Glycosphingolipid-induced relocation of Lyn and Syk into detergent-resistant membranes results in mast cell activation.

Sphingosine, sphingosine-1-phosphate, and the more complex sphingolipid ceramide exert strong immunomodulatory effects on a variety of leukocytes. However, little is known regarding such a potential of glycosphingolipids, a class of sugar derivatives of sphingosine. Here we demonstrate that galactosylsphingosine, one of the smallest representatives of this group, accumulates in the detergent-resistant membranes resulting in the relocation of the tyrosine kinases Lyn and Syk into this compartment. The result of this is an enhanced tyrosine phosphorylation and kinase activity leading to priming and activation of mast cells by conveying a weak yet significant activation of the mitogen-activated protein kinase pathway(s). In comparison to IgE/Ag triggering, galactosylsphingosine stimulates the mitogen-activated protein kinase pathway more rapidly and favors c-Jun NH2-terminal kinase 1 activation over extracellular signal-regulatory kinase 1 and 2. At the transcription factor level, this "ultratransient signaling event" results in an activation of JunD as the predominant AP-1 component. In this respect, the effects of galactosylsphingosine are clearly distinct from the signaling elicited by other sphingolipids without the sugar moiety, such as sphingosine-1-phosphate.

The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.

Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells.

Patients with recurrent malignant brain cancer, who were receiving gene therapy by intracerebral injection of murine retroviral vector producer cells (VPCs), were monitored for the presence of replication-competent retrovirus (RCR). RCR sequences were not detected by polymerase chain reaction (PCR) in any of the 608 peripheral blood leukocyte (PBL) samples analyzed. Vector DNA sequences were detected transiently in PBL samples from a subset of 34 patients. Humoral immune responses to a retroviral core protein p30 and murine VPC were detected in some patients, most frequently in patients receiving repeated administrations of VPC. RCR was not detected in biological assays of PBLs from 41 patients who had either anti-retroviral antibodies in sera and/or vector DNA in PBLs. Our data suggest that in situ generation of RCR was not detected following intracerebral inoculation of VPCs in any of the 128 patients evaluated.

Development of amphotropic murine retrovirus vectors resistant to inactivation by human serum.

Replication-deficient amphotropic retrovirus vectors (RV) or RV-producer cells are being developed for a variety of human gene therapy strategies. One of the hurdles to in vivo use of these agents is their inactivation by components of human serum. Murine leukemia viruses (MLV), from which most current RV are derived, are known to be inactivated by human serum via activation of the classical complement cascade. Other type C retroviruses, e.g., RD114 and BaEV, are resistant to inactivation by human serum when derived from infection of human and mink cells but not murine cells. We hypothesized that amphotropic RV could be made resistant to human serum inactivation if a more appropriate producer cell could be found. To test this hypothesis, RV were made using a variety of human (293, HOS, TE671) and murine (NIH-3T3) cell types as the producer cell. The parental cell lines, RV-producer cells, and RV themselves were evaluated for sensitivity to inactivation by human serum. Results showed that the murine NIH-3T3 cell line, the NIH-3T3-derived PA317 producer cell line, and RV derived from it were all sensitive to human serum inactivation. In contrast, all human cell lines tested were resistant to lysis. RV and RV-producer cells derived from 293 cells were also resistant; RV derived from HOS cells were resistant. Surprisingly, while TE671 cells were resistant, TE671-derived RV were sensitive to inactivation. To test whether expression of the amphotropic envelope protein was responsible for conferring this serum sensitivity to the RV, env was expressed in the absence of gag and pol in TE671 cells. However, TE671 cells expressing env were resistant to human serum inactivation. These observations have important implications for use of RV and RV-producer cells for human gene therapy.

Multiple cis-acting elements are required for proper transcription of the mouse V delta 1 T cell receptor promoter.

