Secretory IgA accumulated in the airspaces of idiopathic pulmonary fibrosis and promoted VEGF, TGF-ß and IL-8 production by A549 cells.

Secretory IgA (SIgA) is a well-known mucosal-surface molecule in first-line defense against extrinsic pathogens and antigens. Its immunomodulatory and pathological roles have also been emphasized, but it is unclear whether it plays a pathological role in lung diseases. In the present study, we aimed to determine the distribution of IgA in idiopathic pulmonary fibrosis (IPF) lungs and whether IgA affects the functions of airway epithelial cells. We performed immunohistochemical analysis of lung sections from patients with IPF and found that mucus accumulated in the airspaces adjacent to the hyperplastic epithelia contained abundant SIgA. This was not true in the lungs of non-IPF subjects. An in-vitro assay revealed that SIgA bound to the surface of A549 cells and significantly promoted production of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß and interleukin (IL)-8, important cytokines in the pathogenesis of IPF. Among the known receptors for IgA, A549 cells expressed high levels of transferrin receptor (TfR)/CD71. Transfection experiments with siRNA targeted against TfR/CD71 followed by stimulation with SIgA suggested that TfR/CD71 may be at least partially involved in the SIgA-induced cytokine production by A549 cells. These phenomena were specific for SIgA, distinct from IgG. SIgA may modulate the progression of IPF by enhancing synthesis of VEGF, TGF-ß and IL-8.

Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA).

Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurologic disorder characterized by variable combinations of myoclonus, epilepsy, cerebellar ataxia, choreoathetosis and dementia. By specifically searching published brain cDNA sequences for the presence of CAG repeats we identified unstable expansion of a CAG in a gene on chromosome 12 in all the 22 DRPLA patients examined. A good correlation between the size of the CAG repeat expansion and the ages of disease onset is found in this group. Patients with earlier onset tended to have a phenotype of progressive myoclonus epilepsy and larger expansions. We propose that the wide variety of clinical manifestations of DRPLA can now be explained by the variable unstable expansion of the CAG repeat.

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene.

Friedreich ataxia (FRDA), the most common autosomal recessive neurodegenerative disease among Europeans and people of European descent, is characterized by an early onset (usually before the age of 25), progressive ataxia, sensory loss, absence of tendon reflexes and pyramidal weakness of the legs. We have recently identified a unique group of patients whose clinical presentations are characterized by autosomal recessive inheritance, early age of onset, FRDA-like clinical presentations and hypoalbuminemia. Linkage to the FRDA locus, however, was excluded. Given the similarities of the clinical presentations to those of the recently described ataxia with oculomotor apraxia (AOA) linked to chromosome 9p13, we confirmed that the disorder of our patients is also linked to the same locus. We narrowed the candidate region and have identified a new gene encoding a member of the histidine triad (HIT) superfamily as the 'causative' gene. We have called its product aprataxin; the gene symbol is APTX. Although many HIT proteins have been identified, aprataxin is the first to be linked to a distinct phenotype.

Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded CAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids-with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

Effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma total homocysteine concentrations in young Japanese male subjects.

OBJECTIVE: To investigate the effects of short-term folic acid and/or riboflavin supplementation on serum folate and plasma plasma total homocysteine (tHcy) concentrations in young Japanese male subjects. DESIGN: In a double blind, randomized controlled trial. INTERVENTION: Subjects were randomly assigned to one of four groups and received a placebo (control group), 800 microg/day folic acid (FA group), 8.4 mg/day riboflavin (R group), or both (FAR group) for 2 weeks. SETTING: Tokyo, Japan. SUBJECTS: In total, 32 healthy male volunteers aged 20-29 years. RESULTS: At the end of the 2 week supplementation period, the tHcy concentration decreased significantly in the FA group. Serum folate concentrations had increased between 2.7 and 2.0-fold in the FA and FAR groups, respectively, but the mean within-group changes in serum folate and plasma tHcy concentrations did not differ between these two groups. At the end of the study, alanine amino transferase was decreased in the R and FAR groups, while alanine amino transferase was increased in the FA group. CONCLUSION: Supplementation with folic acid, 800 microg/day, for 2 weeks, increased the serum and red blood cell folate concentrations and decreased the plasma tHcy concentrations in healthy young male subjects. Riboflavin supplementation may have blunted the effect of folic acid, which resulted in a diminished reduction of tHcy in our subjects.

Chimeric anti-ganglioside GM2 antibody with antitumor activity.

Ganglioside GM2, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We have previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.

The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation.

Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2µM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5µM, while in normal primary Schwann cells at 7.1µM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

Analysis of the mechanism for reversion of a disrupted gene.

