Identification of Ras farnesyltransferase inhibitors by microbial screening.

A microbial screen using a yeast strain with conditional deficiency in the GPA1 gene was carried out to search for inhibitors of protein farnesyltransferase (PFT). A strain of Streptomyces was found to produce active compounds named UCF1-A, UCF1-B, and UCF1-C. Structural determination of these compounds revealed that UCF1-C is identical to the known antibiotic, manumycin, whereas UCF1-A and UCF1-B are structurally related to manumycin. All three UCF1 compounds suppress the lethality of gpa1 disruption, with UCF1-C exhibiting the strongest activity. UCF1 inhibits yeast as well as rat brain PFT. Fifty percent inhibition of yeast PFT activity is observed with 5 microM UCF1-C. Kinetic analyses of the inhibition suggest that UCF1-C acts as a competitive inhibitor of PFT with respect to farnesyl pyrophosphate, exhibiting a Ki of 1.2 microM, whereas the same compound appears to act as a noncompetitive inhibitor of PFT with respect to the farnesyl acceptor, the Ras protein. UCF1-C shows significant activity to inhibit the growth of Ki-ras-transformed fibrosarcoma, raising the possibility of its use as an antitumor drug.     

A missense mutation in Caenorhabditis elegans prohibitin 2 confers an atypical multidrug resistance

Hemiasterlin is a potent antimitotic peptide that interferes with microtubule dynamics at picomolar concentrations in cell culture. The molecule largely eludes P glycoprotein-mediated drug efflux, and an analog is currently being evaluated in clinical trials as cancer chemotherapy. From a nonclonal genetic screen in Caenorhabditis elegans we isolated eight independent mutants resistant to a synthetic hemiasterlin analog. In one recessive mutant, phb-2(ad2154), a point mutation in prohibitin 2 (E130K) protects worms from drug-induced injury. Data indicate that direct binding of hemiasterlin to prohibitin 2 is unlikely. In fact, C. elegans phb-2(ad2154) was also found to be resistant to numerous other drugs that bind tubulin and to camptothecin, yet this mutant was sensitive to nocodazole and phalloidin. Thus, prohibitin 2 is implicated in a previously uncharacterized pathway of multidrug resistance.     

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABA_AR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.

Innate immunity, an evolutionally conserved behavior, constitutes the first defense line of multiple organisms to prevent microbial infections (1). The nematode Caenorhabditis elegans has been used as a model host for human opportunistic pathogen Pseudomonas aeruginosa infection (2) to identify evolutionarily conserved mechanisms of innate immunity. Typically, p38/PMK-1 mitogen-activated protein kinases (MAPKs) (3) and insulin/insulin-like signaling (IIS)/DAF-2 signaling cascades are recognized as two key components of the C. elegans intestinal innate immune response upon P. aeruginosa strain PA14 infection (4), as they are in mammals (3, 4). Moreover, increasing evidence has revealed several neural mechanisms as also being involved in the regulation of innate immunity. For example, G protein–coupled receptor (GPCR) NPR-1– and soluble guanylate cyclase GCY-35–expressing sensory neurons actively suppress the immune response of nonneuronal tissues (5). Additionally, a putative octopamine GPCR, OCTR-1, which is expressed and functions in the C. elegans sensory neurons ASH and ASI (6), down-regulates the unfolded protein response genes pqn/abu to further suppress the immune response of nonneuronal tissues (5, 6).Recent studies demonstrate that dopaminergic signaling inhibits innate immunity (7) whereas neuronal acetylcholine stimulates muscarinic signaling in the epithelium and activates the epithelial canonical Wnt pathway to promote the ability to defend against bacterial infection (8). Moreover, insulin-like peptide INS-7 secreted by the nervous system functions in a cell-nonautonomous manner to activate the IIS/DAF-2 pathway and modulate the intestinal innate immunity of C. elegans (9).GABAergic signaling constitutes a major inhibitory neurotransmission system that plays crucial roles in the central nervous system, especially for maintaining the balance between excitation and inhibition of neuronal networks (10). Disruption of this balance is not only linked to several neuropsychiatric disorders including schizophrenia, autism, and epilepsy (11) but also implicated in autoimmune disease (12). Up to date, multiple lines of evidence have shown that GABAergic signaling cell-autonomously modulates the immune response in immune cells (13–15). However, the roles of GABAergic synapses in innate immunity remain unknown.Here, we found that the nematode C. elegans harboring a deficiency in GABAergic neuromuscular junctions (NMJs) exhibits enhanced resistance to pathogens. P. aeruginosa PA14 infection increases synaptic expression of GABAergic synaptic components at the nerve cord of worms and enhances the strength of GABAergic transmission. Moreover, we identified an insulin-like peptide, INS-31, acting downstream of GABAergic NMJs and in body wall muscle (BWM) to control intestinal innate immunity in a cell-nonautonomous manner. This work reveals a signaling axis of synapse–muscular insulin–intestinal innate immunity in vivo.     