To gain insight into the developmentally regulated expression of the mouse TCR V delta-gene segments, we have investigated the role of the 5' promoter region of the V delta 1-gene. Transient transfection assays showed that a construct encompassing 267 nucleotides upstream from the mapped transcriptional start site was capable of driving promoter activity when transfected into V delta 1+ T cells. The inclusion of an additional 459-bp 5' segment to this construct did not affect promoter activity. However, a deletion of 222 5' nucleotides from the same construct dramatically decreased promoter activity. In vivo genomic footprinting localized several protein-DNA interactions to the stretch of DNA shown to have transcriptional activity. A computer analysis revealed that the segments of DNA participating in these protein-DNA interactions were identical to the previously described cyclic AMP response element (CRE), E box, and leukemia virus E26 cis-acting elements. Transient transfection assays performed with -267 bp constructs containing mutations at each of the localized cis-acting elements revealed that the CRE, E box, and Ets elements work together in driving promoter activity and that the CRE and Ets elements are the most important for driving transcription. Gel mobility shift analyses showed that each of these cis-acting elements is capable of binding specific nuclear factors present in V delta 1-expressing cells. These data indicate that multiple transcription factors acting in concert are responsible for V delta 1 gene expression.

Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer.

Soluble derivatives of T-cell antigen receptors (TCRs) should prove invaluable for studying the interaction of these receptors with antigens and major histocompatibility complex molecules, for structural studies, and for the identification of unknown ligands. We have engineered chimeric proteins, containing the extracellular domains of the mouse V gamma 1.1-C gamma 4 and V delta 6.2-C delta (V, variable; C, constant) TCR chains fused to the hinge region, CH2 (H, heavy), and CH3 domains of human IgG1 heavy chain, and expressed them by transient transfection in COS cells. We show here that TCR gamma-IgH and TCR delta-IgH chimeric chains are produced intracellularly in significant amounts, that the two chains can assemble correctly to form disulfide-linked, glycosylated heterodimers, and that a selective mechanism allows secretion of correctly paired receptor chains into the medium. Identity of the chimeric secreted TCR gamma delta-IgH heterodimer was confirmed by immunoblot analysis using V gamma 1-specific anti-peptide antiserum and immunoprecipitation analysis using the monoclonal antibody UC7, which is shown to be specific for the TCR delta chain. In addition, the soluble TCR gamma delta-IgH heterodimer can be immunoprecipitated with the anti-clonotypic monoclonal antibody F10/56, which suggests that the fusion protein likely has a structural conformation similar to that of the native TCR. The COS cell expression system may prove useful for the production of additional TCR-IgH fusion proteins.

Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.

Mouse autoreactive gamma/delta T cells. I. Functional properties of autoreactive T cell hybridomas.

A minor population of dendritic epidermal T cells (DETC) express the V gamma 1.1C gamma 4V delta 6 T cell receptor and T cell clones and hybridomas derived from this subset constitutively secrete cytokines in culture secondary to recognition of an autoantigen. Activation of these autoreactive cells requires the use of the vitronection receptor (VNR) as an accessory molecule which interacts with the Arg-Gly-Asp-Ser (RGDS) sequence of extracellular matrix (ECM) proteins. We have compared the functional properties of C gamma 4+ hybridomas derived from newborn thymocytes and from adult spleen with the DETC hybridomas/lines in terms of their ability to secrete cytokines spontaneously and for the use of the VNR as an accessory molecule. Almost all the C gamma 4+ thymocyte hybridomas secreted cytokines spontaneously and in the majority of lines the most prominent cytokine secreted was granulocyte-monocyte colony-stimulating factor. In contrast, none of the four splenic C gamma 4+ hybridomas secreted cytokines spontaneously although all were capable of cytokine production following activation via the T cell receptor. Although the thymocyte hybridomas did not grow as adherent cell lines in culture, constitutive cytokine production required engagement of the VNR by its ligand in ECM proteins. In all cases, cytokine production could be inhibited by an anti-VNR monoclonal antibody as well as by soluble RGDS. The strong correlation of functional and molecular properties between thymocyte C gamma 4+ hybridomas and C gamma 4+ DETC suggests that the C gamma 4+ DETC may be of thymic origin and that cells with potential for autoreactivity residing in the thymus at birth may populate other peripheral locations in the mouse. The data also support the concept that the VNR, and possibly other integrins, play a role as accessory elements for autoreactive cells and may be essential for the regulation of such activity.

