Molecular genetic changes found in human lung cancer and its precursor lesions.
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Genes, Dominant↗
Biomedical subjects
Y Kishimoto
Publications and source records attributed to Y Kishimoto.
Binding of cerebrosides and sulfatides to saposins A-D.
Saposins are a family of four small glycoproteins, all of which are derived from prosaposin, and are involved in the lysosomal hydrolysis of various sphingolipids. Results from this investigation demonstrate that saposins A-D bind to galactosyl- and glucosylceramide. The binding was highly dependent on the solution pH; maximum binding of glucosylceramide to all saposins occurred at pH 7. Maximum binding of galactosylceramide to saposins B and D occurred at a more basic pH (8.5). The binding of glucosylceramide to saposins was significantly inhibited by Mg2+, Ca2+, or Zn2+. Although maximum binding of sulfatide to saposins A, C, and D occurred at acidic pH, the binding to saposin B was maximum at pH 8.5. Saposin A also bound sphingomyelin or phosphatidylcholine at neutral pH. No significant binding was evident between these lipids and saposins B-D at any pH value. The existence of saposin-lipid complexes was further confirmed in selected samples by gel filtration, isoelectric focusing, and a TLC binding assay. We have also shown that galactosylceramide bound to saposins A-D was efficiently transported to a rat brain microsomal fraction. This result suggests that saposins and possibly their precursor, prosaposin, may be involved in membrane biogenesis such as the assembly of myelin and plasma membranes.
Animals↗
Musculotopic organization of the facial nucleus of the reeler mutant mouse.
The migration of facial motoneurons is affected by the reeler gene, and the facial nucleus of the reeler mutant is cytoarchitecturally abnormal. The present study was undertaken to compare the musculotopic organization of the reeler facial nucleus with that of the normal mouse by the retrograde horseradish peroxidase method. In the normal mouse, motoneurons supplying the nasolabial muscle were located in the lateral and dorsolateral subnuclei, those supplying the posterior auricular muscle in the ventromedial and dorsomedial subnuclei, those supplying the mentalis/platysma muscle in the ventral intermediate subnucleus of the facial nucleus, and those supplying the posterior belly of the digastric muscle in the accessory facial nucleus. This musculotopic representation on the main facial nucleus and accessory facial nucleus also appears in the reeler mouse. The musculotopic representation of the facial nucleus of the reeler mouse is thus identical to that of the normal mouse in spite of the former's cytoarchitectonic abnormalities.
Animals↗
Structural study of the oligosaccharide moieties of sphingolipid activator proteins, saposins A, C and D obtained from the spleen of a Gaucher patient.
We have determined and compared the structures of the oligosaccharide moieties of saposin A, C and D purified from the spleen of a patient with Gaucher disease. These saposins, together with saposin B, are small glycoproteins, derived from separate domains of a single precursor, prosaposin, and are required for the lysosomal hydrolysis of various sphingolipids. The characteristic features of the oligosaccharide moieties of saposin A are (a) the predominance of a fucosylated trimannosyl core structure and (b) the occurrence of several different oligomannose-type and N-acetyllactosamine-type oligosaccharides. Saposin C contains (a) a predominance of oligomannose-type oligosaccharides and monoantennary oligosaccharides and (b) the presence of four different oligosaccharides having bisecting N-acetylglucosamine residues (found only in this saposin). Saposin D is distinguished by the occurrence of oligomannose-type oligosaccharides, which comprise nearly 90% of its total oligosaccharides. The possible reasons for the unique glycosylation of each saposin is discussed.
Carbohydrate Sequence↗
Effect of saposins on acid sphingomyelinase.
The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes.
Detergents↗
Beam divergence with harmonic gyroresonance in focusing wiggler and axial field.
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Journal Article↗
The effect of carbohydrate removal on stability and activity of saposin B.
