[The Ca(2+) sensitizers CGP 48506 and EMD 57033, but not the Na(+) channel modulator BDF 9148, prolong relaxation in isolated cardiomyocytes of the guinea pig]. / Die Ca(2+)-Sensitizer CGP 48506 und EMD 57033, nicht aber der Na(+)-Kanal-Modulator BDF 9148, verlängern die Relaxation an isolierten Kardiomyocyten des Meerschweinchens.

The sodium channel modulator DPI 201-106 has been described to posses Ca(2+)-sensitizing properties. Therefore, the present study investigated the inotropic effect of the Na(+)-channel modulator BDF 9148 (1 microM), a congener of DPI 201-106, in comparison with the Ca(2+)-sensitizers CGP 48506 (1-50 mumol/l) and EMD 57033 (1-30 mumol/l) in electrically driven left ventricular cardiomyocytes isolated from guinea pigs. The changes of the contraction amplitude in comparison to the basal cell shortening (cell shortening in micron and %) were continuously recorded with a one-dimensional high speed camera. BDF 9148, CGP 48506, and EMD 57033 exerted a significant increase in the contraction amplitude (p < 0.05 vs. control). The maximal positive inotropic effects of CGP 48506 (50 mumol/l) and EMD 57033 (30 mumol/l) were +249 +/- 30% and +226 +/- 28%, respectively. The corresponding value for BDF 9148 (1 mumol/l) was +176 +/- 16%. However, only the Ca(2+)-sensitizers CGP 48506 and EMD 57033, but not BDF 9148, prolonged the contractile twitch. Especially in patients with an already enhanced intracellular myocardial Ca(2+)-concentration, Ca(2+)-sensitizers, which impair relaxation, may be disadvantageous for therapeutical use despite their positive inotropic effect.

Force/shortening-frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts.

BACKGROUND: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. METHODS AND RESULTS: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5-2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). CONCLUSIONS: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.

Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells.

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.

IL-6 receptor independent stimulation of human gp130 by viral IL-6.

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.

The enhanced contractility in phospholamban deficient mouse hearts is not associated with alterations in (Ca2+)-sensitivity or myosin ATPase-activity of the contractile proteins.

Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e.g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sensitivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 +/- 0.2 micromol/l; tension: 1.7 +/- 0.3 micromol/l; p < 0.001) and wild-type controls (1.1 +/- 0.01 micromol/l; 2.2 +/- 0.4 micromol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/ force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban.

Regional expression and functional characterization of the L-type Ca2+-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts.

The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.

Potent vasodilatory with minor cardiodepressant actions of mibefradil in human cardiac tissue.

1. The present study compared the cardiovascular effects of mibefradil (MIB), a novel Ca2+-channel antagonist with high selectivity for T-type Ca2+-channels to the effect of the L-type Ca2+-channel-antagonists nifedipine (NIF) and diltiazem (DIL) in left ventricular myocardium and coronary arteries of hearts obtained from patients suffering from dilated cardiomyopathy (NYHA IV). Right atrial myocardium from patients undergoing aortocoronary bypass surgery without signs of cardiac failure was studied as well. 2. NIF and DIL (100 micromol l(-1)) completely depressed force of contraction (FOC) in electrically driven left ventricular myocardium (NIF 6.5+/-1.4% and DIL 7.1+/-1.2% of control), whereas a similar concentration of MIB only reduced force of contraction to 55.1+/-4.0% of the basal FOC. The negative inotropic potency as measured by the concentration needed to reduce basal FOC for 25% was NIF (0.0095 micromol l(-1))>DIL (0.041 micromol l(-1))>MIB (9.47 micromol l(-1)). 3. All three Ca2+-channel antagonists were more potent in human atrial compared to human left ventricular myocardium to reduce FOC. 4. The rank order of Ca+-antagonistic moiety as measured by the decrease of the intracellular Ca2+-transient (fura-2 ratio method) was NIF>DIL>MIB. 5. All Ca2+-channel antagonists completely relaxed human coronary arteries (% of papaverine effect: MIB 81.7+/-5.5%, DIL 91.3+/-0.9%, NIF 96.4+/-3.7%) precontracted with PGF2alpha (0.3 micromol l(-1)). The rank order of vasodilatory potency was NIF (EC50; 0.02 micromol l(-1))>DIL (0.13 micromol l(-1))>MIB (2.05 micromol l(-1)). 6. The vasoselectivity measured by the ratio of the concentration needed to achieve a 25% decrease in force and the concentration needed for 25% vasodilatation was 316 for MIB, 1.5 for NIF and 1.0 for DIL. 7. The present study provides evidence that blockade of T-type Ca2+-channels (e.g. mibefradil) results in potent vasodilatory properties with only minor cardiodepressant effects.