Cross-linking studies show that herpes simplex virus type 1 glycoprotein C molecules are clustered in the membrane of infected cells.

Chemical cross-linking using ethylene glycol succinimidyl succinate (EGS) and dithiobispropionimidate (DTBP) was performed to determine the association of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) with its nearest neighbours. Human embryonic lung (HEL) cells were infected with HSV-1 strain KOS, treated with EGS, lysed with Nonidet P40, immunoprecipitated with monoclonal antibodies specific for gC, and analysed by SDS-PAGE. These analyses demonstrated the presence of cross-linked complexes that migrated with an apparent Mr in the range 150,000 to 260,000. Two-dimensional SDS-PAGE (non-reduced and then reduced) analyses of HSV-1-infected HEL cells treated with the cleavable cross-linker DTBP demonstrated that molecules that comigrated with gC were the only components of these high Mr complexes. Immunoelectroblot (Western blot) analyses using polyclonal rabbit antiserum specific for gC verified that the high Mr complexes contained gC. These results indicated that gC molecules may be localized in the infected cell membrane as dimers.

Delta-chains of dendritic epidermal T cell receptors are diverse but pair with gamma-chains in a restricted manner.

To assess the diversity of TCR delta-gene expression in dendritic epidermal T cells (DETC), we characterized the delta-gene used by a panel of cell lines that express the gamma delta TCR on the cell surface. Northern hybridization analyses with a panel of V delta probes representing each of the six known V delta families showed that each of these lines expressed either V delta 1 or V delta 6 containing transcripts. Southern hybridization analysis with the V delta probes gave rearrangement patterns consistent with Northern hybridization results. The correlation of V delta expression with V gamma expression revealed that not only may V delta usage be restricted in DETC cells, but that the pairing of gamma- and delta-chains may not be random. In the DETC cells examined, V delta 1 chains paired exclusively with V gamma 3C gamma 1 chains and V delta 6 chains paired with C gamma 2 and C gamma 4 chains. Sequence analysis of delta-cDNA clones corresponding to the expressed delta-chains from one of the V delta 1 expressing cell lines and two of the V delta 6 expressing cell lines revealed that, although the V delta 1 gene segment sequence and one of the V delta 6 gene segment sequences were identical to previously published V delta sequences, considerable variable region diversity was generated by complex V-D-J rearrangement patterns that utilized various combinations of N-additions, D delta 2 and possibly D delta 1 segments, and J delta 1 or J delta 2 segments. These complex rearrangement patterns distinguish these DETC lines from early thymocytes and suggest that, if DETC are thymic derived, they originate from relatively mature thymic cells.

Deoxyuridine triphosphate nucleotidohydrolase: distribution of the enzyme in various rat tissues.

The distribution of deoxyuridine triphosphate nucleotidohydrolase (dUTPase) [EC 3.6.1.23] in the cytosol of various rat tissues was investigated by measuring the enzyme activity and by immunochemical analyses. Among nine rat tissues, thymus, and spleen showed the highest activities of the enzyme per gram of tissue, while intestine, stomach, lung and liver showed very low levels. Rabbit antibodies directed against purified dUTPase of anemic rat spleen showed reactivity with partially purified dUTPases from other rat tissues such as thymus, testis, or regenerating liver. Immunotitration and immunoblot experiments also indicated that the dUTPases in various rat tissues had very similar antigenicity and apparently the same subunit molecular size (Mr = 19,500), suggesting that the enzyme lacks organ-specificity. Immunoblot analysis of dUTPase protein with crude extracts from various rat tissues showed a similar distribution to that of the enzyme activity. No immuno-reactive band corresponding to the dUTPase was detected in intestine, although intestinal mucosa has been recognized as an actively proliferating tissue.

