Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1->>)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

Afrotropical malaria vectors of the Anopheles gambiae complex (Diptera: Culicidae), particularly An. gambiae sensu stricto, are attracted mainly to human hosts. A major source of human volatile emissions is sweat, from which key human-specific components are the carboxylic acids (E)- and (Z)-3-methyl-2-hexenoic acid and 7-octenoic acid. Electrophysiological studies on the antennae of An. gambiae s.s. showed selective sensitivity to these compounds, with a threshold at 10(-6) g comparable to that of known olfactory stimulants 1-octen-3-ol, p-cresol, isovaleric acid, and lower than threshold sensitivity to L-lactic acid and the synthetic mosquito repellent N,N-diethyltoluamide (DEET). A combination of the acids released at concentrations>> 10(-5) g in wind tunnel bioassays significantly reduced the response to CO2, the major attractant released by human hosts, for strains of An. gambiae s.s. originating from East and West Africa. Field trials with odour-baited entry traps (OBETs) in Burkina Faso showed that 7-octenoic acid significantly increased (by 1.7-fold) the catch of females of An. gambiae sensu lato (comprising two sibling species: An. arabiensis Patton and An. gambiae s.s.) in OBETs baited with CO2, whereas combinations of the acids significantly reduced the catch in CO2-baited traps (by 2.1-fold) and in whole human odour-baited traps (by 1.5-fold). The pure (E) and (Z) geometric isomers of 3-methyl-2-hexenoic acid gave comparable results to the (EIZ) isomer mixture. These results provide the first experimental evidence that human-specific compounds affect the behaviour of highly anthropophilic An. gambiae s.l. mosquitoes. The compounds appear to inhibit the upwind flight' response to known long-range attractants, and may serve either to mask' the attractants present or, more probably, to 'arrest' upwind flight when mosquitoes arrive at a host under natural conditions. In the final approach to hosts, vectors are known to reduce their flight speed and increase their turning rate, to avoid overshooting the source. In our experimental apparatus, these changes in flight behaviour would reduce the number of mosquitoes entering the ports of the collection devices.

Elderly patients with diabetes and peripheral neuropathy are more likely to experience falls. However, the information available on how such falls can be prevented is scarce. We investigated the effects of whole-body vibration (WBV) combined with a balance exercise program on balance, muscle strength, and glycosylated hemoglobin (HbA1c) in elderly patients with diabetic peripheral neuropathy. Fifty-five elderly patients with diabetic neuropathy were randomly assigned to WBV with balance exercise group, balance exercise (BE) group, and control group. The WBV and BE groups performed the balance exercise program for 60 min per day, 2 times per week, for 6 weeks. Further, the WBV group performed WBV training (up to 3 × 3 min, 3 times per week, for 6 weeks). The control group did not participate in any training. The main outcome measures were assessed at baseline and after 6 weeks of training; namely, we assessed the postural sway and one leg stance (OLS) for static balance; Berg balance scale (BBS), timed up-and-go (TUG) test, and functional reach test (FRT) for dynamic balance; five-times-sit-to-stand (FTSTS) test for muscle strength; and HbA1c for predicting the progression of diabetes. Significant improvements were noted in the static balance, dynamic balance, muscle strength, and HbA1c in the WBV group, compared to the BE and control groups (P < 0.05). Thus, in combination with the balance exercise program, the short-term WBV therapy is beneficial in improving balance, muscle strength and HbA1c, in elderly patients with diabetic neuropathy who are at high risk for suffering falls.