Saposin B is involved in the hydrolysis of sulfatides, GM1 ganglioside, globotriaosylceramide, and several other sphingolipids and glycerolipids by lysosomal hydrolases. Saposin B is one of four small glycoproteins (saposins) derived from prosaposin. The carbohydrate chain of saposin B was removed and deglycosylated saposin B was characterized and compared with native saposin B. Deglycosylated saposin B stimulated the enzymatic hydrolysis of ganglioside GM1 by acid beta-galactosidase and sulfatide by arylsulfatase A to the same extent as native saposin B. In addition deglycosylated saposin B bound sulfatide and GM1 ganglioside identical to native saposin B. The stability of native saposin B to proteolytic digestion was unchanged by deglycosylation. Neither native saposin B nor deglycosylated saposin B were hydrolyzed by trypsin, endoproteinase Glu-C (V-8), chymotrypsin, or a mixture of acid proteases isolated from human testis. Unlike its effect on metabolic stability, the carbohydrate chain appears to affect folding of saposin B. When native and deglycosylated saposin B were reduced under denaturing conditions and refolded under identical conditions examination of the refolded products indicated that each protein was refolded in a qualitatively different way. A human mutation in saposin B-deficient metachromatic leukodystrophy, in which its glycosylation site is eliminated, has been reported. Our observations suggest that instability of the mutated saposin B is not due to the absence of a protective effect of the carbohydrate chain on proteolysis, but is likely due to aberrant folding resulting from the absence of a carbohydrate chain.
Carbohydrates↗
Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins).
Prosaposin contains separate domains in tandem for four saposins, A, B, C, and D. These mature saposins are produced by limited proteolysis of prosaposin. They are involved in lysosomal hydrolysis of GM1 ganglioside, gluco- and galactocerebrosides, sulfatides, and sphingomyelin and other sphingolipids. Prosaposin also exists as a secretory protein in body fluids. In this investigation prosaposin was expressed in Spodoptera frugiperda cells (Sf9) by infection with baculovirus containing a full length cDNA coding for human prosaposin. Prosaposin was isolated and purified from spent culture medium of the recombinant Sf9 cell cultures as well as from human seminal plasma and milk. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of both native human prosaposins is estimated to be 66 kDa and that of recombinant prosaposin as 58 kDa. Deglycosylation of native and recombinant prosaposins yielded a protein with a molecular weight of 54 kDa and isoelectric point of 5.4. The N-terminal sequence of both native and recombinant prosaposins was identical (G-P-V-L-L-G-L-K). Like mature saposins, all prosaposins possessed stimulative activity for cerebroside beta-glucosidase (saposins A and C activity), GM1 ganglioside beta-galactosidase (saposin B activity), and sphingomyelinase (saposin D activity) but not sulfatide sulfatase (saposin B activity). Partially proteolyzed products derived from prosaposins were isolated and identified. From seminal plasma, two proteins of 48 and 29 kDa and from Sf9 culture media, two proteins of 39 and 26 kDa were characterized. N-terminal amino acid sequencing and Western blot analysis of each protein indicated that the 39-and 48-kDa proteins are cleavage products containing domains for saposins B, C, and D (trisaposins), and the 26- and 29-kDa proteins are cleavage products containing domains for saposins C and D (disaposin). These observations suggest that proteolysis of prosaposin in these tissues occurs sequentially from the N-terminal region. Proteins involved in the initial proteolysis of prosaposin were partially characterized in human testis.
Amino Acid Sequence↗
Establishment of highly metastatic tumor clones derived from a skin squamous cell carcinoma (SqC-NH), D-1 and F-3, with distinct features of pulmonary metastases.
We have established two highly metastatic tumor clones, D-1 and F-3, which have the distinct features of undergoing pulmonary metastases. Both clones were derived from a skin squamous cell carcinoma (SqC-NH), which had spontaneously occurred in DS-Nh mice. F-3 was morphologically spindle-shaped in tumor mass and adherent in culture, while D-1 was round and non-adherent. In in vitro growth, there was no significant difference between them. When the clonal tumor cells were implanted intradermally into syngeneic mice, however, the growth of D-1 was slower than that of F-3. As for the metastatic ability, both D-1 and F-3 were highly metastatic compared with the original tumor. Interestingly, the pulmonary metastases of these two clones were apparently different. In F-3-bearing mice, some round nodules were detected and no tumor cells were seen in the surrounding area around the nodules (nodule-type). In contrast, D-1 cells had initially grown from the periphery of lobes, and interstitially infiltrated the lung parenchyma area without forming nodules (diffuse-type) and the appearance was similar to lymphagitis carcinomatosa. Histologically, a remarkably smaller number of metastatic foci formed around the blood vessel for D-1 as compared with F-3. It should also be noted that some localized nodules (nodule-type both visually and histologically) were seen in the lung, when D-1 cells were inoculated in the tail vein. In addition, D-1 showed markedly higher lymphatic metastasis than F-3. These observations show that the pulmonary metastases of D-1 and F-3 differ in the mechanisms underlying the processes of tumor dissemination. We suppose a possibility that D-1 may preferentially metastasize via the lymphatics and F-3 may metastasize via the bloodstream.
