DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity.

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.

Simultaneous determination of berberine in rat blood, liver and bile using microdialysis coupled to high-performance liquid chromatography.

High-performance liquid chromatography coupled to microdialysis was used for the simultaneous determination of unbound berberine in rat blood, liver and bile for a pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium, the median lobe of the liver, and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of berberine (10 mg/kg) through the femoral vein. Berberine and dialysates were separated using a Zorbax SB-phenyl column and a mobile phase comprised of acetonitrile-methanol-20 mM monosodium phosphate (pH 3.0) (35:20:45, v/v) together with 0.1 mM 1-octanesulfonic acid. The detection limit for berberine was 10 ng/ml. The concentration-response relationship was linear (r2 > 0.995) over the concentration range 0.05-50 microg/ml; intra-assay and inter-assay precision and accuracy for berberine fell within predefined limits. The disposition of berberine in the blood, liver and bile fluid suggests that berberine might be metabolized in the liver and undergo hepatobiliary excretion.

Retinoic acid receptor-alpha messenger RNA expression is increased and retinoic acid receptor-gamma expression is decreased in Barrett's intestinal metaplasia, dysplasia, adenocarcinoma sequence.

BACKGROUND: Expression levels of the retinoic acid receptors (RAR-alpha, RAR-beta, and RAR-gamma) are significantly different in neoplastic tissues compared with non-neoplastic tissues for some tumors. This study investigated whether retinoic acid receptor messenger RNA (mRNA) expression levels are altered in Barrett's esophagus and Barrett's adenocarcinoma tissues. METHODS: Relative mRNA expression levels of the RARs were quantified by using the ABI 7700 Sequence Detector (Taqman) system in Barrett's intestinal metaplasia (n = 15), dysplasia (n = 6), adenocarcinoma (n = 17), and matching normal esophagus tissues (n = 36). RESULTS: RAR-alpha expression was significantly increased, and RAR-gamma expression was significantly decreased, at higher stages in the Barrett's sequence. There was almost complete loss of RAR-gamma expression (relative expression level < or = 1) in a majority (70%) of the dysplasia and adenocarcinoma tissues. There were significant differences in RAR-alpha and RAR-gamma expression in histopathologically normal tissues in patients with cancer versus patients without cancer. RAR-beta expression levels were significantly elevated in adenocarcinoma versus normal esophagus tissues. The RAR expression profile was similar for cancers arising within the esophagus and for cancers arising at the gastroesophageal junction. CONCLUSIONS: RAR mRNA expression levels are significantly different in Barrett's tissues compared with normal esophagus tissues, and these levels are significantly different in Barrett's dysplasia and adenocarcinoma tissues compared with nondysplastic tissues. These results suggest that RAR mRNA levels may be useful biomarkers for this disease and that gastroesophageal junction adenocarcinomas are genetically similar to esophageal adenocarcinomas. These results also suggest that a cancer field is present in the esophagus in patients with cancer and that genetic alterations can precede histopathologic alterations in this disease.

Laparoscopic ultrasonic epithelial ablation of the lower esophagus after nissen fundoplication in a porcine model: assessment of tissue damage and healing process.

A substantial population of patients with Barrett's esophagus has undergone antireflux surgery but still requires annual surveillance endoscopy. These patients would benefit from a definitive ablation of the Barrett's mucosa, which would remove the malignant potential of this disease. This study evaluates the efficacy of applying ultrasonic energy to remove the epithelium of the lower esophagus in a porcine model with prior Nissen fundoplication. Four Yakutan minipigs underwent laparoscopic Nissen fundoplication. After 2 weeks they underwent transgastric Cavitron ultrasonic surgical aspirator (CUSA; Valleylab, Boulder, CO) ablation of the lower esophageal epithelium. Healing of the mucosa was assessed by endoscopy at 2 weeks and pathological examination at 4 weeks after ablation. All pigs underwent successful laparoscopic Nissen fundoplication. Complete lower esophageal epithelial ablation was accomplished through the fundoplication in three animals. One pig developed a bezoar that prohibited ablation. At 2 weeks endoscopy showed patchy squamous epithelial regeneration, which was confirmed histologically. Esophageal specimens at 4 weeks showed complete regeneration of squamous epithelium with a partially healed small ulcer in one animal. No stricture formation was seen. We conclude that the CUSA technique can completely ablate Barrett's mucosa in the setting of a prior antireflux procedure. Healing with squamous mucosal regeneration is rapid and complete.