EBV相关胃癌ras基因突变及法尼基转移酶β-亚单位基因的表达

目的:探讨EBV相关胃癌(EBVaGC)、EBV阴性胃癌(EBVnGC)及相应癌旁组织中ras基因突变和法尼基转移酶(FTase)的表达意义.方法:应用PCR-RFLP技术检测EBVaGCs13例、EBVnGCs45例以及相应癌旁组织58例中H-ras12和K-ras12,13密码子点突变情况;RT-PCR技术检测FTaseβ亚单位mRNA的表达水平.结果:共检测到H-ras12位点突变2例,突变率为3.45%(2/58),均发生在EBVnGCs,未发现K-ras12和13密码子点突变.58例癌组织中FTaseβ亚单位mRNA表达水平为0.93±0.39,癌旁组织中FTaseβ亚单位mRNA表达水平为0.78±0.26,两者有显著性差异(t=2.44,P=0.02).EBVaGCs组织中FTaseβ亚单位mRNA表达水平为0.80±0.19,EBVnGCs组织中FTaseβ亚单位mRNA表达水平为0.96±0.43,两者无显著性差异(t=1.93,P=0.06);EBVaGCs和癌旁组织中FTaseβ亚单位mRNA表达水平无显著性差异(t=0.54,P=0.60);EBVnGCs和癌旁组织中FTaseβ亚单位mRNA表达水平有显著性差异(t=2.39,P=0.02);2例BHRF1阳性和11例BHRF1阴性EBVaGCs组织中FTaseβ亚单位mRNA表达水平无显著性差异(t=0.26,P=0.80);6例BARF1阳性和7例BARF1阴性EBVaGCs组织中FTaseβ亚单位mRNA表达水平亦无显著性差异(t=1.59,P=0.14).结论:胃癌组织ras基因突变率较低,胃癌组织中FTaseβ亚单位mRNA明显高于癌旁组织.EBVaGC组织中ras基因突变、FTaseβ亚单位表达与EBV感染无明显相关性.     

A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans

The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.     

A mutation in mitochondrial complex I increases ethanol sensitivity in Caenorhabditis elegans

BACKGROUND: The gene gas-1 encodes the 49-kDa subunit of complex I of the mitochondrial electron transport chain in Caenorhabditis elegans. A mutation in gas-1 profoundly increases sensitivity to ethanol and decreases complex I-dependent metabolism in mitochondria. METHODS: Mitochondria were isolated from wild-type and gas-1 strains of C. elegans. The effects of ethanol on complex I-, II-, and III-dependent oxidative phosphorylation were measured for mitochondria from each strain. Reversibility of the effects of ethanol was determined by measuring oxidative phosphorylation after removal of mitochondria from 1.5 M ethanol. The effects of ethanol on mitochondrial structure were visualized with electron microscopy. RESULTS: We found that ethanol inhibited complex I-, II-, and III-dependent oxidative phosphorylation in isolated wild-type mitochondria at concentrations that immobilize intact worms. It is important to note that the inhibitory effects of ethanol on mitochondria from either C. elegans or rat skeletal muscle were reversible even at molar concentrations. Complex I activity was lower in mitochondria from gas-1 animals than in mitochondria from wild-type animals at equal ethanol concentrations. Complex II activity was higher in gas-1 than in wild-type mitochondria at all concentrations of ethanol. No difference was seen between the strains in the sensitivity of complex III to ethanol. CONCLUSIONS: The difference in ethanol sensitivities between gas-1 and wild-type nematodes results solely from altered complex I function. At the respective concentrations of ethanol that immobilize whole animals, mitochondria from each strain of worms displayed identical rates of complex I-dependent state 3 respiration. We conclude that a threshold value of complex I activity controls the transition from mobility to immobility of C. elegans.     

Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library of normal human fibroblasts. Each revertant harbors 1-10 recombinant plasmids per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-Ki-ras. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-ras-transformed NIH 3T3 cells. From two of the revertant lines, plasmids capable of giving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.     

