Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines.

Nucleoside analogs (NAs) represent an important class of anticancer agents that induce cell death after conversion to triphosphate derivatives. One of their most important mechanisms of action is the activation of p53, leading to apoptosis through the intrinsic pathway. Classically, the activation of p53 also induces p21 accumulation, which leads to cell cycle arrest at the G1/S transition. In previous work, we observed that 2-chloro-2'-deoxyadenosine (CdA), a NA with high activity in lymphoid disorders, including chronic lymphocytic leukemia (CLL), promotes the G1/S transition in the CLL cell line EHEB at cytotoxic concentrations. This finding led us to investigate the p21 response to NAs in these cells. We show here that CdA, but also fludarabine, gemcitabine, and cytarabine, strongly reduced the p21 protein level in EHEB cells as well as in JVM-2 cells, another CLL cell line. This p21 depletion occurred despite induction of p53 and increase of p21 mRNA and was prevented by proteasome inhibitors. Increase of proteasomal degradation caused by NAs appeared to be ubiquitin-independent. Also, NAs induced in these cells an increase of cyclin-dependent kinase (Cdk2) activity and a monoubiquitination of cell proliferating nuclear antigen (PCNA), two processes that are negatively regulated by p21. These changes were not observed with other p53 activators, like etoposide and nutlin-3a that increased the p21 protein level. In conclusion, our study reveals that NAs can induce an alternative pattern of cellular response in some cell models.

Influence of phosphorylation of THR-3, SER-11, and SER-15 on deoxycytidine kinase activity and stability.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.

Familial large vestibular aqueduct syndrome.

The large vestibular aqueduct syndrome (LVAS) is a distinct clinical entity characterized by stepwise progressive sensorineural hearing loss associated with isolated enlargement of the vestibular aqueduct. A correlative clinical, audiologic, vestibular, cytogenetic, and radiographic analysis of a family with inherited LVAS was performed. The male proband and his affected brother are offspring of unaffected parents, and have no other abnormalities. Pedigree analysis suggests autosomal recessive or X-linked inheritance with variable expressivity of LVAS in this family. This study is the first description of familial inheritance of LVAS. LVAS may account for a significant number of patients with nonsyndromal, genetic sensorineural hearing loss. Future molecular analyses of this study family may identify the causative gene(s) in LVAS.

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue.

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218-225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°-180° (Z-helical), 0°-30° (S-helical); oblique: 110°-150° (Z-helical), 30°-70° (S-helical); transverse: 90°-110° (Z-helical), 70°-90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.

Orientation of cellulose microfibrils in cortical cells of tobacco explants : Effects of microtubule-depolymerizing drugs.

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations - all given with respect to the longitudinal axis of the cell -, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.

Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli.

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.

EPR of a novel high-spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728-3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 spin/mol. In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 spin/mol) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with geff = 5.0 which, under the assumptions that S = 2 and [delta ms] = 2, quantitates to roughly one spin/mol. Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin-state transition of an iron-sulfur cluster.

Application of hydrogenase in biotechnological conversions.

Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented.

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough).

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.