Preferential changes of skeletal muscle echogenicity in myotonic dystrophy type 1.

BACKGROUND AND PURPOSE: In myotonic dystrophy type 1 (DM1), weakness of distal limb muscles affects quality of life. Non-invasive evaluation of muscular involvement by muscle sonography could be useful for characterizing muscle-specific involvement. METHODS: Sonography of the lower leg and forearm was performed in 19 patients with DM1 and 10 control subjects. The mean echo intensities (EIs) of seven limb muscles were obtained by computer-assisted histogram analysis and compared within DM1 according to the overall clinical severity. RESULTS: The EIs of the muscles were significantly higher in DM1 than in the controls (P < 0.01), except for the soleus (P = 0.4). Comparison of adjacent muscles showed the following: (i) greater EIs in flexor digitorum profundus than flexor carpi ulnaris (P < 0.01) and flexor digitorum superficialis (P = 0.02), and (ii) greater EIs in the medial head of the gastrocnemius than the soleus (P < 0.00001). In a subgroup analysis of DM1 according to the modified Rankin Scale (mRS), the more severe subgroup (mRS = 4-5) had lower mean EIs than the less severe subgroup (mRS from 1-3) (P = 0.01) in the flexor digitorum superficialis but not in other muscles. CONCLUSIONS: Preferential high echogenicity in the medial gastrocnemius and deep finger flexors is suggestive of DM1. Muscle echogenicity is not generally related to functional dysfunction in DM1.

Intramuscular dissociation of echogenicity in the triceps surae characterizes sporadic inclusion body myositis.

BACKGROUND AND PURPOSE: Differential diagnosis of sporadic inclusion body myositis (s-IBM) and polymyositis (PM)/dermatomyositis (DM) is difficult and can affect proper disease management. Detection of heterogeneous muscular involvement in s-IBM by muscle sonography could be a unique diagnostic feature. METHODS: Sonography of the lower leg and forearm was performed in patients with s-IBM, PM/DM and control subjects (n = 11 each). Echo intensities (EIs) of the adjacent muscles [medial head of the gastrocnemius versus soleus and the flexor digitorum profundus (FDP) versus flexor carpi ulnaris (FCU)] were scored by three blinded raters. The mean EIs of these muscles were compared using computer-assisted histogram analysis. RESULTS: Both evaluation methods showed high echoic signals in the gastrocnemius of patients with s-IBM. EIs were significantly different between the gastrocnemius and soleus in patients with s-IBM, but not in those with DM/PM and the controls. In the forearm, although the EI of the FDP was higher in the s-IBM group than in the other groups, the EI differences between the FDP and FCU did not differ significantly between disease groups. The difference in area under the curves to differentiate between s-IBM and DM/PM was greatest between the gastrocnemius-soleus EIs (0.843; P = 0.006). CONCLUSIONS: High echoic signals in the medial gastrocnemius compared with those of the soleus are suggestive of s-IBM over PM/DM.

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration.

Axonal transport is critical for neuronal development and function, and defective axonal transport has been implicated in neurodegenerative diseases. However, how axonal transport is regulated, or how defective transport leads to neuronal degeneration, remains unclear. Here, we report that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3 (JIP3)) and JNK-associated leucine zipper protein (JLP) are essential for postnatal brain development. Mice with a double-knockout (dKO) in Jsap1 and Jlp in the dorsal telencephalon developed progressive neuron loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JLP regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JLP to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. Furthermore, we describe a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jlp dKO neurons causes axonal degeneration and subsequent neuronal death. JNK hyperactivation because of increased intra-axonal Ca(2+) in the Jsap1:Jlp dKO neurons was found to mediate both the axonal degeneration and neuronal death, in cooperation with the Ca(2+)-dependent protease calpain. Our results indicate that axonal JNK may relocate to the nucleus in a dynein-dependent manner, where it activates the transcription factor c-Jun, resulting in neuronal death. Taken together, our data establish JSAP1 and JLP as positive regulators of kinesin-1-dependent axonal transport, which prevents neuronal degeneration.

Clinical significance of CADM1/TSLC1/IgSF4 expression in adult T-cell leukemia/lymphoma.