Genetic analysis of life-span in Caenorhabditis elegans.

Crosses between Bristol and Bergerac strains of the self-fertilizing hermaphroditic nematode Caenorhabditis elegans do not show the heterosis effects for life-span that complicate analysis of interstrain crosses with Drosophila or mice. Instead they yield F1 progeny with life-spans similar to those of the parent strains. By analysis of life-span variation among progeny F2 populations from such crosses and by two independent analyses of life-spans among recombinant inbred lines derived from F2 individuals by 18 rounds of self-fertilization, we estimate that the heritability of life-span in C. elegans is between 20% and 50%. Recombinant inbred lines show a range in mean life-spans of 10 days to 31 days compared to life-spans of about 18 days for each of the two parental strains. We conclude that life-span variation in C. elegans has a substantial genetic component and that this organism offers promising opportunities for selective breeding of longer-lived strains and genetic analysis of senescence.     

The Tc2 transposon in Caenorhabditis elegans.

A second family of transposons, named Tc2 elements, has been identified in the nematode Caenorhabditis elegans. Tc2 elements are polymorphic in sequence and are present in different numbers in different strains. Like the transposon Tc1, Tc2 is active in the germ line of some C. elegans strains. A high rate of transposition has been observed in the progeny of certain interstrain crosses, where transposition events are frequent enough to be detected in blot hybridization experiments, without the use of a genetic screen. Our data suggest that transposition of Tc1 and Tc2 may be regulated by the same genomic factors.     

Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.

When grown at a temperature from 16 degrees to 25 degrees and placed on a thermal gradient, the nematode Caenorhabditis elegans migrates to its growth temperature and then moves isothermally. Behavioral adaptation to a new temperature takes several hours. Starved animals, in contrast, disperse from the growth temperature. Several mutants selected for chemotaxis defects have thermotaxis defects as well; these behaviors depend on some common gene products. New mutants selected directly for thermotaxis defects have unusual phenotypes which suggest mechanisms for thermotaxis.     

Transposition of Tc1 in the nematode Caenorhabditis elegans.

We have identified a strain of Caenorhabditis elegans in which the transposable element Tc1 is genetically active. Most spontaneous mutations affecting the unc-54 myosin heavy chain gene of C. elegans variety Bergerac are due to insertions of Tc1 within unc-54. The Bergerac genome contains an unusually high number of Tc1 elements, but this is not responsible for transpositional activity. Another variety of C. elegans, strain DH424, contains an equally high number of Tc1 elements, but transpositions are not detected. Tc1 insertion mutations are genetically unstable. They revert to unc-54+ in both germ-line and somatic cells. Germ-line revertants are wild type and contain precise or nearly precise excisions of Tc1. Somatic revertants are genetic mosaics; they contain small patches of revertant muscle tissue in otherwise mutant animals. The pattern of mosaicism often allows us to know when and where during muscle development the excisions occur. Somatic reversion can be over 1000-fold more frequent than germ-line reversion.     

Dietary restriction in the nematode Caenorhabditis elegans

The first observation of the positive effect of reduced food intake on mammalian life span was made 70 years ago. In the decades that followed, researchers successfully applied this method to increase the life span of a very wide range of animals. The nematode Caenorhabditis elegans is an excellent model organism for studying the aging process. However, relatively little effort has been made to study the effects of dietary restriction in C. elegans. In this review we discuss the difficulties of subjecting C. elegans to dietary restriction, the effects of dietary restriction on metabolism and stress defense, and the potential role of different signaling pathways in DR-induced life extension. Recent experiments suggest that the TOR (target of rapamycin) pathway, rather than insulin-like signaling, might be involved in mediating the life-extending effect of dietary restriction.     

Metabolism in the Caenorhabditis elegans Mit mutants

In many eukaryotes oxidative phosphorylation via the mitochondrial electron transport chain provides the major means of ATP production. Complete removal of this capacity often results in premature death. Recent studies using the nematode Caenorhabditis elegans are surprising because they have revealed that disruption of many of the key components of the normal mitochondrial energy-generating machinery do not result in death, rather they result in adult life span extension. Such mutants have been collectively termed Mit mutants. In this short review, the potential use of alternate metabolic pathways for energy generation by Mit mutants will be considered. The effects of using such pathways on residual mitochondrial functionality, reactive radical species production, and longevity will also be explored.     

Analysis of a transposable element in Caenorhabditis elegans.

