Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.     

Heterogeneous nuclear ribonucleoproteins C1 and C2 associate with the RNA component of human telomerase

Here we demonstrate that heterogeneous nuclear ribonucleoproteins (hnRNPs) C1 and C2 can associate directly with the integral RNA component of mammalian telomerase. The binding site for hnRNPs C1 and C2 maps to a 6-base uridylate tract located directly 5' to the template region in the human telomerase RNA (TR) and a 4-base uridylate tract directly 3' to the template in the mouse TR. Telomerase activity is precipitated with antibodies specific to hnRNPs C1 and C2 from cells expressing wild-type human TR but not a variant of the human TR lacking the hnRNPs C1 and C2 binding site, indicating that hnRNPs C1 and C2 require the 6-base uridylate tract within the human TR to associate with the telomerase holoenzyme. In addition, we demonstrate that binding of hnRNPs C1 and C2 to telomerase correlates with the ability of telomerase to access the telomere. Although correlative, these data do suggest that the binding of hnRNPs C1 and C2 to telomerase may be important for the ability of telomerase to function on telomeres. The C proteins of the hnRNP particle are also capable of colocalizing with telomere binding proteins, suggesting that the C proteins may associate with telomeres in vivo. Therefore, human telomerase is capable of associating with core members of the hnRNP family of RNA binding proteins through a direct and sequence-specific interaction with the human TR. This is also the first account describing the precise mapping of a sequence in the human TR that is required to associate with an auxiliary component of the human telomerase holoenzyme.     

Interaction of vasomotor and exocrine neurons in bullfrog paravertebral sympathetic ganglia

A 2 min sample of an intracellular recording of in vivo synaptic activity from a vasomotor C-neuron in a bullfrog sympathetic ganglion was converted to a series of stimulus pulses. This physiologically derived activity was used to stimulate preganglionic C-fibres of similar ganglia studied in vitro. Intracellular recordings were made from exocrine B-cells within the ganglia. Although they do not receive fast, nicotinic synaptic input from preganglionic C-fibres, B-cell excitability was profoundly increased by stimulation of C-fibres with physiologically derived activity. Also, subthreshold depolarizing current pulses that failed to generate action potentials in B-cells under control conditions almost always generated action potentials whilst C-fibres were activated. These effects were attenuated or prevented by the luteinizing hormone releasing hormone antagonist, [D-pyro-Glu1,D-Phe2,D-Trp3,6]-LHRH (70 microM). The physiological release of luteinizing hormone releasing hormone from C-fibres therefore causes an interaction between vasomotor and exocrine outflow within a paravertebral sympathetic ganglion.     

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.     

The hydrogenation and hydroformylation of alkenes as catalyzed by polymer-anchored rhodium trichloride under water gas shift reaction conditions

The ‘heterogenized’ water gas shift catalyst Rh/P4VP, prepared from the reaction of RhCl₃ with poly(4-vinylpyridine), is also active for hydrogenation and hydroformylation of 1-hexene and cyclohexene in aqueous ethoxyethanol under mild shift reaction conditions (typically 0.9 atm. P_CO at 100°C). The catalytic activities for these systems were studied as functions of several experimental variables. Hydroformylation rates increased with the P_CO but exhibited saturation behavior in the 1.5 atm. range. Rates for cyclohexane and hexane production were inhibited by CO at higher pressures. Cyclohexene hydroformylation and hydrogenation turnover frequencies were independent of the polymer-loading (50–150 μM RhCl₃/1.0 g P4VP) indicating that the active species are of the same nuclearity as the principal species present. The temperature dependence did not follow simple Arrhenius behavior, but appeared segmented. These data are discussed in terms of possible mechanisms.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

The induction of taxol production in the endophytic fungus—Periconia sp from Torreya grandifolia

A Periconia sp was isolated from Torreya grandifolia (a relative of yew that does not synthesize taxol) near Huangshan National Park in the People’s Republic of China. This fungus, not previously known as a tree endophyte, was isolated from the inner bark of a small lower limb. When freshly isolated from the tree and placed in a semi-synthetic medium, the fungus produced readily detectable quantities of the anticancer drug taxol. Other taxol-producing endophytes were also isolated from this source. The production of taxol by Periconia sp was demonstrated unequivocally via spectroscopic and immunological methods. However, successive transfers of the fungus in semi-synthetic medium resulted in gradual attenuation until low production occurred even though fungal growth was relatively unaffected. Several compounds, known previously as activators of microbial metabolism, including serinol, p-hydroxybenzoic acid, and a mixture of phenolic acids, were capable of fully or partially restoring taxol production to otherwise taxol-attenuated cultures. The compound with the most impressive ability to activate taxol production was benzoic acid at 0.01 mM. Benzoic acid was not a taxol precursor. Received 19 December 1997/ Accepted in revised form 19 February 1998