Cell adhesion molecule 1 (CADM1/TSLC1) was recently identified as a novel cell surface marker for adult T-cell leukemia/lymphoma (ATLL). In this study, we developed various antibodies as diagnostic tools to identify CADM1-positive ATLL leukemia cells. In flow cytometric analysis, the percentages of CD4(+)CADM1(+) double-positive cells correlated well with both the percentages of CD4(+)CD25(+) cells and with abnormal lymphocytes in the peripheral blood of patients with various types of ATLL. Moreover, the degree of CD4(+)CADM1(+) cells over 1% significantly correlated with the copy number of the human T-lymphotropic virus type 1 (HTLV-1) provirus in the peripheral blood of HTLV-1 carriers and ATLL patients. We also identified a soluble form of CADM1 in the peripheral blood of ATLL patients, and the expression levels of this form were correlated with the levels of soluble interleukin 2 receptor alpha. Moreover, lymphomas derived from ATLL were strongly and specifically stained with a CADM1 antibody. Thus, detection of CD4(+)CADM1(+) cells in the peripheral blood, measurement of serum levels of soluble CADM1 and immunohistochemical detection of CADM1 in lymphomas would be a useful set of markers for disease progression in ATLL and may aid in both the early diagnosis and measurement of treatment efficacy for ATLL.

Xenopus W-linked DM-W induces Foxl2 and Cyp19 expression during ovary formation.

The molecular mechanisms of vertebrate ZZ/ZW-type sex-determining systems remain unclear. We recently indicated that a W-linked gene, DM-W is a likely ovary-determining gene in Xenopus laevis. We first examined whether Cyp19 for estrogen-synthesizing enzyme P450 aromatase and Foxl2 showed female-specific expression in developing gonads. Both genes showed much higher expression in ZW than in ZZ gonads during and after sex determination. Importantly, transgenic ZZ gonads expressing exogenous DM-W at the sex-determining stage showed a ZW-type pattern of Cyp19 and Foxl2 expression. These results suggest that DM-W up-regulates Cyp19 and Foxl2 expression to guide primary ovary development in X. laevis.

Negative regulation of MEKK1/2 signaling by serine-threonine kinase 38 (STK38).

Mitogen-activated protein kinases (MAPKs) are activated through the kinase cascades of MAPK, MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). MAPKKKs phosphorylate and activate their downstream MAPKKs, which in turn phosphorylate and activate their downstream MAPKs. MAPKKK proteins relay upstream signals through the MAPK cascades to induce cellular responses. However, the molecular mechanisms by which given MAPKKKs are regulated remain largely unknown. Here, we found that serine-threonine protein kinase 38, STK38, physically interacts with the MAPKKKs MEKK1 and MEKK2 (MEKK1/2). The carboxy terminus, including the catalytic domain, but not the amino terminus of MEKK1/2 was necessary for the interaction with STK38. STK38 inhibited MEKK1/2 activation without preventing MEKK1/2 binding to its substrate, SEK1. Importantly, STK38 suppressed the autophosphorylation of MEKK2 without interfering with MEKK2 dimer formation, and converted MEKK2 from its phosphorylated to its nonphosphorylated form. The negative regulation of MEKK1/2 was not due to its phosphorylation by STK38. On the other hand, stk38 short hairpin RNA enhanced sorbitol-induced activation of MEKK2 and phosphorylation of the downstream MAPKKs, MKK3/6. Taken together, our results indicate that STK38 negatively regulates the activation of MEKK1/2 by direct interaction with the catalytic domain of MEKK1/2, suggesting a novel mechanism of MEKK1/2 regulation.

The tree squirrel HP-25 gene is a pseudogene.

The gene for the hibernation-specific protein HP-25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP-25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP-25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP-25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP-25 gene, the 80-bp 5' flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides -67 to -51, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5' flanking sequence had almost no promoter activity in HepG2 cells, and HNF-4 did not bind to the corresponding region of the tree squirrel HP-25 gene. Furthermore, a tree squirrel-type G to A mutation at -57 in the chipmunk HP-25 gene promoter context abolished its binding to and transactivation by HNF-4. Thus, the point mutation in the HNF-4-binding site is likely to be involved in the lack of HP-25 gene expression in the tree squirrel.

HNF-1 regulates the liver-specific transcription of the chipmunk HP-20 gene.

The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.

Human jejunal permeability of two polar drugs: cimetidine and ranitidine.

PURPOSE: To determine the human jejunal permeability of cimetidine and ranitidine using a regional jejunal perfusion approach, and to integrate such determinations with previous efforts to establish a baseline correlation between permeability and fraction dose absorbed in humans for soluble drugs. METHODS: A sterile multi-channel perfusion tube, Loc-I-Gut, was inserted orally and positioned in the proximal region of the jejunum. A solution containing cimetidine or ranitidine and phenylalanine, propranolol, PEG 400, and PEG 4000 was perfused through a 10 cm jejunal segment in 6 and 8 subjects, respectively. RESULTS: The mean Peff (+/- se) of cimetidine and ranitidine averaged over both phases were 0.30 (0.045) and 0.27 (0.062) x 10(-4) cm/s, respectively, and the differences between the two were found to be statistically insignificant. The mean permeabilities for propranolol, phenylalanine, and PEG 400 averaged over both phases and studies were 3.88 (0.72), 3.36 (0.50), and 0.56 (0.08) x 10(-4) cm/s, respectively. The differences in permeability for a given marker were not significant between phases or between the two studies. CONCLUSIONS: The 10-fold lower permeabilities found for cimetidine and ranitidine in this study, compared to propranolol and phenylalanine, appear to be consistent with their less than complete absorption in humans.

