Di(isononyl) phthalate (DINP) and di(isodecyl) phthalate (DIDP) are not mutagenic

Recently there have been reports of liver and kidney tumors in rodents following long-term exposure to di(isononyl) phthalate (DINP). Mechanistic studies suggested that the liver tumors were a consequence of peroxisomal proliferation, whereas the kidney tumors (found only in male rats) were associated with induction of alpha(2u)-globulin. Because both peroxisomal proliferation and alpha(2u)-globulin are considered to be non-genotoxic carcinogenic processes, it seemed appropriate to investigate the genotoxic potential of DINP. Additional studies were also conducted on di(isodecyl) phthalate (DIDP), a structurally related substance that also induces peroxisomal proliferation, although it has not been tested in a carcinogenicity bioassay. The DINP was tested in Salmonella, in vitro cytogenetics and mouse micronucleus assays, whereas DIDP was evaluated in a mouse micronucleus test. All of these tests produced negative results, i.e. neither phthalate was mutagenic in any of the test systems. These data are consistent with results of other published and unpublished genotoxicity tests and provide support for the hypothesis that the liver and kidney tumors induced by DINP were the result of non-genotoxic processes.     

Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors

In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.     

Developmental toxicity of di-(C(7)-C(9) alkyl) phthalate and di-(C(9)-C(11) alkyl) phthalate in the rat

Two phthalate esters, di-(C(7)-C(9) alkyl) phthalate (D79P) and di-(C(9)-C(11) alkyl) phthalate (D911P), have been assessed for their potential to cause developmental toxicity in the rat. Groups of 22 timed-mated Sprague-Dawley rats were administered 250, 500, or 1000 mg/kg D79P or D911P daily by oral gavage (5 ml/kg) between gestation days (GD) 1 and 19. Control animals received the vehicle (olive oil) alone. On GD20, the animals were sacrificed and the fetuses examined. Treatment resulted in no signs of maternal toxicity, as assessed by adjusted maternal bodyweight gain throughout gestation and clinical examinations, and no effects upon litter size, fetal survival or bodyweight. Pups of the high dose D79P and intermediate and high dose D911P groups showed increased incidences of supernumerary lumbar ribs. There was a significant increase in dilated renal pelves in pups of the low dose D79P and high dose D911P groups, but only for D911P was there a significant trend. Consequently, the no observed adverse effect level (NOAEL) for maternal toxicity for both D79P and D911P is 1000 mg/kg/day. The NOAEL values for developmental toxicity are 500 mg/kg/day D79P and 250 mg/kg/day D911P.     

Serotonin(7) receptors (5-HT(7)Rs) and their ligands

Serotonin (5-HT) is involved in various physiological and pathological processes by interaction with 14 distinct receptor subtypes, grouped in seven classes of receptors (5-HT(1-7)) on the basis of amino acid sequence, pharmacology, and signal transduction pathways. The 5-HT(7)R is a G-protein coupled receptor with seven transmembrane domains. It was found by the application of molecular cloning and has been identified in rat, mouse, human, pig, and guinea pig. Although the biological functions of the 5-HT(7)Rs are poorly understood, preliminary evidence suggests that it may be involved in depression, control of circadian rhythms, and relaxation of vascular smooth muscles. For these reasons, the 5-HT(7)R has become a target for the development of novel drugs. This review will give a brief introduction of the pharmacology of 5-HT(7)R (molecular structure, distribution of 5-HT(7)R mRNA, localization of the 5-HT(7)R protein, functional correlates, and therapeutic potential) and a detailed survey of the 5-HT(7)R ligands, which have appeared in the literature in both papers and patents. Structure-activity relationships (SAR) of these ligands will also be described.     

