Search

Dimensional tunability from two dimensions to one dimension is demonstrated for the first time using an artificial superlattice method in synthesizing 1D stripes from 2D layered materials. The 1D confinement of layered Sr 2 IrO 4 induces distinct 1D quantum-confined electronic states, as observed from optical spectroscopy and resonant inelastic X-ray scattering. This 1D superlattice approach is generalizable to a wide range of layered materials., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autoimmune hemophilia-like disease (hemorrhaphilia) due to anti-factor XIII (FXIII) antibodies (AH13) is a very rare, life-threatening bleeding disorder. A 77-year-old woman developed macrohematuria and a right renal pelvic hematoma. The coagulation times were not prolonged, but FXIII activity and antigen levels were severely and moderately reduced to 9 and 29% of normal values, respectively. Accordingly, the FXIII-specific activity turned out to be low. FXIII inhibitor and anti-FXIII-A subunit autoantibodies were detected by a 1:1 crossmixing test and immunoblot and immunochromatographic assays. She was therefore diagnosed with "definite AH13" and treated with plasma-derived FXIII concentrates to arrest the hemorrhage. In addition to a highly compressed inferior vena cava by a huge renal pelvic hematoma, deep vein thrombosis (DVT) and pulmonary thromboembolism (PE) were identified by systemic computed tomography. The patient was immediately started on anticoagulation therapy with low-dose heparin. Emboli disappeared quickly, probably because under-crosslinked thrombi caused by severe FXIII deficiency are vulnerable to fibrinolysis. After about 1.5 years, anti-FXIII-A subunit autoantibodies still remained despite the use of rituximab, steroid pulse therapy, oral prednisolone, and oral cyclophosphamide treatments. In conclusion, an extremely rare AH13 case complicated by DVT and PE was successfully managed by balancing anticoagulation therapy with hemostatic therapy., (© 2017 S. Karger AG, Basel.)

The double hydrogen bond interaction between 7-azaindole (7AI) and protic solvents including methanol (MeOH), formamide (FM), formic acid (FA), pyridone (PY) and 7AI has been investigated as a proper model of mutations generated by tautomeric shifts in hydrogen-bonded bases of DNA. The results confirm electrostatic nature for all considered hydrogen bonds except for NH hydrogen bond in 7AI-FA. The largest interaction and polarization energies are obtained for 7AI-FA by means of energy decomposition analyses (EDA). The EDA results show an inverse competitive correlation between polarization and electrostatic components of attraction energy to determine the nature of hydrogen bond. The red-shifted hydrogen bonds are identified for all complexes as a result of hyperconjugation, except for 7AI-MeOH, which its blue-shift effect is attributed to the rehybridization dominating of CH bond. Investigation of aromaticity indices for 7AI complexes represents an increase in aromaticity of pentagonal ring and a decrease in hexagonal ring. Finally, the double hydrogen bond between 7AI and FA is identified as dominant interaction to achieve the tautomerization of 7AI in all applied approaches., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background : Social, occupational and emotional adjustment of Oncology nurses were assessed and compared with other nurses in this study. Subjects and Methods: One hundred nurses including Oncology nurses (n=50) and non-Oncology nurses (n=50) participated in cross-sectional study conducted in Shariati Hospital. Bell's Adjustment Inventory was used to measure social, emotional and occupational adjustment. Survey data were entered into SPSS statistical software, version 18 and the Kruskal-Wallis test was used for data analysis. Results: The study included nurses from Women's Internal Medicine ward (14%), Men's Internal Medicine ward (13%); Midwifery unit (17%), Operating room (15%) and Hematology-Oncology ward (41%). The mean age of the participants was 36.98 ± 8.28 years. In group of Hematology-Oncology nurses, the mean scores for occupational, social and emotional adjustment were 13.23 ± 1.99, 12.47 ± 1.79 and 18.19 ± 2.52, respectively. Data analysis showed that there is a statistically significant difference in the mean score of three areas of adjustment between Oncology nurses and their colleagues working in general wards (p-value=0.002, p-value<0.001, p-value<0.001 for occupational, social and emotional adjustment, respectively). Conclusion: The results of the study indicated that Oncology nurses had significantly lower social, occupational and emotional adjustment compared with nurses working in other wards.

A 64-year-old man first developed ligneous conjunctivitis at the age of 58 years after right pulmonary resection because of suspected cancer; otherwise, he had been healthy. Since then, he began to suffer from various forms of chronic pseudomembranous mucositis. Laboratory tests demonstrated that he had 7.8 % of plasminogen activity and 5.9 % of the normal antigen level. Thus, he was diagnosed as having severe type I plasminogen deficiency, making him the third case in Japan. DNA sequencing and PCR-restriction fragment length polymorphism analyses revealed that this patient was a compound heterozygote of a G-to-A missense mutation (G266E) in exon VIII and a g-to-a mutation at the obligatory splicing acceptor site in intron 12 (IVS12-1g>a). These two mutations were confirmed to be novel. Molecular modeling and splice site strength calculation predicted conformational disorder(s) for the Glu266 mutant and a drastic decrease in splicing efficiency for intron 12, respectively. Western blot analysis demonstrated that the patient contained a small amount of the normal-sized plasminogen protein. Mass spectrometric analysis of the patient's plasminogen revealed a peptide containing the wild-type Gly266 residue and no peptides with mutations at Glu266. However, he had never suffered from thrombosis. Low levels of fibrinogen/fibrin degradation products (FDP), D-dimer, and plasmin-α2-plasmin inhibitor complex clearly indicated a hypo-fibrinolytic condition. However, his plasma concentration of elastase-digested crosslinked FDPs was 4.8 U/mL, suggesting the presence of an on-going plasmin(ogen)-independent "alternative" fibrinolytic system, which may protect the patient from thrombosis. The patient has been free from recurrence of ligneous conjunctivitis for approximately 2.5 years.