Animals↗
Synthesis and characterization of a bioactive 82-residue sphingolipid activator protein, saposin C.
The sphingolipid activator protein, saposin C (also termed SAP 2), was chemically synthesized, purified, and characterized. The fully protected 82-residue protein was synthesized by automated solid-phase methods, with multiple recoupling steps resulting in a high average coupling efficiency of 98.8%. The overall yield was estimated to be approx 40%. Deprotection and cleavage of the peptide from the resin was followed by folding in the absence of chaotropic agents at pH 8.5. The protein was purified by reversed-phase high pressure liquid chromatography (HPLC) and its purity determined by capillary electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The composition of the synthetic saposin C was determined by amino acid analysis. Its sequence was verified by Edman sequence analysis of overlapping peptide fragments generated by chymotryptic and Staphylococcus aureus V8 digestions. The sequence at the C-terminus was determined by digestion with carboxypeptidase P, followed by phenylthiohydantoin (PTH) derivitization and HPLC analysis of the released amino acid residues. Deglycosylated native saposin C appeared as a lower molecular-weight species than synthetic saposin C on SDS-PAGE. This has been explained by amino acid and C-terminal analysis showing native saposin C to be two amino acids shorter at the C terminus than a deduced sequence (from cDNA) previously published. Synthetic saposin C displayed 85% of full biological activity as determined by its ability to stimulate glucocerebrosidase activity in vitro: Synthetic and native saposin C increased glucocerebrosidase catalyzed hydrolysis of 4-methylumbelliferyl beta-D-glucoside by factors of 6.0 and 7.1, respectively. Furthermore, synthetic and native saposin C share similar K(act) values (0.5 and 1.5 microM respectively) indicating that they bind to glucocerebrosidase with similar affinities.
Amino Acid Sequence↗
Expression of platelet alpha-granule proteins in a human megakaryocytic leukemia cell line (CMK 11-5).
We investigated the expression of three platelet alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fbg), in a human megakaryocytic cell line (CMK 11-5) by immunocytochemical staining, using the alkaline phosphatase anti-alkaline phosphatase (APAAP) complex method and immunoelectron microscopy of ultrathin-frozen sections. CMK 11-5, established from a Down's syndrome patient with acute megakaryoblastic leukemia, has characteristics closely resembling those of normal megakaryocytes. Under basal conditions, TSP expression was observed in the alpha-granules, whereas the other two proteins were not detected. When the cells were cultured with 12-o-tetradecanoylphorbol-13-acetate (TPA), the expression of TSP was enhanced and vWF was also detected, but not Fbg. Cells cultured in the presence of plasma for 24 h took up Fbg and stored it in their alpha-granules. These findings suggest that TSP and vWF are synthesized by CMK 11-5 cells, with the former being produced more rapidly than the latter, whereas Fbg is endocytosed from the plasma.
Cytoplasmic Granules↗
Ultrastructural analysis of a human megakaryocytic leukemia cell line (CMK11-5) following prostaglandin E1 stimulation.
We examined the ultrastructural changes occurring in a human megakaryocytic leukemia cell line, CMK11-5, in response to stimulation with prostaglandin E1 (PGE1). The addition of PGE1 to cultured cells dose-dependently caused vacuole enlargement, the development of cell surface projections, cell surface vacuole formation, and the release of a tracer protein (horseradish peroxidase) which had been incorporated into cytoplasmic granules. These morphological changes, which occurred parallel with [Ca2+]i mobilization in the CMK11-5 cells, were similar to those observed in CMK cells in response to stimulation with ADP, thrombin, and collagen. Our present findings suggest that PGE1 has a stimulatory effect on CMK11-5 cells through the induction of [Ca2+]i mobilization, and that the PGE1-induced transmembrane signaling system in megakaryocytes is completely different from that in platelets.
Alprostadil↗
Ultrastructural localization of myeloperoxidase activity in acute monoblastic leukemia.
