Galpha12 requires acylation for its transforming activity

The alpha subunit of the heterotrimeric G protein G12, harboring a mutation in the GTP binding domain (Q229L), behaves as a potent oncogene in NIH 3T3 cells. This alpha subunit, like most other G protein alpha subunits, undergoes palmitoylation, the reversible posttranslational addition of palmitate to cysteine residues. We investigated the role of palmitoylation of alpha12 in membrane localization and transformation efficiency and whether another lipid modification, myristoylation, could substitute for palmitoylation. NIH 3T3 cells were stably transfected with plasmids that expressed the wild-type alpha12, the constitutively active Q229L (QL) mutant, and mutants in which C11 was changed to S (C11S) and S2 and R6 were changed to G and S, respectively (S2G). Incorporation of [3H]palmitate was found in the endogenous and expressed alpha12 but not in the C11S mutants. Incorporation of [3H]myristate was found only in the S2G mutants. The wild type, QL mutant, and all the acylation mutants were found in the particulate fraction. Cells expressing the nonpalmitoylated C11S,QL mutant did not undergo transformation. The S2G mutation in the nonpalmitoylated C11S,QL mutant restored the transformation efficiency to a greater level than that of the palmitoylated QL mutant as measured by foci formation, growth in soft agar, and growth rate. Palmitoylation was critical for the transformation efficiency of alpha12 but not specifically required because myristoylation could substitute for these functions.     

Characterization of adhesive interactions between human endothelial cells and megakaryocytes

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.     

Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.     

Binocular processes in vernier acuity

The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabolites of various radiolabeled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phospholipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, steric, and oleic acids. [3H]oleic acid, [14C]linoleic acid, [14C]gamma-linolenic acid and [14C]eicosatrienoic acids were incorporated into salivary gland phospholipids but underwent little change including elongation and/or desaturation to arachidonate. Ingested [3H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arachidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal.     

A dinucleotide deletion results in defective membrane anchoring and circulating soluble glycoprotein Ib alpha in a novel form of Bernard-Soulier syndrome

The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIb alpha, GPIb beta, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIb alpha was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIb alpha. This mutation (GPIb alpha deltaAT) causes a frame shift that alters the amino acid sequence of GPIb alpha within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIb alpha deltaAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIb alpha. Transient expression of recombinant GPIb alpha deltaAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIb alpha deltaAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GP beta-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIb alpha starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIb alpha polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIb alpha.     

Contribution of phosphoinositides and phosphatidylcholines to the production of phosphatidic acid upon concanavalin A stimulation of rat thymocytes

Stimulation of rat thymocytes by concanavalin A (Con A) results in a very early increase of the cellular level of phosphatidic acid (PA), while that of diacylglycerol (DAG) was not affected. As the biological activity of PA is very likely to be determined by its molecular species composition, the present study aims to investigate the pathways leading to the production of PA in Con A-stimulated rat thymocytes. Prelabeling the cells with [3H]arachidonic acid, [3H]myristic acid, [3H]choline, or [14C]lysophosphatidylcholine allowed us to determine that PA is formed by both phosphoinositide (PIs) and phosphatidylcholine (PC) hydrolysis. We then investigated whether PA derived from PC was formed by phospholipase C (PLC) or phospholipase D (PLD) hydrolysis. In the presence of 1-butanol, the production of phosphatidylbutanol was only observed in tetradecanoyl phorbol acetate (TPA)-stimulated cells. The use of a specific PC phospholipase C inhibitor resulted in a decrease of Con A-stimulated PA production in cells labeled with [3H]myristate. When cells were labeled with [3H]choline, only TPA stimulation induced a release of labeled choline. All together, these experiments suggest that PA is originated from two phospholipid sources, predominantly PI via PLC hydrolysis and to a lesser extent PC, by PLC hydrolysis also. Molecular species analyses by reverse phase HPLC are in agreement with this hypothesis, as diacyl-GP molecular species composition is similar to that of diacyl-GPC and DAG in resting cells, but resembles that of diacyl-GPI in Con A-treated cells. Thus, in stimulated cells, the amount of 18:0/20:4 species doubled while those of saturated and monounsaturated species decreased.     

Origin of platelet-derived growth factor in megakaryocytes in guinea pigs

Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte.     

Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1alpha

Eukaryotic elongation factor 1alpha (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

Measles virus fusion protein is palmitoylated on transmembrane-intracytoplasmic cysteine residues which participate in cell fusion

[3H]palmitic acid was metabolically incorporated into the viral fusion protein (F) of Edmonston or freshly isolated measles virus (MV) during infection of human lymphoid or Vero cells. The uncleaved precursor F0 and the F1 subunit from infected cells and extracellular virus were both labeled, indicating that palmitoylation can take place prior to F0 cleavage and that palmitoylated F protein was incorporated into virus particles. [3H]palmitic acid was released from F protein upon hydroxylamine or dithiothreitol treatment, indicating a thioester linkage. In cells transfected with the cloned MV F gene, in which the cysteines located in the intracytoplasmic and transmembrane domains (Cys 506, 518, 519, 520, and 524) were replaced by serine, a major reduction of [3H]palmitic acid incorporation was observed for F mutated at Cys 506 and, to a lesser extent, at Cys 518 and Cys 524. We also observed incorporation of [3H]palmitic acid in the F1 subunit of canine distemper virus F protein. Cell fusion induced by cotransfection of cells with MV F and H (hemagglutinin) genes was significantly reduced after replacement of Cys 506 or Cys 519 with serine in the MV F gene. Transfection with the F gene with a mutation for Cys 518 abolished cell fusion, although less mutant protein was detected on the cell surface. These results suggest that the F protein transmembrane domain cysteines 506 and 518 participate in structures involved in cell fusion, possibly mediated by palmitoylation.     

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.     

Non-smoking as risk factor for Alzheimer''s disease?

The short-term effect of dexamethasone, a synthetic glucocorticoid, on both fatty acid and cholesterol synthesis has been investigated in rat hepatocyte cultures. Within 4h following hormone addition to the cultures, a noticeable stimulation of labelled acetate incorporation into fatty acids was observed. Similar behaviour was noticed when [3H]H2O was used as an independent index of the lipogenic activity. In the same cultures, however, cholesterol synthesis from both [14C]acetate or [3H]H2O was significantly reduced by dexamethasone addition. In these conditions, no significative variation of cholesterol synthesis starting from labelled mevalonate was observed.     

Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Noradrenaline- and ionomycin-stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H-fatty acids showed that two different classes of phosphatidylcholine, separated by two-dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P-phospholipids extracted from [32P]Pi-labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.     

Evaluation of ploidy of mature canine megakaryocytes, using Feulgen staining and microspectrophotometry

OBJECTIVE: To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count. ANIMALS: Group A contained 8 and group B contained 11 adult dogs. PROCEDURE: Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry. RESULTS: Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P < 0.05) different from each other for both groups. The model ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N. CONCLUSIONS: Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value. Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis.     

A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass culture-derived exoantigens

The supernatants of in vitro cultures of Babesia divergens Rouen 1987 in human erythrocytes, obtained by using a semidefined medium based on human high-density lipoproteins, were fractionated by gel filtration chromatography into four fractions, F1 to F4. The crude supernatant as well as each fraction adjuvanted with Quil-A protected gerbils from mortality due to a homologous infectious challenge. Analysis of the humoral response of the 10 protected gerbils with fraction F4, containing major proteins with molecular masses lower than 50 kDa, showed that a few antigens (from 50 to 17 kDa) could be important candidates for an improved vaccine against B. divergens babesiosis. As an immunodominant response was directed against the 37-kDa antigen (Bd37) in two different B. divergens strains tested, a polyclonal antibody directed against Bd37 was produced in a rabbit. In an immunofluorescence assay, the anti-Bd37 antiserum strongly labelled small internal vesicles of the merozoites and the cell surface was diffusely labelled after fixation, whereas on live merozoites, this labelling was not observed. [3H]glucosamine-radiolabelling experiments demonstrate that Bd37 is a glycoprotein. The Bd37 protein can also be labelled with [14C]palmitate but not with [3H]myristic acid. In Triton X-114 temperature phase partitioning of B. divergens-infected erythrocyte extracts, Bd37 was exclusively found into the detergent phase, indicating that the palmitoylated Bd37 protein was in the membrane fraction. In the in vitro supernatant, the glycoprotein Bd37 was found in a nonpalmitoylated form, indicating excretion and/or release of the glycoprotein from the merozoite.     

Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) is unique among the nitric oxide synthase family of proteins due to the presence of an N-myristoylation consensus sequence elucidated from the cloning of its cDNA. Although eNOS was metabolically labeled with [3H]myristic acid and mutation of glycine 2 in the N-myristoylation consensus sequence changed the particulate localization of the enzyme to a cytosolic form, the definitive characterization of eNOS as an N-myristoylprotein has not been demonstrated. Therefore, the purpose of the present study was to determine the nature of the fatty acid incorporated into eNOS. Wild-type or G2A mutant (mutation of glycine 2, the myristic acid acceptor site, to alanine) eNOS-transfected COS cells and bovine aortic endothelial cells (BAEC) were metabolically labeled with [3H]myristic acid for 5 h. The radiolabel was primarily incorporated into membrane-associated eNOS from wild-type transfected COS cells and cultured BAEC but not into the mutant eNOS from G2A-transfected COS cells. Qualitatively similar amounts of immunoreactive protein were found in wild-type and G2A-transfected cells. In addition, linkage of the radiolabel to eNOS was insensitive to hydroxylamine treatment, and incorporation of the radiolabel into eNOS was abolished by cyclo-heximide. Chemical analysis of the fatty acid released by acid methanolysis of labeled eNOS verified the 3H-labeled fatty acid as protein-bound myristic acid. These results unequivocally demonstrate that eNOS incorporates myristic acid via an amide linkage with the amino-terminal glycine of the enzyme as a co-translational modification.     

Interferon-gamma enhances megakaryocyte colony-stimulating activity in murine bone marrow cells

We have demonstrated previously that interferon-gamma (IFN-gamma) accelerates platelet recovery in mice with 5-FU induced-marrow aplasia in vivo. However, the mechanism for the regulation of megakaryocyte development induced by IFN-gamma in bone marrow cells in vivo remains unknown. To further study the effects of IFN-gamma on megakaryocyte development, various steps during IFN-gamma-mediated accelerated differentiation of the megakaryocytes were investigated in serum-free cultures of murine bone marrow cells in vitro. IFN-gamma markedly induced acetylcholine esterase (AChE) activity, a marker of murine megakaryocytic cells, accompanied by increased colony formation of the megakaryocyte lineage. A prominent increase in megakaryocyte number was observed after IFN-gamma treatment. All of these effects were dependent on the presence of IL-3, and, therefore, these results suggest that IFN-gamma acts as a megakaryocyte potentiator (Meg-POT). However, IFN-gamma did not enhance megakaryocyte maturation with respect to increase in cell size. The effects of IFN-gamma on megakaryocyte maturation were similar to those observed after treatment with higher doses of IL-3 alone. Meg-POT is defined as a factor that induces megakaryocyte maturation. Since IFN-gamma enhanced IL-3-dependent megakaryocyte colony formation and proliferation rather than megakaryocyte maturation, the effects on megakaryocyte development, which were induced by IFN-gamma treatment, seem to be different from the effects of a Meg-POT. We, therefore, propose a new function for IFN-gamma as an enhancer of megakaryocyte colony-stimulating factor activity. The effect of IFN-gamma in vitro appears to correlate well with the acceleration of platelet recovery in vivo.     

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.     

Juvenile psoriatic arthritis: followup and evaluation of diagnostic criteria

Platelet counts in newborns are similar to those of adults and children. However, newborn infants admitted to intensive care nurseries have a high prevalence of thrombocytopenia. The mechanisms responsible for the increased susceptibility to thrombocytopenia are not known. In addition, some studies have documented functional abnormalities in newborn platelets. In an effort to understand differences between platelets in newborns and in adults, we examined megakaryocyte ploidy in bone marrow from fetuses and compared it with bone marrow from adults, using a modified Feulgen stain to measure DNA of individual megakaryocytes. Faced with small fixed tissue samples, we developed a technique for use on bone marrow biopsies to estimate megakaryocyte ploidy and compared the results obtained with this method to those obtained from bone marrow aspirates. This study demonstrated that the overall mean ploidy of fetal megakaryocytes is decreased compared with adults. Additionally, fetal megakaryocyte ploidy increases as megakaryocyte maturation increases, but not to the same extent that adult megakaryocyte ploidy increases with megakaryocyte maturation. Over the gestational period studied, there was no relationship between gestational age and mean ploidy. The small size, shift to a less mature population, and decreased ploidy of fetal megakaryocytes indicate that there are differences in the post mitotic phase of megakaryocyte development in the fetus. Such differences may be related to quantitative and qualitative platelet abnormalities in the newborn. Understanding the physiology and regulation of megakaryocytopoiesis in the fetus and newborn will be valuable in determining the pathophysiologic basis of platelet dysfunction in the newborn.