Engineering of IF1 -susceptive bacterial F1 -ATPase.

IF1 , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F1 . One of the unique features of IF1 is the selective inhibition in mitochondrial F1 (MF1 ); it inhibits catalysis of MF1 but does not affect F1 with bacterial origin despite high sequence homology between MF1 and bacterial F1 . Here, we aimed to engineer thermophilic Bacillus F1 (TF1 ) to confer the susceptibility to IF1 for elucidating the molecular mechanism of selective inhibition of IF1 . We first examined the IF1 -susceptibility of hybrid F1 s, composed of each subunit originating from bovine MF1 (bMF1 ) or TF1 . It was clearly shown that only the hybrid with the ß subunit of mitochondrial origin has the IF1 -susceptibility. Based on structural analysis and sequence alignment of bMF1 and TF1 , the five non-conserved residues on the C-terminus of the ß subunit were identified as the candidate responsible for the IF1 -susceptibility. These residues in TF1 were substituted with the bMF1 residues. The resultant mutant TF1 showed evident IF1 -susceptibility. Reversely, we examined the bMF1 mutant with TF1 residues at the corresponding sites, which showed significant suppression of IF1 -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF1 residues in α and γ subunits that further enhanced the IF1 -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF1 specifically recognizes F1 with mitochondrial origin, based on the present result and the structure of F1 -IF1 complex. These findings would help the development of the inhibitors targeting bacterial F1 .

Rotary properties of hybrid F1-ATPases consisting of subunits from different species.

F1-ATPase (F1) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FoF1-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and ß, F1 shows diversity in the maximum catalytic turnover rate Vmax and the number of rotary steps per turn. To study the design principle of F1, we prepared eight hybrid F1s composed of subunits from two of three genuine F1s: thermophilic Bacillus PS3 (TF1), bovine mitochondria (bMF1), and Paracoccus denitrificans (PdF1), differing in the Vmax and the number of rotary steps. The Vmax of the hybrids can be well fitted by a quadratic model highlighting the dominant roles of ß and the couplings between α-ß. Although there exist no simple rules on which subunit dominantly determines the number of steps, our findings show that the stepping behavior is characterized by the combination of all subunits.

Usefulness of automatic detection of fragmented red cells using a hematology analyzer for diagnosis of thrombotic microangiopathy.

We previously reported an automatic method for quantitative analysis of schistocytes or fragmented red cells using an automatic hematology analyzer, XE-2100. In the study reported here, we evaluated the accuracy of this detection method in patients with thrombotic microangiopathy (TMA). A follow-up study was performed on 14 patients with two types of TMA, thrombotic thrombocytopenic purpura or hemolytic uremic syndrome. Schistocyte percent was evaluated both with an automatic counter and by means of microscopic observation. Total activity and isoenzyme pattern of lactate dehydrogenase (LD) were also determined. In these patients, schistocyte percent determined by automatic counting correlated highly with that determined by manual counting under microscopic observation (r = 0.852, P < 0.0001). Schistocyte percent was shown to correlate significantly with isoenzyme fractions 1 and 2 of LD (r = 0.732, P < 0.02), reflecting hemolysis. Nine of 11 patients tested had high concentrations of LD isoenzyme five without distinct liver damage, and schistocyte percent did not relate to fraction 5 of LD. Automatic detection of schistocyte percent using a hematology analyzer was useful for an accurate diagnosis and follow-up of thrombotic microangiopathy. The origin of LD fraction 5 remains to be determined.

Fluctuations in plasma macrophage colony-stimulating factor levels during autologous peripheral blood stem cell transplantation for haematologic diseases.

Plasma macrophage colony-stimulating factor (M-CSF) levels were measured in 13 haematologic patients treated with autologous peripheral blood stem cell transplantation (PBSCT). Six of the patients showed an increase in M-CSF peak levels (>3000 pg/ml) during the conditioning and stem cell infusion period. The peak levels of M-CSF in this phase correlated with thrombomodulin levels, indicating the endothelial origin of plasma M-CSF. However, the M-CSF levels were not influenced by TNFalpha. More patients with high M-CSF levels (>5000 pg/ml) suffered from organ failure than those with lower M-CSF levels. These results suggest that high M-CSF levels may correlate with cellular or organ damage in patients treated with PBSCT.

Estimation of stem cell fractions in peripheral blood stem cell harvest by using an SE-9000 hematology analyzer.

We inquired whether stem cell fractions in peripheral blood stem cell harvest could be precisely detected by using a hematopoietic progenitor cell (HPC) counting system applied to an automated hematology analyzer, SE-9000. Although there was an apparent increase in the HPCs 20 h after storage in nondiluted conditions, samples diluted with RPMI-1640 containing 1.0 mg/ml EDTA-2K showed a relatively stable number of HPCs. There was a significant relationship between HPCs and CD34 cells (n = 75, r = 0.769). This method may represent the least expensive and most time-effective way for stem cell estimation in harvest products.

