Geles KG et al. (2001) Development "Germline and developmental roles of the nuclear transport factor importin alpha3 in C. elegans."

Geles KG, Adam SA

[Development, 2001]

The importin alpha family of transport factors mediates the nuclear import of classical nuclear localization signal-containing proteins. In order to understand how multiple importin alpha proteins are regulated both in individual cells and in a whole organism, the three importin alpha (ima) genes of Caenorhabditis elegans have been identified and studied. All three IMAs are expressed in the germline; however, only IMA-3 is expressed in the soma. RNA interference (RNAi) experiments demonstrate that IMA-3 is required for the progression of meiotic prophase I during oocyte development. Loss of IMA-3 expression leads also to a disruption of the nuclear pore complex accompanied by the mis-localization of P granules. A range of defects occurring in ima3(RNAi) F-1 progeny further supports a role for IMA-3 during embryonic and larval development. The functional association of IMA-3,vith distinct cellular events, its expression pattern and intracellular localization indicate that regulation of the nuclear transport machinery is involved in the control of developmental pathways.

Geles KG et al. (2002) Mol Biol Cell "A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis."

Johnson JJ, Geles KG, Adam SA, Jong S

[Mol Biol Cell, 2002]

The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype, ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope

Geles KG et al. (1998) Midwest Worm Meeting "DEVELOPMENTAL EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF THE NUCLEAR LOCALIZATION SEQUENCE RECEPTOR, IMPORTIN ALPHA FROM C. elegans."

Geles KG, Adam SA

[Midwest Worm Meeting, 1998]

The transport of proteins into the nucleus occurs at the nuclear pore complex and requires cytosolic factors: a heterodimeric nuclear localization sequence (NLS) receptor complex known as Importin alpha/beta and the small nuclear Ras-like GTPase, Ran/TC4. Members of the Importin alpha family bind classical-type NLSs of nuclear proteins. Importin beta mediates the association of Importin alpha with the nuclear pore. Ran mediates the interaction of Importin beta with Importin alpha and the nuclear pore in a nucleotide-specific manner. Five Importin alpha homologs have been identified in mammalian cells, two have been identified in X. laevis and one in D. melanogaster. Sequence analysis of the S. cerevisiae genome identifies a single Importin alpha homolog. To determine the functional significance and developmental regulation of multiple Importin alpha homologs, the nematode C. elegans was chosen as a model system to study. We have cloned and characterized three putative Importin alpha (ima) homologs from C. elegans and have termed them ima-1, ima-2 and ima-3. All Importin alpha proteins, including those from C. elegans, contain three distinguishing sequence characteristics: a central domain composed of eight degenerate armadillo repeats, an Importin beta binding domain (IBB) and a carboxyl-terminal phenylalanine residue. The C. elegans Importin alpha proteins cannot be categorized into the two defined groups of vertebrate Importin alpha homologs, the Rch1-subtype and the hSRP1-subtype. All three are equally homologous to the Rch-1 and hSRP1 subtypes. Despite their sequence divergence, each C. elegans Importin alpha can interact with human Importin beta in defined biochemical assays. Treatment of Importin beta with the sulfhydryl-alkylating reagent N-ethylmaleimide abolishes Importin alpha binding. The association of Ran-GTP with Importin beta dissociates the Importin alpha/beta heterodimer. Direct fluorescence in situ hybridization of antisense cRNA to whole worms indicates that ima-1 and ima-2 are developmentally and tissue-specifically expressed. Northern blot analysis confirms the developmental expression pattern and reveals the predominance of ima-1 during development. Each C. elegans Importin alpha gene encodes a protein of approximately 523 amino acids. Northern blot analysis indicates that each gene is expressed as a distinct mRNA transcript. Characterization of the multiple C. elegans Importin alpha proteins will address many aspects of their function and further define a role for nuclear transport in development.

