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Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of ∼200 μm simply by mixing F-CMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated F-CMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Even though a number of microfluidic systems for particle/cell sorting have been proposed, facile and versatile platforms that provide sufficient sorting throughput and good operability are still under development. Here we present a simple but effective numbering-up strategy to dramatically increase the throughput of a continuous-flow particle/cell sorting scheme based on hydrodynamic filtration (HDF). A microfluidic channel equipped with multiple branches has been employed as a unit structure for size-based filtration, which realizes precise sorting without necessitating sheath flows. According to the concept of resistive circuit models, we designed and fabricated microdevices incorporating 64 or 128 closely assembled, multiplied units with a separation size of 5.0/7.0 μm. In proof-of-concept experiments, we successfully separated single micrometer-sized model particles and directly separated blood cells (erythrocytes and leukocytes) from blood samples. Additionally, we further increased the unit numbers by laminating multiple layers at a processing speed of up to 15 mL min-1. The presented numbering-up strategy would provide a valuable insight that is universally applicable to general microfluidic particle/cell sorters and may facilitate the actual use of microfluidic systems in biological studies and clinical diagnosis.

Aim: To assess whether any form of osteopontin (OPN) is correlated with bone resorption markers or treatment effects in rheumatoid arthritis (RA)., Method: Subjects comprised 119 patients with RA. RA disease activity was evaluated by Disease Activity Score (DAS) 28, erythrocyte sedimentation rate (ESR), and levels of C-reactive protein (CRP), rheumatoid factor (RF) and matrix metalloproteinase (MMP)-3. OPN levels in plasma and urine were measured by enzyme-linked immunosorbent assay (ELISA). Levels of tartrate-resistant acid phosphatase (TRACP) 5b in serum and C-terminal telopeptide of type 1 collagen (CTX)-1 in urine were measured by ELISA. Patients were divided into responder and nonresponder groups, and OPN levels were compared at baseline and after treatment., Results: Levels of full-length OPN in plasma (P-fOPN) were significantly correlated with levels of TRACP 5b (r = 0.44, P < 0.001), urine CTX-1 (r = 0.26, P = 0.004) and MMP-3 (r = 0.34, P < 0.001). Levels of TRACP 5b were significantly correlated with age (r = 0.25, P = 0.007), but levels of P-fOPN were not. After treatment, plasma OPN levels were significantly decreased in responders (P = 0.003). Levels of full-length or thrombin-cleaved forms of OPN in urine were not correlated with TRACP 5b or CTX-1., Conclusion: Our results suggest that plasma OPN may reflect inflammatory bone destruction in RA patients., (© 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.)

Aim: To identify autoantibodies useful in the diagnosis of primary vasculitides., Methods: The presence of antibodies against proteins in the lysate of mouse blood vessels was examined by two-dimensional electrophoresis followed by Western blotting for the pooled serum sample from patients with various forms of vasculitis: polyarteritis nodosa (PAN), microscopic polyangiitis (MPA), Wegener's granulomatosis (WG) and Takayasu's arteritis (TA). Autoantigenicity in patients with vasculitides was examined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Clinicopathological correlations between the positivity of the autoantibodies and clinical status of patients with the vasculitis were examined., Results: The autoantigen detected in the lysate of pooled sera from patients with vasculitides was identified by mass spectrometry as carbonic anhydrase III (CAIII). ELISA showed significantly higher prevalence of anti-CAIII antibodies in MPA patients (MPA, 11/23 [47.8%]; healthy controls, 2/32 [6.3%]; P < 0.001). Further, anti-CAIII antibody-positive MPA patients had higher vasculitis activity scores compared to anti-CAIII antibody-negative patients, and a weak and not significant negative correlation was observed between anti-CAIII antibody levels and myeloperoxidase - anti-nuclear cytoplasmic antibody (MPO-ANCA) levels. No significant differences were found in anti-CAIII autoantibody levels between MPA and the other primary vasculitides., Conclusion: We found significantly high prevalence of anti-CAIII antibody levels in sera from MPA patients. Although the number of samples available in this study is small and anti-CAIII autoantibodies display weak specificity for MPA, anti-CAIII antibodies may be useful for diagnosing MPA in patients who have no ANCA, as well as for assessing disease activity., (© 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.)

