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.

Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson's disease.

Inhibitory PAS domain protein (IPAS), a repressor of hypoxia-inducible factor-dependent transcription under hypoxia, was found to exert pro-apoptotic activity in oxidative stress-induced cell death. However, physiological and pathological processes associated with this activity are not known. Here we show that IPAS is a key molecule involved in neuronal cell death in Parkinson's disease (PD). IPAS was ubiquitinated by Parkin for proteasomal degradation following carbonyl cyanide m-chlorophenyl hydrazone treatment. Phosphorylation of IPAS at Thr12 by PTEN-induced putative kinase 1 (PINK1) was required for ubiquitination to occur. Activation of the PINK1-Parkin pathway attenuated IPAS-dependent apoptosis. IPAS was markedly induced in the midbrain following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and IPAS-deficient mice showed resistance to MPTP-induced degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). A significant increase in IPAS expression was found in SNpc neurons in patients with sporadic PD. These results indicate a mechanism of neurodegeneration in PD.

Pro-apoptotic activity of inhibitory PAS domain protein (IPAS), a negative regulator of HIF-1, through binding to pro-survival Bcl-2 family proteins.

Inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) is a dominant negative transcription factor that represses hypoxia-inducible factor 1 (HIF-1) activity. In this study, we show that IPAS also functions as a pro-apoptotic protein through binding to pro-survival Bcl-2 family members. In a previous paper, we reported that NF-κB-dependent IPAS induction by cobalt chloride repressed the hypoxic response in PC12 cells. We found that prolonged incubation under the same conditions caused apoptosis in PC12 cells. Repression of IPAS induction protected cells from apoptosis. Furthermore, knockdown of IPAS recovered cell viability. EGFP-IPAS protein was localized in both the nucleus and the cytoplasm, with a large fraction associated with mitochondria. Mitochondrial IPAS induced mitochondria depolarization and caspase-3 activation. Immunoprecipitation assays revealed that IPAS is associated with Bcl-x(L), Bcl-w and Mcl-1. The association of IPAS with Bcl-x(L) was also observed in living cells by the FLIM-based FRET analysis, indicating direct binding between the two proteins. IPAS contributed to dysfunction of Bcl-x(L) by inhibiting the interaction of Bcl-x(L) with Bax. These results demonstrate that IPAS functions as a dual function protein involved in transcription repression and apoptosis.

Increased circulating cell signalling phosphoproteins in sera are useful for the detection of pancreatic cancer.

BACKGROUND: Intracellular phosphoprotein activation significantly regulates cancer progression. However, the significance of circulating phosphoproteins in the blood remains unknown. We investigated the serum phosphoprotein profile involved in pancreatic cancer (PaCa) by a novel approach that comprehensively measured serum phosphoproteins levels, and clinically applied this method to the detection of PaCa. METHODS: We analysed the serum phosphoproteins that comprised cancer cellular signal pathways by comparing sera from PaCa patients and benign controls including healthy volunteers (HVs) and pancreatitis patients. RESULTS: Hierarchical clustering analysis between PaCa patients and HVs revealed differential pathway-specific profiles. In particular, the components of the extracellular signal-regulated kinase (ERK) signalling pathway were significantly increased in the sera of PaCa patients compared with HVs. The positive rate of p-ERK1/2 (82%) was found to be superior to that of CA19-9 (53%) for early stage PaCa. For the combination of these serum levels, the area under the receiver-operator characteristics curves was showing significant ability to distinguish between the two populations in independent validation set, and between cancer and non-cancer populations in another validation set. CONCLUSION: The comprehensive measurement of serum cell signal phosphoproteins is useful for the detection of PaCa. Further investigations will lead to the implementation of tailor-made molecular-targeted therapeutics.

Apolipoprotein C-1 maintains cell survival by preventing from apoptosis in pancreatic cancer cells.

