Febs Letters

doi: 10.1016/S0014-5793(01)02868-Xpmid: N/A

Thuman-Commike, Pamela A

doi: 10.1016/S0014-5793(01)02804-6pmid: 11566176

Three‐dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three‐dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.

Kamarajan, Pachiyappan; Sun, Nian-Kang; Sun, Chun-Ling; Chao, Chuck C.-K

doi: 10.1016/S0014-5793(01)02817-4pmid: 11566177

Inhibition of caspase‐3‐mediated apoptosis has been hypothesized to be associated with chemoresistance. Investigations of apoptosis revealed that cytosolic cytochrome c is associated with a complex of apoptotic protease activating factor‐1 (Apaf‐1), an adapter molecule, and caspase‐9 to activate caspase‐3. However, whether these apoptotic molecules are involved in acquired cisplatin resistance is not understood. The present work shows reduced activation of caspase‐3 and apoptosis in a cisplatin‐selected HeLa cell line. Ac‐DEVD‐CHO, a caspase‐3 inhibitor, inhibited cisplatin‐induced apoptosis about 60–70% in both cell lines. Ac‐LEHD‐CHO, a caspase‐9 inhibitor or Ac‐IETD‐CHO, a caspase‐8 inhibitor, inhibited cisplatin‐induced caspase‐3 activation and apoptosis similarly in both cell lines. In addition, cisplatin induced the activation of caspase‐9, the upstream activator of caspase‐3, in a dose‐dependent manner, and the activation of caspase‐9 was less induced in resistant cells. The accumulation of cytosolic cytochrome c, an activator of caspase‐9, and the induction of the mitochondrial membrane‐associated voltage‐dependent anion channel were also reduced in cisplatin‐resistant cells. However, the concentration of Bcl‐2 family proteins in cisplatin‐resistant cells was normal. The concentration of Apaf‐1 was unaltered in both cell lines. Increasing the cellular concentration of Apaf‐1 through the transient expression of the gene increased the induction of apoptosis in resistant cells, associated with enhanced activation of caspase‐9, caspase‐3 and DNA fragmentation factor. Regression analysis reveals that the modification factor, the ratio of the slope in the linear range of the dose–response curve with Apaf‐1 to the slope without Apaf‐1, is 1.5 and 4.75 in the HeLa and cisplatin‐resistant HeLa cells, respectively. These results indicate that apoptosis and caspases are less induced in cisplatin‐selected HeLa cells. They also suggest that ectopic overexpression of Apaf‐1 may partially reverse the acquired cisplatin resistance.

Reitinger, Stephan; Müllegger, Johannes; Lepperdinger, Günter

doi: 10.1016/S0014-5793(01)02813-7pmid: 11566178

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH‐20‐like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore‐assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane‐bound hyaluronan‐degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.

Higuchi, Satoshi; Tanimoto, Akihide; Arima, Nobuyuki; Xu, Hui; Murata, Yoshitaka; Hamada, Tetsuo; Makishima, Kazumi; Sasaguri, Yasuyuki

doi: 10.1016/S0014-5793(01)02823-Xpmid: 11566179

We investigated the localization of histidine decarboxylase (HDC), which is the rate‐limiting enzyme that generates histamine from histidine, in human aorta/coronary artery. RT‐PCR and immunohistochemical staining revealed that the HDC gene was expressed in monocytes/macrophages and T cells in the arterial intima but not in smooth muscle cells in either the arterial intima or the media. A luciferase promoter assay with U937 and Jurkat cells demonstrated that interleukin‐4 (IL‐4) inhibited the expression of the HDC gene. In contrast, among a scavenger receptor family, IL‐4 as well as histamine up‐regulated U937 cells to express the LOX‐1 gene but not the SR‐A gene, which genes encode receptors that scavenge oxidized lipids. These findings suggest that histamine synthesized in the arterial wall participates in the initiation and progression of atherosclerosis and that IL‐4 can act as an important inhibitory and/or stimulatory factor in the function of monocytes/macrophages modulated by histamine in relation to the process of atherosclerosis.

Kohroki, Junya; Fujita, Sayaka; Itoh, Norio; Yamada, Yukiko; Imai, Harue; Yumoto, Noboru; Nakanishi, Tsuyoshi; Tanaka, Keiichi

doi: 10.1016/S0014-5793(01)02829-0pmid: 11566180

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C‐terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N‐terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL‐60 cells, and demonstrated that ASB‐2, one of the ASB proteins, was rapidly induced by all‐trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb‐2 gene. We showed that RARα, one of the RARs, binds to the RARE/RXRE in the Asb‐2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB‐2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.

Fréchet, Mathilde; Canitrot, Yvan; Cazaux, Christophe; Hoffmann, Jean-Sébastien

doi: 10.1016/S0014-5793(01)02834-4pmid: 11566181

Oxidative stress has been proposed to be one of the major causes leading to the accumulation of mutation that is associated with the initiation and progression of cancers. Elevated expression of DNA polymerase β, an event found in many human tumors, has been shown to generate a mutator phenotype. Here, we demonstrated that overexpression of DNA polymerase β strengthens the mutagenicity of oxidative damages, concomitantly with a higher cellular sensitivity and increased apoptosis. Deregulated expression of DNA polymerase β could represent a predisposition factor for mutagenic effects of oxidative stress and thus have implication in the generation and/or evolution of cancer.

Dreyer, Ingo; Michard, Erwan; Lacombe, Benoı̂t; Thibaud, Jean-Baptiste

doi: 10.1016/S0014-5793(01)02832-0pmid: 11566182

Among the Shaker‐like plant potassium channels, AKT2 is remarkable because it mediates both instantaneous ‘leak‐like’ and time‐dependent hyperpolarisation‐activated currents. This unique gating behaviour has been analysed in Xenopus oocytes and in COS and Chinese hamster ovary cells. Whole‐cell and single‐channel data show that (i) AKT2 channels display two distinct gating modes, (ii) the gating of a given AKT2 channel can change from one mode to the other and (iii) this conversion is under the control of post‐translational factor(s). This behaviour is strongly reminiscent of that of the KCNK2 channel, recently reported to be controlled by its phosphorylation state.

Borecký, Jirı́; Maia, Ivan G.; Costa, Alexandre D.T.; Ježek, Petr; Chaimovich, Hernan; de Andrade, Paula B.M.; Vercesi, Anı́bal E.; Arruda, Paulo

doi: 10.1016/S0014-5793(01)02835-6pmid: 11566183

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid‐induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K i (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH‐dependent. Kinetics of AtPUMP1‐mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

Kim, Jinoh; Rusch, Sharyn; Luirink, Joen; Kendall, Debra A.

doi: 10.1016/S0014-5793(01)02784-3pmid: 11566184

In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)‐dependent pathway or the Sec‐dependent pathway. Membrane proteins utilize the SRP‐dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery. To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes. We show that depletion of SRP, but not trigger factor, affects all the mutants examined. These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export.