cDNA structure, tissue-specific expression, and chromosomal localization of the murine band 7.2b gene

Band 7.2b is an integral phosphoprotein absent from the erythrocyte membranes of patients with hydrocytosis, an autosomal, dominantly inherited, hemolytic anemia characterized by stomatocytic red blood cells with abnormal permeability to Na+ and K+. The role of this protein in the erythrocyte membrane is not well understood. To gain additional insight into the structure and function of this protein, we have cloned the murine band 7.2b cDNA and studied its tissue-specific expression. 2,873 bp of cDNA with an open reading frame of 852 bp were isolated. This fragment encodes a protein of 284 amino acids with a predicted molecular weight of 31 kD. The band 7.2b gene had a wide pattern of expression, with high levels of mRNA in heart, liver, skeletal muscle, and testis and low levels in lung, brain, and spleen. Using fluorescent in situ hybridization, the murine band 7.2b gene was mapped to chromosome 2, at the border of the distal region of 2B and proximal region of C1, syntenic to 9q33-q34, the location of the human homologue. Models of the predicted protein structure showed a short NH2- terminal head, a strongly hydrophobic 28-amino acid stretch presumably encoding a single membrane-spanning domain, and a large domain composed of beta sheet and alpha helix. Database searching showed no significant homology of other known proteins to murine or human band 7.2b.     

Identification of a restriction fragment length polymorphism involving the oncogene ETS-1 on chromosome 11q23

Twenty four samples of DNA from 23 unrelated individuals were analyzed for the presence of a novel restriction fragment length polymorphism (RFLP) involving the proto-oncogene ETS-1 at an Xba I site. Four samples from unrelated individuals lacked an Xba I site, giving rise to a longer restriction fragment detectable by Southern analysis; two samples were from normal tissue, and two were from acute myelogenous leukemic blasts. Thus, no association could be found between the RFLP and disease among the individuals studied. Pedigree analysis of another cohort demonstrated Mendelian inheritance consistent with a somatic polymorphism. The practical applications of RFLP analysis in clinical and research settings, and the usefulness of this Xba I RFLP in the study of hematologic malignancies because of its location in 11q23, are discussed.     

Liver transplantation in hemophilia A

Four patients with hemophilia A have undergone liver transplantation in our institution, three successfully. The first was a 21-year-old man with chronic active hepatitis (CAH) in whom the effects of previous abdominal operations prevented the satisfactory technical insertion of the new liver. He died intraoperatively. The second patient was a 15- year-old boy with CAH who began to synthesize factor VIII coagulant activity (F VIII:C) within 18 hours of successful liver transplantation and has continued to do so for almost 2 years (F VIII:C range 0.89 to 3.20 U/mL). The first 2 months of his postoperative course were complicated by infections, but since that time he has done well and has returned to school. The third patient was a 48-year-old man with portal fibrosis and severe ascites. He synthesized F VIII:C (range 0.96 to 1.50 U/mL) within six hours after reestablishment of circulation through the new liver. His postoperative course was complicated by numerous infections, and he died with sepsis and an acquired immunodeficiency-like syndrome 4 months after transplantation. The fourth patient was a 47-year-old mild hemophiliac with CAH who produced adequate factor VIII:C levels following transplantation (range 0.79 to 2.80 U/mL). These patients demonstrate that liver transplantation in hemophiliacs with end-stage liver disease may be lifesaving and results in correction of the F VIII:C deficiency and associated hemorrhagic tendency.     

Loss of 111Indium as indicator of platelet injury

We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.     

Reticulocytes. I. Isolation and in vitro maturation of synchronized populations

Studies of reticulocyte maturation have been limited by the inability to obtain pure populations of age-synchronized reticulocytes and by the absence of well-defined methods for the maturation of reticulocytes in vitro. Many of these problems were overcome using temporary suppression of erythropoiesis with thiamphenicol and phlebotomy resulting in a highly reproducible reticulocyte response, Percoll density gradient separation of cells yielding essentially pure populations of age- synchronized reticulocytes, and liquid culture techniques where cell lysis is minimal. The system allows reproducible study of well-defined cohorts of reticulocytes as they mature into erythrocytes. During in vitro maturation we serially monitored changes in reticulocyte count, glucose consumption, 125I-transferrin binding, fluorescein (FITC)- labeled transferrin binding, the activities of four erythrocyte enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, phosphofructokinase, and lactate dehydrogenase) and the appearance of cells on scanning electron microscopy. These variables changed at different rates suggesting that multiple mechanisms underlie these maturational events. Transferrin binding and reticulocyte count decreased most rapidly and reached values near zero after three to four days in culture. The four enzyme activities decreased much more slowly, and only two reached pretreatment values after seven days in culture. In contrast to the findings of others, scanning electron microscopy suggested that cells do not assume the normal biconcave shape in this system. The methods described make it feasible to study the process of reticulocyte maturation in vitro. The data presented represent a first step in the study of the coordination and interrelationships of various maturational processes.     