KG Geles et al. (1999) International C. elegans Meeting "Two Distinct Importin a's, IMA1 and IMA2, Differentially Regulate Germ Line Cell Fates"

KG Geles, SA Adam

[International C. elegans Meeting, 1999]

Metazoans express multiple nuclear localization signal (NLS) receptors, or Importin a 's, some within the same cell type. The ability of distinct Importin a 's to regulate the nuclear localization of cell fate determinants during development has not been elucidated. In order to identify specific functions for different Importin a homologs during development and cell fate specification, C. elegans was chosen as a model system. Nuclear protein import requires a heterodimer of the classical Importins, a and b . Members of the Importin a family bind classical-type NLSs, while Importin b mediates their association with the nuclear pore. Sequence data from the C. elegans genome project has identified three Importin a (IMA) proteins which we have termed IMA-1, -2 and -3. Based on phylogenetic analysis, IMA-2 and -3 are novel members of the Importin a family. IMA-1 is the C. elegans homolog of the human Qip1 protein. As evidenced by northern blot analysis and immunofluorescence, IMA-1 is expressed in multiple cell types including the germ line, while IMA-2 is exclusive to the adult germ line. IMA-1 and -2 are expressed in all embyronic cells until L1 and have a dynamic subnuclear localization during the cell cycle. IMA-1 and -2 associate with the Nuclear Pore Complex (NPC) during G1 as evidenced by co-localization with the anti-NPC antibody, MAb414, and are found within the nucleus during mitosis. RNAi was used to investigate the role of a specific IMA during development. Surprisingly, distinct germ line defects were observed in the injected animals. RNAi of ima-1 results in P o germ lines that lack nuclei in pachytene and diakinesis of meiosis I, but instead contain more condensed nuclei from the distal to proximal region. Interestingly, germ lines from ima-2 RNAi'ed adults contain an abnormal mitotic region containing meiotic-like nuclei. Based on tissue expression and nuclear localization, these results suggest distinct roles for two Importin a 's during germ line cell fate specification. We hypothesize that IMA-1 and IMA-2 transport a specific subset of nuclear proteins within the syncytium of the adult germ line.

Xu W et al. (2011) Cancer Cell "Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases."

Wang P, Liu J, Xu YH, Yang H, Xu W, Liu LX, Zhao SM, Wang B, Kim SH, Xiao MT, Xiong Y, Zhang Y, Ito S, Frye S, Jiang WQ, Lei QY, Zhang JY, Guan KL, Liu Y, Yang C, Yang Y

[Cancer Cell, 2011]

IDH1 and IDH2 mutations occur frequently in gliomas and acute myeloid leukemia, leading to simultaneous loss and gain of activities in the production of alpha-ketoglutarate (alpha-KG) and 2-hydroxyglutarate (2-HG), respectively. Here we demonstrate that 2-HG is a competitive inhibitor of multiple alpha-KG-dependent dioxygenases, including histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases. 2-HG occupies the same space as alpha-KG does in the active site of histone demethylases. Ectopic expression of tumor-derived IDH1 and IDH2 mutants inhibits histone demethylation and 5mC hydroxylation. In glioma, IDH1 mutations are associated with increased histone methylation and decreased 5-hydroxylmethylcytosine (5hmC). Hence, tumor-derived IDH1 and IDH2 mutations reduce alpha-KG and accumulate an alpha-KG antagonist, 2-HG, leading to genome-wide histone and DNA methylation alterations.

Abuzar S et al. (1986) J Med Chem "Studies in potential filaricides. 18. Synthesis of 2,2'-disubstituted 5,5'-dibenzimidazolyl ketones and related compounds as potential anthelmintics."

Gupta S, Fatma N, Sen AB, Sharma S, Abuzar S, Katiyar JC, Murthy PK, Chatterjee RK

[J Med Chem, 1986]

A series of 2,2'-disubstituted 5,5'-dibenzimidazolyl ketones and related compounds have been synthesized of which 2,2'-bis(carbomethoxyamino)-5,5'-dibenzimidazolyl ketone exhibited a broad spectrum of anthelmintic activity in experimental animals. At doses of 10-50 mg/kg given intraperitoneally, 5 killed 100% of the adult worms of Litomosoides carinii, Dipetalonema viteae, and Brugia malayi. By the oral route the macrofilaricidal efficacy of 5 was 97-100% at 100-200 mg/kg X 5 days. The treated animals showed gradual disappearance of microfilariae and before autopsy they became amicrofilariaemic. Some of the compounds also showed 100% efficacy against the human hookworms and tapeworm, Ancylostoma ceylanicum in hamsters, and Hymenolepis nana in rats at a single oral dose of 50-250 mg/kg. Compound 5 was also effective against Syphacia obvelata in mice at a single oral dose of 100 mg/kg and was found to be well tolerated by mice up to an oral dose of 2500 mg/kg.

Zahner H et al. (2001) Int J Parasitol "Filaricidal efficacy of anthelmintically active cyclodepsipeptides."

Harder A, Taubert A, Zahner H, von Samson-Himmelstjerna G

[Int J Parasitol, 2001]

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.

Awadzi K et al. Trans R Soc Trop Med Hyg "The effects of high-dose ivermectin regimens on Onchocerca volvulus in onchocerciasis patients."

Opoku NO, Awadzi K, Attah SK, Quartey BT, Addy ET

[Trans R Soc Trop Med Hyg]

Ivermectin, at the standard dose of 150 micrograms/kg bodyweight, does not kill the adult worms of Onchocerca volvulus and does not disrupt embryogenesis or spermatogenesis. Repeated standard doses, if maintained, arrest microfilarial production but result in only a mild-to-modest macrofilaricidal effect. We investigated whether high doses would effectively kill the adult worms, and whether cessation of microfilarial production could be reproduced by an equivalent, single, high dose. One hundred men participated in a double-blind placebo-controlled trial and received increasing doses of ivermectin from 150 micrograms/kg to 1600 micrograms/kg bodyweight. Nodules were excised at day 180 and examined by histopathology. Total doses of ivermectin up to 1600 micrograms/kg were not significantly more effective than 150 micrograms/kg. Moreover, they did not reproduce the marked inhibitory effects of the repeat standard-dose regimens on embryogenesis, nor the modest effect on adult worm viability, at comparable total doses. These effects may be functions of multiplicities of dosages rather than of the total dose. Our findings also suggest that repeated high-dose regimens are unlikely to be more effective than a similar number of 150 micrograms/kg doses. This deficiency of ivermectin requires that the search for macrofilaricides remains a top priority.

Brown KR et al. (2000) Parasitology "Ivermectin: effectiveness in lymphatic filariasis."

Ottesen EA, Brown KR, Ricci FM

[Parasitology, 2000]

This detailed review of the published studies underlying ivermectin's recent registration for use in lymphatic filariasis (LF) demonstrates the drug's single-dose efficacy (over the range of 20-400 microg/kg) in clearing microfilaraemia associated with both Wuchereria bancrofti and Brugia malayi infections of humans. While doses as low as 20 microg/kg could effect transient microfilarial (mf) clearance, higher dosages induced greater and more sustained mf reduction. The single dose of 400 microg/kg yielded maximal responses, but a number of practical considerations suggest that either 400 microg/kg or 200 microg/kg doses would be acceptable for use in LF control programmes. Associated safety assessments indicate that adverse events, which occur commonly following treatment of microfilaraemic individuals, develop not because of drug toxicity but because of host inflammatory responses to dying microfilariae killed by the ivermectin treatment. Ivermectin is, therefore, a highly effective and generally well tolerated microfilaricide that may soon become an essential component of many public health initiatives to interrupt transmission of lymphatic filarial infection in an effort to eliminate LF globally.

Chin RM et al. (2014) Nature "The metabolite -ketoglutarate extends lifespan by inhibiting ATP synthase and TOR."

Wang JX, Reue K, Monsalve GC, Chang HR, Godwin HA, Trauger SA, Fazlollahi F, Whelan SA, Kaweeteerawat C, Chin RM, Krall AS, Jiang M, Petrascheck M, Nili M, Christofk HR, Hu E, Hwang H, Vergnes L, Quach A, Pai MY, Meli VS, Fu X, Faull KF, Frand AR, Guo F, Huang J, Diep S, Jung G, Braas D, Teitell MA, Saghatelian A, Clarke CF, Jung ME, Deng G, Solis GM, Lomenick B

[Nature, 2014]

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that -ketoglutarate (-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit is identified as a novel binding protein of -KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that -KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by -KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by -KG requires ATP synthase subunit and is dependent on target of rapamycin (TOR) downstream. Endogenous -KG levels are increased on starvation and -KG does not extend the lifespan of dietary-restricted animals, indicating that -KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.