A positive selection system for intrachromosomal recombination in Saccharomyces cerevisiae has been developed. This was achieved by integration of a plasmid containing an internal fragment of the HIS3 gene into its chromosomal location. This resulted in two copies of the HIS3 gene one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end. Reversion of the gene disruption could be brought about by plasmid excision, unequal sister chromatid exchange or sister chromatid conversion. The purpose of this study was to define the mechanisms involved in reversion of the gene disruption. The frequency of plasmid excision could be determined by placing a yeast sequence bearing an origin of replication onto the plasmid that was subsequently integrated into the yeast genome. Unequal sister chromatid exchange and conversion could be distinguished by determining the nature of the reciprocal product by Southern blotting. The results indicate that reversion might occur mainly by conversion between sister chromatids. This is because the frequency of plasmid excision was about two orders of magnitude lower than the overall frequency of reversion and no reciprocal product indicative of sister chromatid exchange was found. The findings of this presentation suggest that conversion might be an important mechanism for recombination of sister chromatids and possibly for repair of damaged DNA in S or G2.

Corneal advanced glycation end products increase in patients with proliferative diabetic retinopathy.

OBJECTIVE: To evaluate corneal advanced glycation end product (AGE) fluorescence in patients with diabetes and in healthy control subjects. RESEARCH DESIGN AND METHODS: Corneal autofluorescence was measured in 26 eyes of 26 patients with type 2 diabetes (mean age 57.0 years; mean disease duration 12.2 years; mean HbA1c 7.1%) and 13 eyes of 13 healthy age-matched control subjects (mean age 57.9 years). The patients with type 2 diabetes were divided into the following groups: patients without diabetic retinopathy (DR), patients without proliferative diabetic retinopathy (PDR), and patients with PDR. Corneal autofluorescence was measured by fluorophotometry with the wavelength that is characteristic of AGE fluorescence (excitation and emission 360-370 nm and 430-450 nm, respectively). We defined peak corneal autofluorescence levels as corneal AGE fluorescence values. We compared the corneal AGE fluorescence values in the four groups. RESULTS: In the PDR group (11.9 +/- 3.9 arbitrary units [mean +/- SD]), the corneal AGE fluorescence values were significantly higher compared with the control subjects (6.9 +/- 1.3 arbitrary units), the patients without DR (7.4 +/- 2.1 arbitrary units), and the patients without PDR (6.9 +/- 2.2 arbitrary units) (P < 0.05). CONCLUSIONS: We found that corneal AGEs may increase in patients with diabetes and PDR compared with control subjects, patients without DR, and patients without PDR. In the patients with PDR, increased corneal AGEs may play a role in diabetic keratopathy.

Determination of carbonic anhydrase-III by enzyme-immunoassay in liver, muscle and serum of male rats with streptozotocin-induced diabetes mellitus.

Insulin has a plethora of metabolic effects but its action on carbonic anhydrase-III (CA-III), a key enzyme in acid-base regulation, has been little studied. The present studies examined the effects of streptozotocin induced diabetes on the concentrations of CA-III. The concentration of CA-III in the liver, muscles and serum of rats with experimental diabetes mellitus was measured by the method of enzyme-immunoassay. Streptozotocin-induced diabetes mellitus resulted in a reduction in concentration of CA-III in the liver and serum, but not in skeletal muscles, of adult male rats. A 98% reduction in hepatic CA-III content relative to control values was observed. The reduction in CA-III content in the liver was restored to control value by administration of insulin. The CA-III content in serum of diabetic rats declined to approx. 25% of control values, but the reduction was unaffected by administration of insulin. The concentration of CA-III in the liver and serum of diabetic rats was not influenced by administration of methyltestosterone. Although the content of CA-III in m. rectus femoris, m. tibialis craniaris and m. soleus differed, no significant difference of CA-III content was found between diabetes mellitus and control rats. The effect of chronic diabetes mellitus on CA-III content was obviously different between liver and muscle, suggesting that the regulation of CA-III biosynthesis differs between these two tissues. These results suggest that biosynthesis of CA-III in hepatocytes of rats is influenced by irregular patterns of GH secretion brought about by diabetes mellitus.

Effect of activin on luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in granulosa cells.

