Intra-gastrointestinal amyloid-ß1-42 oligomers perturb enteric function and induce Alzheimer's disease pathology.

KEY POINTS: Alzheimer's disease (AD) patients and transgenic mice have beta-amyloid (Aß) aggregation in the gastrointestinal (GI) tract. It is possible that Aß from the periphery contributes to the load of Aß in the brain, as Aß has prion-like properties. The present investigations demonstrate that Aß injected into the GI tract of ICR mice is internalised into enteric cholinergic neurons; at 1 month, administration of Aß into the body of the stomach and the proximal colon was observed to partly redistribute to the fundus and jejunum; at 1 year, vagal and cerebral ß-amyloidosis was present, and mice exhibited GI dysfunction and cognitive deficits. These data reveal a previously undiscovered mechanism that potentially contributes to the development of AD. ABSTRACT: Alzheimer's disease (AD) is the most common age-related cause of dementia, characterised by extracellular beta-amyloid (Aß) plaques and intracellular phosphorylated tau tangles in the brain. Aß deposits have also been observed in the gastrointestinal (GI) tract of AD patients and transgenic mice, with overexpression of amyloid precursor protein. In the present studies, we investigate whether intra-GI administration of Aß can potentially induce amyloidosis in the central nervous system (CNS) and AD-related pathology such as dementia. We micro-injected Aß1-42 oligomers (4 µg per site, five sites) or vehicle (saline, 5 µl) into the gastric wall of ICR mice under general anaesthesia. Immunofluorescence staining and in vivo imaging showed that HiLyte Fluor 555-labelled Aß1-42 had migrated within 3 h via the submucosa to nearby areas and was internalised into cholinergic neurons. At 1 month, HiLyte Fluor 555-labelled Aß1-42 in the body of the stomach and proximal colon had partly re-distributed to the fundus and jejunum. At 1 year, the jejunum showed functional alterations in neuromuscular coupling (P < 0.001), and Aß deposits were present in the vagus and brain, with animals exhibiting cognitive impairments in the Y-maze spontaneous alteration test (P < 0.001) and the novel object recognition test (P < 0.001). We found that enteric Aß oligomers induce an alteration in gastric function, amyloidosis in the CNS, and AD-like dementia via vagal mechanisms. Our results suggest that Aß load is likely to occur initially in the GI tract and may translocate to the brain, opening the possibility of new strategies for the early diagnosis and prevention of AD.

On the analytical formulation of excess noise in avalanche photodiodes with dead space.

Simple, approximate formulas are developed to calculate the mean gain and excess noise factor for avalanche photodiodes using the dead-space multiplication theory in the regime of small multiplication width and high applied electric field. The accuracy of the approximation is investigated by comparing it to the exact numerical method using recursive coupled integral equations and it is found that it works for dead spaces up to 15% of the multiplication width, which is substantial. The approximation is also tested for real materials such as GaAs, InP and Si for various multiplication widths, and the results found are accurate within ∼ 15% of the actual noise, which is a significant improvement over the local-theory noise formula. The results obtained for the mean gain also confirm the recently reported relationship between experimentally determined local ionization coefficients and the enabled non-local ionization coefficients.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. MATERIALS AND METHODS: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. RESULTS: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. CONCLUSION: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.

Involvement of TRPV1 and TRPA1 in the modulation of pacemaker potentials in the mouse ileum.

BACKGROUND: The roles of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and subfamily A, member 1 (TRPA1) in mechanisms of gastrointestinal motility are complex. This study aimed to clarify the effects of several TRPV1 and TRPA1 ligands on the electrical potentials generated by pacemaker cells in the mouse-isolated ileum. METHOD: The pacemaker potentials of ileal segments of mice were recorded extracellularly using a 60-channel microelectrode array. The dominant frequencies, average waveform periods and propagation velocities were quantified. The effects of TRPV1 and TRPA1 agonist and antagonist were compared with the baseline recordings. RESULTS: The electrophysiological recordings showed that capsaicin (30 µM to 3 mM), resiniferatoxin (300 µM), capsazepine (100-300 µM), allyl isothiocyanate (300 µM), isovelleral (300 µM), icilin (300 µM), A-967,079 (10 µM), AP18 (20 µM) and HC-030,031 (50 µM) significantly reduced the pacemaker frequency and increased the waveform period relative to the baseline. Conversely, ruthenium red (300 µM) significantly increased the pacemaker frequency and reduced the waveform period. Capsaicin (3 mM) and AP18 (20 µM) also significantly reduced the propagation velocity. However, all tested antagonists failed to inhibit the effects of agonists. AMG9810 (300 µM), but not A-967,079 (300 µM), significantly inhibited the increases in pacemaker frequency caused by increased temperatures. CONCLUSION: Our findings suggest that TRPV1 and TRPA1 play a minor role in regulating pacemaker potentials and that at non-specific actions at other TRP and ion channels most likely contributed to the overall effects on the electrophysiological recordings that we observed.