T- and L-type Ca2+-channel antagonists reduce contractility in guinea pig cardiac myocytes.

The aim of this study was to investigate the influence of L- and T-type Ca2+-channel blockade on myocardial contractility in guinea pig cardiomyocytes. Left ventricular myocardium from guinea pig contains both L- and T-type Ca2+ channels. The T-type Ca2+ influx was inhibited with mibefradil (1-100 microM), a novel compound with a threefold higher affinity for T- compared with L-type Ca2+ channels. In comparison, L-type Ca2+ influx was reduced by the benzodiazepine diltiazem (1-100 microM). The effect of mibefradil and diltiazem on electrically driven (0.5 Hz) isolated cardiomyocytes (n = 12) was studied in a concentration-dependent manner. The change of the contraction amplitude (percentage of cell shortening) was continuously recorded with an one-dimensional high-speed camera. Both mibefradil and diltiazem concentration-dependently reduced (p < 0.05 vs. control) the contraction amplitude in isolated myocytes from guinea pig. The concentration at which the contraction amplitude of guinea pig cardiomyocytes was reduced by 50% (EC50) was 31.6 microM for diltiazem and 6.3 microM for mibefradil, indicating that the T-type Ca2+-channel blocker mibefradil is more potent in reducing contractility in guinea pig cardiac myocytes in comparison with the L-type Ca2+-channel antagonist diltiazem. Mean values for cell shortening in percentage +/- SEM for mibefradil (0, 1, 10, 100 microM) were 100%, 78 +/- 9.2%, 36 +/- 5.4%, and 24 +/- 3.6%. The corresponding values for diltiazem were 100%, 92 +/- 12.5%, 79 +/- 8.9%, and 35 +/- 2.6%. In contrast, the increase of the extracellular Ca2+ concentration (2-7.5 mM) resulted in a significant increase of the contraction amplitude (+213 +/- 14%). Therefore, blockade of the Ca2+ influx through voltage-dependent T- or L-type Ca2+ channels decreases contraction in isolated cardiac myocytes from guinea pigs containing L- and T-type Ca2+ channels.

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium.

The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.

Effect of cyclopiazonic acid on the force-frequency relationship in human nonfailing myocardium.

The present study investigated the functional role of the sarcoplasmic reticulum Ca++-ATPase in contraction and relaxation, intracellular Ca++-transients, as well as on the force-frequency relationship in human myocardium. The Ca++-ATPase activity of membrane vesicles isolated from sarcoplasmic reticulum (SR) obtained from nonfailing donor hearts (n = 7) was measured in the presence of cyclopiazonic acid (CPA, 0-30 microM), a highly specific inhibitor of the Ca++-ATPase of the SR (SERCA). The effects of CPA on parameters of contraction and relaxation, force-frequency relationship and [Ca++]i transients (with fura-2) were studied on isolated left ventricular muscle strips from human nonfailing myocardium. CPA concentration-dependently inhibited SERCA activity of isolated SR vesicles. In the presence of CPA (30 microM) the former positive force-frequency relationship in human left ventricular nonfailing myocardium became negative. Especially at high frequencies of stimulation, CPA decreased developed tension, peak rate of tension rise and systolic fura-2-light emission, whereas time to peak tension, time to peak [Ca++]i, time to 95% relaxation, diastolic tension and diastolic Ca++ levels were increased. Peak rate of tension decay and time to half-relaxation and half-decay of [Ca++]i were not altered significantly after treatment with CPA. These findings provide evidence that the SERCA plays a functional role in the frequency-dependent increase in force of contraction in human myocardium. Because an impaired function of the SERCA is predominantly followed by alterations of inotropic and to a lesser degree of lusitropic function, other important factors to lower [Ca++]i and influence relaxation may be present in human myocardium to compensate for the reduced SERCA activity, e.g., Na+-Ca++ exchanger.

