Circadian rhythms in microalgae.

Circadian rhythms have been described in a variety of microalgae. In each group, some model organisms arose and most detailed studies have been done with them. They include the cyanobacterium ("blue-green alga") Synechococcus and eukaryotic microalgae Gonyaulax polyedra (Dinophyta), Chlamydomonas reinhardtii (Chlorophyta), and Euglena gracilis (Euglenophyta). This review focuses on recent approaches to depict molecular components of the circadian system and the mechanisms of regulation in these organisms. In Synechococcus, the identification of the kailocus, which represents a central part of its oscillatory system, is discussed, as well as diverse approaches based on a luminescent reporter gene, which is driven by a clock-controlled cyanobacterial promoter. In eukaryotic microalgae, the diversity of genes/proteins that are controlled by the circadian clock is described and the kind of regulation (transcriptional and translational control) is emphasized. The role and function of conserved clock-controlled RNA-binding proteins such as CCTR from Gonyaulaxor Chlamy 1 from Chlamydomonas are discussed.

Differential translational initiation of lbp mRNA is caused by a 5' upstream open reading frame.

Expression of the luciferin-binding protein (LBP) from Gonyaulax polyedra is regulated by the circadian clock at the translational level. Here we report that in vitro translation of lbp mRNA results in the synthesis of two LBP variants of different sizes, which is shown to be due to translational initiation at different in-frame AUG codons on lbp mRNA. Differential initiation is caused by a small open reading frame (ORF, situated in the 5' untranslated region of lbp mRNA), which gives rise to a leaky scanning mechanism. In Gonyaulax, only one of these variants, which is produced by initiation from the first AUG of the lbp ORF, exhibits a circadian rhythm and is far more abundant during night phase.

Serum levels of soluble Fas/APO-1 receptor are increased in systemic sclerosis.

It has been suggested that rheumatic diseases may result from a deficit in Fas-mediated T-cell apoptosis. Recent studies have demonstrated increased soluble Fas in sera from lupus erythematosus patients. We were interested to determine whether elevated soluble Fas levels are associated with systemic sclerosis. Soluble Fas levels were retrospectively assayed using a sandwich enzyme-linked immunosorbent assay in serum from 30 patients with systemic sclerosis and 15 normal controls. Hospital medical records were retrospectively reviewed for clinical and laboratory characteristics of the patients. Soluble Fas levels were analysed in subsets of patients with limited (lcSSc) versus diffuse cutaneous systemic sclerosis (dcSSc) and correlated with inflammatory activity. In systemic sclerosis soluble Fas serum levels (lcSSc, 2.19 +/- 0.71 ng/ml, dcSSc 2.53 +/- 1.37 ng/ml) were significantly higher than in normal controls (1.26 +/- 0.36 ng/ml). However, there were no significant differences in soluble Fas levels between lcSSc and dcSSc and poor correlation between soluble Fas levels and inflammatory activity status. Detection of elevated soluble Fas might serve as a clinical marker for immunological dysregulation in systemic sclerosis, but not for inflammatory disease activity.

The mRNA level of the circadian regulated Gonyaulax luciferase remains constant over the cycle.

The expression of luciferin-binding protein (LBP) and luciferase (LCF), two proteins that are involved in bioluminescence in Gonyaulax polyedra, is controlled by a cellular circadian clock. In the case of LBP, its temporal expression is reported to be regulated at the translational level, involving both 5' and 3' untranslated regions (UTRs) of lbp mRNA. Here, we show that the amounts of lcf mRNA are constant throughout the day-night cycle, indicating that the circadian expression of LCF is also regulated at the translational level.

Cutaneous breast angiosarcoma after conserving treatment of breast cancer.

Cutaneous angiosarcoma is a rare malignancy that sometimes occurs as a late sequela of breast conservation therapy. We report on a 79-year-old female who developed well-differentiated angiosarcoma in a lymphedematous left breast 5.5 years after surgery and radiotherapy for early invasive ductal breast cancer. The initial appearance was very similar to late radiation dermatitis, and histologically interpreted as scar tissue with atypical vascular lesion. The lesion progressed further, and was clinically suspicious for angiosarcoma. Thus, a second biopsy was taken which confirmed the diagnosis. A complete mastectomy removed all the tumor with clear margins. However, within a period of 16 months she presented four local recurrences which were treated by wide local excision. At present, the patient is free of locally recurrent tumour for 7 months. The few cases of breast angiosarcoma after breast conservation therapy reported so far demonstrate that these lesions are difficult to diagnose due to their rarity and their highly variable and benign appearance, which sometimes may mimic radiation-induced cutaneous changes. Since chronic lymphedema possibly contributes to the development of angiosarcoma, long-term clinical surveillance of these patients is recommended. Biopsies should be taken if new skin lesions occur.

