Is hexamerin receptor a GPI-anchored protein in Achaea janata (Lepidoptera: Noctuidae).

The process of uptake of hexamerins during metamorphosis from insect haemolymph by fat body cells is reminiscent of receptor-mediated endocytosis. Previously, we had identified a hexamerin-binding protein (HBP) and reported for the first time that uptake of hexamerins is dependent on the phosphorylation of HBP partly by a tyrosine kinase, which is, in turn, activated by 20-hydroxyecdysone (20E). However, the exact nature of HBP and the mechanism of interaction are still unknown. Here we report the possibility of HBP being a GPIanchored protein in the fat body of Achaea janata and its role in the tyrosine-kinase-mediated phosphorylation signalling. Digestion of fat body membrane preparation with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the hexamerin binding to HBP was inhibited after digestion. Hexamerin overlay assay (HOA) of co-immunoprecipitated in vitro phosphorylated HBP showed exclusive binding to ~120 kDa protein. Lectin-binding analysis of hexamerins revealed the presence of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GluNAc), whereas HBP showed the presence of GalNac alone. Mild chemical deglycosylation studies and binding interaction in the presence of sugars revealed that glycan moieties are possibly not involved in the interaction between HBP and hexamerins. Taken together, these results suggest that HBP may be a GPI-anchored protein, and interaction and activation of HBP is through lipid-linked non-receptor src tyrosine kinases. However, additional studies are needed to prove that HBP is a GPI-anchored protein.

Study on the clinical features of patients with aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome and typical paroxysmal nocturnal hemoglobinuria / 中国实用内科杂志

Objective To explore the differences of clinical features and relationship between aplastic anemia paroxysmal nocturnal hemoglobinuria syndrome(AA PNH syndrome)and typical paroxysmal nocturnal hemoglobinuria(t PNH).Methods A case control study on the discrepancies of clinical and laboratory features between patients with AA PNH syndrome and t PNH was carried out.Results Compared with t PNH,AA PNH syndrome showed following features:①Lower frequencies of venous thrombosis,jaundice and enlarged liver or spleen.②Higher percentages of pancytopenia and bone marrow hypoplasia.③Lower percentages of positive hemolysis tests.The percentages of CD55 and CD59 of peripheral blood cells were not significantly different in most cases of both groups.④Immunoglobulins and subgroups of T lymphocytes were normal in cases of both groups.⑤Adrenocortical hormone was effective in cases of both groups.Conclusion AA PNH syndrome shares a same pathophysiology with t PNH;CD55 and CD59 tests can improve the diagnosis of AA PNH syndrome.

CD59 Analysis and Screening of PIG-A Gene Mutation in Aplastic Anemia, Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria / 대한임상병리학회지

BACKGROUND: Aplastic anemia(AA), myelodysplastic syndrome(MDS) and paroxysmal nocturnal hemoglobinuria(PNH) are hematopoietic stem cell disorders. To investigate the pathogenetic links, we performed CD59 analysis and screened PIG-A gene mutation in the patients with AA, MDS, and PNH developed from AA or MDS. METHODS: We analyzed the proportion of the patients with CD59-deficient cells by flow cytometry for CD59 in 42 patients with AA or MDS and eight patients with PNH developed from AA or MDS. The mutations of PIG-A gene were screened with dideoxy fingerprinting(ddF). RESULTS: In normal controls, the proportion of the RBCs normally expressing CD59 was 97.2+/-1.9% and that of the granulocytes was 98.4%+/-1.5%. In patients with AA or MDS, 9.5%(4/42) had CD59 deficiency on RBCs and 10.3%(3/29) on granulocytes. In patients whose CD59 on both RBCs and granulocytes were analyzed, 17.2%(5/29) showed reduced CD59 in at least one cell lineage. Screening test using ddF revealed abnormal band shifts in three patients with PNH developed from AA or MDS. CONCLUSION: We found the presence of PNH clones in the patients with AA or MDS. And it was indirectly confirmed by ddF that PNH arisen from AA or MDS is also associated with the mutations of PIG-A gene as classical PNH. CD59 analysis in AA or MDS will be helpful for the early diagnosis of PNH.

Mutation Analysis of PIG-A Gene in Korean Patients with Paroxysmal Nocturnal Hemoglobinuria / 대한혈액학회지

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in hemopoietic stem cells. Mutation of phosphatidyl inositol glycan class A (PIG-A) gene, an X-linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. Characteristics of somatic mutation of PIG-A gene in the Korean patients with PNH and their relationships to clinical characteristics were analyzed. METHODS: Twenty five patients with PNH and a donor of bone marrow transplantation were selected. Ham tests, sucrose hemolysis tests and CD59 expressions of erythrocytes and granulocytes were performed to confirm diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterize the mutations. RESULTS: The mutations of PIG-A gene were found in twelve cases and ten of them were novel mutations. There were five deletions, six substitutions and a insertion. Therewere six premature terminations, three abnormal splicings, a missense and two nonsense mutations. There were six point mutations and six frameshift mutations. Five cases of hypoplastic PNH showed mutations only in exons, but three in seven cases of hemolytic PNH showed mutations in introns. Two cases with symptoms of venous thrombosis showed mutations in exon 3. CONCLUSION: There were ten novel mutations among twelve mutations in the Korean patients with PNH and characteristics of the mutations were variable without any remarkable hot spot in sites and types. The characteristics of mutation were not correlated with the results of clinical courses of the patients with PNH.