Lepidopteran-active variable-region sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein.

A unique, coleopteran-active protein, termed eCry3.1Ab, was generated following variable-region exchange of a Bacillus thuringiensis lepidopteran-active protein, Cry1Ab, with a Cry3A region. Our results support the hypothesis that this variable-region exchange is responsible for imparting strong bioactivity against the larvae of western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte), a pest species which is not susceptible to either parent protein sequence. This study demonstrates the potential of successfully engineering a portion(s) of a lepidopteran-active B. thuringiensis sequence so that it has activity against coleopterans. Further elucidation of the eCry3.1Ab activity indicated the importance of variable regions 4 to 6 that were derived from Cry1Ab instead of Cry1Ac. There was some flexibility in making domain III of engineered hybrid insecticidal proteins even more Cry1Ab-like and retaining activity, while there was less flexibility in making domain III more Cry3A-like and retaining activity. In vitro binding studies with brush border membrane vesicles demonstrated that there was specific binding of chymotrypsin-processed modified Cry3A (mCry3A), which was not diminished by addition of a 100-fold molar excess of chymotrypsin-processed eCry3.1Ab or unprocessed eCry3.1Ab. In addition, in the converse experiment, specific binding of chymotrypsin-processed eCry3.1Ab was not diminished by the presence of a 75-fold molar excess of chymotrypsin-processed mCry3A. These data support the hypothesis that eCry3.1Ab can interact with different binding sites than the activated form of mCry3A in the WCR brush border and may provide a different mode of action from the standpoint of resistance management.

Sulprostone-Induced Gastric Dysrhythmia in the Ferret: Conventional and Advanced Analytical Approaches.

Nausea and emesis resulting from disease or drug treatment may be associated with disrupted gastric myoelectric activity (GMA). Conventional analytical techniques can determine the relative degrees of brady-, normo-, and tachygastric power, but lose information relative to the basic slow wave shape. The aim of the present study was to investigate the application of advanced analytical techniques in the analysis of disrupted GMA recorded after administration of sulprostone, a prostaglandin E3 / 1 agonist, in ferrets. Ferrets were implanted with radiotelemetry devices to record GMA, blood pressure, heart rate (HR) and core body temperature 1 week before the administration of sulprostone (30 µg/kg) or vehicle (saline, 0.5 mL/kg). GMA was initially analyzed using fast Fourier transformations (FFTs) and a conventional power partitioning. Detrended fluctuation analysis (DFA) was also applied to the GMA recordings to reveal information relative to the fluctuation of signals around local trends. Sample entropy (SampEn) analysis was used for examining the regularity of signals. Conventional signal processing techniques revealed that sulprostone increased the dominant frequency (DF) of slow waves, with an increase in the percentage power of the tachygastric range and a decrease in the percentage power of the normogastric range. DFA revealed that sulprostone decreased the fluctuation function, indicative of a loss of the variability of GMA fluctuations around local trends. Sulprostone increased SampEn values, indicating a loss of regularity in the GMA data. Behaviorally, sulprostone induced emesis and caused defecation. It also increased blood pressure and elevated HR, with an associated decrease in HR variability (HRV). Further analysis of HRV revealed a decrease in both low-frequency (LF) and high-frequency (HF) components, with an overall increase in the LF/HF ratio. Sulprostone did not affect core body temperature. In conclusion, DFA and SampEn permit a detailed analysis of GMA, which is necessary to understand the action of sulprostone to modulate gastric function. The action to decrease HRV and increase the LF/HF ratio may be consistent with a shift toward sympathetic nervous system dominance, commonly seen during nausea.

An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against Western corn rootworm larvae.

The western corn rootworm remains one of the most important pests of corn in the United States despite the use of many pest management tools. Cry3A, the first coleopteran-active Bacillus thuringiensis toxin isolated, has not been useful for control of the corn rootworm pest complex. Modification of Cry3A so that it contained a chymotrypsin/cathepsin G protease recognition site in the loop between alpha-helix 3 and alpha-helix 4 of domain I, however, resulted in consistent activity of the toxin ("mCry3A") against neonate western corn rootworm. In vitro chymotrypsin digests showed that there was a substantial difference between the enzyme sensitivity of mCry3A and the enzyme sensitivity of Cry3A, with mCry3A rapidly converted from a 67-kDa form to a approximately 55-kDa form. The introduced protease site was also recognized in vivo, where the approximately 55-kDa form of mCry3A toxin was rapidly generated and associated with the membrane fraction. After a point mutation in mcry3A that resulted in the elimination of the native domain I chymotrypsin site (C terminal to the introduced chymotrypsin/cathepsin G protease site of mCry3A), the in vitro and in vivo digestion patterns remained the same, demonstrating that the introduced site was required for the enhanced activity. Also, 55-kDa mCry3A generated by cleavage with chymotrypsin exhibited specific binding to western corn rootworm brush border membrane, whereas untreated 67-kDa mCry3A did not. These data indicate that the mCry3A toxicity for corn rootworm larvae was due to the introduction of a chymotrypsin/cathepsin G site, which enhanced cleavage and subsequent binding of the activated toxin to midgut cells.