P53 expression, p53 and Ha-ras mutation and telomerase activation during nitrosamine-mediated hamster pouch carcinogenesis.

Squamous cell carcinomas (SCC) induced in hamster buccal pouch (HBP) by 22 weeks of topical N-methyl-N-benzylnitrosamine (MBN) treatment (twice-weekly, 10 mg MBN/ml propylene glycol) were evaluated for: (i) altered expression of p53 using immunohistochemistry (IHC); (ii) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP); (iii) telomerase activity using the telomerase repeat amplification protocol (TRAP). Precancerous lesions were also evaluated using p53 IHC. Hamsters were killed for lesion analysis at either 3 days (group A, eight hamsters, 89 carcinomas) or 7 weeks (group B, six hamsters, 105 carcinomas) following the final MBN application. Between 3 days and 7 weeks post-treatment the proportion of tumors exhibiting p53 IHC activity (at least 10% of nuclei stained using D07 antibodies for detection of both mutant and wild-type p53) fell from 91 to 50%. However, during this same post-treatment period the frequency of tumors analyzed exhibiting confirmed sequence alterations in the conserved exons (E5-E8) of p53 remained constant (5/15 = 33% in group A versus 14/45 = 31% in group B). Heightened expression of wild-type p53 resulting from DNA damage in the immediate post-treatment period is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. Nearly 80% of the identified p53 mutations were G-->A and C-->T transitions. The identified p53 point mutations occurred at or near (within three codons) of the corresponding hot-spot codons (175, 245, 248 and 273) of human oral SCC. The proportion of group A and group B tumors analyzed exhibiting Ha-ras mutations was 1/15 (7%) and 7/45 (16%), respectively. Only four of the observed eight Ha-ras mutations occurred in codons known to result in activation of this gene. Telomerase activation was demonstrated in 11 of 13 group A tumors (85%) and in 23 of 24 (96%) group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in this HBP-MBN model are similar in many respects to those observed in the analogous human lesions of the head and neck. This model may be particularly useful for development of cancer chemoprevention regimens and multimodality cancer therapies.

Telomerase reverse transcriptase expression is increased early in the Barrett's metaplasia, dysplasia, adenocarcinoma sequence.

Barrett's esophagus is a multistage polyclonal disease that is associated with the development of adenocarcinoma of the esophagus and esophagogastric junction. Telomerase activation is associated with cellular immortality and carcinogenesis, and increased expression of the telomerase reverse transcriptase catalytic subunit (hTERT) has been used for the early detection of malignant diseases. To identify biomarkers associated with each stage of the Barrett's process, relative mRNA expression levels of hTERT were measured using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector (TaqMan system) in Barrett's intestinal metaplasia (n = 14), Barrett's dysplasia (n = 10), Barrett's adenocarcinoma (n = 14), and matching normal squamous esophagus tissues (n = 32). hTERT expression was significantly increased at all stages of Barrett's esophagus, including the intestinal metaplasia stage, compared to normal tissues from patients without cancer (intestinal metaplasia vs. normal esophagus, P <0.0001; dysplasia, P = 0.001; adenocarcinoma, P = 0.007; all Mann-Whitney U test ). hTERT expression levels were significantly higher in adenocarcinoma tissues than in intestinal metaplasia tissues (P = 0.003), and were higher in dysplasia compared with intestinal metaplasia tissues (P = 0.056). hTERT levels were also significantly higher in histologically normal squamous esophagus tissues from cancer patients than in normal esophagus tissues from patients with no cancer (P = 0.013). Very high expression levels ([hTERT x 100: beta-actin] >20) were found only in patients with cancer. These findings suggest that telomerase activation is an important early event in the development of Barrett's esophagus and esophageal adenocarcinoma, that very high telomerase levels may be a clinically useful biomarker for the detection of occult adenocarcinoma, and that a widespread cancer "field" effect is present in the esophagus of patients with Barrett's cancer.

Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.