[M-CSF levels during peripheral blood stem cell harvest and autologous stem cell transplantation].

Plasma macrophage colony-stimulating factor(M-CSF) levels were determined during peripheral blood stem cell harvest and autologous stem cell transplantation(PBSCT). Plasma of 10 patients were analyzed by using ELISA system. The average peak values during PBSCT were quite higher than those during harvest(20,092 vs 1,681 pg/ml). Peak values were observed mainly around leukocyte nadir(Phase II) during harvest. On the other hand, they were detected just after pretreatment(Phase I) during PBSCT courses. Moreover, samples showing extremely high M-CSF values(Phase I) were associated with increase in serum LDH levels. These data suggest that plasma M-CSF in Phase I are mainly derived from chemotherapy-induced cellular damage during PBSCT courses, and there might be different mechanisms to raise M-CSF around the nadir of leukocytes. It is necessary to elucidate the biological meanings of M-CSF.

[G-CSF administration following autologous peripheral blood stem cell transplantation--the effect of G-CSF level on neutrophil recovery].

We studied the usefulness of rhG-CSF (filgrastim) administration in patients who received autologous peripheral blood stem cell transplantation (PBSCT) combined with super-high dose chemotherapy. Twenty patients received 0-8.3 micrograms/kg/day filgrastim after PBSCT. There was a significant relationship between G-CSF dose and the neutrophil recovery rate, and the highest levels of serum G-CSF tended to correlate with neutrophil recovery rate. The highest G-CSF level after 75 micrograms injection in normal volunteers is reported to be 1,500 pg/ml. On the other hand, as one patient in our series exhibited extremely high endogenous G-CSF of 11,500 pg/ml, measurements of G-CSF might reduce the over-administration of rhG-CSF.

Transfusion-related acute lung injury in a patient with acute myelogenous leukaemia having anti-IgA2m(1) antibody.

A 69-year-old man with acute myelogenous leukaemia developed a transfusion-related acute lung injury (TRALI). He had anti-IgA2m(1) antibody rather than other antibodies that have previously been reported to be related to TRALI. This case suggests that the pre-existing condition of patients may be important in the development of TRALI.

Treatment of post-transfusion graft-versus-host disease with nafmostat mesilate, a serine protease inhibitor.

BACKGROUND: Cytotoxic T lymphocytes from donors are thought to injure the target organs in post-transfusion graft-versus-host disease (PT-GVHD) through perforin-granzyme- and Fas-dependent cell killings. The protease involved is a serine protease, and nafmostat mesilate (NM), a serine protease inhibitor, has been found to inhibit the in vitro allocytotoxicity of the T cell clone established from a patient with PT-GVHD, thus suggesting the usefulness of NM for treatment of PT-GVHD. CASE REPORT: A 47-year-old male with esophageal cancer, who received 3 units of packed red cells and 20 units of platelet concentrates from 5 unrelated donors, was diagnosed as having PT-GVHD on the basis of typical clinical features, HLA typing of the patient and the responsible donor, and a mixed chimera of CD8+ lymphocytes on microsatellite DNA polymorphism analysis. NM was administered to inhibit the activity of the serine proteases, thought to be granzymes; a liver dysfunction and thrombocytopenia with leukocytopenia simultaneously improved. Subsequently, a high-dose methylprednisolone pulse therapy and monoclonal anti-CD3 were administered to reduce the donor's proliferating lymphocytes, which resulted in lymphopenia accompanied by elimination of the donor's lymphocytes and normalization of the CD4/CD8 ratio. However, recurrence of the proliferation of the responsible donor's lymphocytes developed after cessation of NM administration, probably because of excessive immunosuppression caused by steroids and the monoclonal anti-CD3. CONCLUSION: This case indicates that administration of a serine protease inhibitor may improve PT-GVHD symptoms by inhibiting cytotoxic T-cell-mediated killing of target cells in fatal PT-GVHD. Steroids and monoclonal anti-CD3 were probably responsible for the transient clinical improvements. More studies are required, however, on mechanisms to eliminate the donor's lymphocytes.

Response to granulocyte colony-stimulating factor in an autoimmune neutropenic adult.

Clinical value of granulocyte colony-stimulating factor (G-CSF) for autoimmune neutropenia (AIN) has not been well established. We experienced an adult case of AIN which showed an excellent response to G-CSF. A 75-year-old female was admitted with high-grade fever. Her neutrophil count was remarkably low (neutrophil 0.09 x 10(9)/l). Antigranulocyte autoantibody was demonstrated in her serum by an immunofluorescence method and she was diagnosed as AIN. Administration of G-CSF (filgrastim 5 microgram/kg) gave a rapid increase of neutrophils (from 0.11 x 10(9)/l to 2.10 x 10(9)/l on the second day), which has enabled us to preserve the use of G-CSF for emergency, that is, for overt serious infection.