Ablation of advanced tongue or base of tongue cancer and reconstruction with free flap: functional outcomes.

AIM: To evaluate the functional outcomes of patients who underwent total or nearly total glossectomy for advanced tongue or base of tongue cancer. MATERIAL AND METHODS: We used the radial forearm free flap (RFFF), anterior lateral thigh flap (ALTF) or fibular osteocutaneous flap (FOCF) to reconstruct the oral defect after radical resection in 39 patients undergoing total or nearly total glossectomy with laryngeal preservation. RESULTS: Good functional outcomes, measured by independent feeding, speech and swallowing were achieved in 35, 36 and 35 patients, respectively. The cumulative 4-year survival rates were 63.8% for tongue cancer and 42.9% for base of tongue cancer. CONCLUSION: Reconstruction with free flaps is a feasible method to restore the functional outcomes in speech and deglutition among patients who undergo total or nearly total glossectomy with laryngeal preservation.

Modification of flower colour by suppressing ß-ring carotene hydroxylase genes in Oncidium.

Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The ß-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow.

Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.

A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.

Characterization of lysosomal enzymes from cultured cynomolgus monkey trabecular meshwork cells.

The current study characterizes selected properties of lysosomal enzymes associated with cynomolgus monkey trabecular meshwork (MTM) cells. These proteins may participate in the turnover of macromolecules involved in regulating the aqueous outflow. Intracellular levels of lysosomal enzymes in MTM cells were similar to those found in cultured human fibroblasts. The presence of ammonium chloride increased the secretion rate of certain lysosomal enzymes from 47 to 122% of normal. Column chromatography of the secreted enzymes on the galactose-specific lectin Ricinus communis I demonstrated an increase in the number of accessible galactose residues on lysosomal enzymes secreted in the presence of ammonium chloride. The presence of mannose-6-phosphate receptors on the trabecular meshwork cells was demonstrated by the specific uptake of purified 125I-beta-D-glucosidase. This uptake represented 20% of that observed with cultured human fibroblasts and was inhibited only 50% by the presence of mannose-6-phosphate.

The extracellular matrix of the human optic nerve.

The nerve fibers of the optic nerve are enclosed and segmented by extracellular matrix. With immunostains, we localized collagen types I through VI, laminin, and fibronectin in frozen sections of the extracellular matrix of the prelaminar, laminar, and retrolaminar human optic nerve. The internal limiting lamina of the optic nerve has an extracellular composition similar to the thicker adjacent retinal internal limiting lamina. We confirmed that the lamina cribrosa contains type IV collagen and laminin, whereas the sclera does not. The septa of the retrolaminar optic nerve appear as vascular inward extensions of the pia mater. The glial tube that lines the optic nerve extends forward from the retrolaminar optic nerve through the lamina cribrosa to end anteriorly at the retinal pigment epithelium. It does not separate the optic nerve from the adjacent sensory retina.

Interaction of [3H]arylazido aminopropionyl ATP ([3H]ANAPP3) with P2-purinergic receptors in the smooth muscle of the isolated guinea-pig vas deferens.

Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP3, intact vasa deferentia were treated with [3H]ANAPP3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [3H]ANAPP3 (10(-5) M; 3.0 mu Ci/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54-66 and 43-57 kilodaltons. The photoincorporation of [3H]ANAPP3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [3H]ANAPP3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP3, and dipyridamole had no effect on the contractile response to [3H]ANAPP3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P2-purinergic receptors.

Lysosomal enzyme activity in human aqueous humor.

Activity of the lysosomal enzymes N-acetyl-beta-D-hexosaminidase, alpha-D-mannosidase, beta-D-glucuronidase, and beta-D-galactosidase was detected in aqueous humor from eyes undergoing intraocular surgery. There was no correlation between lysosomal enzyme activity and age. Lysosomal enzyme activity in human released by ocular tissues surrounding the anterior chamber including the cornea, trabecular meshwork, ciliary body, iris, and lens. Their release into aqueous humor may have a role in regulating aqueous outflow in normal and glaucomatous eyes.

Occurrence of a new toxin and tetrodotoxin in two species of the gastropod mollusk Nassariidae.