The hyperosmolarity-induced response of the ocular standing potential in mature rabbits.

The hyperosmolarity-induced response of the ocular standing potential (SP) provides a method of testing the function of the retinal pigment epithelium (RPE) without using light stimulation. In this study, the changes in potential level occurring after a short-term intravenous injection of 10 ml of 20% mannitol were determined by means of a direct current amplifier for the following groups: Group 1, normal rabbit eyes; Group 2, rabbit eyes in which the RPE was damaged by sodium iodate; Group 3, rabbit eyes in which the photoreceptors were damaged by monoiodoacetic acid; Group 4, rabbit eyes with uveoretinitis experimentally induced by Arthus-type inflammation. The following results were obtained: 1. The hyperosmolarity-induced SP response consisted of a transient increase in potential level (positive wave) in Group 1. 2. For Group 2 a transient decrease in potential level (negative wave) was obtained, i.e., a reversal of the normal positive response to negative wave. 3. A positive wave and no reversal was found for Group 3. 4. For Group 4 a negative wave and a reversal of the normal response was obtained. These hyperosmolarity-induced SP responses provide additional information concerning the possibilities of the method for studying the function of the RPE.

Deoxyuridine triphosphate nucleotidohydrolase activity and its correlation with multiplication of erythroid cells in rat spleen.

The administration of phenylhydrazine to rats brought about a marked increase in the dUTPase activity in the cytosol fractions of spleen and red blood cells; the activity began to increase with a two-day lag and reached the maximum at the 5th or 6th day of the phenylhydrazine treatment (13 and 5 times the control values in total activity in the spleen and red blood cells, respectively), and then the activity decreased. The activities of thymidine kinase and sigma-aminolevulinate synthase in the spleen and red blood cells also changed in parallel with that of dUTPase. The increases of these activities were suppressed completely by methotrexate, an inhibitor of DNA synthesis. The time courses of the enzyme activity changes in the red blood cells, however, were slightly behind those in the spleen. Thus, a close correlation was assumed between the dUTPase activity and the multiplication of erythroid cells in rat spleen.

Purification and structural characterization of herpes simplex virus glycoprotein C.

Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17% alpha-helix, 24% beta-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% alpha-helix, 10% beta-sheet, and 81% disordered structures. These data were in good agreement with the 11% alpha-helix, 18% beta-sheet, 61% beta-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.

An immunochemical study of delta-aminolevulinate synthase and delta-aminolevulinate dehydratase in liver and erythroid cells of rat.

The relationship between erythroid delta-aminolevulinate (ALA) synthase and hepatic ALA synthase in rat was analyzed immunochemically, using antibodies directed against rat liver ALA synthase and against chicken liver ALA synthase. Rat erythroid ALA synthase showed no cross-reactivity with anti-liver ALA synthase antibodies, but hepatic ALA synthases from rat, mouse, and chicken share substantial cross-reactivity with one another. These results clearly distinguish the isozyme relationship between erythroid ALA synthase and hepatic ALA synthase in rat and suggest that there may be at least two different ALA synthase genes in rat. ALA dehydratase in rat liver, on the other hand, could not be immunochemically distinguished from ALA dehydratase in rat erythroid cells when antibody against rat erythroid ALA dehydratase was used. The finding that erythroid ALA synthase is an entity different from hepatic ALA synthase may provide a clue to understanding the different features in hepatic and erythropoietic porphyrias.

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.

Relation of the extra-sequence of the precursor form of chicken liver delta-aminolevulinate synthase to its quaternary structure and catalytic properties.