Introduction: Coagulation factor XIII (FXIII) is a plasma fibrin-stabilizing factor comprising A and B subunits (FXIII-A and FXIII-B, respectively) in the form of a heterotetramer (FXIII-A2B2). A humanized monoclonal antibody to the interleukin-6 receptor (tocilizumab, TCZ) has emerged as an effective treatment for rheumatoid arthritis (RA), because it drastically reduces the inflammation of RA. We previously reported that two TCZ-treated RA patients with acquired FXIII deficiency developed pelvic hemorrhage., Methods: Because TCZ treatment had been shown to be related to low FXIII ammonia release activity and FXIII antigen in the two RA cases, we further examined FXIII-related parameters in 36 TCZ-treated RA patients and compared to 29 healthy controls by employing functional and immunologic assays for FXIII., Results: FXIII-A antigen and FXIII amine incorporation and ammonia release activities were significantly lower in the TCZ-treated group than the control group. The TCZ-treated group also showed mildly low FXIII-A2B2 and FXIII-B levels, and their fibrinogen levels were the lower limit of normal. A significant correlation between FXIII-B and fibrinogen was observed in the control and the TCZ groups, suggesting a common metabolic mechanism(s) for these two hepatic proteins. Because the specific activities of FXIII were normal and neither anti-FXIII-A nor anti-FXIII-B antibody was detected, the overall low FXIII level may have resulted from its impaired synthesis under an unbalanced cytokine milieu caused by TCZ treatment., Conclusion: Concomitant deficiencies in multiple hemostatic factors, including FXIII, may lead to an increased risk for hemorrhage in TCZ-treated RA patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Autoimmune hemorrhaphilia due to anti-factor XIII (FXIII) antibodies (AH13) is a life-threatening disease associated with high risk of surgical bleeding. Since AH13 occurs mainly in the elderly, patients of AH13 tend to be complicated with other life-threatening diseases that may require surgical procedures. During our nation-wide survey on AH13, supported by the Japanese Ministry of Health, Labor, and Welfare, patients with unexplained bleeding were examined for FXIII-related parameters and anti-FXIII autoantibodies. A 64-year-old man had previously been tentatively diagnosed with AH13 and received immunosuppressive therapies, as FXIII inhibitor was detected by functional cross-mixing studies. About 2 years later, he was definitively diagnosed with AH13, because our immuno-chromatographic test and enzyme-linked immuno-sorbent assay detected FXIII-bound anti-FXIII-A subunit autoantibodies. Since routine endoscopic examination revealed suspected esophageal carcinoma, a preparatory FXIII pharmacokinetic (PK) analysis was performed by infusing FXIII concentrates prior to biopsy. Consequently, biopsy of this lesion was done without bleeding complications. One month later, a second PK study was carried out before surgery, and esophageal bypass surgery was completed successfully under FXIII replacement therapy. Our experience with this case suggests that operations can be performed safely and with confidence even in patients with such life-threatening hemorrhagic diseases.

Background: Hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for acute leukemia. As HSCT improves the long-term survival, it is necessary to assess the late-onset complications affecting the quality of life following HSCT., Subjects and Methods: The study included 122 patients (65 male, 57 female) with leukemia (72 AML and 50 ALL) who received transplants from fully- matched siblings, unrelated donors and unrelated cord blood donors between February 2013 and August 2014 in Shariati Hospital. All study participants were over 18 years of age and had the minimum and maximum survival of 2 and 5 years, respectively. Patients who received HLA-haploidentical SCT were excluded from the study. All allogeneic recipients received busulfan and cyclophosphamide as conditioning regimen. Nobody received TBI-based conditioning regimen in this study. Patients were evaluated for cardiovascular, vision, psychological, endocrine, fertility problems and secondary malignancies one year after transplantation. Results : Data were analyzed using SPSS 15.0. Mitral and tricuspid regurgitation (TR/MR) were the most common cardiac complications (n=12, 10.5%).Thirty-nine percent of patients had psychological problems, especially depression (34%). Cataract was observed in 13% of patients and 34% complained of dry eye. Symptomatic pulmonary changes were found in 13 patients (10.6%). None of the HSCT survivors had experienced fertility before study entry. According to LH and FSH levels, 15% and 9% of females had ovarian failure, respectively. Testosterone level was less than normal in 49(84%) men and, according to their FSH and LH level, 20 (41%) had secondary hypogonadism and 29 (59%) had primary gonadal dysfunction., Conclusion: The results showed that patients who received Bu/Cy conditioning regimen experienced fewer late side effects such as cataract formation and hypothyroidism, compared to previous studies using TBI-based conditioning regimen.