Experiments were done to determine whether the strength of reciprocal inhibition from ankle flexors to extensors can be controlled independently of the level of ongoing motor activity in a task-dependent manner. In this paper we use the term reciprocal inhibition in the functional sense--inhibition of the antagonist(s) during activity of the agonist(s)--without reference to specific neural pathways that may be involved. The strength of reciprocal inhibition of the soleus alpha-motoneurons was determined by measuring the amplitude of the H reflex during voluntary, postural, and locomotor tasks requiring activity of the ankle flexor tibialis anterior (TA). Differences in the strength of reciprocal inhibition between tasks were determined from plots of the soleus H reflex amplitude versus the mean value of the TA electromyogram (EMG). Additionally, in tasks involving movement, the correlation between the H reflex amplitude and the joint kinematics was calculated. In most subjects (15 of 22) the soleus H reflex decreased approximately linearly with increasing tonic voluntary contractions of the TA. The H reflex also decreased approximately linearly with the TA EMG activity when subjects where asked to lean backward. There were no statistical differences between the regression lines obtained in these tasks. In some subjects (7 of 22), however, the H reflex amplitude was independent of the level of TA EMG activity, except for a sudden drop at high levels of TA activity (approximately 60-80% of maximum voluntary contraction). The type of relation between the soleus H reflex and the TA EMG activity in these tasks was not correlated with the maximum H reflex to maximum M wave (Hmax/Mmax) ratio measured during quiet standing. In marked contrast, during the swing phase of walking--over the same range of TA EMG activity as during the tonic voluntary contraction task--the H reflex was reduced to zero in most subjects (24 of 31). In seven subjects the H reflex during the swing phase was reduced to some 5% of the value during quiet standing. The same result was found when subjects were asked to produce a stepping movement with one leg (OLS) in response to an auditory "go" signal. Additionally, in the OLS task it was possible to examine the behavior of the H reflex during the reaction time and thus to evaluate the relative contribution of central commands versus movement-related afferent activity to the inhibition of the soleus H reflex. In 11 of 12 subjects the H reflex attained its minimum value before either the onset of EMG activity or movement of any of the leg joints. It is significant that the H reflex was most powerfully inhibited during the swing phase of walking and the closely related OLS task. The H reflex was also measured during isolated ankle dorsiflexion movements. The subjects were asked to track a target displayed on a computer screen with dorsiflexion movements of the ankle. The trajectory of the target was the same as that of the ankle during the swing phase of walking. The soleus H reflexes were intermediate in size between the values obtained in the tonic contraction task and the walking or OLS tasks. A negative, but weak, correlation (r2 < 0.68) between the soleus H reflex and the TA EMG was found in 3 of 10 subjects. Furthermore, there was no correlation between the H reflex amplitude and the ankle angular displacement or angular velocity. In this task, as in the OLS task, the H reflex began to decrease during the reaction time before the onset of TA EMG activity. We conclude that the strength of reciprocal inhibition of the soleus alpha-motoneuron pool can thus be controlled independently of the level of motor activity in the ankle flexors. The strength of the inhibition of the antagonist(s) depends on the task, and for each task the strength of the inhibition is not necessarily proportional to the level of motor activity in the agonist(s). (ABSTRACT TRUNCATED)

The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratriene-<em>ol</em> (CBT-<em>ol</em>) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-<em>ols</em>, and verified the function of a P450 gene responsible for conversion of CBT-<em>ols</em> to CBT-di<em>ols</em>. CBT-di<em>ols</em> are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense co-suppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-<em>ols</em>/CBT-di<em>ols</em>) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-<em>ol</em> cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding regions of both genes in AS and S orientations were </=20%. Our results identify the function of a CBT-<em>ol</em> cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.

In this report we describe studies on the structures of the O-linked carbohydrate units in cell-surface glycoproteins of epimastigote forms of the G-strain of Trypanosoma cruzi. Mild alkaline reductive degradation of the 38/43 kDa glycoproteins resulted in beta-elimination of glycosylated threonine and/or serine residues, and the liberation of N-acetylglucosaminitol, galactobiosyl-, galactotriosyl-, galactotetraosyl- and galactopentaosyl-N-acetylglucosaminitol. The structures of these oligosaccharide alditols were established by n.m.r. spectroscopy and methylation analysis as: Galf beta 1-4(Galp beta 1-6)GlcNAc-ol; Galp beta 1-3Galp beta 1-6(Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galp beta 1-2Galf beta 1-4)GlcNAc-ol.