Activin (the dimer of inhibin beta-subunit) is involved in the modulation of granulosa cell function. Recent reports have indicated that activin had an effect on LH/human CG (hCG) receptor induction and steroidogenesis in granulosa cells. To characterize the regulation inducing LH/hCG receptor by activin, we investigated messenger RNA (mRNA) levels, the expression of the LH/hCG receptor, and intracellular cAMP accumulation in cultured rat granulosa cells. Northern blot analysis showed an increase in the LH/hCG receptor mRNA level with FSH (30 ng/ml) and activin (100 ng/ml) cotreatment, whereas activin alone could not augment LH/hCG receptor mRNA at all. After the addition of actinomycin D to the culture medium, LH/hCG receptor mRNA was more stable in the presence of FSH plus activin than in the presence of FSH alone. Similarly, a receptor binding assay revealed that the cotreatment with FSH and activin induced more LH/hCG receptor than FSH alone 96 h after exposure to hormone, but that activin (100 ng/ml) alone could not induce the LH/hCG receptor. Since the primary, if not the sole, second messenger mediating the action of FSH in granulosa cells has been shown to be cAMP, intracellular cAMP accumulation was measured in granulosa cells in the presence of FSH (30 ng/ml) and/or activin (100 ng/ml). Although FSH-stimulated cAMP accumulation reached a peak 15 min after incubation, activin did not significantly alter cAMP accumulation in either control nor FSH-stimulated granulosa cells, indicating that the effects of activin on the LH/hCG receptor in granulosa cells are not mediated by the increase in cAMP. These results demonstrate that activin enhances the FSH-induced LH/hCG receptor mRNA, LH/hCG receptor mRNA stability, and LH/hCG binding sites not due to the stimulation of the adenylate cyclase system. Although the signal pathway from the activin receptor has not been elucidated upon yet, activin is capable of increasing LH/hCG receptor levels through the accumulation of LH/hCG receptor mRNA levels.

Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells.

Activin, a dimer of beta-subunits of inhibin, has been found to induce FSH receptor on cultured rat granulosa cells. The effect of activin on FSH receptor messenger RNA (mRNA) levels has not been elucidated. To study the effect of activin on FSH receptor mRNA levels, we used a specific complementary RNA probe to evaluate changes in FSH receptor transcripts in cultured rat granulosa cells. Granulosa cells obtained from immature diethylstilbestrol-treated rats contained two predominant FSH receptor mRNA transcripts (5.5 and 2.4 kilobases). Compared to the control, the treatment of granulosa cells with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum of about a 4-fold increase at 24 h. FSH receptor mRNA markedly decreased after 48 h and maintained a level comparable to that found in the control. The FSH receptor expression was also increased by activin. Scatchard analysis of the binding of rat FSH to granulosa cells showed that the increase in FSH binding after activin treatment was due to an increase in the receptor number and not the affinity of binding. Treatment of granulosa cells for 24 h with activin (20-300 ng/ml) increased FSH receptor mRNA in a dose-dependent manner to a maximum of about a 4-fold increase at a concentration of 100-300 ng/ml. We analyzed rat type II activin receptor mRNA transcripts in cultured rat granulosa cells with a specific complementary RNA probe to study the action of activin on granulosa cells. Granulosa cells contained two predominant rat type II activin receptor mRNA transcripts (6.0 and 3.0 kilobases). Furthermore, we measured intracellular cAMP production by activin to examine the mechanism by which activin acts on granulosa cells. In result, activin alone did not increase intracellular cAMP accumulation. In conclusion, this study demonstrates that the effect of activin A on the induction of FSH receptor expression is associated with a change in FSH receptor mRNA levels, suggesting that modulation of follicle development occurs.

Hormonal regulation of estrogen receptor alpha and beta gene expression in human granulosa-luteal cells in vitro.

Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ERalpha and ERbeta in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERalpha and ERbeta messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERalpha is expressed at a relatively lower level than ERbeta. Basal expression studies indicated that ERalpha mRNA levels remain unchanged, whereas ERbeta mRNA levels increased with time in culture in vitro, suggesting that ERbeta is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ERalpha and ERbeta expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERalpha (45%; P < 0.01) and ERbeta (40%; P < 0.01) mRNA levels. The hCG-induced decrease in ERalpha and ERbeta expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 micromol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ERalpha and ERbeta mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ERalpha and ERbeta mRNA levels, respectively, were observed after treatment with 0.1 micromol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHa-induced down-regulation of ERalpha and ERbeta expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ERalpha and ERbeta proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 micromol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 micromol/L), or phorbol 12-myristate 13 acetate (10 micromol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17beta-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 micromol/L GnRHa for 24 h before 17beta-estradiol administration. In summary, we observed a differential expression of ERalpha and ERbeta mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ERalpha and ERbeta gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ERalpha and ERbeta expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.

Structures of the human and mouse growth inhibitory factor-encoding genes.

Growth inhibitory factor (GIF) is down-regulated in Alzheimer's disease (AD) brains. To analyze the mechanism of this down-regulation, we isolated the human and mouse GIF genes. These genes consist of three exons, are approx. 1-kb long and show strikingly high homology to metallothionein-encoding genes. A comparison of the human and mouse GIF showed several conserved sequences, including the putative AP-2, SP-1, TATA-binding protein and metal-responsive elements (MRE). A sequence similar to the human gfa common sequence (hgcs), recently identified as the sequence for an astrocyte-specific transcriptional factor, is present in the promoter of these GIF. Characterization of factors associated with the putative regulatory elements in the promoter of GIF should help in determining the mechanism of the down-regulation of GIF in AD brains.

Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene.

Alpha-N-acetylgalactosaminidase (2-acetamido-2-deoxy-alpha-d-galactoside acetamidodeoxy-galactohydrolase, NAGA; EC 3.2.1.49) deficiency is a recently recognized autosomal recessive lysosomal disease. As a prerequisite for the generation of an animal model, the mouse NAGA gene was cloned and characterized. The NAGA gene was assigned to mouse chromosome 15 band E3, syntenic to the region encompassing the human gene, and NAGA-deficient mutant human cells transfected with the cosmid clone containing the mouse NAGA gene expressed NAGA activity. Comparison of the mouse NAGA nucleotide sequence with the human NAGA sequence predicted that the mouse NAGA gene contains an open reading frame of 1245bp, comprising nine coding exons and spanning a genomic region of 8258bp, and a 3' untranslated region of 0.5kb. The 5' untranslated region was determined in primer extension studies to be 235bp in length. Nucleotide identity between the human and mouse NAGA exons ranged from 67.4 to 89.5%, with better matches for exons 1-7 than for 8 and 9. The overall amino acid identity between the mouse and human deduced NAGA polypeptides was 82.0%, between those of mouse and chicken 72.9%. Homology was found to only one other mouse gene, i.e. the alpha-galactosidase A (GALA; EC 3.2.1. 22) gene. The amino acid identity ranged from 51.6 to 62.1% in the polypeptide regions corresponding to NAGA exons 2-7 and GALA exons 1-6, but little, if any, in the remainder. These analyses gave emphasis to the strong conservation of the NAGA gene and its origin from an ancestor common with the GALA gene, with NAGA exons 8 and 9 and GALA exon 7 being the most divergent regions in the evolution of the two genes.

Cloning and sequencing of a rat type II activin receptor.

A full-length cDNA for a rat type II activin receptor was cloned by hybridization from a rat ovary cDNA library. The deduced amino acid sequence (513 residues) containing a single membrane-spanning domain and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 99.8% and 99.4% identical in the coding region with the previously cloned mouse and human type II activin receptor, and only 66.7% identical in the coding region with the previously cloned rat type IIB activin receptor. We examined the effect of PMSG-hCG on the mRNA level of type II activin receptor in immature rat ovaries. Northern blot analysis of ovarian RNA revealed two mRNAs (3.0 kb and 6.0 kb).

Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential.

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) and Griffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha-D-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances has approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.

Substrate adhesiveness and experimental metastatic potential of rat ascites hepatoma AH7974-derived variant sublines.

Cells of an adherent subline (74AD, adhesion greater than 95%) and a floating subline (74FL, adhesion less than 1%) of rat ascites hepatoma AH7974 produced substrates containing fibronectin (FN), laminin (LN) and type IV collagen (CL-IV), with 74AD cells producing higher levels of each component. 74AD cells possessed high adhesion affinities to LN and CL-IV substrates. By contrast, 74FL cells hardly adhered to these purified attachment proteins. The difference in adhesion between the two lines in vitro tended to increase on incubation of the cells in medium containing fetal bovine serum. However, 74FL and 74AD cells adhered avidly to the extracellular matrix (ECM) of vascular endothelial cells. Although the cell-ECM adhesion apparently was not inhibited by pretreatment of the ECM with anti-FN, anti-LN and anti-CL-IV antibodies, the 74FL cell-ECM adhesion was inhibited considerably by pretreatment of the ECM with a mixture of these antibodies, especially with a combination of anti-FN and anti-LN antibodies. The lung-colonizing potential of 74FL cells was greater than that of 74AD cells, but the liver-colonizing potential of 74FL cells was less than that of 74AD cells. These results suggest that rat ascites hepatoma cells with extremely reduced substrate adhesiveness retain an adhesion mechanism that binds to FN and LN in the ECM of vascular endothelial cells. This mechanism may be a minimum essential unit of tumor cell-ECM adhesion in lung colonization, but the unit is insufficient for liver colonization of these cells.

Strong correlation between the number of CAG repeats in androgen receptor genes and the clinical onset of features of spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), a motor neuron disease associated with androgen insensitivity, is caused by androgen receptor gene mutations with an increased number of tandem CAG repeats in exon 1. We investigated the increased number of CAG repeats in androgen receptor genes of 19 SBMA patients and found that this correlated strongly with the age at onset of muscle weakness. Thus, SBMA is the first genetic disease in which a strong correlation between the degree of genetic abnormality (number of CAG tandem repeats) and clinical phenotypic expression is demonstrable. The results further indicate that androgen gene mutation is directly involved in the degeneration of motor neurons.