Introduction: Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor that efficiently inhibits activated factor X when ZPI is in complex with PZ. We previously reported significantly higher concentrations of plasma ZPI (and PZ) in women during normal pregnancy than in non-pregnant women., Methods: We explored the possible contribution of estrogen to the ZPI levels in patients with or without bilateral oophorectomy (OVX), which induces artificial menopause where blood estrogen levels drastically decrease. One hundred ninety-one pre-menopausal Japanese women who underwent open hysterectomy owing to neoplasms participated in this study and were divided into two groups: 98 OVX and 93 Non-OVX cases. Plasma ZPI was measured by ELISA., Results and Conclusion: Contrary to our working hypothesis, plasma ZPI levels increased significantly in the OVX group after surgery when compared with the pre-operation levels. When these patients were individually analyzed, their ZPI value also rose significantly from pre-operation to post-operation levels. In contrast, plasma PZ levels remained unchanged. The significantly increased ZPI and unchanged PZ levels were also observed in the Non-OVX group. The increased ZPI levels were not significantly related to 17β-estradiol, luteinizing hormone or follicular stimulating hormone levels, clearly indicating that estrogen did not contribute to the plasma ZPI concentrations. Typical acute phase reactants fibrinogen and C-reactive protein (CRP) were also significantly elevated after surgery in both OVX and Non-OVX groups. However, only weakly significant linear relationships were observed between ZPI and fibrinogen or CRP, indicating the presence of alternative regulatory mechanisms underlying their plasma concentrations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Autoimmune haemorrhaphilia XIII/13 (AH13) is an acquired life-threatening bleeding disorder due to anti-factor XIII (FXIII) autoantibodies (auto-Abs). AH13 patients may die of haemorrhage without correct diagnosis and proper treatment because of lack of awareness and the absence of rapid easy-to-use tests specific for this disease. Currently, the definitive diagnosis is established by cumbersome and time-consuming laboratory tests such as dot-blot assays and enzyme-linked immunosorbent assays (ELISA), and therefore these tests are generally not carried out. To save AH13 patients' lives, there is an urgent necessity for developing a rapid test for FXIII auto-Abs. We first generated and characterised mouse monoclonal antibodies (mAb) against human FXIII A subunit (FXIII-A), and then developed a rapid immunochromatographic test (ICT) for detection of anti-FXIII-A auto-Abs using one mAb with a dissociation constant of 9.3 × 10⁻¹¹ M. The auto-Ab-FXIII-A complex was captured by the mAb on a nitrocellulose membrane and visualised by Au-conjugated anti-human IgG Ab. Mixing with healthy control plasma improved the detection of auto-Abs in patients having extremely low levels of FXIII-A. The specificity and sensitivity of the ICT were 87 % and 94 %, respectively. We also detected auto-Abs against activated FXIII (FXIIIa) in three patients by pre-converting FXIII to FXIIIa by thrombin treatment. ICT values were significantly inversely correlated with FXIII activity levels, indicating an association between the quantity of anti-FXIII autoantibodies and AH13. This reliable rapid ICT assay can be applied to a point-of-care test to detect anti-FXIII-A auto-Abs, and will contribute to early diagnosis and treatment of AH13.

Although the incidence of autoimmune hemorrhaphilia due to anti-Factor XIII (FXIII, not FVIII or FXII to avoid confusion) antibodies (AH13) or hemorrhagic "acquired FXIII deficiency due to anti-FXIII autoantibodies" was previously considered rare, it has been on the increase in the twenty-first century, at least in Japan. An 83-year-old woman with an unexplained hemorrhage was admitted to our hospital for intramuscular hematoma and severe anemia. Her FXIII activity was reduced to 10 % of normal; since FXIII inhibitors and anti-FXIII-A subunit autoantibodies were detected, she was definitively diagnosed with AH13. Despite developing cardiac tamponade due to pericardial hemorrhage, she clinically recovered from AH13 after hemostatic therapy with FXIII-concentrates and immunosuppressive treatment with rituximab and cyclophosphamide. However, her FXIII activity remained low and she died of hemorrhage 3.5 years after admission. AH13 patients should be monitored for a prolonged period, as this disease is very likely a chronic intractable hemorrhagic disorder.

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

This experiment was conducted to investigate the seasonal pattern of hair follicle activity, wool growth and fibre diameter (FD) in Sanjabi sheep in west Iran, Kermanshah (34° 18' N and 47° 3' E, elevation 1420 m). Ten male and 10 female Sanjabi sheep with an initial live weight of 32.1 ± 1.3 and 32.7 ± 1.5 (means ± SD), respectively, were used in a 365-day study. A diet was offered with an estimated concentration of 2.18 Mcal metabolizable energy and 130.0 g/kg DM crude protein. Body weight, average daily gain (ADG) and dry matter intake (DMI) were recorded weekly. The percentages of active primary and secondary wool follicles (PAP and PAS), follicle density and the ratio of secondary to primary follicles (S/P) were determined from skin biopsies, taken from the right mid-side of the sheep at monthly intervals. Raw and clean fibre growth rates and FD were measured from left mid-side patches (10 × 10 cm) harvested at the end of every month. There was a gradual increase in live weight throughout the experiment, while ADG and DMI changed in concert with day length. The greatest values for PAP and PAS were observed in summer, whereas lowest were obtained in winter (p < 0.001). Clean wool growth rate and FD were greatest (p < 0.001) in summer and lowest (p < 0.001) in winter. It is concluded that a seasonal cycle of feed intake, body growth, fibre follicle activity, wool growth and FD occur in fat-tailed Sanjabi sheep.

A 65-year-old woman was admitted with acute intramuscular hemorrhage of the left gluteus medius and piriformis muscles and associated anemia. Blood tests showed low plasma factor XIII (FXIII) antigen and activity. A cross-mixing test revealed a concave "inhibitor" pattern and anti-FXIII-A subunit antibody was detected. The patient was diagnosed with autoimmune hemorrhaphilia resulting from anti-FXIII antibody. The bleeding has not recurred since the initiation of treatment with oral immunosuppressive agents. Although hemorrhagic acquired FXIII deficiency is a rare disorder, prompt recognition of the underlying mechanism can save lives.

A 62-year-old man had a history of acute aortic dissection (Stanford type A) and had been diagnosed with polycystic kidney disease three years earlier, and then developed end-stage renal failure. He was referred with chief complaints of difficult hemostasis and consecutive hemorrhagic episodes at the puncture site of the shunt soon after dialysis introduction. We suspected chronic disseminated intravascular coagulation (DIC) due to mild thrombocytopenia and a fibrinolytic system abnormality. Plasma factor XIII activity was decreased, but no inhibitor was detected. In addition, contrast-enhanced computed tomography showed exacerbation of a dissecting aortic aneurysm. We finally diagnosed chronic DIC and secondary factor XIII deficiency associated with the aortic aneurysm. We selected treatment involving recombinant human soluble thrombomodulin (rTM) because he was on maintenance dialysis and required long-term follow-up bofore the operation. Hemostatic function improved with regular administration of rTM, and was well-controlled preoperatively.