腹腔镜在直肠癌手术治疗中的临床应用

目的:探讨腹腔镜手术治疗直肠癌的方法和可行性．方法:2005-02／12共开展腹腔镜直肠癌手术31 例,其中Dixon手术20例,Miles手术9例,乙状结肠袢式造口术2例．结果:手术均获成功,无中转开腹,手术时间平均200 min,术中平均出血量80 mL,术后肠功能恢复时间平均30 h,术后1例Dixon手术患者出现吻合口瘘,经保守治疗痊愈．结论:腹腔镜直肠癌手术具有创伤小、手术出血少、术后痛苦小、肠道功能恢复快等特点,是可行的、安全有效的手术方法．     

Interleukin-1 (22-K factor) induces release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes

An electrophoretically pure preparation of natural human interleukin-1 (IL-1) was shown to stimulate in vitro colony formation in human bone marrow cultures. Day 4 myeloid cluster-forming cells (CFC), as well as early (day 7) and late (day 10) granulocyte-macrophage colony-forming units (CFU-GM) were stimulated in a dose-dependent fashion. At optimal concentrations of IL-1, the number of day 4 CFC reached 72%, the number of day 7 CFU-GM reached 32%, and the number of day 10 CFU-GM reached 80% of the respective numbers of colonies obtained by addition of crude leukocyte-conditioned medium (LCM). The IL-1-induced stimulatory effect on CFU-GM growth could be completely neutralized by a rabbit anti-IL-1 antiserum. Colony growth was abrogated by depleting the marrow cell suspensions of phagocytic cells prior to IL-1 addition. Conversely, the effect could be reintroduced by addition of marrow-derived adherent cells to bone marrow cell suspensions that had been depleted of both phagocytic and E rosetting T cells. Furthermore, media conditioned by bone marrow-derived adherent cells or by peripheral blood mononuclear phagocytes in the presence but not in the absence of IL-1, stimulated in vitro colony growth of phagocyte-depleted bone marrow cell suspensions. These results indicate that IL-1 induces release of granulocyte-macrophage colony-stimulating activity (GM-CSA) from human mononuclear phagocytes.     

Effects of fresh-frozen plasma and its cryosupernatant fraction on von Willebrand factor multimeric forms in chronic relapsing thrombotic thrombocytopenic purpura

Remission plasma samples of some patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP) contain unusually large von Willebrand factor (vWF) multimers similar to those produced by normal human endothelial cells in culture. The infusion of the cryosupernatant fraction of normal plasma is as effective as normal fresh-frozen plasma (FFP) in the treatment or prevention of TTP episodes in patients with the chronic relapsing form of TTP. Three patients with chronic relapsing TTP during remission have unusually large vWF multimers present in their plasma. Two of the patients were transfused once with FFP, one of the two received cryosupernatant on three occasions, and the third patient was studied before and immediately after plasma exchange. Unusually large vWF multimers decreased or disappeared from patient plasma samples within 1/2 to 1 1/2 hours following the transfusion of FFP (on two occasions) or cryosupernatant (on two of three occasions), and immediately after plasma exchange (on one occasion). The patient who received cryosupernatant was studied serially after the infusions. Unusually large vWF multimers returned to her plasma within ten to 24 hours and persisted thereafter. Unusually large vWF multimers did not disappear from patient remission plasma samples, or from the culture medium removed from normal human endothelial cells, when these fluids were incubated in vitro with either normal FFP or cryosupernatant. We conclude that an activity in FFP, and its cryosupernatant fraction, promoted the rapid in vivo disappearance of unusually large vWF multimers from the plasma of two patients with chronic relapsing TTP in remission, and plasma exchange reversed the abnormality in a third patient who was in partial remission. Neither FFP nor cryosupernatant directly converted unusually large multimers to smaller vWF forms in vitro in the fluid phase. These results indicate that an activity in the cryosupernatant fraction of normal plasma is involved in vivo in controlling the metabolism of unusually large vWF multimers, and that this process is defective in some chronic relapsing TTP patients.     

Successful allogeneic cryopreserved marrow transplantation

Cryopreservation has been used extensively in autologous marrow transplantation (BMT), but there has been limited use in allogeneic BMT. We describe here 6 cases of successful engraftment following allogeneic BMT with cryopreserved marrow. Patients suffered from Wiscott-Aldrich syndrome, osteopetrosis, aplastic anemia, and acute lymphocytic, acute non-lymphocytic, and chronic myelogenous leukemia, and ranged in age from 5 mos to 35 yrs. Marrow was collected using standard techniques. In one case T-cells were removed to prevent graft-vs-host disease. Marrow was frozen for a variety of reasons. Buffy coat cells were frozen at controlled rate in 10% DMSO, and stored in liquid nitrogen for 6 to 49 d. Engraftment (WBC greater than 1000/uL x 3 d) occurred from 13 to 37 d post BMT. In 4 of 4 cases in which data are available, donor origin of engraftment was documented, 1 with cytogenetics, 2 with red cell typing, and 4 with restriction fragment length polymorphisms. 3 patients are alive and well 21, 21, and 42 months post BMT. These results suggest frozen marrow can be successfully used for allogeneic BMT.