Expression of JNK cascade scaffold protein JSAP1 in the mouse nervous system.

The mitogen-activated protein kinase (MAPK) cascades consist of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). The specificity of activation of MAPK cascades may be determined, in part, by scaffold proteins that organize multi-enzyme complexes. We have earlier reported a scaffold protein JSAP1 (also known as JIP3) in the JNK MAPK cascade. We also showed that, of the adult mouse tissues tested, JSAP1 mRNA was predominantly expressed in brain. Here we report the localization of JSAP1 protein in mouse embryos and adult brain by immunohistochemical analysis. In embryos (E11-16), JSAP1 immunoreactivity was mainly found in the central and peripheral nervous systems, where it was localized to the cell bodies and/or axons of developing neurons, but not neural precursor cells. In the adult brain, immunoreactive JSAP1 was localized mostly to cell bodies in almost all neurons. We also showed that the expression of JSAP1 transcripts and proteins gradually increased during the neural differentiation of mouse P19 embryonal carcinoma (EC) cells. Furthermore, we showed that overexpressed JSAP1 facilitated the efficient activation of JNK by MEKK1 in P19 cells. These results suggest that JSAP1 may function as a scaffold protein for the JNK signaling module in neuronal cells.

Decreased absorption and retention rates of magnesium in the rats fed on spinach-supplemented diets: possible explanations.

We studied the bioavailability of magnesium (Mg) in spinach after boiling with distilled water, using Mg-deficient growing male rats. The rats were fed a semipurified diet (Mg:0.063 per cent (w/w)) for 3 days. then a Mg-deficient diet (Mg:0.001 per cent (w/w)) for 5 days. They were then divided randomly into 7 groups of 6 rats each, and fed the semipurified diet (Mg: 0.063, 0.045 or 0.027 per cent (w/w)), or the spinach-supplemented diet (10 per cent (w/w) dried and powdered spinach after boiling with distilled water for 3 min at 100 degrees C). The Mg content of the diets supplemented with spinach grown on chemical nutrients, and on manure from pigs, cattle and fowl, was 0.069, 0.051, 0.043 and 0.036 per cent (w/w), respectively. Water intake and volumes of urine and faeces were significantly greater in the rats fed the spinach-supplemented diets than in those fed the semipurified diets. Apparent absorption of Mg, and urinary and faecal excretions of Mg were directly related to Mg intake: no significant difference was observed amongst the groups. Both the ratios of Mg absorption and retention were significantly lower in the rats fed diets supplemented with spinach than in those fed semipurified diets. The plasma Mg level was directly related to Mg intake in the rats fed the semipurified diets and the spinach-supplemented diets. However, the plasma Mg level in the rats fed spinach grown organically on manure from fowl tended to be higher than in the other groups. From these results, it was concluded that bound Mg in spinach was effectively utilized by Mg-deficient rats, however, the absorption and retention rates of Mg in rats fed diets supplemented with spinach were decreased. Possible explanations were discussed.

Nitric oxide-mediated antitumor activity induced by the extract from Grifola frondosa (Maitake mushroom) in a macrophage cell line, RAW264.7.

We have investigated D-fraction (MDF) extracted from Grifola frondosa (Maitake mushroom) on the inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 (RAW) cells, a murine monocyte/macrophage cell line, with special reference to antitumor activity of MDF against human hepatoma-derived huH-1 cells. MDF could induce iNOS mRNA expression in RAW cells in a dose range of more than 30 microg/ml, but the effect of 10 microg/ml of MDF was negligible. The iNOS mRNA expression induced by 100 microg/ml of MDF was 6 hrs later, but lasted for a longer time than that of lipopolysaccharide (LPS), a representative iNOS inducer. Although iNOS mRNA levels in MDF-stimulated cells were almost equal to LPS-stimulated cells at the peak time, the cumulative amount of nitrite was only about 50% compared with that of LPS-treated cells. When huH-I cells were cultured in MDF containing media in a 24-well plate with inserted porous bottom in the presence or absence of RAW cells, the viability of huH-1 cells decreased significantly only in the presence of RAW cells in MDF dose-dependent manner. This antitumor activity of RAW cells in the presence of MDF was abolished or attenuated by the addition of L-NAME, a NOS inhibitor, confirming that this phenomenon is due to iNOS-mediated NO production by RAW cells, but not direct cytotoxic activity of MDF against huH-1 cells. These data suggest that MDF is a novel inducer for iNOS which contributes at least in part to antitumor activity of MDF.