A transposable element, designated Tc1, has been characterized in Caenorhabditis elegans. Tc1 is 1.7 kilobases long, has an inverted terminal repeat of less than 100 base pairs, and is repeated as a highly conserved element. The copy number and genomic positions of Tc1 are extremely variable among strains, implying that Tc1 is mobile. However, progeny of interstrain crosses did not show hybrid dysgenic traits that might be due to Tc1 transposition.     

Genetic interactions affecting touch sensitivity in Caenorhabditis elegans.

At least 13 genes (mec-1, mec-2, mec-4-10, mec-12, mec-14, mec-15, and mec-18) are needed for the response to gentle touch by 6 touch receptor neurons in the nematode Caenorhabditis elegans. Several, otherwise recessive alleles of some of these genes act as dominant enhancer mutations of temperature-sensitive alleles of mec-4, mec-5, mec-6, mec-12, and mec-15. Screens for additional dominant enhancers of mec-4 and mec-5 yielded mutations in previously known genes. In addition, some mec-7 alleles showed allele-specific, dominant suppression of the mec-15 touch-insensitive (Mec) phenotype. The dominant enhancement and suppression exhibited by these mutations suggest that the products of several touch genes interact. These results are consistent with a model, supported by the known sequences of these genes, that almost all of the touch function genes contribute to the mechanosensory apparatus.     

Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice.

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its transforming activity. Cell-active inhibitors of the enzyme that catalyzes this reaction, protein farnesyltransferase, have been shown to selectively block ras-dependent transformation of cells in culture. Here we describe the protein farnesyltransferase inhibitor 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl] pentyloxy-3-phenylpropionylmethioninesulfone methyl ester (L-739,749), which suppressed the anchorage-independent growth of Rat1 cells transformed with viral H-ras and the human pancreatic adenocarcinoma cell line PSN-1, which harbors altered K-ras, myc, and p53 genes. This compound also suppressed the growth of tumors arising from ras-transformed Rat1 cells in nude mice by 66%. Under the same conditions, doxorubicin inhibited tumor growth by 33%. Control tumors formed by v-raf- or v-mos-transformed Rat1 cells were unaffected by L-739,749. Furthermore, mice treated with L-739,749 exhibited no evidence of systemic toxicity. This is a demonstration of antitumor activity in vivo using a synthetic small molecule inhibitor of protein farnesyltransferase.     

Characterization of a life-extending mutation in age-2, a new aging gene in Caenorhabditis elegans

We have generated a life-extending mutation, yw23, in Caenorhabditis elegans. The mutation is in what appears to be a new aging gene, which we have designated age-2. When homozygous, yw23 produces an increase of mean and maximum life span of about 20% over that of the wild-type strain, N2. Strain HG23 [age-2(yw23)] was obtained by screening for longer life spans among 430 lines of nematodes two generations after exposure to the mutagen ethylmethanesulfonate. Strain HG231 [age-2(yw23)] was obtained after a single out-crossing of HG23 to N2. When compared with N2, HG231 exhibits normal motility, slightly higher swimming rates, reduced fertility (especially at higher temperatures), somewhat longer development times, and a slightly larger size at the time of first egg laying. A Gompertz analysis suggests that HG231 extends life span by reducing the initial mortality rate. In genetic crosses, yw23 complements other known aging mutants in C. elegans genes-age-1, daf-2, spe-26, clk-1, clk-2, clk-3, and gro-1. A double-mutant strain, HG284, combining mutations in age-1 and age-2, lives longer than animals with individual mutations in either age-1 or age-2, and exhibits a longer life span at 25 degrees C than at 20 degrees C.     

A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans.

After light touch to its nose, the nematode Caenorhabditis elegans halts forward locomotion and initiates backing. Here we show that three classes of neurons (ASH, FLP, and OLQ) sense touch to the nose and hence are required for this avoidance response. ASH, FLP, and OLQ have sensory endings that contain axonemal cilia. Mutant animals that have defective ciliated sensory endings as well as laser-operated animals that lack ASH, FLP, and OLQ fail to respond to touch to the nose. Together with the previous work of others, these results demonstrate that C. elegans has at least five morphologically distinct classes of mechanosensory neurons. Interestingly, the ASH neuron also acts as a chemosensory neuron; it mediates the avoidance of noxious chemicals. Since ASH possesses both chemosensory and mechanosensory modalities, this neuron might be functionally analogous to vertebrate nociceptors, which mediate the sensation of pain.