Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex.

Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex.

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex.

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.

Translocational rearrangements of 11q23 in acute monoblastic leukemia.

We report the rearrangements of 11q23 in the form of t(6;11)(q27;q23) and t(11;16)(q23;q24) in three cases of acute monoblastic leukemia. The former translocation had only previously been reported in five cases of acute myeloid leukemia, while the latter is hitherto undescribed. In addition to describing a new chromosomal locus 16q24, which may participate in translocational exchanges with 11q23, this report also confirms the close association between 11q23 rearrangement and the involvement of the monocytic lineage in acute myeloid leukemia.

Central nervous system involvement in non-Hodgkin's lymphoma.

Fifty-eight Hong Kong Chinese patients with CNS lymphoma were reviewed (primary seven, secondary 51). The incidence of secondary CNS lymphoma in patients with non-Hodgkin's lymphoma was estimated to be 9.4%. The Working Formulation separated subtypes which had a special propensity to involve the CNS. Significant proportions of our patients with secondary CNS lymphoma had other features which were known to be associated with a high risk of CNS disease including stage IV (48/51, 91.4%), bone marrow (26/51, 50.9%), peripheral blood (7.51, 13.7%), nasal (7/51, 13.7%), orbital (3/51, 5.9%), testicular (2/51, 3.9%) and bulky retroperitoneal (6/51, 11.8%) disease. 82% of patients with secondary CNS lymphoma had concurrent systemic disease and a further 12% had systemic relapse shortly afterward. CNS lymphoma is associated with poor prognosis and only 29% and 14% of the patients with primary and secondary CNS lymphoma respectively survived beyond 1 year. Patients responding to therapy had significantly better survival. 69.9% of the deaths were related to progressive systemic disease.

Hypogonadotropic hypogonadism in severe beta-thalassemia: effect of chelation and pulsatile gonadotropin-releasing hormone therapy.

We studied pituitary-gonadal function in 11 male and 5 female patients, aged 12-30 yr, with severe beta-thalassemia and chronic iron overload. All had normal basal serum cortisol, T4, and PRL concentrations and normal serum cortisol and GH responses to insulin-induced hypoglycemia and TSH responses to TRH. Of the 11 male patients (all over 17 yr of age), only 3 attained full pubertal development and 4 had subnormal serum LH and FSH responses to GnRH. As a group, their mean basal serum testosterone (T) level was low [11.7 +/- 4.9 (+/- SE) nmol/L; normal, 10-40 nmol/L], and 9 of the 11 male patients responded to hCG with a rise in serum T. Two of the 3 female patients over 17 yr of age were prepubertal with undetectable serum estradiol (E2) levels and absent serum LH and FSH responses to GnRH; the other female patient had regular menstrual cycles and normal serum E2 levels and LH and FSH responses to GnRH. Six of the prepubertal patients (4 males and 2 females, aged 17-30 yr) were studied serially for 3 yr after the start of chelation therapy. Despite a fall of median serum ferritin from 11,910 to 1,303 pmol/L, there was no progression of puberty, and their basal and GnRH-stimulated serum LH and FSH and serum T or E2 levels did not change. Three of these patients (1 male and 2 female) then received pulsatile sc GnRH therapy in addition to chelation therapy for 6 months with no improvement. We conclude that chronic iron overload in patients with severe thalassemia leads to variable degrees of hypogonadotropic hypogonadism, which do not respond to chelation therapy given late in the course of the disease. The hypogonadism in most patients was due to pituitary hyporesponsiveness to GnRH.

Acute lymphoblastic leukemia in Chinese adults in Hong Kong.

Forty-four previously untreated Chinese adult patients with acute lymphoblastic leukemia (ALL) were treated with vincristine, adriamycin and prednisone with or without L-asparaginase. The clinical features and prognostic factors were similar to those reported in Caucasian series. Overall complete remission (CR) rate was 52%. Duration of first remission and overall median survival were nine and 12 months respectively. The addition of L-asparaginase did not improve CR rate or duration of remission and was associated with clotting dysfunction and other adverse reactions. Factors associated with a higher CR rate include age less than 40 years, blast count less than 10 x 10(9)/l and CALLA + phenotype at presentation. Sex, platelet count and FAB morphology did not affect CR rate. Bone marrow relapse occurred in 11 patients and was associated with short survival after relapse (median two months; mean two months; range 0.5-7 months). Central nervous system relapse occurred in four patients and was compatible with relatively long survival after relapse (median 13 months; mean 12 months; range 6-12 + months). The poor CR rate and short median survival in this study could not be adequately explained by an increase in risk factors and is likely to be due to what is currently regarded as suboptimal chemotherapy.

Combination of three alpha-globin gene loci deletions and hemoglobin New York results in a severe hemoglobin H syndrome.

A chinese patient with hemoglobin (Hb) H and Hb New York is described. Unlike classical Hb H disease, the additional beta New York chain defect resulted in a more marked alpha/beta-globin chain imbalance in this patient with only one alpha-gene, accounting for her severe anemia (3.4-6.8 g/dl) during the steady state.