[Livedo racemosa with ulcerations. Cyclic therapy with iloprost infusions]. / Livedo racemosa mit Ulzerationen. Zyklische Therapie mit Iloprost (Ilomedin)-Infusionen : Zyklische Therapie mit Iloprost (Ilomedin)-Infusionen.

A 43 year old woman suffered from an intermittent painful livedo racemosa at the back since her childhood. Her clinical course was complicated by ulcerations. In careful clinical investigations no signs of an underlying disease could be found, in particular a Sneddon syndrome could be excluded. By means of both conservative and surgical treatments, initial healing of the ulcerations was achieved but relapses occurred. Cyclic infusions of iloprost achieved long-term clearing of the ulcerations and disappearance of the pain. To the best of our knowledge the effectiveness of this treatment has not been described for this disease in the literature.

Conserved circadian elements in phylogenetically diverse algae.

Circadian expression of the luciferin-binding protein (LBP) from the dinoflagellate Gonyaulax polyedra is regulated at the translational level. A small interval in the lbp 3'-untranslated region, which contains seven UG-repeats, serves as a cis-acting element to which a trans-acting factor (CCTR) binds in a circadian manner. Its binding activity correlates negatively with the circadian expression of LBP. Here I report the identification of a protein in the green alga Chlamydomonas reinhardtii that represents a CCTR analog. It binds both specifically and under control of the circadian clock to the UG-repeat region. The data show for the first time that circadian cis-elements implicated in translational regulation have been conserved during evolution.

In vitro mutagenesis of binding site elements for the clock-controlled proteins CCTR and Chlamy 1.

The luciferin-binding protein (LBP) from the dinoflagellate, Gonyaulax polyedra, is regulated by a circadian clock at the translational level. A 22-nucleotide long interval in the lbp 3' untranslated region, which contains seven UG-repeats, was characterized as a circadian cis-acting element, to which a clock controlled factor (CCTR) binds. Recently we have found that the phylogenetically distant green alga, Chlamydomonas reinhardtii, contains a CCTR analog, called Chlamy 1. Here we show that the flanking nucleotides surrounding the UG-repeats are required for high binding activity of CCTR and Chlamy 1. The absence of three or more UG-repeats abolishes binding with both proteins.

[Progressive systemic scleroderma--prognosis determining involvement of internal organ systems]. / Die progressive systemische Sklerodermie--prognosebestimmender Befall innerer Organsysteme.

Prognosis of systemic sclerosis (scleroderma, Ssc) is largely depending on involvement of internal organs. Abnormalities of the gastrointestinal tract are found most frequently (85%), especially decreased motility of the oesophagus, which has little impact on the longterm clinical course of Ssc. Pulmonary manifestations can be demonstrated in 40-90% of patients; one must distinguish between pulmonary hypertension or fibrotic lung disease. The heart is affected in 50% of cases. Patchy or diffuse myocardial fibrosis, as well as pericarditis and pericardial effusions can induce symptoms of arrhythmia or congestive heart failure. Renal involvement is associated with increased mortality and occurs in 45% of Ssc, producing proteinuria, hypertension, scleroderma renal crisis and renal failure. In conclusion, involvement of the lungs, heart and kidneys are determining factors for the longterm course of systemic sclerosis.

A novel function of yeast fatty acid synthase. Subunit alpha is capable of self-pantetheinylation.

The prosthetic group of yeast fatty acid synthase (FAS), 4'-phosphopantetheine, is covalently linked to Ser180 of subunit alpha. It originates from coenzyme A and is transferred to the enzyme by a specific phosphopantetheine:protein transferase (PPTase). The present study demonstrates that the FAS-activating PPTase of yeast represents a distinct catalytic domain of the FAS complex and resides within the C-terminal portion of subunit alpha. The autoactivation capacity of yeast FAS became evident from in vitro pantetheinylation studies using purified apo-FAS preparations. These were readily converted to pantetheinylated holo-FAS simply upon addition of free coenzyme A. Pantetheinylation-competent apo-FAS was prepared in vitro by constructing hybrid oligomers containing alpha-subunits from two different pantetheine-less FAS-mutants. The respective mutants were selected according to their ability to complement each other, in vivo. In vitro formation of hybrid apo-FAS complexes was achieved by dimethylmaleic anhydride (DMMA) -induced reversible dissociation of mixtures of the two constituent mutant enzymes. This treatment was both necessary and sufficient to produce pantetheinylation-competent apo-FAS. Specific FAS activities were comparable independent of whether the apo-enzymes were pantetheinylated in vivo or in vitro. Apart from the induction of overall FAS activity, incorporation of phosphopantetheine into apo-FAS was also demonstrated by the use of 3H-labelled coenzyme A, leading to the formation of radioactively labelled FAS. It is concluded that pantetheinylation of yeast FAS is performed by an intrinsic catalytic activity of the apo-enzyme proper. The endogenous PPTase acts in trans between different subunits alpha in the alpha6beta6 oligomer. The self-pantetheinylation of yeast FAS represents the first example of an apo-enzyme being capable of post-translational autoactivitation.