Green tea containing 634 micromol of flavan-3-ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0-24 h after ingestion were analysed by HPLC-MS. The ileal fluid contained 70% of the ingested flavan-3-ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O-linked sulphates and methyl-sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan-3-ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101-256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan-3-ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8+/-0.6% of total flavan-3-ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (-)-epicatechin and (+)-catechin.

The Taiwanese health insurance industry is just over 30 years old. Originally private and domestic, the industry underwent substantial institutional changes when it opened to foreign competition between 1987 and 1994 and when the Taiwanese government established national health insurance (NHI) coverage in 1995. Congruent with these changes, rapid growth occurred in the Taiwanese demand for private health insurance. In order to better understand the recent performance of the Taiwanese health insurance industry, the structure of the NHI system is described and then household decisions to purchase private health insurance are analyzed using a two-part (hurdle) model on 1998 Survey of Family Income and Expenditure data. Logistic and OLS regressions are used to examine the factors influencing the probability and amount of private health insurance purchased. Generally, factors affecting the probability of having insurance also influence the amount of insurance coverage purchased. Higher income and education levels are associated with increased probabilities and larger quantities of private insurance purchases. Married females, the employed, and household heads working in state-run enterprises are more likely to purchase private insurance than their counterparts. The probability of private insurance purchases varies by region, with northern Taiwanese households having higher odds of owning private insurance than non-northern households. Compared to those in rural villages, households in cities and towns are more likely to have private insurance. The likelihood of private insurance purchase also tends to rise with advancing age and larger family sizes. In addition, one important implication in the private health insurance market is highlighted. There is no complementarity between the public and private systems.

2-((1E,3E)-4-(6-((11)C-methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol ((11)C-PBB3) is a clinically useful PET probe that we developed for in vivo imaging of tau pathology in the human brain. To ensure the availability of this probe among multiple PET facilities, in the present study we established protocols for the radiosynthesis and quality control of (11)C-PBB3 and for the characterization of its photoisomerization, biodistribution, and metabolism.

METHODS

(11)C-PBB3 was synthesized by reaction of the tert-butyldimethylsilyl desmethyl precursor ( 1: ) with (11)C-methyl iodide using potassium hydroxide as a base, followed by deprotection. Photoisomerization of (11)C-PBB3 under fluorescent light was determined. The biodistribution and metabolite analysis of (11)C-PBB3 was determined in mice using the dissection method.

RESULTS

(11)C-PBB3 was synthesized with 15.4% ± 2.8% radiochemical yield (decay-corrected, n = 50) based on the cyclotron-produced (11)C-CO2 and showed an averaged synthesis time of 35 min from the end of bombardment. The radiochemical purity and specific activity of (11)C-PBB3 were 98.0% ± 2.3% and 180.2 ± 44.3 GBq/μmol, respectively, at the end of synthesis (n = 50). (11)C-PBB3 showed rapid photoisomerization, and its radiochemical purity decreased to approximately 50% at 10 min after exposure to fluorescent light. After the fluorescent light was switched off, (11)C-PBB3 retained more than 95% radiochemical purity over 60 min. A suitable brain uptake (1.92% injected dose/g tissue) of radioactivity was observed at 1 min after the probe injection, which was followed by rapid washout from the brain tissue. More than 70% of total radioactivity in the mouse brain homogenate at 5 min after injection represented the unchanged (11)C-PBB3, despite its rapid metabolism in the plasma.

CONCLUSIONS

(11)C-PBB3 was produced with sufficient radioactivity and high quality, demonstrating its clinical utility. The present results of radiosynthesis, photoisomerization, biodistribution, and metabolite analysis could be helpful for the reliable production and application of (11)C-PBB3 in diverse PET facilities.

Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels.

Epigallocatechin gallate (EGCG) is a flavonoid belonging to the chemical class of falvan-3-ols (catechins) esterified with gallic acid. It is the main catechin found in green tea (Camellia sinensis L.) accounting for about 50% of its total polyphenols. Extensive research performed in recent years has revealed that green tea demonstrates a wide range of positive biological activities against serious chronic diseases such as cardiovascular and neurodegenerative pathologies, cancer, metabolic syndrome and type 2 diabetes. These protective properties can be traced back to the potent antioxidant and anti-inflammatory activities of EGCG. Recent studies have suggested that it may exert its beneficial effects by modulating mitochondrial functions impacting mitochondrial biogenesis, bioenergetic control (ATP production and anabolism), alteration of the cell cycle, and mitochondria-related apoptosis. This review evaluates recent evidence on the ability of EGCG to exert critical influence on the above mentioned pathways.