In five patients with acute monoblastic leukemia (AMoL), the ultrastructural localization of myeloperoxidase (MPO) activity was investigated by two methods, one generally used for the detection of MPO and the other for the detection of platelet peroxidase. The MPO-positive rate achieved was lower with the former method than with the later, indicating that MPO is degraded during the fixation of AMoL cells for electron microscopy. If the ultrastructural MPO positivity of leukemic cells varies when different detection methods are used, the possibility of monocytic leukemia should be considered.
Adult↗
Ultrastructural analysis of a human megakaryocytic leukemia cell line (CMK11-5) in response to platelet agonists.
To further characterize the ultrastructure of the human megakaryocytic leukemia cell line, CMK, and its subclone CMK11-5, was observed. Stimulation with platelet agonists, ADP, thrombin, and collagen caused enlargement of vacuoles, projections of the cell surface, appearance of fibrous material in the enlarged vacuoles, and loss of alpha granules. We also found that horseradish peroxidase (HRP) incorporated into the granular structures of the cell could be transported out of the cell. The activation of CMK cells by platelet agonists indicates that human megakaryocytes may be activated in thrombotic states. CMK with particular megakaryocytic properties is a useful cell model for studying megakaryocytic function.
Blood Platelets↗
Binding and transport of gangliosides by prosaposin.
Prosaposin, the precursor of saposins A, B, C, and D, which activate lysosomal hydrolysis of sphingolipids, exists in various tissues and body fluids and is especially abundant in the nervous system. Prosaposin and saposins A,B, C, and D formed stable complexes with 13 different gangliosides as measured by an assay using column chromatography. Gangliosides of the gangliotetraose type (a series) were bound with high affinity, whereas b series gangliosides, O-acetylated gangliosides, and gangliosides with shorter carbohydrate chains, were bound with lower affinity. Prosaposin and saposins transferred gangliosides from donor liposomes to erythrocyte ghost membranes. Prosaposin also stimulated ganglioside GM1 beta-galactosidase more than mature saposins. Prosaposin exists as a secretory protein and as an integral membrane protein, and we propose that prosaposin is active as a ganglioside binding and transport protein in vivo.
Biological Transport↗
Heterogeneity of antigens related to 7S domain of type IV collagen in serum: application to patients with chronic liver disease.
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Antibody Specificity↗
Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung.
Aberrations of the p53 gene in 115 surgical specimens of non-small cell carcinomas of the lung were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products. Structural abnormalities of the p53 gene were observed in 60 tumors (52%), i.e., 8 of 14 large cell carcinomas, 24 of 58 adenocarcinomas, 25 of 37 squamous cell carcinomas, and 3 of 6 adenosquamous carcinomas. Direct sequencing of abnormal DNA fragments revealed 45 single-base substitutions, 9 deletions or insertion of a short nucleotide sequence, and 3 two-base substitutions in 57 tumors. In the other 3 tumors, loss of one of the p53 alleles was observed, with no mutation in the other allele. Allelic loss of the p53 gene was observed in 14 of 43 informative cases (33%), and in 11 of the 14 cases the remaining allele was mutated. The aberrations of the p53 gene were not limited to a particular histological type or clinical stage. Their high frequency suggests that they were involved in the genesis of non-small cell carcinomas of the lung. The mutation frequency (46%) of the p53 gene in tumors carrying mutated ras genes was essentially the same as the overall frequency in lung cancers, suggesting that accumulation of mutations in these two genes in a tumor is a random phenomenon.
Base Sequence↗
The medial dorsal nucleus is one of the thalamic relays of the cerebellocerebral responses to the frontal association cortex in the monkey: horseradish peroxidase and fluorescent dye double staining study.
To reveal the thalamic relay nucleus of the cerebellocerebral responses in the frontal association cortex, simultaneous labeling of the cerebellothalamic (C-T) terminals and the thalamocortical (T-Cx) neurons was performed in three monkeys. Horseradish peroxidase (HRP) was injected into the deep cerebellar nuclei and small doses of HRP or fluorescent dye were injected into the prefrontal cortex. The distribution of anterogradely labeled C-T terminals and retrogradely labeled T-Cx neurons was examined in the same sections. In addition to being distributed in the ventral thalamic nuclei and nucleus X, as previously reported, anterogradely labeled terminals were distributed in the ventrolateral part of the medial dorsal (MD) nucleus where retrogradely labeled thalamo-frontal projection neurons were localized. This study revealed that the ventrolateral parts of the MD together (MDmf, MDpc and MDdc) form one of the thalamic relays of the cerebelloprefrontal responses.
Animals↗