Pancreatic cancer still remains one of the most lethal diseases and establishment of new therapy is needed. The purpose of this study is to find novel factors involved in pancreatic cancer progression by proteomic approach. We compared pre- and postoperative serum protein profiling obtained from pancreatic cancer patients who had curative pancreatectomy using surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The peak intensity levels of both 6630 and 6420 Da were significantly higher in the preoperative serum than in the postoperative serum (P<0.002). Sequential amino acid analysis identified these proteins to be apolipoprotein C-1 (ApoC-1). The high level of ApoC-1 in preoperative serum significantly correlated with poor prognosis. Furthermore, ApoC-1 was abundantly expressed in pancreas neoplastic epithelium, and was detected in the culture medium of the pancreatic cancer cell line in vitro, which suggests that cancer cells secrete ApoC-1. Inhibition of ApoC-1 expression by short interfering RNA suppressed cell proliferation and induced apoptosis of pancreatic cancer cells. The specific expression of ApoC-1 and its role in preventing from spontaneous apoptosis in pancreatic cancer cells suggest that ApoC-1 contributes to the aggressiveness of pancreatic cancer and will be useful as a new therapeutic target.

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

Keratin mRNA for detecting micrometastasis in cervical lymph nodes of oral cancer.

We studied three keratin (K) gene candidates, K13, K19, and K20 mRNAs, for detecting micrometastases in cervical lymph nodes (LNs) by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 166 histologically metastasis-negative nodes, 24 micrometastatic LNs (14. 4%) were detected based on K13 gene expression. Keratin 19 mRNA is an inadequate marker for the genetic diagnosis due to not only illegitimate gene expression from lymphatic tissue but also gene expression from the ectopic salivary gland. Keratin 20 mRNA showed low sensitivity. It is suggested that K13 mRNA may be a promising tumor marker among these keratin genes for detecting the micrometastases in cervical LNs of oral cancer.

Histological study on pN upgrading of oral cancer.

The International Union Against Cancer (UICC) does not define the number of sections required from each regional lymph node to record pTNM classification. This study was designed to clarify the incidence of occult metastasis and to assess the pN upgrading of patients with oral cancer. Ultimately, this study led to a proposal for appropriate semiserial sectioning guidelines. Five hundred fifty-four nonmetastatic cervical lymph nodes taken from 73 patients with oral cancer were subjected to hematoxylin-eosin (HE) staining and keratin immunohistochemistry. Micrometastases, defined as foci < or =3 mm, were detected in 29 sites of 23 lymph nodes (4.2%) of 16 patients (21.9%). In 9 patients (12.3%) pN upgrading was needed: in 6 from pN0 to pN1, in 1 from pN0 to pN2b, and in 2 from pN1 to pN2b. The remaining 13 lymph nodes with occult metastasis were found in 5 pN2b and 2 pN2c patients, resulting in no pN upgrading. Occult metastasis was also detected in 6 small lymph nodes < or =5 mm in diameter. The average minor axis of the micrometastasis was 1.36-/+0.85 mm. We propose that the lymph nodes should be cut and examined at 1-mm intervals to detect micrometastatic foci and to evaluate the pN classification accurately.

Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3.

BACKGROUND: The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin. RESULTS: We investigated the transcriptional mechanism of mArnt3/mClock heterodimer. While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C-terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock. It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer. Deletion proceeding from amino acids 559-492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock-delta 19 mutant. Yeast and mammalian two-hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain. The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay. CONCLUSION: Taken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.

Induction of apoptosis and inhibition of DNA topoisomerase-I in K-562 cells by a marine microalgal polysaccharide.

We have previously purified an extracellular polysaccharide, D-galactan sulfate associated with L(+)-lactic acid, produced from a marine microalga Dinoflagellate Gymnodinium sp. A3 (GA3). The GA3 polysaccharide, irrespective of presence or absence of lactic acid, exhibited significant cytotoxicity, which is based on an induction of apoptotic cell death, toward human myeloid leukemia K562 cells. Furthermore, we found that the GA3 polysaccharide with or without lactic acid possesses an inhibitory effect on topoisomerase-I (topo-I). The potent cytotoxic effect of GA3 polysaccharide may result from its inhibitory effect on topo-I, because the topo-I inhibition is known to trigger apoptotic cell death.

Clinical usefulness of telomerase assay for the detection of lymph node metastasis in patients with oral malignancy.