4（R）—（6—氨基—9H—嘌呤—9—基）—2（R）—（羟甲基）四氢呋喃—3（R）—醇的合成

为寻找抗HIV化合物,我们以D－核糖为原料,经甲基化、硅烷基化、还原裂解反应制得重要中间体1－脱氧核糖（5）,再通过形成环状亚砜化合物,与NaN3发生反应后,经过还原、缩合、环合、氨化、脱保护基反应制得异脱氧腺嘌呤核苷（1）,各步反应收率均超过70％。其抗HIV活性测定尚在进行中。     

Hydrophilized poly(lactide-co-glycolide) nanospheres with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer

A novel method for preparing the PLGA nanospheres with hydrophilic surface has been designed and characterized. Because of good solubility of tetraglycol in water, PLGA (poly(lactide-co-glycolide)) nanospheres were formed by spraying the PLGA/tetraglycol solution into water. The size of PLGA nanospheres was manipulated by changing the concentration of PLGA/tetraglycol solution. Based on the hydrophobic interaction between PLGA and poly(propylene oxide) domain of F-127 (one of Pluronics, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, F-127-coated PLGA nanospheres was prepared to enhance the stability of PLGA nanospheres in the aqueous media. For the application as a drug delivery vehicle, it was characterized by measuring the loading amount, the encapsulation efficiency and the release pattern of drug. Paclitaxel used as a potent anti-cancer drug was selected as a model drug.     

4（R）—（6—氨基—9—嘌呤基）—2（R）—（羟甲基）四氢呋喃—3（R）—醇的合成

为寻找抗免疫缺陷病毒化合物,以D－核糖为原料,经甲基化、硅烷基化、还原裂解反应制得重要中间体1－脱氧核糖（5）,再通过形成环状亚砜化合物,与NaN3发生反应后,经过还原、缩合、环合、氨化、脱保护基反应制得异脱氧腺嘌呤核苷（1）,各步反应收率均超过70％。     

Mixtures of triblock copolymers E(62)P(39)E(62) and E(137)S(18)E(137) potential for drug delivery from in situ gelling micellar formulations

The gelation behaviour of concentrated micellar solutions of mixtures of a block copolymer of ethylene oxide and styrene oxide (E(137)S(18)E(137)) with one of ethylene oxide and propylene oxide (E(62)P(39)E(62)) has been investigated. Over a wide range of compositions, up to 90 wt.% E(137)S(18)E(137) in the mixture, gelation resembled that of solutions of E(62)P(39)E(62) alone, i.e. they gelled on heating from ambient to body temperature. In related experiments, using the aromatic drug griseofulvin as a comparative standard, it was demonstrated that solubilisation efficiency of dilute micellar solutions of the mixtures with 80 wt.% or more E(137)S(18)E(137) approached that of solutions of E(137)S(18)E(137) alone. Thus it was shown that the mixed system could have both the satisfactory solubilisation capacity of micellar solutions of E(137)S(18)E(137) and the desirable gelation characteristics of E(62)P(39)E(62), and so have potential for use in drug release applications involving in situ gelation.     

Hydrophilized poly(lactide-co-glycolide) nanospheres with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer

A novel method for preparing the PLGA nanospheres with hydrophilic surface has been designed and characterized. Because of good solubility of tetraglycol in water, PLGA (poly(lactide-co-glycolide)) nanospheres were formed by spraying the PLGA/tetraglycol solution into water. The size of PLGA nanospheres was manipulated by changing the concentration of PLGA/tetraglycol solution. Based on the hydrophobic interaction between PLGA and poly(propylene oxide) domain of F-127 (one of Pluronics, poly(ethylene oxide)-poly(propylene oxide)poly(ethylene oxide) triblock copolymer, F-127-coated PLGA nanospheres was prepared to enhance the stability of PLGA nanospheres in the aqueous media. For the application as a drug delivery vehicle, it was characterized by measuring the loading amount, the encapsulation efficiency and the release pattern of drug. Paclitaxel used as a potent anti-cancer drug was selected as a model drug.     

A single amino acid residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.     