Circadian expression of the luciferin-binding protein correlates with the binding of a protein to the 3' untranslated region of its mRNA.

The circadian-expressed luciferin-binding protein from the dinoflagellate Gonyaulax polyedra is regulated at the translational level. We detected a protein, apparently a dimer, that binds specifically to the 3' untranslated region of its mRNA. Its binding site was localized within a 22-nt region in the 3' untranslated region containing seven UG repeats. The binding activity of this protein cycles on a daily basis, decreasing at the beginning of the night when synthesis of luciferin-binding protein starts and increasing at the end of the night when synthesis of luciferin-binding protein stops. This suggests that it functions as a clock-controlled repressor, preventing the translation of lbp mRNA during the day.

[Biological age in patients with progressive scleroderma]. / Biologisches Alter bei Patienten mit progressiver Sklerodermie.

In 25 patients (10 male, 15 female) with systemic sclerosis aged between 28 and 79 years the biological age was determined. Alterations in the sense of "premature aging" were observed in the mean and developed stronger in male patients. They have to be counted in a greater part on the real process of disease. Compared to a healthy reference group, the expected diminishing of hand grip strength and tendon extensibility, vital capacity and partial oxygen pressure in the arteries could be confirmed. The latter correlated to an increase in rest pulse. Auditory acuity and the condition of the teeth were also reduced. Beyond that, noteworthy differences in psychological and social parameters not presumed at the beginning point to a decrease in social activities and to psychic changes. They underline the necessity of psychological counseling of the patients.

Systemic sclerosis-related Raynaud's phenomenon: effects of iloprost infusion therapy on serum cytokine, growth factor and soluble adhesion molecule levels.

Microvascular damage occurs in systemic sclerosis and is associated with increased serum levels of endothelial adhesion molecules and endothelium-associated cytokines, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, endothelin-1 and vascular endothelial growth factor (VEGF). Iloprost, a prostacyclin analogue, induces clinical benefit in patients suffering from scleroderma-related Raynaud's phenomenon. This study was performed to investigate the effect of iloprost infusions on endothelium activation. Serum samples from 12 patients with systemic sclerosis were examined using specific enzyme-linked immunoassays. The serum levels of sICAM-1, sVCAM-1 and soluble E-selectin were initially elevated and significantly reduced after iloprost infusions. The serum concentrations of VEGF and endothelin-1 revealed decreased levels after therapy too. These results indicate that the well-known clinical benefit of iloprost infusions on Raynaud's phenomenon is serologically detectable by a reduction of serum levels of endothelium-associated adhesion molecules, cytokines and growth factors reflecting an improvement in endothelial function.

[Cellular mechanisms of circadian clocks]. / Zelluläre Mechanismen circadianer Uhren.

The mechanisms of the biological clock are today being investigated in single neurons in cell culture or in unicellular and in other microorganisms. The results show that all components of this "endogenous clock" can be found at the cellular level. The cellular circadian program is controlled by a complex system of biochemical reactions, which can contain more than one circadian pacemaker and which comprises several feed-back loops at the input and the output side. This complex temporal program is a prerequisite for specialization and survival within the chrono-ecological niches of the "temporal space" day. It enables organisms on the one hand to adaptively react to environmental changes and thereby reaching transient independence of the external, physical time course; on the other side, it ensures that the endogenous day never runs out of synchrony with the solar day of the environment.

Substrate and product binding sites of yeast fatty acid synthase. Stoichiometry and binding kinetics of wild-type and in vitro mutated enzymes.

The four known substrate binding sites of yeast fatty acid synthase (FAS), Ser819 (acetyltransferase, OHAC) and Ser5421 (malonyl/palmitoyl transferase, OHMa1) of subunit beta and Ser180 (pantetheine binding site, SHc) and Cys1305 (3-oxoacyl synthase, SHp) of subunit alpha were replaced, by targeted in vitro mutagenesis, by the non-acylatable amino acids glutamine, glycine or alanine. The four mutated FAS proteins together with two pairs of double mutants (OHAc/OHMa1 and SHc/SHp) were episomally expressed in appropriate delta fas1 or delta fas2 deletion strains. The purified enzymes isolated from these transformants were used for comparative acyl binding studies with the substrates [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Malonate was found to be transacylated to enzyme-bound pantetheine (SHc) exclusively by the Ser5421 hydroxyl group of malonyltransferase (OHMa1) while acetate could use both the acetyl (Ser819) and the malonyl (Ser5421) transferase active sites on its way to the SHc and SHp binding sites. Acylation of SHc with either substrate was unaffected by the absence of the 'peripheral' SH group (SHp) while binding of acetate to SHp was dependent on enzyme-bound pantetheine (SHc). These genetic data support a revised model regarding the intra-molecular channeling of acetate and malonate within yeast fatty acid synthase. Quantitative acyl binding studies revealed a maximum of 2-3 mol rather than the expected 12 mol of malonate and of 6-7 mol rather than 24 mol of acetate bound/mol hexameric yeast FAS. Only 20-30% of the malonyl-enzyme and 35-50% of the acetyl enzyme represented performic-acid-labile thioester bonds. The binding characteristics of both substrates, exhibiting Hill coefficients distinctly lower than 1, as well as their non-linear Lineweaver-Burk and Scatchard plots, point to a marked negative cooperativity among the 12 yeast FAS subunits. The observed sub-stoichiometric substrate binding characteristics of the enzyme are ascribed to this effect. An a priori asymmetry of the complex appears unlikely since the coenzyme-A:FAS transacylation equilibrium may be shifted towards the fully acetylated enzyme in the presence of N-ethylmaleimide. In contrast to the limited acylation capacity of the 'resting' enzyme, complete acylation of yeast FAS at all of its 12 SHc and SHp sites is observed under steady-state conditions of fatty acid biosynthesis. Under these conditions, the enzyme exhibits full-site reactivity at its SHp, SHc and OHAc sites, but a concomitant 18-fold increase in Km of the coenzyme-A:OHAc transacylation reaction keeps the acyl-O-ester content of the acylated enzyme at less than 5% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and genomic organization of a gene for luciferin-binding protein from the dinoflagellate Gonyaulax polyedra.

The circadian expressed luciferin-binding protein (LBP) gene from the marine bioluminescent alga Gonyaulax polyedra represents the first dinoflagellate gene that has been cloned and sequenced at both cDNA and genomic levels. Starting with a fragment from the 3'-end of the LBP cDNA that was found by immunoscreening of a cDNA library, genomic clones were obtained by the inverse polymerase chain reaction technique. Full-length cDNA clones were selected by screening a cDNA library by plaque hybridizations and by polymerase chain reaction amplifications. The LBP sequence has a 2004-nucleotide open reading frame coding for a protein of 668 amino acids (approximately 75 kDa). The reading frame and identity of the clone were confirmed by the sequence of an octapeptide obtained from a purified fragment of CNBr-treated LBP. A variant LBP cDNA was found to differ in sequence by approximately 11% at the DNA level. The untranslated regions of the mRNA are 111 nucleotides (5'-untranslated region) and 158 nucleotides (3'-untranslated region) long, respectively. The LBP gene contains no introns and exhibits certain features not typical for a eukaryotic gene. Its promoter does not include the typical TATA box within approximately 50 nucleotides upstream of the transcription start site, and the usual poly(A+) signal (AAUAAA) is not present on the end of the LBP mRNA. The copy number of the gene is very high (approximately 1000 copies/cell). However, the universal genetic code and conserved positions relevant for the translational apparatus are maintained.

[ANCA-positive vasculitis of the skin and kidneys associated with acne conglobata]. / cANCA-positive Vaskulitis der Haut und Nieren im Verlauf einer Acne vulgaris conglobata.

A 19 year old man with severe acne conglobata and ulcerated pyoderma gangraenosum-like skin lesions on the face was first treated with isotretinoin (Roaccutan((R))), then immunosuppressively with prednisolone, diaminodiphenylsulfone (Dapson-Fatol((R))) and mycophenolate mofetil (Cellcept((R))). Under a daily maintenance dose of immunosuppressive treatment with 2.5 mg prednisolone and 1 g mycophenolate mofetil, weakness, muscle and joint aches appeared. Due to proteinuria, haematuria and an elevated antineutrophil cytoplasmic antibody (cANCA) and the histological detection of a leukocytoclastic vasculitis, the diagnosis of cANCA positive vasculitis of the skin and kidneys was established. Therapy with cyclophosphamide (Endoxan((R))) along with prednisolone was effective. An exact classification of this disease process was not possible.