The proanthocyanidins in three foods (pinto beans, plums and cinnamon) were studied with electrospray ionization (ESI) mass spectrometry (MS) in the negative mode following separation by normal-phase high-performance liquid chromatography. The MS/MS analysis demonstrated that the major ions derived from heterocyclic ring fission and retro-Diels-Alder reaction of flavan-3-ol provided information about the hydroxylation pattern and type of interflavan bond. The connection sequence of the oligomers was identified through diagnostic ions derived from quinone methide (QM) cleavage of the interflavan bond. Novel heterogeneous B-type proanthocyanidins containing (epi)afzelechin as subunits were identified in pinto beans. Proanthocyanidins with interestingly different A-type linkages were identified in plums and cinnamon. In efforts aimed at extending the identification capacity of ESI-MS to polymers, we found that the polymeric procyanidins fragmented readily instead of forming multiply charged ions in the negative ESI mode. Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS and ESI time-of-flight MS.

On the Chinese mainland, betel quid (BQ) chewing is common in the Hunan and Hainan provinces. The BQ chewing habit in Hunan consists of dried husks and betel nuts, which are sold as industrially packaged, areca nut-based products. In Hainan, the fresh nut is chewed. Tobacco is not added. Reported prevalence of BQ chewing in Hunan province is high (64.5-82.7%). Oral diseases associated with BQ chewing are oral submucous fibrosis (OSF), oral leukoplakia (OL) and oral cancer. Reported prevalence of OSF among BQ chewers ranges from 0.9% to 4.7%. People most commonly affected are between the ages of 30 and 39 years, and 40 and 49 years. The reported prevalence of OL in Hainan ranges from 2.1% to 2.5%. In BQ chewers who also smoke, the reported prevalence is 20.3%. The prevalence of OL in Hunan province ranges from 0.1% to 0.5%. The prevalence of oral cancer among BQ chewers is low, ranging from 0.02% to 0.05%. In cases of OSF, reported prevalence is 2.6% and 1.2%. Presently, data on prevalence of BQ chewing in southern provinces of Mainland China is limited. BQ chewing habits, however, seem to differ between geographic areas. Future case-control studies are necessary to evaluate the risk for oral cancer and other associated oral mucosal diseases resulting from variations in BQ chewing habits.

Orphanin FQ/nociceptin (OFQ/N) is the endogenously occurring peptide ligand for the nociceptin opioid receptor (NOP) that produces anxiolytic-like effects in mice and rats. The present study assessed the anxiolytic-like activity of 8-[bis(2-methylphenyl)-methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510), a novel potent piperidine NOP agonist (EC(50) = 12 nM) that binds with high affinity (K(i) = 0.3 nM) and functional selectivity (>50-fold over the mu-, kappa-, and delta-opioid receptors). The anxiolytic-like activity and side-effect profile of SCH 221510 were assessed in a variety of models and the benzodiazepine, chlordiazepoxide (CDP), was included for comparison. The effects of chronic dosing of SCH 221510 were also assessed. Furthermore, the specificity of the anxiolytic-like effect of SCH 221510 was investigated with the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and the opioid receptor antagonist naltrexone. Like CDP (1-30 mg/kg i.p.), SCH 221510 (1-30 mg/kg p.o.) produced anxiolytic-like effects in the elevated plus-maze (rat and gerbil), Vogel conflict (rat), conditioned lick suppression (rat), fear-potentiated startle (rat), and pup separation-induced vocalization (guinea pig) assays. In the Vogel conflict, the anxiolytic-like effect of SCH 221510 (10 mg/kg) was attenuated by J-113397 (3-10 mg/kg p.o.), but not naltrexone (3-30 mg/kg i.p.). Additionally, the anxiolytic-like effects of SCH 221510 did not change appreciably following 14-day b.i.d. dosing in rats (10 mg/kg). Furthermore, unlike CDP, SCH 221510 (3-30 mg/kg) produced anxiolytic-like activity at doses that did not disrupt overt behavior. Collectively, these data suggest that NOP agonists such as SCH 221510 may have an anxiolytic-like profile similar to benzodiazepines, with a reduced side-effect liability.

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.

Flavonoids have anti-inflammatory and antioxidative effects and thus may protect against diabetes. Therefore, we hypothesized that consumption of flavonoids and specific food and beverage sources of flavonoids would be associated with reduced risk of incident diabetes. At baseline (1986), diet (by food frequency questionnaire) and health information were collected from 35,816 postmenopausal women free of diabetes. Self-reported incident diabetes was ascertained 5 times during the study (1987, 1989, 1992, 1997, and 2004). Cox proportional hazards regression was used to calculate hazard ratios for incident diabetes according to categories of total flavonoids and anthocyanidins, flavones, flavanones, flavonols, flavan-3-ol monomers, isoflavones, and proanthocyanidins. Hazard ratios according to intake categories of flavonoid-rich foods and beverages were also calculated (apples, pears, berries, broccoli, bran, citrus, tea, and red wine). Flavonoid consumption was not associated with diabetes risk after multivariable adjustment. Although other flavonoid-rich foods and beverages were not associated, red wine was inversely associated with diabetes. Women who reported drinking red wine>>or=1 time/wk had a 16% reduced risk of diabetes than those drinking wine <1 time/wk [HR (95% CI): 0.84 (0.71, 0.99)], with parallel findings for white wine, beer, and liquor. In conclusion, these data do not support a diabetes-protective effect of flavonoids. The suggestive evidence of a protective effect of regular red wine consumption is shared with an inverse association between alcohol drinks in general and diabetes risk and may reflect the effects of nonflavonoid constituents that are common to all alcohol drinks.

Therapeutic intervention for human succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) has been limited to vigabatrin (VGB). Pharmacologically, VGB should be highly effective due to 4-aminobutyrate-transaminase (GABA-transaminase) inhibition, lowering succinic semialdehyde and, thereby, gamma-hydroxybutyric acid (GHB) levels. Unfortunately, clinical efficacy has been limited. Because GHB possesses a number of potential receptor interactions, we addressed the hypothesis that antagonism of these interactions in mice with SSADH deficiency could lead to the development of novel treatment strategies for human patients. SSADH-deficient mice have significantly elevated tissue GHB levels, are neurologically impaired, and die within 4 weeks postnatally. In the current report, we compared oral versus intraperitoneal administration of VGB, CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid, a GABA(B) receptor antagonist], and the nonprotein amino acid taurine in rescue of SSADH-deficient mice from early death. In addition, we assessed the efficacy of the specific GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-[H]benzocycloheptene-5-ol-6-ylideneacetic acid) using i.p. administration. All interventions led to significant lifespan extension (22-61%), with NCS-382 being most effective (50-61% survival). To explore the limited human clinical efficacy of VGB, we measured brain GHB and gamma-aminobutyric acid (GABA) levels in SSADH-deficient mice receiving VGB. Whereas high-dose VGB led to the expected elevation of brain GABA, we found no parallel decrease in GHB levels. Our data indicate that, at a minimum, GHB and GABA(B) receptors are involved in the pathophysiology of SSADH deficiency. We conclude that taurine and NCS-382 may have therapeutic relevance in human SSADH deficiency and that the poor clinical efficacy of VGB in this disease may relate to an inability to decrease brain GHB concentrations.

Nociceptin activation of ORL1 (opioid receptor-like 1 receptor) has been shown to antagonize mu receptor-mediated analgesia at the supraspinal level. ORL1 and mu-opioid receptor (muR) are co-expressed in several subpopulations of CNS neurons involved in regulating pain transmission. The amino acid sequence of ORL1 also shares a high degree of homology with that of mu receptor. Thus, it is hypothesized that ORL1 and muR interact to form the heterodimer and that ORL1/muR heterodimerization may be one molecular basis for ORL1-mediated antiopioid effects in the brain. To test this hypothesis, myc-tagged ORL1 and HA-tagged muR are co-expressed in human embryonic kidney (HEK) 293 cells. Co-immunoprecipitation experiments demonstrate that ORL1 dimerizes with muR and that intracellular C-terminal tails of ORL1 and muR are required for the formation of ORL1/muR heterodimer. Second messenger assays further indicate that formation of ORL1/muR heterodimer selectively induces cross-desensitization of muR and impairs the potency by which [D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin (DAMGO) inhibits adenylate cyclase and stimulates p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. These results provide the evidence that ORL1/muR heterodimerization and the resulting impairment of mu receptor-activated signaling pathways may contribute to ORL1-mediated antiopioid effects in the brain.

Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

This study evaluated the inhibitory effects of plant-based extracts (grape seed, green tea, and white tea) and their constituent flavan-3-ol monomers (catechins) on α-amylase and α-glucosidase activity, two key glucosidases required for starch digestion in humans. To evaluate the relative potency of extracts and catechins, their concentrations required for 50 and 90% inhibition of enzyme activity were determined and compared to the widely used pharmacological glucosidase inhibitor, acarbose. Maximum enzyme inhibition was used to assess relative inhibitory efficacy. Results showed that grape seed extract strongly inhibited both α-amylase and α-glucosidase activity, with equal and much higher potency, respectively, than acarbose. Whereas tea extracts and catechin 3-gallates were less effective inhibitors of α-amylase, they were potent inhibitors of α-glucosidase. Nongallated catechins were ineffective. The data show that plant extracts containing catechin 3-gallates, in particular epigallocatechin gallate, are potent inhibitors of α-glucosidase activity and suggest that procyanidins in grape seed extract strongly inhibit α-amylase activity.

The central nucleus of the amygdala (CeA) and the nucleus of the accumbens shell (NAc) have been shown to be involved in opioid-mediated feeding behavior. The present study examined whether mu-opioid signalling between the CeA and NAc affected feeding. Male Sprague-Dawley rats were fitted with one cannula placed in the CeA and two cannulae placed in the NAc, which allowed for coadministration of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in one site and the opioid antagonist naltrexone (NTX) in the other site. Single injection of DAMGO (2.4 nmol) into the CeA and bilateral injections of DAMGO (2.4 nmol) into the NAc stimulated feeding (P<0.05). The DAMGO-induced increase of food intake following injection into the CeA was decreased by bilateral injection of NTX (13.2 and 26.5 nmol) into the NAc at 2- and 4-h postinjections (P<0.05). In the reverse situation, the DAMGO-induced increase of food intake following injection into the NAc was decreased by injection of NTX (13.2 and 26.5 nmol) into the CeA at 1-, 2-, and 4-h postinjections (P<0.05). These results suggest that a bi-directional mu-opioid-opioid signalling pathway exists between the CeA and the NAc, which influences feeding.

OBJECTIVE

Hypoxia is an important negative prognostic factor for radiation treatment of head and neck (H&N) cancer. The focus of this study was to evaluate the feasibility of 18F-HX4 (3-[18F]fluoro-2-(4-((2-nitro-1Himidazol- 1-yl)methyl)-1H-1,2,3,-triazol-1-yl)-propan-1-ol) on hypoxia imaging compared with 18F-fluoromisonidazole (18F-FMISO) mainly in human H&N cancer.

METHODS

18F-HX4 precursor, standards, and methods were provided by Siemens Molecular Imaging Inc. 18F-HX4 was prepared in an automated module. Twelve patients with H&N cancer were recruited into this study. Each patient underwent 18F-HX4 PET/CT imaging, followed by 18F-FMISO and 18F-fluorodeoxyglucose (18F-FDG) PET/CT on separate days. 18F-HX4 and 18F-FMISO images of the H&N areas were acquired 1.5 and 2 h after injection, respectively. Standard uptake values and tumor-to-muscle (T/M) ratios were calculated. Immunohistochemical analysis of the hypoxia-associated marker CA-IX was carried out to investigate the relationship with PET uptake.