Two-generation reproduction toxicity studies of di-(C(7)-C(9) alkyl) phthalate and di-(C(9)-C(11) alkyl) phthalate in the rat

Di-(C(7)-C(9) alkyl) phthalate (D79P) and di-(C(9)-C(11) alkyl) phthalate (D911P), based on high-normality linear oxo-alcohols, have been assessed for their impact upon reproductive performance in Sprague-Dawley rats. Rats were continuously exposed to either D79P or D911P at dietary levels of 0%, 0.1%, 0.5%, or 1.0% over two generations. Selected F(0) offspring (F(1) generation) were exposed to the same dietary concentration of D79P or D911P as the respective F(0) animals, and were mated to produce F(1) offspring. Both D79P and D911P markedly reduced body weight gain in F(0) and F(1) adult males at the highest dose, but females were affected to a lesser extent. There was no impairment of fertility, fecundity, or development in either generation, but body weights of offspring in the 1.0% D79P and 1.0% D911P groups were slightly and transiently reduced over the weaning period. Although decreases in the weight of several organs were accounted for by depressed body weight, ovary weights were reduced in both generations exposed to 1.0% D79P, and epididymidal weights were slightly reduced in adults of both generations exposed to 1.0% D911P. However, ovarian function-assessed by the oestrus cycle and mating behaviour-and epididymidal sperm concentration, motility, and morphology were unaffected by either substance. Treatment resulted in liver changes, particularly in males, characterised by increased liver weight in young animals, histopathologic changes and reduced organ weight in mature animals, and an increase in palmitoyl CoA oxidase activity. In conclusion, neither D79P nor D911P impaired reproductive function in rats when administered in the diet at levels that induce systemic toxicity, and the NOAEL for effects on reproduction in the rat is 0.5% for both D79P and D911P.     

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.     

Mechanisms of anabolic androgenic steroid modulation of alpha(1)beta(3)gamma(2L) GABA(A) receptors

Modulation of GABA(A) receptors induced by both anabolic androgenic steroids (AAS) and the benzodiazepine (BZ) site agonist, zolpidem, show equivalent dependence upon gamma subunit composition suggesting that both compounds may be acting at a shared allosteric site. Here we have characterized modulation induced by the AAS, 17alpha-methyltestosterone (17alpha-MeT), for responses elicited from alpha(1)beta(3)gamma(2L) GABA(A) receptors and compared it to modulation induced by the BZ site agonists, zolpidem and diazepam. For responses elicited by brief pulses of 20 microM GABA, both the AAS and the BZ site compounds significantly increased the peak current amplitudes and total charge transfer, although 17alpha-MeT was an appreciably weaker agonist than either diazepam or zolpidem at alpha(1)beta(3)gamma(2L) receptors. Neither class of modulator enhanced peak current amplitudes for responses elicited by mM concentrations of GABA. BZ site compounds altered time constants of deactivation, desensitization, and recovery from desensitization, however 17alpha-MeT had no overall effect on these parameters. Experiments in which 17alpha-MeT and BZ site ligands were applied concomitantly indicated that potentiation elicited by 17alpha-MeT and zolpidem were additive and that potentiation by 17alpha-MeT could be elicited in the presence of concentrations of flumazenil that blocked BZ potentiation. Finally, kinetic modeling suggests that while effects of 17alpha-MeT can be simulated by altering receptor affinity, the data for these alpha(1)beta(3)gamma(2L) receptors were best fitted by simulations in which 17alpha-MeT increases transitions into the singly liganded open state. Taken together, our results suggest that 17alpha-MeT does not act at the high-affinity BZ site, but may elicit some of its effects at the low affinity BZ site or at a novel site.     

3-(R)-(碱基)-4-(S)-羟基-5-(R)-羟亚甲基四氢呋喃的合成及抗肿瘤活性

目的：研究一系列３（Ｒ）单脱氧异核苷的合成和抗肿瘤活性。方法和结果：由Ｌ木糖出发,合成了环氧化物５（Ｒ）二甲氧甲基３（Ｓ）,４（Ｓ）环氧四氢呋喃４;在碱性条件下,利用嘌呤的Ｎ９位或嘧啶的Ｎ１位对环氧化物进行亲核进攻,得到一系列３（Ｒ）单脱氧异核苷５ａｄ和６ａｄ;并进行了体外抗肿瘤活性筛选。结论：其中３（Ｒ）单脱氧异核苷５ａｄ为首次报道;同已报道的３（Ｓ）单脱氧异核苷合成方法相比,路线缩短,收率提高。在体外抗肿瘤和端粒酶抑制活性筛选中,只有化合物６ａ显示了对ＢＩＵ细胞较弱的抑制活性,其余均未显示有意义的抗肿瘤活性和端粒酶抑制活性。     