Objective: We aimed to define the clinical features of liver dysfunction in patients with systemic lupus erythematosus (SLE)., Methods: The frequency and causes of liver dysfunction were examined in 206 patients with SLE., Results: Liver dysfunction was evident in 123 (59.7%) of the 206 patients. Liver dysfunction in patients with SLE can be drug-induced (30.9%) or caused by SLE itself (28.5%), fatty liver (17.9%), autoimmune hepatitis (AIH) (4.9%), primary biliary cirrhosis (2.4%), cholangitis (1.6%), alcohol (1.6%) or viral hepatitis (0.8%), and it tends to be mild except when caused by AIH. Values for aminotransferase were significantly increased when AIH was the cause, whereas alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GTP) were significantly increased when AIH or drugs were the cause. The liver was already dysfunctional at the time of SLE onset in 56 (45.5%) of 123 patients with liver dysfunction. Neurological involvement was more common among patients with than without liver dysfunction, whereas SLE activity and prognosis did not significantly differ between the two groups., Conclusion: Liver dysfunction in the presence of SLE can be caused by many factors, but when extant at the time of SLE onset, either SLE itself or drugs can be the cause. Autoimmune hepatitis should be considered when liver dysfunction is relatively severe.

Objective: L-ficolin plays an important role in innate immunity and is involved in apoptosis. The objective of this study was to investigate the relationship between serum L-ficolin levels and clinical manifestations in patients with systemic lupus erythematosus (SLE)., Methods: Serum L-ficolin levels were determined by enzyme-linked immunosorbent assay in 66 SLE patients and 50 healthy controls., Results: Median serum L-ficolin levels were 5.0 and 8.7 μg/ml in SLE patients and controls, respectively (p = 0.0001). There were no significant differences in serum L-ficolin levels between the active disease group [SLE Disease Activity Index (SLEDAI) >6] and the inactive disease group (SLEDAI <5). Decreased serum L-ficolin levels were associated with thrombocytopenia (median of with vs. without thrombocytopenia 3.4 vs. 5.3 μg/ml, p = 0.008). There were no correlations between serum L-ficolin levels and SLEDAI, serum C3, or serum C4 levels., Conclusion: The association between L-ficolin and thrombocytopenia suggests a pathogenic role for L-ficolin in thrombocytopenia in SLE.

A 40-year-old man with acute meningitis, nephrotic syndrome, elevated antinuclear antibody (2560×), and liver dysfunction was referred to our hospital. He was antimitochondrial antibody positive, and renal and liver biopsy findings revealed membranous nephropathy (MN) and primary biliary cirrhosis (PBC), respectively. This extremely rare MN accompanied by PBC was detected while the patient was undergoing treatment for acute meningitis.

We have built a combined scanning tunneling microscope-atomic layer deposition (STM-ALD) tool that performs in situ imaging of deposition. It operates from room temperature up to 200 °C, and at pressures from 1 × 10(-6) Torr to 1 × 10(-2) Torr. The STM-ALD system has a complete passive vibration isolation system that counteracts both seismic and acoustic excitations. The instrument can be used as an observation tool to monitor the initial growth phases of ALD in situ, as well as a nanofabrication tool by applying an electric field with the tip to laterally pattern deposition. In this paper, we describe the design of the tool and demonstrate its capability for atomic resolution STM imaging, atomic layer deposition, and the combination of the two techniques for in situ characterization of deposition.

A 27-year-old woman exhibited progressive pancytopenia during cyclophosphamide pulse therapy for lupus nephritis and low-dose methotrexate therapy for severe arthralgia. Bone marrow aspiration revealed highly abnormal cell morphology, indicating therapy-related myelodysplastic syndrome. Pancytopenia and bone marrow cell morphology improved 3 months after discontinuation of cyclophosphamide. It is necessary to promptly examine bone marrow cell morphology and chromosomal aberration in cases with connective tissue diseases complicated by sudden cytopenia during immunosuppressive therapy with chemotherapeutic agents.