OBJECTIVE: Telomerase is considered a diagnostic marker of malignancy. We investigated the usefulness of telomerase assay for the detection of lymph node micrometastasis. METHODS: Sixteen cervical lymph nodes with metastasis of oral cancer and 20 benign lymph nodes were studied. The oral cancer cell line was used to estimate the sensitivity for telomerase assay. Telomerase activity was measured by semiquantitative telomeric repeat amplification protocol. RESULTS: There was a significant difference between malignant and benign lymph nodes. The telomerase activity of 50 mg of lymph nodes with 103 or more cancer cells differed from that of control lymph nodes. Lymph nodes with 102 or fewer tumor cells expressed similar levels as benign lymph nodes. CONCLUSIONS: In addition to routine histologic examination, telomerase assay is considered a useful tool for the detection of lymph node metastasis in patients with oral malignancy.

[Regulatory mechanism of genes by the Ah receptor which mediates toxic effects of dioxins].

The aryl hydrocarbon receptor(AhR) plays a central role in the metabolic pathways involved in the detoxification of important environmental carcinogens, most of which act as ligand for the receptor, although no endogeneous ligand has not yet been known. Activation of the AhR is responsible for a variety of toxic responses in animals and humans. The activation mechanisms become clear that include binding of ligand to receptor, transfer to the nucleus, formation of a ternary complex with Arnt, followed by binding to response elements upstream of the relevant target genes. However, the specific mechanisms responsible for the toxic responses of dioxins are unknown.

Increased polyploid incidence is associated with abnormal copper accumulation in the liver of LEC mutant rat.

Long-Evans Cinnamon (LEC) rats are deleted at the p-type copper transport ATPase gene (Atp7b), so that they exhibit abnormal hepatic copper concentration. In this study, it was confirmed that LEC rat liver possesses a feature of increase in polyploid. Furthermore, a segregation analysis using backcrosses between LEC and F344 normal rats showed that the increased polyploid incidence is strongly associated with excessive copper content in their liver. These results should demonstrate that copper cytotoxicity leads to the impairment of mitotic progression, resulting in the increase of polyploid in the liver of LEC rats.

Chapter 14. Ah receptor gene polymorphisms and human cancer susceptibility.

The Ah receptor (Ahr) gene occupies a central role in the metabolic pathways involved in the detoxification of important environmental carcinogens. The structures of the rodent and human genes have been elucidated, and the molecular details of the receptor function, including its interaction with other proteins such as Arnt and hsp90, have been thoroughly investigated. The Ahr gene is polymorphic in mice and in humans. In mice, good correlations have been found between structural polymorphisms in the gene and functional variants in various genetic strains. In humans, work on polymorphisms and their possible role in gene function as well as cancer susceptibility is just beginning.

Telomerase activation and cell proliferation during 7,12-dimethylbenz[a]anthracene-induced hamster cheek pouch carcinogenesis.

Telomerase is a ribonucleoprotein complex intimately involved in cell immortalization and carcinogenesis. This enzyme is activated and stabilizes telomere length in almost all types of cancer. Telomerase may be necessary for continuous cell proliferation. In this study, we analyzed telomerase activity in hamster experimental oral lesions (starting from epithelial hyperplasia through dysplasia, carcinoma in situ, and invasive carcinoma) evoked by 7,12-dimethylbenz[a]anthracene, and in normal mucosa. We also analyzed proliferative activity in these lesions by using immunohistochemical analysis and flow cytometry. Histologically normal epithelium expressed weak telomerase activity. The telomerase activity count increased rapidly in the early stage of carcinogenesis and gradually in the late stage. Cell-proliferative activity closely correlated with progression of disease. These findings indicate that telomerase activation is an early event and that increases in telomerase activity upregulate cell proliferation in chemically induced hamster oral carcinogenesis.

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Species specificity of cytokeratin polypeptide expression in the submandibular gland.

Using nine monospecific and seven polyspecific monoclonal antibodies (MoAbs) against cytokeratin (CK), we immunohistochemically studied the species specificity of CK localization in human, rat, mouse, hamster and guinea pig submandibular glands (SMGs). All species showed different staining patterns with various degrees of intensity. The pattern of immunostaining was broadly classified into three groups. Group I showed positive reactivity to the rodent salivary gland, but not to human SMGs (6B10 and 34betaE12). Group 2 showed the reverse staining pattern (M20, A53-B/A2 and Ks19.1). Group 3, for which almost all species were positive, showed interspecific diversity in the staining pattern (CY-90, K8.12, K8.13, C-11 and KH-1). Species specificity of CK should always be taken into consideration when immunohistochemically examining CK expression during development or during carcinogenesis in rodents.