Comparison of the radiorecovery activity of copper(II)2(3,5-diisopropylsalicylate)4 and copper(II) (chloride)2

Copper(II)₂(3,5-diisopropylsalicylate)₄ [Cu(II)₂(3,5-DIPS)₄] and copper(II)(chloride)₂ [Cu(II)Cl₂ were used to treat γ-irradiated female C57BL/6 mice after irradiation at levels LD50/30 to compare their efficacy in facilitating recovery from radiation-induced systemic inflammatory disease accompanied by loss of body mass and in increasing survival of irradiated mice. Doses of 5, 10 or 20 μmol Cu(II)Cl₂ or 5, 10 or 20 μmol [Cu(II)₂(3,5-DIPS)₄]/kg were administered subcutaneously 3 h after LD_50/30 irradiation and body mass and survival determined throughout the 30-day post-irradiation period compared with controls. Treatment with Cu(II)₂(3,5-DIPS)₄ or Cu(II)Cl₂ facilitated recovery of radiation-induced systemic inflammatory disease, recovery of body mass, and increased survival. Treatment with 5, 10 or 20 (μmol [Cu(II)₂(3,5-DIPS)₄]/kg produced a 44%, 67% or 44% increase in survival, respectively, compared with the vehicle-treated control group. Treatment with 5,10 or 20 μmol Cu(II)Cl₂/kg produced a 7%, 21% or 29% increase in survival, respectively, compared with the vehicle-treated control group. The recovery of radiation-induced loss in body mass and an increase in survival document that both Cu(II)₂(3,5-DIPS)₄ and Cu(II)Cl₂ are effective radiorecovery agents. In addition, Cu(II)₂(3,5-DIPS)₄ is a more effective radiorecovery agent than Cu(II)Cl₂.     

Synthesis and antiviral activity of (E)-5-(2-bromovinyl)uracil and (E)-5-(2-bromovinyl)uridine

(E)-5-(2-Bromovinyl)uracil (BVU) and (E)-5-(2-bromovinyl)uridine (BVRU) were synthesized starting from 5-formyluracil via (E)-5-(2-carboxyvinyl)uracil or starting from 5-iodouridine via (E)-5-(2-carbomethoxyvinyl)uridine and (E)-5-(2-carboxyvinyl)uridine, respectively. Depending on the choice of the cell system, BVU and BVRU exhibited a marked activity against herpes simplex virus type 1 (HSV-1) in vitro. Although BVU and BVRU were less potent than the reference compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), their antiviral activity spectrum was remarkably similar to that of BVDU. The latter findings suggest that BVU and BVRU are metabolically converted to BVDU or a phosphorylated product thereof. In vivo, BVU protected mice against a lethal disseminated HSV-1 infection.     