Aim:   Liver dysfunction is not rare in patients with collagen disease. We sought to elucidate the clinical features of liver dysfunction in the presence of collagen disease., Methods:   We analyzed the frequency and causes of liver dysfunction in 607 patients (rheumatoid arthritis [RA], n = 220; systemic lupus erythematosus [SLE], n = 164; systemic sclerosis [SSc], n = 47; Sjögren's syndrome [SjS], n = 44; Behçet's disease, n = 43; polymyositis/dermatomyositis [PM/DM], n = 27; vasculitis syndrome, n = 25; mixed connective tissue disease [MCTD], n = 21; and adult-onset Still's disease [AOSD], n = 16)., Results:   Liver dysfunction was observed in 238 (39.2%) of 607 patients showing collagen disease. Patients with AOSD (81.3%), PM/DM (51.9%) and vasculitis syndrome (48.0%) frequently displayed liver dysfunction. Liver dysfunction in collagen diseases results from many causes; drug-induced liver injury (26.1%), fatty liver (7.6%), viral hepatitis (1.3%), autoimmune hepatitis (4.2%), primary biliary cirrhosis (15.9%) and the collagen disease itself (15.5%). Conversely, primary biliary cirrhosis was a leading cause in SSc (76.1%) and SjS (70.0%). Liver dysfunction in collagen disease tended to be mild. In addition, alanine aminotransferase levels correlated positively with ferritin levels in AOSD (R = 0.708, P < 0.05). Moreover, alkaline phosphatase levels correlated positively with C reactive protein levels in vasculitis syndrome (R = 0.833, P < 0.05)., Conclusion:   Liver dysfunction in the presence of collagen disease has various causes, and dysfunction associated with collagen disease reflects the activity of the collagen disease itself., (© 2010 The Japan Society of Hepatology.)

Rheumatoid arthritis (RA) is a systemic autoimmune disease that is characterized by chronic synovial inflammation. Patients with RA have increased risk of infection; this is related to RA itself or the adverse effects of medication. In this report, we describe a case of emphysematous pyelonephritis in a patient with RA associated with AA amyloidosis and steroid-induced diabetes mellitus who was taking corticosteroid and low-dose methotrexate.

Objective: To measure concentrations of the thrombin-cleaved isoform of osteopontin (OPN) in urine and plasma of patients with rheumatoid arthritis (RA), and to assess whether levels of thrombin-cleaved OPN are associated with measures of RA., Methods: Subjects comprised 70 patients with RA, 20 patients with osteoarthritis (OA), and 46 healthy controls. RA disease activity was evaluated by tender joint count, swollen joint count, patient's global assessment of disease activity, erythrocyte sedimentation rate (ESR), and levels of C-reactive protein (CRP), matrix metalloproteinase-3 (MMP-3), and rheumatoid factor (RF), as well as 28-joint count Disease Activity Score (DAS28). OPN levels in plasma and urine were measured by ELISA., Results: Median levels of thrombin-cleaved OPN in urine (U-half) were significantly higher in RA patients (143.5 pmol/mmol Cr) than in healthy controls (67.9 pmol/mmol Cr) or OA patients (69.8 pmol/mmol Cr). Thrombin-cleaved OPN was not detected in plasma. U-half levels correlated significantly with levels of CRP (r = 0.26, p = 0.03), ESR (r = 0.26, p = 0.03), and RF (r = 0.28, p = 0.03). Median U-half levels were significantly higher in patients with stage III (249.9 pmol/mmol Cr) and IV (251.6 pmol/mmol Cr) disease than in patients with stage I (98.6 pmol/mmol Cr) disease., Conclusion: Our results suggest that urine levels of the thrombin-cleaved isoform of OPN may reflect the severity of active inflammatory arthritis in patients with RA.

Synovial vascularization in metacarpophalangeal joints of a patient with rheumatoid arthritis treated with leukocytapheresis (LCAP) was evaluated by Doppler sonography. After the treatment with LCAP, evaluation with American College of Rheumatology core set showed improvement, and the levels of C-reactive protein and serum amyloid A protein decreased. Power Doppler sonography demonstrated a reduction of color flow signals of the joints, and spectral Doppler sonography demonstrated an increase in vascular resistant, indicating a reduction of vessel's permeability. This is the first report evaluating a synovial vascularization and blood flow of the joints by Doppler sonography before and after LCAP therapy. Doppler sonography might be one of the useful methods for evaluating the therapeutic response of LCAP.

We report a case of phlegmonous gastritis in a 36-year-old man presenting with anorexia, epigastralgia, and high fever. Endoscopy showed an edematous lesion suggesting a submucosal lesion. Endosonography revealed thickening of the gastric wall, mainly involving the submucosa, with a few anechoic areas. The separation between the submucosa and the muscularis propria was unclear. The patient was treated with antibiotics and endoscopic mucosal resection. Culture of the aspirated pus from the lesion revealed Streptococcus pyogenes. Follow-up endosonography performed 1 week later showed significant improvement., ((c) 2007 Wiley Periodicals, Inc.)

