-FU. Studies are underway to determine if stimulation of pyrimidine synthesis by excess ammonia improves therapy with 5-FU alone or combined with PALA.

Animals↗

Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N-acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N-acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl-phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.

Cells, Cultured↗