Precursor and mature forms of delta-aminolevulinate (ALA) synthase were purified to near homogeneity from chicken liver mitochondria and cytosol, respectively, and their properties were compared. The enzyme purified from mitochondria had apparently the same subunit molecular weight (65,000) as that of the native mitochondrial enzyme. The enzyme purified from the cytosol fraction, however, showed a subunit molecular weight of about 71,000 which was somewhat smaller than that estimated for the native cytosolic enzyme (73,000). The enzyme purified from liver cytosol seems to have been partially degraded by some endogenous protease during the purification, but may have the major part of the signal sequence. On sucrose density gradient centrifugation, the purified mitochondrial and cytosolic ALA synthases showed an apparent molecular weight of about 140,000, indicating that both enzymes exist in a dimeric form. The ALA synthase synthesized in vitro was also shown to exist as a dimer. Apparently the extra-sequence does not interfere with the formation of dimeric form of the enzyme. The purified cytosolic ALA synthase had a specific activity comparable to that of the purified mitochondrial enzyme. Kinetic properties of the two enzymes, such as the pH optimum and the apparent Km values for glycine and succinyl-CoA, were quite similar. The extra-sequence does not appear to affect the catalytic properties of ALA synthase. The isoelectric point of the cytosolic ALA synthase was 7.5, whereas that of the mitochondrial enzyme was 7.1. This suggests that the extra-sequence in the cytosolic enzyme may be relatively rich in basic amino acids.

Identification of a high affinity growth hormone receptor in rat adipocyte membranes.

Affinity cross-linking techniques have been used to identify a growth hormone (GH) receptor in rat adipocyte membranes. Adipocytes were incubated with 125I-human GH (125I-hGH) for 2 h at 37 degrees C and washed once to remove unbound hGH. The bivalent cross-linking reagent disuccinimidyl suberate (0.4 mM) was added to the cells for 15 min at 15 degrees C. A plasma membrane-enriched fraction was prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoradiographs of gels containing samples treated with reductant revealed an intense band of apparent Mr = 134,000 which was not present when cells were incubated with an excess of hGH or bovine GH. In contrast, the intensity of the Mr = 134,000 band was not altered by the presence of a similar excess of insulin or rat prolactin during the binding step. Multiple lower molecular weight species displaying the same hormone sensitivity as the Mr = 134,000 species were also present but at much lower levels. In the absence of reductant, the affinity-labeled GH receptor migrated as a broad band of Mr = 116,000-125,000 and as a less intense band of Mr = 230,000. At low reductant concentrations, both of the hGH-labeled complexes exhibited larger apparent molecular weights (Mr (X 10(3) ) = 135 and 270), indicating the presence of intrachain disulfide bonds. At higher reductant concentrations, the Mr = 270,000 species disappeared as the Mr = 134,000 band increased in intensity. Use of a cleavable cross-linking reagent, ethylene glycol bis(succinimidyl succinate), in conjunction with two-dimensional gel electrophoresis, revealed that the Mr = 134,000 complex is composed of iodinated monomeric hGH (Mr = 22,000) bound to a membrane protein. The molecular weight of the reduced hGH receptor protein itself was calculated to be 112,000, assuming a 1:1 stoichiometry of hormone to receptor.

Nonketotic hyperglycinemia: two patients with primary defects of P-protein and T-protein, respectively, in the glycine cleavage system.

The glycine cleavage system was investigated in the livers and brains of two patients with typical nonketotic hyperglycinemia who died in the neonatal period. The overall activity of the glycine cleavage system was found to be extremely low in both the liver and brain of each patient. In one patient, the disturbance of the glycine cleavage system was due to absence of activity of the P-protein. Immunochemical analysis indicated that this resulted from an absence of the enzyme protein. In the other patient, the activity of the T-protein was undetectable in the brain and was extremely low in the liver. Clinically classic nonketotic hyperglycinemia resulted from molecular defects in two different protein components of the glycine cleavage system.