Therapeutic strategy for post-transfusion graft-vs.-host disease.

An effective treatment for post-transfusion graft-vs.-host disease (PT-GVHD), a fatal complication of blood transfusion, has not yet been identified. In this review, we propose a treatment for PT-GVHD based on the mechanism of its onset. First, we briefly review the findings that PT-GVHD is induced by cytotoxic T-lymphocyte (CTL)-mediated tissue injuries through the Fas/Fas ligand system, the perforin/granzyme system, and alloantigen-specific antibodies, as well as through inflammatory cytokines. Secondly, we emphasize the usefulness of a serine protease inhibitor for the inhibition of CTL-mediated cytotoxicity in the earlier stages of onset. Subsequent administration of methylprednisolone and 2-chlordeoxyadenosine is recommended for elimination of the donor's lymphocytes. The usefulness of chloroquine for the suppression of CTL activity and the production of tumor necrosis factor as well as the efficiency of pentoxyfylline for the suppression of the production of tumor necrosis factor are also discussed. Therapeutic strategies for PT-GVHD should also be useful for treating acute GVHD secondary to allogeneic bone marrow transplantation, and to prevent the host's rejection of transplanted organs as well as tissue damage in autoimmune diseases.

[Giant platelet-like cell fragments produced from abnormal promyelocytes in acute myelogenous leukemia].

A 38-year old man was transmitted to our hospital because of his pneumonia and disconsciousness. Laboratory data showed leukocytosis (32,500/microliter), mild anemia, and decreased platelet count (6.7 x 10(4)/microliter). The bone marrow aspiration revealed the presence of 40% blastoid cells and cytogenetic study showed abnormal karyotype, 45, X, -Y, t(8; 21) (q22; q22), indicating acute myeloid leukemia (AML, M2). Furthermore, on the microscopic observation, cell fragments resembling giant platelets were observed which were positive for myeloperoxidase, and several fragments connected with abnormal promyelocytes through thin cytoplasm. These results suggested these cell fragments may be produced from abnormal promyelocytes in this case.

[Analysis of mechanism of increased platelet counts during preoperative autologous blood donation].

Autologous blood transfusion is advantageous in that it eliminates the risk of transfusion-transmitted infection or complications caused by the immune reaction. Increased platelet counts after repeated phlebotomy are commonly observed. This study was carried out to clarify the mechanism of increased platelets during preoperative autologous blood donation. Eleven patients of elective surgery in which is in good preoperative condition and there is no emergency were selected for this study. Blood cell counts, platelet size distribution(PDW, MPV, P-LCR) and serum concentration of erythropoietin(EPO), interleukin-3(IL-3) and interleukin-6(IL-6)were measured. A transient increase in platelet was seen in almost patients during preoperative autologous blood donation. No marked changes in platelet size distribution and serum concentration of EPO, IL-3 and IL-6. These results suggest that increased platelet counts during preoperative autologous blood donation might be caused by some specific cytokines related to megakaryocyte differentiation such as c-MPL ligand.

The involvement of the rho gene product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK.

The role of rho proteins, which are ras p21-related small GTP-binding proteins, in megakaryocyte endomitosis was examined using a botulinum C3 exoenzyme (C3), a rho inactivating enzyme. The megakaryocytic leukemia cell line CMK expressed high levels of rhoA and rhoC mRNAs, whereas rhoB mRNA was expressed at a very low level. The addition of C3 to the culture medium caused ADP-ribosylation of the rho proteins in CMK cells in a dose- and time-dependent manner. This procedure also induced a higher frequency of polyploid cells with increased glycoprotein (GP) IIb/IIIa antigens on the cells. This effect of C3 on both ploidy and the antigen expression was abolished by prior incubation of C3 with an anti-C3 monoclonal antibody. Cytochalasin B, an actin polymerization inhibitor, also induced polyploid cells; however, it did not stimulate the expression of GP IIb/IIIa antigens in CMK cells. This finding suggests that C3-induced increase in the expression of GP IIb/IIIa antigens was not through the actin microfilament disassembly. The present study suggests that the rho p21 is a partly regulatory component in polyploidization and GP IIb/IIIa antigen expression of a human megakaryocytic cell line, CMK.

[Philadelphia chromosome-negative chronic myelogenous leukemia with trisomy 13].

Trisomy 13, as a sole karyotypic abnormality in acute leukemia, has been reported in several cases. However, in chronic myelogenous leukemia (CML), only two cases with this abnormality were reported so far. We describe herein a 68-year-old case with Philadelphia chromosome-negative CML and trisomy 13. Leukocytosis was pointed out during the treatment for other diseases. After 7 months, abrupt increase in leukocyte count (108,000/microliters) and splenomegaly developed. Decreased neutrophil alkaline phosphatase activity and morphological features fulfilled the diagnostic terms for CML. However, the karyotypic analysis revealed trisomy 13 instead of Philadelphia chromosome, and the BCR gene rearrangement was not detected. In cases with acute leukemia accompanied by trisomy 13, malignant transformation of an immature hematopoietic precursor cell has been suggested by the expression of antigens characteristic of both the myeloid and lymphoid lineage. In a few cases with myelodysplastic syndrome, a multipotent stem cell disorder, trisomy 13 has also been reported. From these standpoints, there might be a possibility that trisomy 13 as a sole abnormality in hematologic disorders would be related to tumorigenesis in the levels of multipotent stem cells.

Tumor necrosis factor alpha-induced up-regulation of interleukin-3 receptor mRNA expression in a CD34-positive hematopoietic cell line.

Regulation of interleukin-3 receptor (IL-3R) gene expression by tumor necrosis factor alpha (TNF alpha) was investigated using an IL-3-dependent CD34-positive hematopoietic cell line (KMT2) and a human megakaryocytic cell line (CMK). KMT2 expressed IL-3R alpha-subunit mRNA, whereas the level of expression of IL-3R beta-subunit mRNA was low. CMK expressed IL-3R beta-subunit mRNA more strongly. The expression of IL-3R mRNA varied in the progenitor cells of different lineages. TNF alpha markedly enhanced expression of IL-3R beta-subunit mRNA in KMT2, whereas it only slightly augmented IL-3R alpha-subunit mRNA level. TNF alpha weakly augmented IL-3R mRNAs in CMK. However, the enhancement of IL-3R beta-subunit mRNA in CMK was hardly detectable. The effects of TNF alpha on IL-3R mRNA expression were completely different in a primitive and in a more committed hematopoietic cell line. Addition of TNF alpha to KMT2 resulted in increased numbers of IL-3R on the cell surface without increased IL-3R affinity. The combination of IL-3 with TNF alpha abolished TNF alpha-induced inhibition of proliferation of KMT2. These results indicate that TNF alpha modulates the IL-3-responsiveness of primitive hematopoietic cells through up-regulation of the expression of IL-3R mRNAs, especially that of IL-3R beta-subunit mRNA. Phorbol ester (TPA) enhanced the IL-3R mRNA expression in KMT2.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hematologic improvement by alpha-interferon in a case of the 5q- syndrome with basophilia and eosinophilia].

An 82-year-old female was diagnosed as having 5q- syndrome accompanied by basophilia, eosinophilia and thrombocytosis. Since cytogenetic abnormalities other than 5q- were also detected, she was considered to be type B of 5q- syndrome. According to the FAB classification a diagnosis of refractory anemia with excess of blasts (RAEB) was made. She was treated with recombinant interferon alpha 2b, because peripheral blood findings resembled those of chronic myelogenous leukemia. Interferon was effective, and resulted in disappearance of peripheral blasts, normalization of platelet numbers, and improvement of basophilia. These changes were interferon-dependent. After 1 year, cytogenetic studies revealed that about 2/3 of metaphases showed normal karyotype. Twenty months after diagnosis, myeloid blastic crisis occurred and eventually the patient died. However, treatment with interferon in this case might support the usefulness of the drugs for this kind of disease.

Diagnosis of post-transfusion graft-versus-host disease after formalin-fixation.

A 72-year-old woman with multiple recurrence of gallbladder cancer was treated by intrahepatic-arterial infusion of doxorubicin using an extracorporeal system of direct hemoperfusion with venovenous bypass. During this treatment, the patient received 600 ml of fresh whole blood and 30 units of platelet concentrate from five unrelated donors. Thereafter, high fever, skin rash over the whole body, and watery diarrhea developed, followed by leukopenia progressing to a fatal sepsis. Post-transfusion graft-versus-host disease (PT-GVHD) was suspected by the clinical manifestations and postmortem pathologic findings. To establish the diagnosis of PT-GVHD, polymerase chain reaction (PCR) amplification of DNA polymorphism associated with length variation in dinucleotide or trinucleotide microsatellite repeats at the loci of D6S89, int-2 protooncogene, and human growth factor with each of the different primer sets was performed using DNA from blood drawn from the patient with clinically established PT-GVHD of a donor origin and formalin-fixed pancreas of recipient origin. Genetic analysis revealed the changes in the patient's lymphocytes from that of the patient to that of donor origin. The present finding that formalin-fixed tissues can be used as a material of patient origin may contribute to accurate diagnosis of PT-GVHD after autopsy.