RESULTS

18F-HX4 and 18F-FMISO in the patients gave similar hot spots well within the 18F-FDG uptake region. At 1.5 h postinjection 18F-HX4 yielded a T/M similar to that of 18F-FMISO at 2 h postinjection (1.94±1.03 vs. 1.85±1.01; P> 0.05). A total of 12 lesions were identified. Among them, eight lesions were positive and two lesions were negative on both 18F-HX4 and 18F-FMISO images; one of the other two lesions was positive only on 18F-HX4, whereas the other one was positive only on 18F-FMISO. The CA-IX expression result correlated with the hypoxia imaging but not with 18F-FDG imaging.

CONCLUSIONS

18F-HX4 is a safe and feasible agent in hypoxia imaging of H&N cancer patients. We could assume that 18F-HX4 may have higher sensitivity and specificity, faster clearance, and shorter injection–acquisition time compared with traditional 18F-FMISO. Additional evaluations need to be carried out to validate the assumption. Further development of 18F-HX4 for eventual targeting of antihypoxia therapies is warranted.

Intrauterine growth-restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in the guinea pig results in long-term deficits in brain myelination and could therefore contribute to altered neural function. CPI was induced by unilateral ligation of the uterine artery at mid-gestation (term~67 days of gestation; dg), producing growth-restricted (GR) foetuses (60 dg), neonates (1 week) and young adults (8 week); controls were from the unligated horn or sham-operated animals. In GR foetuses (n=8) and neonates (n=7), white matter (WM) volume was reduced (p<0.05); this reduction did not persist in young adults (n=11) however the corpus callosum width was reduced (p<0.05). Immunoreactivity (IR) for myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP), all markers of myelinating oligodendrocytes (OL), was reduced in GR foetuses compared to controls. MBP was the most markedly affected with an abnormal retention of protein in the OL soma and a reduction of its incorporation into the myelin sheath. MAG-IR OL density was reduced (p<0.05), while the density of OLs immunoreactive for Olig-2, a transcription factor expressed throughout the entire OL lineage, was increased (p<0.05). MBP-, MAG- and PLP-IR recovered to control levels postnatally. These results suggest that IUGR transiently delays OL maturation and myelination in utero but that myelination and WM volume are restored to control levels postnatally. Long-term deficits in myelination are therefore unlikely to be the major factor underlying the altered neurological function which can be associated with IUGR.

Chinese medicine is a complex system guided by traditional Chinese medicine (TCM) theories, which has proven to be especially effective in treating chronic and complex diseases. However, the underlying modes of action (MOA) are not always systematically investigated. Herein, a systematic study was designed to elucidate the multi-compound, multi-target and multi-pathway MOA of a Chinese medicine, QiShenYiQi (QSYQ), on myocardial infarction. QSYQ is composed of Astragalus membranaceus (Huangqi), Salvia miltiorrhiza (Danshen), Panax notoginseng (Sanqi), and Dalbergia odorifera (Jiangxiang). Male Sprague Dawley rat model of myocardial infarction were administered QSYQ intragastrically for 7 days while the control group was not treated. The differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with QSYQ, followed by constructing a cardiovascular disease (CVD)-related multilevel compound-target-pathway network connecting main compounds to those DEGs supported by literature evidences and the pathways that are functionally enriched in ArrayTrack. 55 potential targets of QSYQ were identified, of which 14 were confirmed in CVD-related literatures with experimental supporting evidences. Furthermore, three sesquiterpene components of QSYQ, Trans-nerolidol, (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol and (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol from Dalbergia odorifera T. Chen, were validated experimentally in this study. Their anti-inflammatory effects and potential targets including extracellular signal-regulated kinase-1/2, peroxisome proliferator-activated receptor-gamma and heme oxygenase-1 were identified. Finally, through a three-level compound-target-pathway network with experimental analysis, our study depicts a complex MOA of QSYQ on myocardial infarction.