Deformation density analysis is performed on fullerene-based anti-HIV agents to investigate the influence of charge redistribution on the capability of binding to HIV enzymes. Two types of HIV inhibitors including malonic acid- and amino acid-type C60 derivatives are considered to study. Total deformation density and its components including orbital relaxation and kinetic energy pressure are obtained for C60 derivatives. The deformation natural orbitals for each component of deformation density are assessed and their amounts of charge displacement are quantified to evaluate the binding affinity of HIV inhibitors. The results show that the orbital relaxation plays a more prominent role in deformation of electron density of studied compounds. Among the considered drugs, the amino acid-type derivatives, N-(carboxymethyl)-2,5-dicarboxylic fulleropyrrolidines, show the most charge displacement. Moreover, the investigation into the deformation density of amino acid-type functional groups on C60 reveals that the connection of functional groups to the 5,6-ring junction results more displaced charge than the connection to the 6,6-ring junction.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Acquired factor XIII (FXIII) deficiency is a common disease and seldom causes bleeding. However, severe FXIII deficiency may result in life-threatening bleeding. Although the inhibitor against FXIII has recently been focused as the cause of haemorrhagic acquired FXIII deficiency, the pathophysiology of inhibitor-negative cases could also be involved. We report a case of an 85-year-old Japanese man with serious subdural haemorrhage showing a remarkable decreased level of FXIII activity. He also manifested complications of compensated disseminated intravascular coagulation (DIC) with chronic renal failure, abdominal aortic aneurysm (AAA) and right renal carcinoma. Despite the successful evacuation of the haemorrhage, acute subdural haemorrhage subsequently developed that necessitated further craniotomies. Plasma cross-mixing studies and dot blot assay revealed no inhibitors against FXIII. We speculated that the decreased FXIII activity could be mainly due to hyperconsumption by DIC and surgery. Because plasma-derived FXIII concentrates are available to stop bleeding, clinicians should be aware of severe acquired inhibitor-negative FXIII deficiency in cases of unexplained excessive bleeding.

Factor XIII (FXIII) is a fibrin-stabilising factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). FXIII-B prevents the fast clearance of FXIII-A from the circulation. Congenital FXIII-A deficiency is a rare bleeding disorder, and congenital FXIII-B deficiency is even rarer. Through our recent nationwide survey on "acquired haemophilia-like disease due to anti-FXIII autoantibodies," we newly diagnosed severe congenital FXIII-B deficiency in a Japanese man. He developed thrombocytopenia and gingival bleedings at the age of 73, and his FXIII activity was as low as 10% of the normal. When he suddenly developed spontaneous intramuscular haematoma, the bleeding was arrested by infusing FXIII concentrates. However, his FXIII activity remained around 10% of the normal. At the age of 74, ELISA and western blotting assay unexpectedly revealed complete absence of FXIII-B in the patient's plasma. A dot blot assay detected anti-FXIII-B alloantibodies for the first time in this disease, which could be attributed to the infusion of exogenous FXIII. He was found to be homozygous for a Japanese founder-effect mutation of F13B. Repeated infusions of exogenous FXIII for hemostasis increased anti-FXIII-B alloantibodies that resisted FXIII substitution. To the best knowledge of the authors, none of the remaining 10 reported cases of congenital FXIII-B deficiency developed alloantibodies to exogenous FXIII-B of plasma FXIII. An originally mild bleeding phenotype of severe congenital FXIII-B deficiency can be exaggerated by additional acquired conditions. Physicians should consider congenital FXIII-B deficiency when they encounter cases of unexplained bleeding disorders.

Factor XIII (FXIII) is a fibrin-stabilizing factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). Congenital FXIII deficiency is a rare bleeding disorder. Acquired FXIII deficiency resulting from FXIII hypo-synthesis and/or hyperconsumption is a relatively common disorder in which patients seldom bleed. On the contrary, 'autoimmune/acquired hemorrhaphilia XIII/13 due to anti-FXIII antibodies (AH13)' is a rare but life-threatening bleeding disorder. Through a nationwide survey of AH13, we diagnosed aggressive AH13 in a 66-year-old woman. She consulted our department because of a spontaneous hematoma in her hand. After 1.5 months, she also developed an intramuscular hematoma but retained approximately half (52%) of the normal FXIII activities. The patient's bleeding symptoms were aggravated to catastrophic massive bleedings in the large abdominal muscles and intrapelvic and intraperitoneal spaces. Two months after the bleeding onset, she died despite undergoing plasma exchange, which was performed because we were deeply suspicious of the presence of an anti-FXIII inhibitor. Seven days after her death, extremely low FXIII activity (6%) and positive data on anti-FXIII inhibitor were reported by a commercial laboratory. Our dot blot assay detected anti-FXIII-A autoantibodies, afterwards. Thus, the diagnosis of aggressive AH13 as early as possible is necessary to save patients' lives.

Factor XIII (FXIII) is the final enzyme in the coagulation cascade. Acquired FXIII deficiency is caused by inhibitors of FXIII or decreased synthesis and/or increased consumption of FXIII, which leads to severe bleeding. Recently, we experienced a case of hemorrhagic-acquired factor XIII deficiency that occurred during treatment with the IL-6 inhibitor tocilizumab for rheumatoid arthritis. A 48-year-old man was referred because of right hip pain due to a hematoma. Laboratory findings showed that routine coagulation tests were normal, while FXIII activity was slightly low (52.4 %). The patient was successfully treated with plasma-derived factor XIII concentrates. The time course of recovery suggests that tocilizumab might have inhibited FXIII production. To our knowledge, this is the first report of acquired factor XIII deficiency associated with administering of tocilizumab. When recurrent bleeding is seen during administering of tocilizumab, acquired factor XIII deficiency may have been induced, thus attending physicians should consider this disease in a differential diagnosis.

Introduction: Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor, which contributes to hemostasis, wound healing, and maintenance of pregnancy. Accordingly, patients with congenital FXIII deficiency manifest a life-long bleeding tendency, abnormal wound healing and recurrent miscarriage. In order to understand the molecular mechanisms of congenital FXIII deficiency, genetic analysis and molecular modeling were carried out in a Japanese male neonate with severe FXIII deficiency., Methods and Results: Two novel mutations, Y204Stop (or Y204X, TAT to TAA) and S708R (AGC to AGG), were heterozygously identified by nucleotide sequencing analysis in exons V and XV of the gene for the A subunit of FXIII (FXIII-A). Y204X and S708R would lead to nonsense mediated mRNA decay and misfolding of the FXIII-A molecule, respectively. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the presence of these mutations was confirmed both together in the proband and one each separately in either the maternal or paternal sides of his family. In addition, moderately decreased FXIII activity was associated with the presence of either mutation. Molecular modeling predicted that the mutant molecule of S708R would be structurally compromised by the substitution of the Ser with the larger extended bulky and positively charged Arg side-chain., Conclusion: It is probable that the impaired tertiary structure of the mutant S708R molecule leads to its instability, which is at least in part responsible for the FXIII deficiency of this patient. This is consistent with the fact that the mutations and the reduced FXIII activities co-segregate among the patient's family members., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor which efficiently inactivates activated factor X, when ZPI is complexed with PZ in plasma. Reduced plasma levels of ZPI and PZ have been reported in association with thrombosis. It has also been reported that PZ increases during pregnancy and that its partial deficiency is related to early pregnancy loss or recurrent miscarriage (RM). However, until now there has been no report on ZPI in pregnancy. To explore the possible role(s) of ZPI in the maintenance of pregnancy, we studied 42 non-pregnant normal women, 32 women with normal pregnancies, and 134 cases of unexplained RM in Japan, as well as 64 non-pregnant normal German females. Plasma ZPI was measured by in-house ELISA. There were significantly higher concentrations of plasma ZPI in normal pregnancies compared to non-pregnant women. The present study also confirmed that both factor X, the major target of ZPI, and protein Z increased during normal pregnancies. This increased ZPI and PZ may counteract the increased activated factor X, which may in turn contribute to the maintenance of normal placental circulation. Plasma ZPI levels were unchanged in non-pregnant RM women, while the plasma PZ level was slightly reduced, a finding consistent with existing reports. The exact relationship between RM and this unaltered ZPI with mild PZ reduction relative to normal pregnancies warrants further investigation.

The present study aims were to determine the profiles of changes in progesterone (P4) and 17-β-estradiol (E2) in the peripheral blood of Markhoz goat (Iranian Angora) during estrous cycle, gestation, and parturition throughout natural breeding season. Gestation length averaged 145.3 ± 0.8 days, and the litter size was 1.1 ± 0.1. Birth weight ranged 2.4-2.8 and 1.5-2.5 kg in male and female kids, respectively. The mean estrous cycle lengths were 20.3 ± 0.4 and 20.9 ± 0.4 days for PGF(2α)-induced and natural cycles, respectively. Blood sampling was carried out daily during estrous cycle and weekly during gestation till parturition. E2 attained higher level (24.7 ± 2.15 pg mL(-1)) at estrus phase and dropped down to the lower level (18.80 ± 1.16 pg mL(-1)) within 3 to 4 days post-estrus. Concomitantly, P4 started to increase from the mean basal value of 0.5 ± 0.03 ng mL(-1) on day 0 to 6.88 ± 0.95 ng mL(-1) on day 6 of estrous cycle and reached the peak value of 12.8 ± 0.61 ng mL(-1) on day 12. From day 15, a decline was observed in P4 values till the end of the cycle. P4 remained at lower concentrations for 20-50 days of gestation, then increased and reached to its maximum level (13.51 ± 0.279 ng mL(-1)) in week 15 and returned again to its basal values within 1-2 weeks before parturition. The results will be discussed in terms of the usage of steroid hormone profile in several assisted reproductive technologies.

Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, alpha(2)-plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. In contrast, there was no CR impairment in the PRP of tissue transglutaminase-knockout mice compared with that of wild-type mice. Furthermore, a transglutaminase inhibitor, cystamine, halted CR in the PRP of wild-type mice. These results indicate that the enzymatic activity of FXIII is necessary for CR, at least in mice.

Protein Z is a vitamin K-dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its gamma-carboxylation was completely blocked. An exchange of the propeptide and/or gamma-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarin-sensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and gamma-carboxyglutamic acid domain to luciferase also demonstrated that the gamma-carboxyglutamic acid domain of protein Z was responsible for its warfarin-sensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their gamma-carboxyglutamic acid domains.

Factor XIII (FXIII) is a heterotetramer composed of two catalytic A subunits (FXIII-A) and two B subunits (FXIII-B). FXIII-B has 10 Sushi domains. To explore the structure-function relationship of FXIII-B, we looked for domains in FXIII-B responsible for its homodimer and heterotetramer assembly with FXIII-A. Full-length recombinant human FXIII-B (rFXIII-B) and truncated rFXIII-Bs with various numbers of Sushi domains (rFXIII-B x- y ) were expressed in a baculovirus expression system. rFXIII-B was indistinguishable from purified human plasma FXIII-B, in terms of the molecular weight (after being deglycosylated by glycosidases) and the ability to form complexes between the two subunits. rFXIII-B was in dimer form and produced a heterotetramer complex with FXIII-A. Gel-filtration and FXIII-A binding analysis of the various truncated forms of rFXIII-B x- y revealed that the first Sushi domain was responsible for the binding of FXIII-B to FXIII-A and that the fourth and ninth Sushi domains were involved in the FXIII-B homodimer assembly. rFXIII-B and rFXIII-B 1-9, which formed a heterotetramer complex with FXIII-A, protected FXIII-A from proteolytic digestion. These findings suggest that only full-length or nearly full-length FXIII-B is large enough to cover the exposed surface of FXIII-A. In conclusion, at least 3 out of the 10 Sushi domains of FXIII-B have the distinct function of forming a homodimer and a heterotetramer, which should be ascribed to the differences in their amino acid sequences. The present studies, however, do not exclude the possibility that additional Sushi domains may also support either or both functions.

This study was made on the effects of a normal diet containing 12.8 mg Cu kg(-1) DM which added gradually molybdenum and sulphur on the copper status and fibre quality in eight 1-year Merghoze goat. One group (n = 4 mean weight 31 +/- 2.0 kg) was treated with Mo and S supplements for 20 weeks, the second group (n = 4 mean weight 32 +/- 2.1 kg) served as controls. In addition of blood sampling for measuring copper status in plasma, the copper content and quality of fleeces were measured every 6 weeks. Mohair measurements were carried out by taking patch samples (10 x 10 cm2) from the mid-side area of the goat. The analytical set consists of plasma copper concentrations (Pl Cu), Trichloroacetic acid soluble copper concentrations (TCA-Cu), and fleece copper content. The results indicated that the addition of 20-30 mg Mo and 2-2.5 g S kg(-1) DM to the normal diet did produce sub clinical copper deficiency in treated goats. One such visual symptom was the loss of fleeces pigmentation and poorer crimp being observed. The Pl Cu minus TCA-Cu plasma became more than 2 microM in the blood of treated goat, indicating that there was a significant thiomolybdate formation in the body. The results showed that there was a significant decrease in Pl Cu (p < 0.05) along with a significant increase in thiomolybdate (MoS) production after 4 months. The sub clinical signs of copper deficiency and mohair quality are likely to be from high molybdenum intake and thiomolybdate formation in the body.

Background: Multiple studies suggest that the hemostatic and innate immune systems functionally cooperate in establishing the fraction of tumor cells that successfully form metastases. In particular, platelets and fibrinogen have been shown to support metastatic potential through a mechanism coupled to natural killer (NK) cell function. As the transglutaminase that ultimately stabilizes platelet/fibrin thrombi through the covalent crosslinking of fibrin, factor (F) XIII is another thrombin substrate that is likely to support hematogenous metastasis., Objective: Directly define the role of FXIII in tumor growth, tumor stroma formation, and metastasis., Methods: Tumor growth and metastatic potential were quantitatively and qualitatively evaluated in wild-type mice and gene-targeted mice lacking the catalytic FXIII-A subunit., Results: Loss of FXIIIa function significantly diminished hematogenous metastatic potential in both experimental and spontaneous metastasis assays in immunocompetent mice. However, FXIII was not required for the growth of established tumors or tumor stroma formation. Rather, detailed analyses of the early fate of circulating tumor cells revealed that FXIII supports the early survival of micrometastases by a mechanism linked to NK cell function., Conclusions: Factor XIII is a significant determinant of metastatic potential and supports metastasis by impeding NK cell-mediated clearance of tumor cells. Given that these findings parallel previous observations in fibrinogen-deficient mice, an attractive hypothesis is that FXIII-mediated stabilization of fibrin/platelet thrombi associated with newly formed micrometastases increases the fraction of tumor cells capable of evading NK cell-mediated lysis.

Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A subunits (FXIII-A) and non-catalytic B subunits (FXIII-B), and acts in haemostasis and wound healing. We generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. A longitudinal study was carried out using the gene-targeted mice to explore the possible effects of FXIII deficiency on aging. Survival rates of FXIII-A(-/-) males decreased to approximately 50% at 10 months after birth, although most FXIII-A(-/-) females and both genders of wild-type mice survived. Four FXIII-A(-/-) males died of severe intra-thoracic haemorrhage, and a large haematoma was found in their hearts. Haemorrhage, haemosiderin deposition and/or fibrosis were observed in the hearts of other dead FXIII-A(-/-) males. Fibrosis together with haemosiderin deposition was also found in the hearts of FXIII-A(-/-) males sacrificed. The in-vivo cardiac function was normal in FXIII-A(-/-) mice when compared with wild-type mice despite the presence of significant cardiac fibrosis. Although survival rates for both genders of the FXIII-B(-/-) and wild-type mice did not differ, mild fibrosis together with haemosiderin deposits were only found in the hearts of the sacrificed FXIII-B(-/-) males. Carditis and fibrosis in FXIII-deficient mice might be caused by a faulty or delayed reparative process that was initiated by abnormal haemorrhagic events within heart tissue. It is important therefore to examine possible cardiac involvement in human patients with congenital FXIII deficiency.

Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A (FXIII-A) and noncatalytic B subunits (FXIII-B), and acts in hemostasis and wound healing. We freshly generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. Mice carrying the disrupted allele were born at the expected Mendelian ratios, and the homozygous mice were viable and fertile under specific pathogen-free conditions. Although all homozygous and heterozygous mice showed no marked difference from the wild-type animals in general appearance, homozygous mice of either FXIII-A- or FXIII-B-deficiency did have prolonged bleeding times. It was confirmed that thrombin-dependent amine incorporation and fibrin-crosslinking in plasma were undetectable in the FXIII-A-deficient mice and markedly reduced in the FXIII-B-deficient mice; however, the gene expression of each subunit was regulated independently. Recombinant human FXIII-B (rFXIII-B) was expressed in a baculovirus expression system. When rFXIII-B was injected into FXIII-B-deficient mice, FXIII-A levels, fibrin crosslinking, and amine-incorporation activities increased in their plasma, indicating that FXIII-B assisted the maintenance of FXIII-A levels in the circulation. These mouse strains will be useful in exploring the possible pathophysiological roles of each subunit in vivo.

Background and Objectives: Protein Z (PZ), which regulates blood coagulation, is mainly synthesized in the liver. Its plasma level varies widely among individuals, and is highly sensitive to Warfarin. The mechanism for the basic transcription of the human PZ gene, however, has not been reported. The aim of this study was to elucidate the mechanism of gene regulation for PZ by characterizing its 5'-flanking region., Methods and Results: A reporter gene assay using the human hepatoma cell line, HepG2, identified a minimal promoter region (site A) and two enhancer regions (sites B and C) in the PZ gene. DNase I footprinting and electromobility shift assays revealed binding of the liver-enriched transcriptional factor hepatocyte nuclear factor (HNF)-4alpha to site A, the ubiquitous transcriptional factor Sp1 to sites A and C, and an unidentified factor to site B. The co-transfection of an HNF-4alpha expression vector with reporter gene constructs to the non-hepatic cell line HeLa resulted in a significant increase of PZ promoter activity., Conclusions: HNF-4alpha plays a crucial role in human PZ gene expression in hepatocytic cells, and Sp1 is also important. These findings provide the first step toward understanding the mechanisms of the varying plasma PZ levels in individuals under physiological and pathological conditions.