In a previous paper we reported a new function of tissue kallikrein: rat urinary kallikrein (RUK) has a marked growth-stimulatory effect on neural stem cells prepared from brains of prenatal rats. We here report that conspicuous differentiation of neural stem cells to neurons and/or glial cells did not occur during stimulation by kallikrein, and this growth-stimulating effect of kallikrein is considerably specific for neural stem cells, i.e., RUK showed no detectable stimulatory effect on rat glial, PC12, GH(3), and HeLa cells. Thus this new function of kallikrein suggests the potential involvement of this enzyme on brain physiology. In addition, this effect of kallikrein for neural stem cells may have value in the treatment of cerebral ischemic stroke-induced injuries etc., i.e., kallikrein administered into the body stimulates neural stem cell proliferation and new neurons may be generated from them.

To evaluate the usefulness of Bb, a split product of complement factor B, as a clinical marker for disease activity of lupus nephritis, we measured the Bb concentration of sera from 42 patients with lupus nephritis. Serum Bb levels were significantly higher in patients with active nephritis (active nephritis group, n= 30) than in patients with nephritis in remission (remission group, n=12) (14.3+/-8.3 versus 7.4+/-5.9 microg/ml; p = 0.012). In contrast, there was no significant difference in serum C3 levels between active nephritis group and remission group (42.5+/-20.9 versus 44.7+/-15.9 mg/dl ; p = 0.77). In the comparison of Bb levels between active nephritis group and remission group, the sensitivity was 66.6%, specificity was 83.3%, and the positive and negative likelihood ratios were 3.95% and 0.41%, respectively. The present results suggest that serum Bb level is a useful clinical marker for disease activity in lupus nephritis.

Tissue kallikrein is expressed in many species and is widely distributed throughout the body, including the brain. In general, this protease is well known to release the vasoactive peptide, kinin, from kininogen. We report here that kallikrein has a prominent growth-stimulating effect on neural stem cells prepared from the brains of prenatal rats. This growth-stimulating effect was suppressed by antiserum against rat tissue kallikrein. Since bradykinin B2-receptor antagonist, Hoe140, did not suppress the growth-stimulating effect, kallikrein-mediated kinin release does not appear to be involved in this effect. Thus, our data suggest a new physiological function of kallikrein, the growth of neural stem cells. Such involvement would suggest that kallikrein is not only potentially involved in an important function of brain development, but also available for studies on regenerative medicine for neurons.

A 46-year-old woman was diagnosed with palmoplantar pustulosis (PPP) at the Department of Dermatology, Fukushima Medical University Hospital in 2000, and was treated with ointment. However, because liver dysfunction developed in 2003, she was referred to our department, where primary biliary cirrhosis (PBC) was also diagnosed on the basis of clinical findings. One year later, at the age of 49, she developed manifestations of Behçet's disease (BD), including erythema nodosum in the lower extremities. Because she had a history of uveitis, recurrent oral ulceration was present, and the HLA typing was positive for B51, BD was additionally diagnosed. Liver function normalized within three months of the start of treatment with ursodesoxycholic acid (UDCA). This is the first case of PBC associated with BD and PPP.

We describe a 49-year-old woman who presented in 2002 with pure red cell aplasia (PRCA), systemic lupus erythematosus (SLE), and idiopathic portal hypertension (IPH) that developed following a thymectomy. She underwent a thymectomy at 40 years of age to treat myasthenia gravis. PRCA developed 3 years after the thymectomy and she was successfully treated with cyclosporin. Systemic lupus erythematosus and IPH were diagnosed 6 years later. We conclude that immunological dysfunction resulting from the thymectomy contributed significantly to the subsequent development of PRCA, SLE, and IPH in this patient. This is the first report to describe this extremely rare occurrence.

In the present study, anti-ribosomal P antibody in sera of patients with systemic lupus erythematosus was assayed using an enzyme-linked immunosorbent assay, and its association with clinical symptoms of the patients was analyzed. The presence of anti-ribosomal P antibody was associated with increased frequency of lupus nephritis in the presence of anti-DNA antibody, and was associated with increased frequency of vascular thrombosis in the presence of anti-beta2 glycoprotein I antibody and/or lupus anticoagulant. The level of anti-ribosomal P antibody correlated inversely with the peripheral lymphocyte counts.

We report a case of HLA-B52-positive Behçet disease accompanied by multiarterial lesions. A 24-year-old woman was suffering from sporadic high fever and recurrent oral and genital ulcers, and laboratory data revealed severe inflammation. A diagnosis of Behçet disease was made. Magnetic resonance angiography, ultrasound study, and computed tomographic angiography demonstrated multiarterial lesions that had caused no symptoms. These noninvasive examinations were extremely useful in evaluating asymptomatic early vascular lesions.

We report a case of ulcerative colitis (UC) that occurred during the course of rheumatoid arthritis (RA). A 29-year-old woman with a 25-year history of RA was hospitalized for high fever, abdominal pain and hematochezia. Colonoscopy revealed erosive and reddish mucosa from the distal transverse colon to rectum. Histology revealed cryptitis (mainly caused by neutrophils), mild crypt abscess and goblet cell depletion. She was diagnosed with left-sided UC and treated with mesalamine enema. The abdominal symptoms and colonoscopic findings were greatly ameliorated. We conclude that the mesalamine enema was effective in this case of UC occurring during the course of RA.

A 23-year-old man was admitted to our department due to hemorrhage from gastric varices. He had been diagnosed as having Wilson's disease at the age of 17. Abdominal ultrasonography and computed tomography (CT) showed portal thrombosis and a large mass occupying most of the right lobe in the liver. The tumor was diagnosed as hepatocellular carcinoma (HCC) by image views and tumor markers. He died 3 months after the diagnosis, and an autopsy was performed. Histologic examination of the tumor showed moderately to poorly differentiated HCC. The nontumorous lesion of the liver revealed cirrhosis. HBX-DNA sequence was not detected in the liver. Hepatic cirrhosis is a well-recognized complication of Wilson's disease, but HCC is extremely rare. We describe the clinical findings of this patient and discuss the relationship of the development of HCC with a review of the relevant literature.

A 56-year-old man suffered from muscle weakness with elevated serum creatine kinase. Under diagnosis of polymyositis, the patient was treated with corticosteroid, methotrexate and cyclosporin A. Eleven months after the first signs of muscle weakness, the patient suffered an abrupt onset of anuria and underwent hemodialysis. The patient died of respiratory insufficiency 14 months after the first signs of muscle weakness. Autopsy findings revealed associated urinary bladder cancer with histological indications of adenosquamous cell carcinoma, liver metastasis and cancerous lymphangitis of the lung.

Tissue kallikrein (EC 3.4.21.35) that hydrolyzes kininogen and releases a physiologically active peptide, kinin, is found in rat brains. Insulin-like growth factor binding proteins (IGFBPs) that modulate IGF actions are also expressed in a variety of tissues including rat brains, and one of the major IGFBPs expressed in brain is known to be IGFBP-5, which is reported to be hydrolyzed in vitro by prostate-specific antigen (PSA) and gamma-nerve growth factor (gamma-NGF), both of which belong to the member of the kallikrein gene family. This study was designed to determine whether or not kallikrein has a potential to hydrolyze IGFBP-5 and their topographic proximity was investigated in rat brain using double immunohistochemical staining method. Immunohistochemically, IGFBP-5 positive cells were numerous and widespread in the cerebral cortex and belonged to neurons in the cell configuration. IGFBP-5 positive cells were negative for S-100 protein and were positive for betaIII tubulin, confirming them to be neurons. In addition, kallikrein positive cells were virtually all IGFBP-5 positive cells. IGFBP-5 was clearly hydrolyzed by kallikrein with cleavage sites of Arg188-Met189 and Arg136-Arg137 of IGFBP-5. Therefore, there is a possibility that kallikrein plays an important role in brain physiology, specifically in the neurons by regulating the actions of IGFBP-5 and IGF.

Two patients with amyopathic dermatomyositis complicated by interstitial lung lesions were effectively treated with a combination of corticosteroids and cyclosporine and/or cyclophosphamide. A 48-year-old female patient was treated with pulse methylprednisolone and cyclosporine 2 months after onset of dermal symptoms. A 45-year-old male patient was treated with oral prednisolone and pulse cyclophosphamide 2 1/2 months after onset of dermal symptoms. Early evaluation of interstitial lung lesions and early extensive therapy may improve prognosis of interstitial lung lesions in patients with amyopathic dermatomyositis.