Effect of inotropic interventions on contraction and Ca2+ transients in the human heart.

The present study investigated the influences of inotropic intervention on the intracellular Ca2+ transient (intracellular Ca2+ concentration ([Ca2+]i)) and contractile twitch. Isometric twitch and [Ca2+]i (fura 2 ratio method) were measured simultaneously (1 Hz, 37 degrees C) after stimulation with Ca2+ (0.9-3.2 mM), the cardiac glycoside ouabain (Oua; 0.1 microM), the beta1- and beta2-adrenoceptor-agonist isoprenaline (Iso; 1-10 nM), and the Ca2+ sensitizer EMD-57033 (30 microM) by using isolated human nonfailing right auricular trabeculae (n = 19). Inotropic interventions increased force of contraction and peak rate of tension rise (+T) significantly. Only Iso stimulated peak rate of tension decay (-T) higher than +T (P < 0.05), thereby reducing time of contraction (Ttwitch). EMD-57033 increased +T more effectively than -T and prolonged Ttwitch (P < 0.05). Ca2+, Oua, and Iso, but not EMD-57033, increased systolic Ca2+. Diastolic Ca2+ increased after stimulation with Oua or Ca2+, but not in the presence of EMD-57033. Iso shortened the Ca2+ transient and did not influence diastolic Ca2+. In conclusion, positive inotropic agents differently affect force and [Ca2+]i depending on their mode of action. Inotropic interventions influence diastolic Ca2+ and thus may be less advantageous in a situation with altered intracellular Ca2+ homeostasis (e.g., heart failure due to dilated cardiomyopathy).

[Effect of changed extracellular K(+) and Mg(2+)-concentration on intracellular Ca(2+) homeostasis, contraction coupling and force-frequency relations in the human myocardium]. / Einfluss einer veränderten extrazellulären K(+)- und Mg(2+)-Konzentration auf die intrazelluläre Ca(2+)-Homöostase, die Kontraktionskopplung und die Kraft-Frequenz-Beziehung am menschlichen Myokard.

The intracellular Ca(2+)-homeostasis may be affected by changes of the extracellular K(+)- and/or Mg(2+)-concentrations. Mg2+ reduces the Ca(2+)-influx via L-type Ca(2+)-channels, facilitates Ca(2+)-uptake into the sarcoplasmic reticulum, modulates the Ca(2+)-induced Ca(2+)-release and the Ca(2+)-binding to troponin C. The extracellular K+ activates the Na+/K(+)-ATPase and changes the membrane potential thereby affecting the mode of action of the Na+/Ca(2+)-exchanger. Especially when intracellular Ca2+ regulation is altered, for example in heart failure, Mg2+ and K+ exert beneficial effects on the frequency-dependent force-generation in human myocardium. Thus, extracellular Mg2+ and K+ influence contraction coupling in the human myocardium.

[A proposal for the MR staging of early rheumatoid arthritis in the metacarpophalangeal and proximal interphalangeal joints]. / Vorschlag einer MR-Stadieneinteilung der frühen rheumatoiden Arthritis an den Metakarpophalangeal- und proximalen Interphalangealgelenken.