Is hypereosinophilic syndrome a malignant disease?

A patient with an abdominal mass for 4 years developed hypereosinophilia and weight loss. Bone marrow aspirations and biopsy specimens showed hypercellularity of eosinophils. No cause for hypereosinophilia was found. Biopsy specimens of several organs, including the abdominal mass, showed eosinophilic infiltration with reactive fibrosis in some. Cytogenetic studies of the bone marrow before treatment with cytoxic drugs showed chromosomal abnormalities. Several of these have been reported in association with acute or eosinophilic leukemias. Based upon these findings, the authors believe that the patient had eosinophilic leukemia and that the abdominal mass probably was an eosinophilic chloroma.

Iron overload in thalassaemic patients in Hong Kong.

Body iron status, as measured by serum ferritin, was studied in 101 adult Chinese thalassaemic patients, 46 males and 55 females. Thirty of them had mild disease (beta thalassaemia trait), 56 disease of intermediate severity (haemoglobin H disease) and 15 severe disease (homozygous beta thalassaemia, Hb E-beta thalassaemia and delta beta-beta thalassaemia). The extent of iron overload correlated with the severity of disease. In severe thalassaemia, iron overload occurred early in life and was independent of multiple transfusions. While significant impairment of left ventricular function was present in only one of six patients studied, evidence of decreased pituitary (especially gonadotrophic) function was observed in six out of seven. Hypocalcaemia, probably due to hypoparathyroidism, was seen in one.

Iron overload in patients with haemoglobin H disease.

Serum ferritin was measured in 46 Chinese patients with Hb H disease (HbHD) and compared with 84 controls according to age group. Ferritin levels were elevated in 14 of 20 males and 20 of 26 females and the extent of the abnormality increased with age. Splenectomy did not appear to be associated with particularly high levels of serum ferritin in this disease.

The effect of oral contraceptives on coagulation and fibrinolytic parameters in the Chinese--a prospective study.

Thirty-two Chinese and 7 Caucasians were studied prospectively for 12 months for the effects of oral contraceptives (OC) on certain coagulation and fibrinolytic parameters. In both ethnic groups there was an increase in alpha 1 antitrypsin level. There was no significant change in antithrombin III concentration measured either by radioimmunoassay (At-III-RIA) or chromogenic assay (anti Xa-chromogenic) in the Chinese, but a significant decrease in anti Xa-chromogenic at 7 months and At-III-RIA at 12 months in the Caucasians. Also, alpha 2 plasmin inhibitor (alpha 2 PI) levels were unchanged in the Chinese but a significant increase occurred in the Caucasians at 12 months. Enhanced fibrinolytic response to venous occlusion was demonstrated in the Chinese at 12 months but not in the Caucasians.

Venous thrombosis in haemoglobin H disease after splenectomy.

While venous thrombosis and pulmonary embolism are rare among the Chinese, two of nine patients with haemoglobin H disease developed these complications after splenectomy. The clinical data of the two patients were reported and the relevant literature reviewed. It was concluded that the persistent thrombocytosis and an intravascular haemolysis, particularly prominent in this form of thalassaemia, are contributory to the hypercoagulable state.

Deep vein thrombosis and changes in coagulation and fibrinolysis after gynaecological operations in Chinese: the effect of oral contraceptives and malignant disease.

Of 154 Chinese patients who underwent gynaecological operations, four showed a positive 125I fibrinogen leg scan for venous thrombosis, and overall incidence of 2 . 6%. In those who were on oral contraceptives and had major pelvic surgery for benign conditions, the incidence was 10 . 5%; in those who had Wertheim hysterectomy for carcinoma of cervix, it was 6 . 7%. This confirms the rarity of post-operative thromboembolism in the Chinese. Fragment E showed a biphasic rise after major operation due to tissue injury and venous thrombosis. In patients with malignancy, the postoperative 'fibrinolytic shutdown', represented by decreased plasminogen activator together with increased alpha 1 antitrypsin and C1- inhibitor levels, was more marked. In addition, alpha 2 macroglobulin level was lower and fell significantly after operation. In patients on oral contraceptives, fragment E levels were higher after surgery and there was no decrease in plasminogen activator levels. Antithrombin III levels did not fall except in three of the four patients with venous thrombosis. A marked increase in fragment E level and a decrease in antithrombin III level might be useful diagnostic markers for postoperative venous thrombosis.

PIP: Of 154 Chinese patients who underwent gynecological operations, 4 showed a positive fibrinogen leg scan for venous thrombosis, an overall incidence of 2.6%. In those who were on (OC) oral contraceptives and had major pelvic surgery for benign conditions, the incidence was 10.5%; in those who had Wertheim hysterectomy for carcinoma for cervix, it was 6.7%. This confirms the rarity of postoperative thromboembolism in the Chinese. Fragment E showed a biphasic rise after major operation due to tissue injury and venous thrombosis. In patients with malignancy, the postoperative fibrinolytic shutdown, represented by decreased plasminogen activator together with increased alpha 1 antitrypsin and C inhibitor levels, was more marked. In addition alpha 2 macroglobulin level was lower and fell significantly after operation. In patients on OCs, fragment E levels were higher after surgery and there was no decrease in plasminogen activator levels. Antithrombin 3 levels did not fall except in 3 of the 4 patients with venous thrombosis. A marked increase in fragment E level and a decrease in antithrombin 3 level might be useful diagnostic markers for postoperative venous thrombosis.

Deep venous thrombosis after strokes in Chinese.

In 35 Chinese patients suffering from strokes resulting in hemiparesis or hemiplegia, deep venous thrombosis, as detected with 125I-fibrinogen scan, occurred in the paralysed leg in five and in the normal leg in one. The total incidence of 17% was lower than that reported in the West. No clinical evidence of pulmonary embolism was seen. This report represents an objective confirmation of the low incidence of venous thrombosis in the Chinese.

Folate status in pregnant Chinese women in Hong Kong.

Hematologic and folate studies were carried out in 116 pregnant Chinese women in Hong Kong. Mean serum folate values fell progressively with pregnancy reaching subnormal levels during the puerperium. Mean red-cell folate values remained normal or high throughout, but showed a significant fall after delivery in women who carried out breast-feeding. Individually, 18 women showed hematologic abnormalities compatible with folate deficiency, but only four had subnormal red-cell folate values satisfying the strict criterion of folate deficiency, giving an overall frequency of 3.4%. Compared to primigravidae and other multiparae, gravida 2 women appeared to be at highest risk of folate deficiency.

The determination of antithrombin III by radioimmunoassay and its clinical application.

A radioimmunoassay (RIA) had been developed for the determination of antithrombin III (AT III) in man. The detection limit was 25 microgram/dl. AT III-RIA level and biological activity (anti-Xa) was significantly correlated (r = 0.737, P less than 0.001). Plasma levels in 36 healthy males (mean +/- SD, 19.9 +/- 2.5 mg/dl) and 21 healthy females (19.1 +/- 2.4 mg/dl) were similar. Serial AT III measurements in normal menstruating females showed lower levels during midcycle and higher concentrations during menstruation. In carcinomas, the AT III levels were lower than normal, particularly in hepatocellular carcinoma. In cirrhosis of liver, the levels were markedly decreased and in some patients were below that found in congenital AT III deficiency. Patients with deep vein thrombosis and patients with heart valve replacement had lower levels than normal, while patients with cerebral vascular occlusion had normal levels. The possible use of AT III as a diagnostic tool of post-operative deep vein thrombosis was demonstrated in one patient after hysterectomy. The increased sensitivity, specificity and precision of this type of assay offer distinct advantages over existing methods of AT III estimation.

Aplastic anaemia: a study of prognosis and the effect of androgen therapy.

One hundred and twenty-nine Chinese patients with aplastic anaemia, were studied. In ten it was induced by drugs, one followed hepatitis and the remainder were of unkown cause. Mortality within the first six months was 47.3 per cent. Features associated with poor prognosis included a short duration of symptoms of three months or less, neutropenia of less than 0.5 x 10(9)/l and severe thrombocytopenia. On the other hand, some preservation of erythroid activity of the bone marrow was associated with long survival. Remission occurred in 47 patients and this was associated with androgen therapy in 33. Remission was complete in 18 and partial in 29. In the latter group, persistent thrombocytopenia was the main abnormality and treatment with calusterone led to an increase in platelets although the effect was not sustained after its withdrawal. Hepatotoxicity was seen in 16.0 per cent of patients treated with androgens and this occurred even with non-17alpha-alkylated compounds. It is concluded that androgen therapy was useful and should be started as early as possible after diagnosis. Hepatic intolerance to androgens may be an indication for bone marrow transplantation.

Drug-induced haemolysis in glucose-6-phosphate dehydrogenase deficiency.

People with the variants of glucose-6-phosphate dehydrogenase (GPD) deficiency common in the southern Chinese (Canton, B(-)Chinese, and Hong Kong-Pokfulam) have a moderate shortening of red-cell survival but no anaemia when they are in the steady state. With a cross-transfusion technique, primaquine, nitrofurantoin, and large doses of aspirin were found to aggravate the haemolysis while sulphamethoxazole did so only in some people. Individual differences in drug metabolism may be the reason for this. Many commonly used drugs reported to accentuate haemolysis in GPD deficiency did not shorten red-cell survival.

Significance of subnormal red-cell folate in thalassaemia.

Subnormal red-cell folate values were encountered in 20 of 63 thalassaemic subjects in a population with a low incidence of megaloblastosis. The abnormality was not associated with haematological changes of megaloblastosis and could not be explained on the basis of incomplete liberation of intra-erythrocytic folates, serum conjugase deficiency of vitamin B12 deficiency. Evidence is presented to indicate that it is unlikely to represent a state of subclinical folate deficiency. The exact cause of the abnormality is unknown.