Immunoglobulin treatment ameliorates murine myocarditis associated with reduction of neurohumoral activity and improvement of extracellular matrix change.

OBJECTIVES: We examined effects of immunoglobulin on murine myocarditis induced by encephalomyocarditis virus, not pathogenic to humans, and analyzed the plasma cytokine and catecholamine levels and the changes of the extracellular matrix with or without the treatment. BACKGROUND: We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis by an antiviral effect. However, it is not yet determined whether beneficial effects of immunoglobulin for myocarditis are due to antiviral effects or to other unknown effects. METHODS: Antiviral activity of human immunoglobulin (Polyglobin-N) against encephalomyocarditis virus was determined in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected mice daily for two weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. RESULTS: Antiviral activity of immunoglobulin could not be detected in the assay of a plaque-reduction method in vitro. The in vivo study showed that immunoglobulin administration ameliorated both myocardial necrosis with interstitial fibrin deposition in experiment I and interstitial fibrosis with the improvement of ventricular remodeling in experiment II by the reduction of plasma catecholamines, interferon-alpha, and soluble intercellular adhesion molecule-1. CONCLUSIONS: Immunoglobulin therapy could suppress myocarditis associated with the improvement of extracellular matrix changes by the reduction of neurohumoral activity.

Isoforms of JSAP1 scaffold protein generated through alternative splicing.

We have identified four isoforms of c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein that participates in JNK mitogen-activated protein kinase cascades, termed JSAP1a, JSAP1b, JSAP1c, and JSAP1d. The previously identified JSAP1 was renamed JSAP1a to avoid confusion. Analyses of the exon-intron structure of the jsap1 gene indicated that the isoforms are generated through alternative splicing involving exons 5 and 6. The mRNA expression levels of the JSAP1 isoforms differed among the mouse tissues examined. We also investigated the region of JSAP1 responsible for its interaction with JNK, and found that the JNK-binding domain is located between aa residues 201 and 217 in JSAP1a, which is encoded by part of exon 6. As all the JSAP1 isoforms contain this binding domain, we examined the binding affinity of the JSAP1 isoforms for JNK1, JNK2, and JNK3. JSAP1c and JSAP1d, which contain a 31-aa sequence not present in JSAP1a or JSAP1b, had a lower binding affinity for the JNKs, especially JNK3. These results suggest that JSAP1c and JSAP1d may attenuate the scaffolding activity of JSAP1a and/or JSAP1b in JNK cascades, especially the JNK3 cascades.

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.

Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis.

SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain. Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen. Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse. Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro. In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV.

Prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival microflora of Japanese patients with adult and rapidly progressive periodontitis.

BACKGROUND, AIMS: This study investigated the prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans among various periodontitis patients and healthy individuals in Japan, and correlated it with clinical parameters. METHOD: Subgingival plaque samples were collected from 21 patients with adult periodontitis (AP), 8 with rapidly progressive periodontitis (RPP) and 15 healthy individuals. RESULTS: The frequency detected in culture was as follows: B. forsythus was found in 47.6% of AP sites and in 37.5% of RPP sites. P. gingivalis was identified in 64.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in 4.8% of AP and 3.1% of RPP sites. The 3 species were detected in only 2 of the healthy individuals. The proportion of B. forsythus in the total microflora in culture was 0.07% in the healthy group, 4.1% in AP and 2.4% in RPP. The proportions of P. gingivalis were 0% in the healthy group, 18.8% in AP and 16.2% in RPP. The proportion of A. actinomycetemcomitans was very low in all 3 groups. A DNA probe detected B. forsythus in 78.6% of AP and 65.6% of RPP sites, as well as P.gingivalis in 58.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in only 1.2% of AP sites. The 3 species were undetectable in the healthy group. CONCLUSIONS: The prevalence and the proportion of B. forsythus and P. gingivalis were significantly correlated with clinical parameters, suggesting that B. forsythus and P. gingivalis are closely related to AP and RPP in the Japanese population.

HNF-4 plays a pivotal role in the liver-specific transcription of the chipmunk HP-25 gene.

The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5' flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.

Barnacle cement proteins. Importance of disulfide bonds in their insolubility.

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.