PURPOSE: MRI can demonstrate pathology of joint disease in the early course of rheumatoid arthritis prior to destructions seen on conventional radiographs. In a prospective study, we tried to develop a systematical classification of joint pathology demonstrated by MRI, which would be essential for scoring the course of the disease. PATIENTS AND METHOD: Metacarpophalangeal and interphalangeal joints of 48 patients suffering from early rheumatoid arthritis (mean disease duration: 6.4 months) were evaluated by MRI using a high-resolution transmitter-receiver coil. Examinations included 2 mm sliced T2-, T1- and gadolinium enhanced T1-SE sequences in coronal and axial orientation. In consideration of pathological findings on MRI and histopathogenetical pathways of destruction in rheumatoid arthritis a MR-score (0-5) was established. RESULTS: This allowed to score each joint examined: score 0 (normal) in 47.8%/49.5%, score 1 in 35.5%/50.5%, score 2 in 4.2%/0%, score 3 in 10.8%/0%, score 4 in 1.5%/0% of the metacarpophalangeal/interphalangeal joints, respectively. CONCLUSIONS: Using the MR-score a relative individual destruction number can be calculated, which may be used to follow up patients in the early course of rheumatoid arthritis (e.g. drug therapy studies). The presented MR scoring system has to be evaluated further in longitudinal studies and must be correlated to radiographical and clinical findings.

Inotropic and coronary vasodilatory actions of the K-adenosine triphosphate channel modulator nicorandil in human tissue.

The present study aimed to characterize the inotropic and vasodilatory properties of the K-ATP channel opener nicorandil (NIC) in isolated human cardiac tissue. For comparison, the Ca+2 channel blockers diltiazem (DIL) and nifedipine (NIF) have been studied. Concentration-dependent effects of NIC, DIL and NIF on the force of contraction (FOC) and the vascular tone have been studied on left ventricular papillary muscle strips (dilated cardiomyopathy, New York Heart Association Class IV, n = 20; nonfailing, donor hearts, n = 4), on right auricular trabeculae (nonfailing, n = 5) and on precontracted (prostaglandin F2 alpha: 0.3, 0.5 or 1 mumol/l) isolated human coronary artery rings (cardiac transplantation, n = 15). NIC, DIL and NIF concentration-dependently reduced the FOC of the papillary muscle preparations. However, the IC25 for the negative inotropic effect was significantly higher for NIC compared to DIL and NIF. The maximal negative inotropic effects of NIC, DIL and NIF (100 mumol/l) were -48.2 +/- 4.1, -92.9 +/- 0.9 and -93.4 +/- 1.4% of the basal FOC. The negative inotropic actions of NIC were similar in the human failing and the nonfailing ventricular and in the right atrial myocardium. Whereas pretreatment with methylene blue, an inhibitor of guanylyl cyclase, had no effect on the negative inotropic action of NIC, it was almost abolished by glibenclamide, a selective antagonist of the ATP-dependent K channels. NIC, DIL and NIF relaxed the coronary artery rings with 97.1 +/- 0.5, 90.7 +/- 0.9 and 96.4 +/- 0.7% of maximal relaxation (papaverine, 100 mumol/l). The rank order of vasodilatory potency was NIF > NIC > DIL. In conclusion, NIC is as effective as DIL and NIF in relaxing human coronary artery rings. However, NIC showed significantly lower negative inotropic effects when compared with the Ca+2 channel antagonists. The negative inotropic action of NIC is probably due to an interaction with the ATP-dependent K channels. In addition, activation of guanylyl cyclase does not seem to exert any negative inotropic action in the human myocardium.

Genomic sequences and structural organization of the human nidogen gene (NID).

Nidogen/entactin is a ubiquitous 150-kDa multidomain basement membrane protein. Since in vitro binding studies indicated that nidogen may function as a major mediator in basement membrane organization and assembly, analysis of gene structure and regulation of gene expression will help us to understand many biological processes that involve degradation and reorganization of the basement membrane zone. An approximately 100-kb region of genomic DNA encoding the human nidogen gene (NID) including 5' and 3' flanking sequences has been cloned and characterized by restriction mapping and sequencing. The entire gene is more than 90 kb in length and contains 20 exons. All introns interrupt protein coding sequences. The size of individual introns varies significantly, ranging from 0.6 to 18 kb. Its exon/intron structure revealed that the protein domains of human nidogen are organized in a domain-specific manner with various subdomains being encoded by individual exons, indicating that exon duplication and shuffling have played an important role in determining the present structure of the protein. Comparison of the exon organization with the recently published ascidian nidogen amino acid sequence strongly suggests that vertebrate nidogen might have evolved from a common ancestral precursor resembling ascidian nidogen.