Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.     

Macamides and their synthetic analogs: Evaluation of in vitro FAAH inhibition

Maca (Lepidium meyenii), a traditional food crop of the Peruvian Andes is now widely touted as a dietary supplement. Among the various chemical constituents isolated from the plant are a unique series of non-polar, long-chain fatty acid N-benzylamides known as macamides. We have synthesized 11 of the 19 reported macamides and have tested each as potential inhibitors of the human enzyme, fatty acid amide hydrolase (FAAH). The five most potent macamides were FAAH inhibitors (IC₅₀ = 10–17 μM). These amides were derivatives of oleic, linoleic and linolenic acids and benzylamine or 3-methoxybenzylamine. Of the three compounds evaluated in a pre-incubation time study, two macamides were not irreversible inhibitors of FAAH. The third, a carbamate structurally related to macamides, was shown to be an irreversible inhibitor of FAAH (IC₅₀ = 0.153 μM).     

Glucocorticoid receptors are involved in the regulation of pulsatile urea excretion in toadfish

The objectives of this study were to characterize the pattern of pulsatile urea excretion in the gulf toadfish in the wake of exogenous cortisol loading and to determine the receptors involved in the regulation of this mechanism. Toadfish were fitted with indwelling arterial catheters and were infused with isosmotic NaCl for 48 h after which fish were treated with cortisol alone, cortisol+peanut oil, cortisol+RU486 (a glucocorticoid receptor antagonist) or cortisol+spironolactone (a mineralocorticoid receptor antagonist). Upon cortisol loading, fish treated with cortisol alone, cortisol+oil or cortisol+spironolactone experienced a two- to threefold reduction in pulsatile urea excretion. This reduction was due to a decrease in urea pulse size with no effect on pulse frequency compared to values measured during the control NaCl infusion period. In addition, these fish showed an increase in plasma urea concentrations upon treatment. These apparent effects of cortisol treatment were abolished in fish treated with cortisol+RU486. In contrast, these fish showed an increase in pulsatile urea excretion mediated by a twofold increase in pulse size with no change in frequency. Likewise, fish treated with cortisol+RU486 showed a significant decrease in plasma urea concentrations over the course of the experiment. The findings of this study indicate that high levels of cortisol reduce pulsatile urea excretion by decreasing pulse size. In addition, it appears that glucocorticoid receptors and not mineralocorticoid receptors are involved in the regulation of the toadfish pulsatile urea excretion mechanism.Communicated by G. Heldmaier     

Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.     

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.     

The impact of the paper of Edsall and Mehl (1940) on actin and actomyosin research

The paper of Edsall and Mehl, ‘The effect of denaturing agents on myosin, II. Viscosity and double refraction of flow’, J. Biol. Chem. 133 (1940) 409–429, inspired our research on actin and actomyosin. It led to the specific purification of actin with magnesium ions and to the demonstration of the central role of the Mg²⁺-activated actomyosin ATPase in contraction of live muscle.     

Ab Initio and DFT Study of the Structures,Vibrations and Energetics of the Urea Dimer and Trimer

Stable structures and electronic properties of small urea clusters are investigated with ab initio calculations. We optimized the cluster geometries and calculated the vibrational frequencies with Hartree–Fock (HF), second-order Møller-Plesset perturbation theory (MP2), and Density Functional Theory (DFT) methods using different basis sets. The most stable dimer was found to consist of two nonplanar urea molecules which are connected by two N–-H...O bonds in a common plane, and the most stable trimer has a flat structure of complex and planar C2 form for each urea molecule, like in the crystal. The interaction energies were corrected for the basis set superposition error (BSSE) using the full Boys*ndash;Bernardi counterpoise correction scheme. The stability of different dimer and trimer structures, the features of formation of H-bonds and presented here are compared to the available experimental data.     

不同种类氮肥对土壤释放N2O的影响

用培养试验模拟研究了在正常水分 ( 2 2 % )、干旱 ( 1 2 % )和高含水量 ( 32 % )条件下 ,普通碳酸氢铵 (普碳 )、尿素及新型肥料长效碳酸氢铵 (长碳 )对土壤释放N2 O的影响 ;同时考察了土壤NO-3 的形成时间和形成量 .结果表明 ,农田中施加的无机氮肥是大气中N2 O的重要来源 ,而长碳与普碳和尿素相比 ,不但可以明显延后N2 O释放高峰期出现时间 ,而且大多数情况下可以显著减少其释放量 (P <0 .0 1 ) .在 5个月的监测期内 ,与普碳和尿素相比其减少N2 O释放的比例分别为 80 .2 3和 88.41 % ( 1 2 %含水量 ) ,40 .0 0和2 7.59% ( 2 2 %含水量 ) ,无减少作用和 45.88% ( 32 %含水量 ) .本研究结果提示长碳具有作为农田生态系统N2 O减排措施的巨大潜力 ,同时暗示在农业中用长碳代替目前普遍应用的普碳 ,可以减少地下水中NO-3 引起的污染 .     

The effects of arginine vasotocin and catecholamines on nitrogen excretion and the cardio-respiratory physiology of the gulf toadfish, Opsanus beta

Simultaneous measurements of cardio-respiratory variables, oxygen uptake and whole body urea/ammonia/tritiated water effluxes were performed on cannulated gulf toadfish, Opsanus beta, before and after intra-arterial injection of the vasoactive agents, adrenaline, isoproterenol and arginine vasotocin. These experiments were conducted to test the hypothesis that the phenomenon of pulsatile urea excretion might reflect sudden changes in the general diffusive properties of the gill for solute transfer. Injection of isoproterenol (final nominal circulating level = 10⁻⁶ mol l⁻¹), was used as a tool to maximise the diffusive and perfusive conditions for branchial solute transfer. This protocol caused a pronounced reduction in arterial blood pressure, an elevation of cardiac frequency and associated increases in whole body urea and tritiated water effluxes; ammonia excretion and oxygen uptake were unaffected. Injection of adrenaline (final nominal circulating level=10⁻⁶ mol l⁻¹), caused a significant increase in arterial blood pressure and a tachycardia, yet nitrogen excretion and oxygen uptake were unaffected. Injection of arginine vasotocin, caused a dose-dependent (final nominal circulating levels = 10⁻¹¹–10⁻⁹ mol l⁻¹) increase in arterial blood pressure without affecting cardiac or ventilation frequency. At the two higher concentrations, arginine vasotocin caused large and transient increases in urea excretion without significantly affecting ammonia, water or oxygen fluxes. These results suggest that increased gill diffusive or perfusive conductance, while capable of augmenting urea efflux, cannot fully explain the sudden and massive increases in urea transfer associated with pulsatile urea excretion in toadfish. It is suggested that pulsatile urea excretion in this species may reflect a specific enhancement of urea excretion under the control of the neurohypophyseal hormone, arginine vasotocin. Accepted: 21 April 1998