Purpose: To evaluate the efficacy of autologous plasmin enzyme as an adjunct to vitrectomy in diabetic macular edema., Methods: Plasmin derived from autologous blood was injected intravitreally into seven eyes 15 min before vitreous surgery. The development and progression of a posterior vitreous detachment (PVD) was followed, and the time required for vitreous removal was measured. Both pre- and postoperative visual acuities and optical coherence tomography (OCT)-determined macular thickness were measured., Results: In the seven eyes in which plasmin was used, a PVD developed approximately 5 min after the injection and was confirmed to extend to the far periphery. In all cases, the removal of the vitreous was completed in a shorter time and no complications were observed. A restoration of the shape of the macula was observed in all cases. The visual acuity improved by two or more lines in four eyes, and remained unchanged in the remaining three eyes., Conclusions: Autologous plasmin alone will create a full PVD, and eliminates the need for a mechanical creation of a PVD. Thus, plasmin is a safe and effective adjunct to vitreous surgery for the treatment of diabetic maculopathy.

Purpose: To determine whether a single intravitreal injection of autologous plasmin or a combination of plasmin and intraocular gas without peeling the internal limiting membrane (ILM) will close idiopathic macular holes., Methods: Eight eyes of seven patients with an idiopathic macular hole were studied. The degree of posterior vitreous detachment (PVD), vitreal liquefaction, closure of the macular hole, visual acuity, and complications following intravitreal plasmin or plasmin with gas were investigated. The removed ILM was examined by electron microscopy., Results: A PVD was created in seven out of eight eyes exposed to plasmin or plasmin with gas, however, the macular hole was not closed by either. Closure occurred in two eyes using conventional vitrectomy after the plasmin with gas injection, but peeling the ILM was required in the remaining six eyes. Vitreal fibers and glial cells were not observed on the vitreal surface of the extracted ILM., Conclusions: A PVD was created safely and reliably although closure of the macular hole did not occur with either plasmin or with plasmin and gas injection. However, vitreous surgery became easier, and it required a shorter time to close the macular hole with intravitreal plasmin.

Protein Z is a vitamin K-dependent glycoprotein that plays a role in the regulation of coagulation. A nucleotide substitution of G by C in exon II of the protein Z gene, resulting in the replacement of Glu-30 with Gln (E30Q), and a G to A transition at the 79th nucleotide in intron F (IntF79G/A) were heterozygously identified in a patient with a severe thrombotic tendency, whose plasma protein Z level was about 15% of normal. Other vitamin K-dependent coagulation factors were within normal ranges. Glu-30 is one of 13 gamma-carboxylation sites in protein Z and is well conserved among vitamin K-dependent proteins. Expression studies revealed that the E30Q mutant was not released from synthesizing cells, although wild-type protein Z was readily secreted in a vitamin K-dependent fashion. The E30Q mutant was N-glycosylated, gamma-carboxylated, and translocated from the endoplasmic reticulum (ER) to the Golgi in the presence of vitamin K, as was the wild type. Coexpression of E30Q with wild-type protein Z interfered with the secretion of the wild type, while only a minor or no effect was observed on the secretion of factor X and plasminogen. The IntF79A allele has been reported to be also associated with lowered protein Z levels.

The clinical significance of diminished protein Z in plasma is controversial. Studies in mice demonstrated that deficiency of protein Z dramatically increases the prothrombotic tendency of factor V Leiden mutation. This finding was confirmed by initial results in humans, indicating that thromboembolism in factor V Leiden patients with lowered protein Z level occurs earlier than in patients with normal protein Z levels. Consequently, the aim of our present study was to find out whether genetic alterations of protein Z were demonstrated in patients with factor V Leiden mutation and early onset of thromboembolic disease. DNA-sequencing of the protein Z gene was performed in two patients with factor V Leiden mutation, early onset of thromboembolism, and lowered protein Z levels. In both patients, R255H substitution of the protein Z gene was identified. Subsequently, the R255H substitution was also found in 12 of 132 additional patients. Patients presenting with the R255H substitution in addition to factor V Leiden mutation showed thromboembolic events more frequently than factor V Leiden patients without R255H substitution of the protein Z gene. In conclusion, R255H substitution of the protein Z gene seems to influence clinical symptoms of thromboembolism in factor V Leiden patients.

Purpose: To report an improved preparation of safer and highly-purified autologous plasmin and to demonstrate its clinical applications., Methods: Prior to clinical application, animal experiments were carried out. In addition, the activity of plasmin in the vitreous cavity after injection was measured serially. In preparation for clinical use, the proteinase inhibitors aprotinin and benzamidine were used for suppression of protein denaturation. The samples were incubated for 48 hours to check for contamination. The preparation was applied to patients with idiopathic macular hole and diabetic macular edema., Results: The plasmin detached the vitreous body from the inner limiting membrane of the retina without any ill effects in animal experiments. The specific activity reached a peak in 5-10 minutes and decreased rapidly thereafter. By adding a suppressor of protein denaturation in the purification process, 0.2 IU (0.1 ml) of high-purity plasmin could be prepared from each patient. No bacterial contamination was noted. Complete vitreous detachment could be induced in two clinical cases. Urokinase was used instead of streptokinase to activate plasminogen., Conclusions: Vitreous detachment is considered to be induced safely and consistently by injection of plasmin prepared using a protein denaturation suppressor and activated with urokinase. This method is expected to be of clinical value.

