Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluidity characteristics of bovine thyroid plasma membranes

Highly purified plasma membranes of bovine thyroid were obtained by differential pelleting followed by discontinuous gradient centrifugation in a swing-out rotor. Subfractions of plasma membranes were prepared by affinity chromatography on Con A-Sepharose. The final membrane fractions were enriched 25-30-fold over homogenate in 5'-nucleotidase and alkaline phosphatase and displayed a protein to phospholipid ratio of 1.67 and a cholesterol to phospholipid molar ratio of 0.55. The phospholipid composition did not deviate appreciably from that of whole tissue except for the higher sphingomyelin level (22.5 vs. 14.0%). The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. The physical state of the membrane was studied by (i) calculation of the lipid structural order parameter SDPH from steady-state fluorescence anisotropy determinations of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH); (ii) estimation of the lateral diffusion coefficient of pyrene following excimer formation. These parameters were determined in native thyroid plasma membranes and in reconstituted vesicles, obtained by detergent dialysis from octylglucoside solubilized membrane components. The presence of membrane protein or neutral lipids induced more restraint on the movements of the fluorophores. The lipid order parameter, SDPH was mainly determined by the neutral lipids. Subfractions of plasma membrane enriched in luminal membranes have a slightly lower fluidity (higher SDPH and lower Ddiff values) than subfractions enriched in basolateral membranes. This difference appears to be due to both differences in lipid as well as protein composition. Under physiological conditions, no significant alterations in probe dynamics could be observed upon addition of thyrotropin or cholera toxin, even at micromolar concentrations.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

毛乌素沙地南缘沙丘生物结皮对凝结水形成和蒸发的影响

在水分极度匮乏的荒漠生态系统,凝结水是除降雨之外最重要的水分来源之一,它对荒漠生态系统结构、功能和过程的维持产生重要的影响。为探明半干旱沙区生物结皮表面的凝结水形成和蒸发特征,采用自制的微型蒸渗计(直径7 cm、高5 cm的PVC管)实验观测了不同类型地表(裸沙、浅灰色藻类结皮、黑褐色藻类结皮和苔藓结皮)对凝结水形成和蒸发的影响。结果表明:(1)观测期间共有20次凝结水形成记录,除降雨天气外,几乎每天都能观测到水分凝结现象;(2)不同类型地表凝结水总量依次为(1.998±0.075),(2.326±0.083),(2.790±0.058)和(3.416±0.068) mm,生物结皮表面的凝结水总量显著大于裸沙(P < 0.05);随生物结皮的发育,不同类型生物结皮表面的凝结水总量呈增加的趋势,凝结水总量之间差异显著(P < 0.05);观测期间不同类型地表日平均凝结水量依次为(0.100±0.003),(0.116±0.004),(0.140±0.002)和(0.171± 0.003) mm,不同类型地表日平均凝结水量之间差异极显著(P < 0.01);(3)凝结水形成过程的观测结果显示,凝结水19:00开始形成,23:00-凌晨1:00形成不明显,1:00-7:00继续形成,除浅灰色藻类结皮外,太阳升出后在黑褐色藻类结皮和苔藓结皮表面继续形成少量的凝结水;凝结水7:30开始蒸发,10:30到11:00之间结束蒸发,凝结水在裸沙和浅灰色藻类结皮中的保持时间显著大于黑褐色藻类结皮和苔藓结皮中的保持时间(P < 0.05);(4)凝结水的形成受大气温度、地表温度、空气相对湿度和大气地表温度差等气象因素的影响,但其形成过程不与某一个气象因素呈简单的线性关系。