2-取代胺甲基-5(6)-(E)-取代亚甲基环戊(己)酮盐酸盐类化合物的设计、合成及其抗肿瘤活性

目的设计合成20个2,5(6)-双取代环戊(己)酮类化合物,进行抗肿瘤活性研究。方法以WB852为先导化合物,设计合成了20个2-取代胺甲基-5(6)-(E)-取代亚甲基环戊(己)酮盐酸盐类化合物。利用MTT法对其中17个化合物进行了体外细胞毒活性筛选,所用肿瘤细胞株为人乳腺癌细胞T47D、MCF-7、MCF-7/Adr;通过Habig的酶动力学方法,测试了部分目标化合物细胞外对GSTπ活性的影响。结果与结论合成了20个2-取代胺甲基-5(6)-(E)-取代亚甲基环戊(己)酮盐酸盐类化合物,其中16个为未见文献报道的新化合物,其结构均经1H-NMR、MS和IR确证。体外抗肿瘤活性筛选结果,17个化合物对3种肿瘤细胞均有不同程度的生长抑制活性,A-16、A-17、A-18、A-19等4个化合物活性显著,值得进行深入研究。9个化合物均有不同程度地抑制GSTπ的活性,其中A-4、8、9、11和15等5个化合物对GSTπ的抑制作用强于WB852。取代胺甲基部分、取代亚甲基侧链的改变以及环的大小对抗肿瘤活性和选择性影响不大,但显著影响对GSTπ的抑制作用。A-16、A-17、A-18、A-19对MCF-7/Adr的生长抑制作用与WB852相当,但均低于对MCF-7细胞的活性,对耐药细胞的活性与GSTπ抑制无关。     

Studies on gamma-glutamyl transpeptidase (GGT) after di(2-ethylhexyl) phthalate (DEHP) exposure

Administration of Di(2-ethylhexyl)phthalate (DEHP) at doses of 250, 500, 1000 and 2000 mg/kg to adult rats was found to significantly increase the activity of gamma-glutamyl transpeptidase (GGT) in liver and serum in a dose-dependent manner. The data indicate that DEHP can be hepatotoxic since an increase in serum GGT levels are indicative of hepatobiliary dysfunction and liver malignancy. An assay of GGT in serum of the individuals exposed to DEHP could be helpful in early detection of liver disorders due to this widely used plasticizer.     

N-(Phosphonoacetyl)amino phosphonates. Phosphonate analogues of N-(phosphonoacetyl)-L-aspartic acid (PALA)

Michaelis-Arbuzov reaction of N-(chloroacetyl)amino phosphonic acids or their esters, followed by acidolysis, gives moderate yields of N-(phosphonoacetyl) derivatives of a variety of (aminoalkyl)phosphonic acids, including analogues of the cytostatic agent PALA, in which the alpha- or beta-carboxylic groups in the aspartate moiety are replaced by a PO3H2 function. Assay of cytostatic activity with human KB cell lines indicates that the substitution of any of the COOH groups in PALA with PO3H2 results in total loss of cytostatic activity. No activity was observed also in the case of other [N-(phosphonoacetyl)amino]alkylphosphonic acids described in this report.     

Molecular analysis of beta(2)-adrenoceptor coupling to G(s)-, G(i)-, and G(q)-proteins

The beta(2)-adrenoceptor (beta(2)AR) couples to the G-protein G(s) to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the beta(2)AR can also interact with G-proteins of the G(i)- and G(q)-family. To assess the efficiency of beta(2)AR interaction with various G-protein alpha-subunits (G(xalpha)), we expressed fusion proteins of the beta(2)AR with the long (G(salphaL)) and short (G(salphaS)) splice variants of G(salpha), the G(i)-proteins G(ialpha2) and G(ialpha3), and the G(q)-proteins G(qalpha) and G(16alpha) in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G(xalpha) because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the beta(2)AR couples to G(s)-, G(i)-, and G(q)-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding. The combined analysis of ternary complex formation, GTPgammaS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the beta(2)AR with the various classes of G-proteins. Comparison of the coupling of the beta(2)AR and formyl peptide receptor to G(ialpha2) revealed receptor-specific differences in the kinetics of GTPgammaS binding. We also detected highly efficient stimulation of GTPgammaS dissociation from G(salphaL), but not from G(qalpha) and G(16alpha), by a beta(2)AR agonist. Moreover, we show that the 1:1 stoichiometry of receptor to G-protein in fusion proteins reflects the in vivo stoichiometry of receptor/G-protein coupling more closely than was previously assumed. Collectively, our data show 1) that the beta(2)AR couples differentially to G(s)-, G(i)-, and G(q)-proteins, 2) that there is ligand-specific coupling of the beta(2)AR to G-proteins, 3) that receptor-specific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation.