eIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction.

Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.

Impaired eIF5A function causes a Mendelian disorder that is partially rescued in model systems by spermidine.

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Stemness and chemotherapeutic drug resistance induced by EIF5A2 overexpression in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies of the digestive tract in East Asian countries. Multimodal therapies, including adjuvant chemotherapy and neo-adjuvant chemotherapy, have become more often used for patients with advanced ESCC. However, the chemotherapy effect is often limited by patients' drug resistance. This study demonstrated that EIF5A2 (eukaryotic translation initiation factor 5A2) overexpression induced stemness and chemoresistance in ESCC cells. We showed that EIF5A2 overexpression in ESCC cells resulted in increased chemoresistance to 5-fluorouracil (5-FU), docetaxel and taxol. In contrast, shRNAs suppressing eIF5A2 increased tumor sensitivity to these chemotherapeutic drugs. In addition, EIF5A2 overexpression was correlated with a poorer overall survival in patients with ESCC who underwent taxane-based chemotherapy after esophagectomy (P < 0.05). Based on these results, we suggest that EIF5A2 could be a predictive biomarker for selecting appropriate chemo-treatment for ESCC patients and EIF5A2 inhibitors might be considered as combination therapy to enhance chemosensitivity in patients with ESCC.

Translation initiation factor 5A in Picrorhiza is up-regulated during leaf senescence and in response to abscisic acid.

Translation initiation, the first step of protein synthesis process is the principal regulatory step controlling translation and involves a pool of translation initiation factors. In plants, from recent studies it is becoming evident that these translation initiation factors impact various aspects of plant growth and development in addition to their role in protein synthesis. Eukaryotic translation initiation factor eIF5A is one such factor which functions in start site selection for the eIF2-GTP-tRNAi ternary complex within the ribosomal-bound preinitiation complex and also stabilizes the binding of GDP to eIF2. In the present study we have cloned and analysed a gene (eIF5a) encoding eIF5A from Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) a medicinal plant of the western Himalayan region. The full length eIF5a cDNA consisted of 838 bp with an open reading frame of 480 bp, 88 bp 5' untranslated region and 270 bp 3' untranslated region. The deduced eIF5A protein contained 159 amino acids with a molecular weight of 17.359 kDa and an isoelectric point of 5.59. Secondary structure analysis revealed eIF5A having 24.53% α-helices, 8.81% ß-turns, 23.27% extended strands and 43.40% random coils. pk-eIF5a transcript was found to be expressing during the active growth phase as well as during leaf senescence stage, however, highest expression was observed during leaf senescence stage. Further, its expression was up-regulated in response to exogenous application of abscisic acid. Both high intensity as well as low intensity light decreased the expression of pk-eIF5a. The findings suggest eIF5a to be an important candidate to develop genetic engineering based strategies for delaying leaf senescence.

Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a "humanized" transgenic mouse model of type 1 diabetes.

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4(+) T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

Proteomic analysis of the effect of triterpenes from Patrinia heterophylla on leukemia K562 cells.

For centuries, Patrinia heterophylla had been used in China to treat many diseases including tumor. Triterpenes has been identified as the major active constituents in Patrinia heterophylla. To elucidate the antitumor mechanism of triterpenes from Patrinia heterophylla1 (TPH), a proteomic analysis is carried out with TPH treatment in K562 cells. The total proteins extracted from TPH treated K562 cells are analyzed by two dimensional gel electrophoresis (2-DE) and compared with those untreated K562 cells. Mass spectrometry is applied to identify the differentially expressed proteins. Twenty-three differentially expressed significant proteins are discovered. Eight proteins are later identified by mass spectrometry (MALDI-TOF-MS) and Mascot software. Among them, four proteins are up-regulated (Aldolase A, Glyceraldehyde-3-phosphate dehydrogenase, Flavin reductase and Hemoglobin subunit) and four proteins were down-regulated (Heat-shock protein 90 〈Alpha〉 (HSP90-〈Alpha〉), Eukaryotic translation initiation factor 5A, Moesin, tublin) by TPH treatment in K562 cells. The identified proteins are associated with energy metabolism, oxidative stress, apoptosis, signal transduction, differential induction, and protein biosynthesis. These findings might provide valuable insights into the antitumor mechanism of TPH in K562 cells.

"Hybrid exercise" prevents muscle atrophy in association with a distinct gene expression pattern.

"Hybrid exercise" utilizing combined electrical stimulation and voluntary muscle contraction has been developed as a muscle exercise method. Although our previous studies have confirmed the effectiveness of the procedure, the mechanisms of its efficacy still remain unclear. In the present study, we identified genes that are specifically expressed in disused muscles, using the semitendinosus muscle from patients who underwent anterior cruciate ligament (ACL) reconstruction. Preoperative exercise was performed by four ACL-injured patients, who were subjected either to hybrid exercise (n=2), electrical stimulation (n=1), or no electrical stimulation (n=1), in addition to standard weight training for 4 weeks. Cross-sectional area (CSA) of the semitendinosus muscle was measured before and after the exercise by magnetic resonance imaging (MRI). A piece of the semitendinosus muscle was isolated during the surgery, and comprehensive analysis of the gene expression in this sample was performed using DNA microarray analysis. CSA increased in size by 4.2 and 14.7%, respectively, after hybrid exercise, and by 1.4% after electrical stimulation. However it shrunk by 7.7% without electrical stimulation. DNA microarray analysis revealed that hybrid exercise was more effective at stimulating the expression of signal transduction-, transcription- and cytoskeleton-related genes in semitendinosus muscles than electrical stimulation alone. In particular, gene ontology analysis revealed that hybrid exercise induced significantly higher expression of eukaryotic translation initiation factor 5A (EIFSA), peroxisomal biogenesis factor 6 (PEX6) and histone cluster 1 H4 (HIST1H4), compared with electrical stimulation alone. The expression of signal transduction-, transcription- and cytoskeleton-related genes may play an important role in muscle bulk increasing mechanisms in hybrid exercise.

Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata mycelia are mediated by down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell.

AIM OF THE STUDY: Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that Antrodia camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities. MATERIALS AND METHODS: Alcohol extracts of Antrodia camphorata mycelia were prepared by the serial extraction with the solvents with increasing polarity and fractionated using HPLC. Cell viability was determined by MTT assay. Apoptosis detection was carried out by subG(1) analysis and annexin V/propidium iodide staining using flow cytometry. The impacts of HPLC fractions on the expression levels of apoptosis- and cancer-related proteins were evaluated by western blotting. RESULTS: Three HPLC fractions, fractions 5-7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6-induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor alpha (RhoGDI-alpha) expression and subsequently activated c-Jun NH(2)-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target. CONCLUSION: These results suggested that the anti-cancer activity of Antrodia camphorata might be due to multiple active metabolites, which work together to induce cell apoptosis via various pathways.

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

[Proteomic analysis of differentially expressed proteins in human hepatoma SMMC-7721 cells induced by Fufang Banmao capsule serum].

OBJECTIVE: To investigate the effects of Fufang Banmao capusle on the proteome of SMMC-7721 cells and discover potential molecular mechanism of anti-cancer at molecular level. METHOD: SMMC-7721 cells were treated by Fufang Banmao capusle serum prepared with serum pharmacological method; proteomic protocol involving 2-DE, image analysis and mass spectrometry were used to detect the proteins in cells influenced by Fufang Banmao capusle. RESULT: Approximately 450 protein spots in SMMC-7721 cells were resolved and detected in 2-D gel maps from pH3-10L IEF. 47 protein plots varied over 2-fold quantitively between treated sample and control sample were uncovered. 13 differentially expressed proteins spots were further identified by MALDI-TOF-MS analysis and four of them were successfully identified. Annexin A5, heatshock 70 x 10(3) protein 8 was significantly up-regulated in treated sample compared with control sample, while Eukaryotic translation initiation factor 5A and Peroxiredoxin-2 was significantly down-regulated in treated sample. CONCLUSION: 4 differently expressed proteins associated with the proliferation, apoptosis, immunity of tumor were detected and they might provide clues for the coming research. The protocol of proteomics combined with serum pharmacological method is an effective platform to research complicated formulas in that it is capable of laying out many proteins associated with Fufang Banmao capusle.

Heat stress-induced loss of eukaryotic initiation factor 5A (eIF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis.

Alterations of intracellular proteins during the process of heat stress-induced cell death of a human pancreatic cancer cell line, MIA PaCa-2, were investigated using two-dimensional gel electrophoresis (2-DE), agarose gel electrophoresis, and cell biology techniques. Incubation of MIA PaCa-2 at 45 degrees C for 30 min decreased the cell growth rate and cell viability without causing chromosomal DNA fragmentation. Incubation at 51 degrees C for 30 min suppressed cell growth and again led to death without DNA fragmentation. The cell death was associated with the loss of an intracellular protein of M(r) 17,500 and pI 5.2 on 2-DE gel. This protein was determined to be eukaryotic initiation factor SA (eIF-5A) by microsequencing of the N-terminal region of peptide fragments obtained by cyanogen bromide treatment of the protein blotted onto a polyvinylidene difluoride (PVDF) membrane. The sequences detected were QXSALRKNGFVVLKGRP and STSKTGXHGHAKVHLVGID, which were homologous with the sequence of eIF-5A from Gln 20 to Pro 36 and from Ser 43 to Asp 61, respectively. Furthermore, the result of sequencing suggested that the protein was an active form of hypusinated eIF-5A, because Lys 46 could be detected but not Lys 49, which is the site for hypusination. These results suggest that loss of the active form of eIF-5A is an important factor in the irreversible process of heat stress-induced death of MIA PaCa-2 cells.

Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato.

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA.

Dendritic cells (DCs), nature's adjuvant, must mature to sensitize T cells. However, although the maturation process is essential, it is not yet fully understood at the molecular level. In this study, we investigated the course of expression of the unique hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A), which is part of a particular RNA nuclear export pathway, during in vitro generation of human DCs. We show that eIF-5A expression is significantly upregulated during DC maturation. Furthermore, an inhibitor of the hypusine modification, GC7 (N(1)-guanyl-1, 7-diaminoheptane), prevents CD83 surface expression by apparently interfering with nucleocytoplasmic translocation of the CD83 mRNA and, importantly, significantly inhibits DC-mediated T lymphocyte activation. The data presented suggest that CD83 mRNA is transported from the nucleus to the cytoplasm via a specific nuclear export pathway and that hypusine formation appears to be essential for the maturation of functional DCs. Therefore, pharmacological interference with hypusine formation may provide a new possibility to modulate DC function.

Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species.

Eukaryotic initiation factor eIF5A is essential for cell viability and contains a characteristic post-translational modification of a specific lysine residue into a hypusine. cDNAs with similarity to eIF5A sequences were derived from Spodoptera exigua and S. frugiperda cDNA libraries. The deduced amino acid sequences are identical for both species and predict a protein with a molecular mass of 17.5 kDa. The Drosophila melanogaster eIF5A cDNA sequence was retrieved from the Drosophila EST Project. The predicted protein is 80% similar to Spodoptera eIF5A. A single eIF5A gene copy is present in the S. frugiperda genome, which is transcribed into four different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decline of all four transcripts already at 12 h after infection. In contrast, the eIF5A protein was fairly stable up to 48 h post infection.

Identification of YHR068w in Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. Expression and characterization of the enzyme.

Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the first step in hypusine biosynthesis. Amino acid sequences of five tryptic peptides from rat deoxyhypusine synthase were found to match partially the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061). In order to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase,a 1.17-kilobase pair cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S. cerevisiae cDNA by polymerase chain reaction and was expressed in Escherichia coli B strain BL21 (DE3). The recombinant protein was found mostly in the E. coli cytosol fraction and comprised approximately 20% of the total soluble protein. The purified form of the expressed protein effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A precursors as well as in human precursor and in those from Chinese hamster ovary cells. The molecular mass of the enzyme was estimated to be 172,000 +/- 4,300 Da by equilibrium centrifugation. The mass of its polypeptide subunit was determined to be approximately 43,000 Da, in close agreement with that calculated for the coding region of the YHRO68w gene. These findings show that this gene is a coding sequence for yeast deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.

Diamine and triamine analogs and derivatives as inhibitors of deoxyhypusine synthase: synthesis and biological activity.

Deoxyhypusine synthase catalyzes the initial step in the posttranslational formation of the amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic initiation factor 5A (eIF-5A). eIF-5A and its hypusine modification are believed to be essential for cell growth. A number of compounds related to diamines and triamines were synthesized and tested as inhibitors of this enzyme. The findings indicate that the long chain triamines 2a and 2b and their guanyl derivatives 3a, 3b, 4a, and 4b exert inhibition by binding to enzyme through only a portion of their structures at any one time. The inhibition exhibited by N-ethyl-1,7-diaminoheptane 20 and its guanyl derivative 21 supports this notion and is evidence for participation of the secondary amino group in binding to enzyme. There is preliminary evidence that amidino and isothiuronium groups may also serve as basic centers for binding to enzyme. Few of the compounds tested here were comparable in inhibitory potency to 1-guanidino-7-aminoheptane (GC7) the most effective known inhibitor of deoxhypusine synthase, and none proved nearly as efficient as GC7 in inhibiting the enzyme in Chinese hamster ovary cells. Hence, unlike the antiproliferative effect of GC7, for which there is evidence of cause by interference with deoxhypusine synthase catalysis (Park, M. H.; Wolff, E. C.; Lee, Y. B.; Folk, J. E. J. Biol. Chem. 269, 1994, 27827-27832), the effective growth arrest exerted by several of the newly synthesized compounds cannot be attributed to inhibition of hypusine synthesis.

Cloning and sequencing of a chick embryo cDNA encoding the 20-kDa hypusine-containing protein, eIF-5A.

Chick embryo contains 18- and 20-kDa isoforms of eukaryotic translation initiation factor 5A (eIF-5A). cDNA clones corresponding to the 20-kDa eIF-5A were isolated and sequenced. A full-length cDNA clone encodes a 153-amino-acid (aa) protein. The deduced aa sequence exactly matches with the partial aa sequence determined for this protein and shows high identity to that of human or rabbit eIF-5A. The results of Southern and Northern hybridization provide evidence for multiple transcripts for chick embryo eIF-5A or an eIF-5A-like protein that presumably derive from more than one gene.