The lethalities of 102 specimens of three species of the gastropod mollusk Nassariidae, collected from fish markets in Taiwan, were examined. The frequency of toxicity in Zeuxis scalaris and Z. castus-like specimens was 94 and 41%, respectively. The range of lethal potency in toxic specimens of Zeuxis scalaris and Z. castus-like was 2-140 and 2-13 mouse units, respectively, while all tissues of Z. castus were non-toxic. The toxins were partially purified from the toxic specimens of Z. scalaris and Z. castus-like. Two toxin fractions were obtained from the extract of each species of Nassariidae by using Bio-Gel P-2 column chromatography. Analyses by thin layer chromatography, electrophoresis, high performance liquid chromatography and ultraviolet spectroscopy, showed that toxin fraction I contained tetrodotoxin while toxin fraction II contained a new neurotoxin.

Variation and secretion of toxins in gastropod mollusc Niotha clathrata.

Nearly 300 specimens of the gastropod mollusc Niotha clathrata were collected in South Taiwan. All specimens were assayed for toxicity by the official method for tetrodotoxin (TTX). The frequency value of toxic specimens in N. clathrata was 30.0%, 68.0% and 80.4% at Anping, Chiating and Tungkang, respectively. The highest lethal potency of a gastropod specimen was 1900 mouse units (MU). The specimens collected from Tungkang showed the highest frequency of toxic specimens and toxicity, followed by those from Chiating and Anping. The specimens collected in autumn and spring showed higher toxicity than those collected in other seasons. Moreover, another 17 specimens of N. clathrata were collected for testing the toxin secretion by electric shock treatment. It is found that the gastropod did not secrete any additional toxin when electric shock treatment was performed twice at approximately 1-hr intervals. The toxicity of secreted toxin was 206 MU.

Occurrence of tetrodotoxin in the gastropod mollusk Natica lineata (lined moon shell).

Paralytic toxicity was detected in 28 of 38 specimens of the gastropod mollusk Natica lineata (lined moon shell). The highest toxicity, expressed as tetrodotoxin (TTX), was 720 mouse units per gram (MU/g) muscle, followed by other parts which included salivary gland, brain and mouth organs (28 MU/g) and digestive gland (12 MU/g). The toxin was partially purified by ultrafiltration and Bio-Gel P-2 column chromatography. The toxin showed a specific toxicity (as TTX) of 620 MU/mg. Results of analyses by thin layer chromatography, cellulose acetate membrane electrophoresis and high performance liquid chromatography showed that the toxin was composed of TTX and anhydrotetrodotoxin. This is the first time that TTX and its related substance have been found in this species of gastropod.

Tetrodotoxin secretion from the lined moon shell Natica lineata in response to external stimulation.

The lined moon shell Natica lineata secretes tetrodotoxin (TTX) in response to an external stimulation such as removal from the seawater. The toxin released from the shellfish contained 14-361 mouse units of TTX per specimen. The shellfish did not secrete any further toxin when seawater removal was repeated over four times at about 1 hr intervals. All specimens recovered TTX secreting ability when they were kept in an aquarium for 5 days.

Gonyautoxin-3 as a minor toxin in the gastropod Niotha clathrata in Taiwan.

Paralytic toxicity was detected in the gastropod mollusc Niotha clathrata collected from South Taiwan in April and November 1993. Each seasonal toxin was partially purified from toxic specimens of N. clathrata by ultrafiltration using a membrane (Diaflo YM-2), followed by chromatography on a column (Bio-Gel P-2). Two toxin fractions (I and II) were then obtained for each seasonal shell toxin. The ratio of fraction I to fraction II for each seasonal shell toxin was about 4:1 according to tetrodotoxin bioassay. Based on analyses by TLC, electrophoresis, and HPLC, fraction I toxin contained tetrodotoxin and its derivative anhydrotetrodotoxin, and fraction II toxin contained gonyautoxin-3 for each seasonal shell toxin.

Toxicity and toxic components of two xanthid crabs, Atergatis floridus and Demania reynaudi, in Taiwan.

Paralytic toxicity was detected by tetrodotoxin bioassay in eight specimens of Atergatis floridus and seven specimens of Demania reynaudi, collected from Taiwan in 1994. The toxicity of crab specimens was 161 +/- 115 (mean +/- S.D.) mouse units (MU) for A. floridus and 640 +/- 273 MU for D. reynaudi. The respective toxins were partially purified from specimens of A. floridus and D. reynaudi by ultrafiltration using a Diaflo YM-1 membrane, followed by chromatography on a Bio-Gel P-2 column. Electrophoresis, thin-layer chromatography, high-performance liquid chromatography, ultraviolet spectrum and gas chromatography-mass spectrometry indicated that the toxin of A. floridus was mainly composed of tetrodotoxin (85%), along with minor gonyautoxin 1-4 (15%), and the toxin of D. reynaudi was mainly composed of tetrodotoxin (88%), along with minor gonyautoxin 2-4 and neosaxitoxin (12%).