Responses of the silkworm tyramine receptor to 2-phenylethylamines and 5-phenyloxazoles

Tyramine (TA), a biogenic amine, attenuates intracellular cAMP production by acting on its receptor in insects. Several non-biogenic amines were examined for their actions on native and heterologously expressed silkworm TA receptors. 5-(4-Hydroxyphenyl)oxazole, which showed an attenuating effect on cAMP production in silkworm-head membranes, did not attenuate forskolin-stimulated cAMP production in HEK-293 cells expressing the silkworm TA receptor, although the compound bound to the cloned receptor. 2-Phenylethylamines (2-PEAs), which showed positive and negative effects on cAMP production in silkworm-head membranes, inhibited [3H]TA binding to the cloned TA receptor. 2-Chloro-2-(4-chlorophenyl)ethylamine was the most potent inhibitor of [3H]TA binding among the 2-PEAs tested, with an IC50 of 30.4 nM. This compound acted as an antagonist and abolished TA-attenuation of forskolin-stimulated cAMP production in the cloned TA receptor. The discrepancy in the effects of the non-biogenic amines on the native and cloned TA receptors remains to be further examined. A newly synthesized 2-PEA, 2-chloro-2-(4-hydroxyphenyl)ethylamine, attenuated forskolin-stimulated cAMP production in the cloned TA receptor, indicating that the para-hydroxy group is important for the agonist action.     

Critical factors influencing the occurrence of Vibrio cholerae in the environment of Bangladesh

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype Vibrio cholerae O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic Vibrio cholerae serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a "reemerging" disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis. 187:96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably V. cholerae). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of V. cholerae is proving useful in developing environmental models for the prediction of cholera epidemics.     

Spasmolytic flavonoids from Syzygium samarangense (Blume) Merr. & L.M. Perry

The hexane extract of Syzygium samarangense (Ss.Hex) dose-dependently (10-1000 microg/ ml) relaxed the spontaneously contracting isolated rabbit jejunum. Four rare C-methylated flavonoids with a chalcone and a flavanone skeleton were isolated from Ss.Hex and were subsequently tested for spasmolytic activity. All flavonoids, identified as 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (1), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (2), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (3), and 7-hydroxy-5-methoxy-6,8-dimethyl-flavanone (4), showed dose-dependent spasmolytic activity in the rabbit jejunum with IC50 values of 148.3 +/- 69.4, 77.2 +/- 43.5, 142.4 +/- 58.6 and 178.5 +/- 37.5 microg/ml (mean +/- SEM), respectively. The dihydrochalcone derivative of compound 1, 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (5), when tested for spasmolytic activity, did not significantly relax the smooth muscle relative to the other compounds. Verapamil, a standard spasmolytic, has an IC50 value of 0.16 +/- 0.04 microg/ml. This is the first report of the relaxant activity of chalcones, specifically of compounds 1-3.     

Gallbladder carcinoma: a retrospective analysis of twenty-two years experience of a single teaching hospital

BACKGROUND: The purpose of this study was to retrospectively evaluate our experience with gallbladder cancer since the establishment of a tumour registry in our institute. METHODS: Between 1975 and 1998, 23 consecutive patients with gallbladder cancer were identified using the tumour registry database. There were 18 females (78%) and 5 (22%) males. The mean age at diagnosis was 70.6 (range 42-85) years. The diagnosis was achieved either intra-operatively or following the histological analysis of the gallbladder (n = 17), following gallbladder or liver biopsy (n = 4) or at autopsy (n = 2). Presenting symptoms included upper abdominal pain, weight loss, nausea, vomiting, fever, painless jaundice, hepatomegaly, upper abdominal mass, upper abdominal tenderness, and gastrointestinal haemorrhage. RESULTS: Histological examination revealed 20 adenocarcinomas (87%), 2 squamous cell carcinomas (9%) and one spindle cell sarcoma (4%). At presentation, 14 (61%) gallbladder cancers were stage IV, 5 (22%) were stage III and 4 (17%) were stage II. Kaplan Meier analysis revealed a mean survival of 3.2, 7.8 and 8.2 months for stage IV, III, and II disease respectively. Out of 14 patients with stage IV disease, 8 patients received adjuvant chemotherapy and survived for 4.6 months whereas six patients who did not receive adjuvant chemotherapy survived for 1.3 months. This difference was statistically significant (p = 0.04). CONCLUSION: The majority of patients with gallbladder cancer presented with advanced stage disease (stage IV) which carries a dismal prognosis. Patients who received chemotherapy with stage IV disease, however, did better than those who did not, but this is probably a reflection of patient selection.     

Determination of Rhodamine 123 in cell lysate by HPLC with visible wavelength detection

Rhodamine 123 (R123) is widely used to quantify P-glycoprotein (P-GP) functional efflux activity in vitro. We developed a rapid and specific high-performance liquid chromatography (HPLC) method to quantify Rhodamine 123 for use in experimental cell culture studies. The R123 standards (2.5-250 ng/mL) and quality controls (QCs) (5, 75, 200 ng/mL) were prepared in cell lysis buffer consisting of 0.75% Triton 100X and 0.2% sodium chloride. The mobile phase consisted of acetonitrile, 1.5 mM tetrabutyl ammonium bromide in 20mM sodium acetate buffer (pH 4.0) (50:20:30) delivered at a rate of 1.0 mL/min. Samples (50 microl) were injected onto a C(18) reversed-phase HPLC column with detection at 500 nm. Analyte retention times were 1.4 and 4.3 min for R123 and internal standard (R6G), respectively. Intra- and inter-day coefficients of variation were < or = 4.2%. Samples were stable for at least three freeze-thaw cycles at room temperature for 24 and 48 h. This method was used to evaluate the functional activity of P-glycoprotein in renal tubule cell models including human kidney (HK-2), Madin-Darby canine kidney (MDCK) and multi-drug resistance gene-transfected MDCK cells (MDR1-MDCK).     

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.     

Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.     

新疆褐家鼠种群生命表及其在干旱区的生存能力分析

从2000年至今,曾于野外捕捉的褐家鼠3 837只,其中雌鼠2 685只,雄鼠1 152只,经鉴定划分为幼年组(Ⅰ)、亚成年组(Ⅱ)、成年Ⅰ组(Ⅲ)、成年Ⅱ组Ⅳ、老年组(Ⅴ)等5个年龄组,其年龄结构探讨种群数量动态,结果表明,种群中Ⅱ龄的平均生命期望值最高1.0357,其次为Ⅲ龄0.9488。该种群中1龄个体占优势地位,种群属于增长性种群,种群内禀增长率rm为0.2789/年,年周限增长率λ为1.3217,世代时间T为2.623年,种群经一世代的净增殖率R0为2.0782。