Factor XIII (XIII) is a heterotetramer consisting of two catalytic A subunits (XIIIA) and two noncatalytic B subunits (XIIIB). We examined the molecular mechanisms of a Y283C mutation which had previously been identified in a patient with XIIIA deficiency. The recombinant Y283C protein was labile when expressed in MEG-01 cells, which can endogenously synthesize XIIIA. We also included two other mutants, G562R and I464stop, previously characterized in a non-XIIIA-producing cell line. All these mutants exhibited decreased thermostability and resistance against proteolytic digestion when compared with the wild-type. Gel-filtration analysis revealed that the mutants were in monomer form, while the wild-type formed a dimer. These results were consistent with the prediction by molecular modeling that the mutant molecules would be misfolded. Although assembly of a heterotetramer with XIIIB was demonstrated for Y283C, its binding ability was 10% that of the wild-type. No complex formation was observed for the G562R or I464stop mutants. The wild-type was stabilized in plasma by complex formation with XIIIB, resulting in an increased resistance against proteolytic digestion. In contrast, the mutants were unstable in plasma even in the presence of XIIIB. Thus, impaired folding, dimer formation, and heterotetramer assembly of the mutant XIIIAs lead to both intra- and extracellular instability, which must be responsible for XIIIA deficiency in the patient.

In an Italian patient with severe factor XIII deficiency, a novel mutation, Y283C (TAT to TGT), was identified heterozygously by nucleotide sequencing analysis in exon VII of the gene for the A subunit. The presence of this mutation was confirmed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in the proband and his brother. Molecular modelling predicts that the mutant molecule would be misfolded. It is probable that the impaired folding of the mutant Y283C A subunit led to its instability, which is at least in part responsible for the factor XIII deficiency of this patient.

Two Japanese patients were newly diagnosed as having B subunit (XIIIB) deficiency of factor XIII (former type I deficiency). Both patients have a previously described one-base deletion at the boundary between intron A/exon II in the XIIIB gene, heterozygously or homozygously. A founder effect was proposed for this mutation because 3 unrelated patients with XIIIB deficiency also share 2 3'-polymorphisms. In one patient heterozygous for the above mutation, a novel mutation was also identified: a deletion of guanosine in exon IX (delG) of the XIIIB gene. To understand the molecular and cellular pathology of the delG mutation, expression studies were performed using a cultured mammalian cell line. Pulse-chase experiments showed that a resultant truncated XIIIB remained inside the cells and could not be secreted into the culture medium. Furthermore, immunocytochemical examinations by epifluorescence, confocal, and electron microscopes indicated impaired intracellular transportation of the truncated XIIIB from the endoplasmic reticulum to the Golgi apparatus. No mutations in the gene for the A subunit (XIIIA) were identified in this patient. Therefore, secretion of the truncated XIIIB must also be impaired in vivo, leading to a secondary XIIIA deficiency. These results support a previous conclusion that genetic defects of XIIIB are the basis for the former type I factor XIII deficiency.

Factor XIII is a proenzyme for a plasma transglutaminase. Factor XIII in plasma is a tetramer (A2B2) held together by noncovalent bonds, and the A subunit contains the active site. Recently, the three-dimensional structure of the A subunit has been determined by x-ray crystallography. To understand the structure-function relationships of the factor XIII molecule and its clinical implications in factor XIII deficiency, we characterized its genetic defects and closely examined its gene products, including mRNA and protein levels. A variety of missense and nonsense mutations (Arg260-Cys, Tyr283-Cys, Gly562-Arg) and deletions/insertions with or without out-of-frame shift/premature termination and splicing abnormalities (4-bp deletion with 464Stop, T insertion at the exon IV/intron D boundary with exon IV-skipping, 20-bp deletion at the exon I/intron A boundary) has been identified in cases demonstrating A subunit deficiency. In some cases, the A subunit mRNA levels were severely reduced. Their molecular and cellular bases have also been explored by expression experiments in mammalian cells and by molecular modeling. In most cases, impaired folding and/or conformational changes of the mutant A subunits lead to both intra- and extracellular instability, which is responsible for the A subunit deficiency in the patients.

Human HADHA and HADHB genes encode the subunits of an enzyme complex, the trifunctional protein, involved in mitochondrial beta-oxidation of fatty acids. Both genes are located in the same region of chromosome 2p23. We isolated genomic clones, including 5' flanking regions, for HADHA and HADHB. Sequencing revealed that both of these genes are linked in a head-to-head arrangement on opposite strands and have in common a 350-bp 5' flanking region. The 5' flanking region has bidirectional promoter activity within this region; two cis elements proved critical for the activity. Transcription factor Sp1 functions as an activator for the bidirectional promoter by binding to both elements. Therefore, expression of trifunctional protein subunits are probably coordinately regulated by a common promoter and by Sp1.

To study the mechanism of gene regulation for coagulation factor XIII A subunit (FXIIIA), we characterized its 5'-flanking region using a monocytoid (U937), a megakaryocytoid (MEG-01), and other cells. Our results confirmed that U937 and MEG-01 contained FXIIIA mRNA. A tentative transcription start site was determined to be 76 bases upstream from the first exon/intron boundary. Reporter gene assays revealed that a 5'-fragment (-2331 to +75) was sufficient to support basal expression in U937 and MEG-01 but not in the other cells. Deletion analysis confined a minimal promoter sequence from -114 to +75. DNase footprinting, electrophoretic mobility shift, and reporter gene assays demonstrated that promoter elements for a myeloid-enriched transcription factor (MZF-1-like protein) and two ubiquitous transcription factors (NF-1 and SP-1) in this region were important for the basal FXIII expression. It was also revealed that an upstream region (-806 to -290) had enhancer activity in MEG-01 but silencer activity in U937. DNA sequences for binding of myeloid-enriched factors (GATA-1 and Ets-1) were recognized in this region, and the GATA-1 element was found to be responsible for the enhancer activity. These transcription factors play a major role in the cell type-specific expression of FXIIIA, which differs from other transglutaminases.