Sera from 93 patients with connective tissue disease, 36 rheumatoid arthritis, 41 systemic lupus erythematosus, 12 polymyositis/dermatomyositis and 4 systemic sclerosis and sera from 12 patients with liver disease, along with sera of 10 healthy subjects, were tested for antikeratin antibodies using enzyme-linked immunosorbent assay in which the wells were coated with human epidermal keratin. Values above the mean+SD of the 10 healthy subjects were found in 8.3% of rheumatoid arthritis, 29.3% of systemic lupus erythematosus, 33.3% of polymyositis/dermatomyositis, 50% of systemic sclerosis, 16.7% of liver disease patients, and 20% of healthy subjects. The results indicated a character of naturally occurring antibody of antikeratin antibodies in human sera.

We report herein the case of a 70-year-old patient with elderly onset rheumatoid arthritis associated with severe muscle pain in shoulder and pelvic girdle. The patient revealed erosive polyarthritis with high titers of rheumatoid factor. Muscle pain started one month after the onset of rheumatoid arthritis followed by muscle weakness and muscle atrophy. Synovial effusion and edema in the soft tissue outside of the articular capsule in the knee joint were confirmed ultrasonographically. Administration of prednisolone at 20 mg/day dramatically abolished the muscular manifestations. The coexistence of an early stage of elderly onset rheumatoid arthritis and polymyalgia rheumatica was considered due to the presence of seropositive erosive arthritis and severe muscle manifestations at the same time.

Immunoreactive kallikrein has been demonstrated in the neurons of both prenatal and adult rat brains. However, the preferential site is quite different between the prenatal and adult brains, i.e., the former is mainly in the nuclei, and the latter is in the cell bodies and processes. In this study, the intensity of kallikrein mRNA expression was investigated in prenatal and postnatal rat brains by reverse transcription polymerase chain reaction (RT-PCR). A marked progressive increase in kallikrein mRNA expression was observed during the prenatal and early postnatal stage, with the maximum level at postnatal days 0 and 1 (P0 to P1). This helps to explain the previous immunohistochemical findings in which a strong immuno-intensity of kallikrein in the neurons of prenatal and newborn rat brains was found as compared with adult rats. The function(s) of kallikrein expressed in the neurons in the developing stages is unclear, but the role(s) must be different from that of the adult because of the unequivocal difference in its location between them. It is thus possible that kallikrein has certain unknown important role(s) in the neuronal physiology acting on nuclear protein(s) in the developing stages, although the possibility of function via kinin receptors cannot be excluded at present.

The ferredoxin:thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N-terminus, one in the middle of the subunit and one near the C-terminus. We have obtained some evidence indicating that the N-terminal consensus domain is possibly involved in the interaction with the catalytic subunit.

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

Ferredoxin:thioredoxin reductase (FTR) is an iron-sulfur protein, which, in the presence of ferredoxin and thioredoxin, catalyses the light-dependent activation of several photosynthetic enzymes. Spinach FTR consists of two dissimilar polypeptide chains, A and B, present in equal amounts. Whereas subunit B seems to be responsible for the catalytic activity, subunit A has no known catalytic function. We found earlier that the N-terminus of subunit A, also called the variable subunit, shows terminal redundancy and that 2-3 of its serine residues are phosphorylated [Tsugita, A. Yano, K., Gardet-Salvi, L. & Schürmann, P. (1991) Protein Sequence Data Anal. 4, 9-13]. We now report the complete amino acid sequence of subunit A, determined by conventional protein sequencing methods. The polypeptide chain with a calculated molecular mass of 12,669 Da consists of 112 amino acids and has a calculated isoelectric point of 5.4. The analysis of the sequence supports the idea that this subunit has no catalytic function. The comparison with a known cyanobacterial FTR reveals about 58% similarity and the striking presence of a N-terminal extension in the spinach protein. This extension may be responsible for the reported size variability of this subunit.

Peptides or proteins were hydrolyzed by vapors of 90% pentafluoropropionic acid or heptafluorobutyric acid at 90 degrees C for various time periods. The hydrolyzate mixtures analyzed by both fast-atom-bombardment and electrospray ionization mass spectrometry showed a series of C-terminal successive degradation molecular ions. The degradation reaction may be due to the selective formation of an oxazolone ring at the C-terminal amino acid, followed by hydrolytic removal of the C-terminal amino acid. The major side reactions were cleavages of the peptide bonds at the C side of the internal aspartic acid residue and the N side of serine residue.