Beta-carotene affects gene expression in lungs of male and female <Emphasis Type="Italic">Bcmo1</Emphasis><Superscript>−/−</Superscript> mice in opposite directions

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^−/−) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1 ^−/− mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1 ^−/− mice. Testosterone levels were higher after BC supplementation only in Bcmo1 ^−/− mice, which had, unlike wild-type (Bcmo1 ^+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.     

Diversity of Cl− Channels

Cl⁻ channels are widely found anion pores that are regulated by a variety of signals and that play various roles. On the basis of molecular biologic findings, ligand-gated Cl⁻ channels in synapses, cystic fibrosis transmembrane conductors (CFTRs) and ClC channel types have been established, followed by bestrophin and possibly by tweety, which encode Ca²⁺-activated Cl⁻ channels. The ClC family has been shown to possess a variety of functions, including stabilization of membrane potential, excitation, cellvolume regulation, fluid transport, protein degradation in endosomal vesicles and possibly cell growth. The molecular structure of Cl⁻ channel types varies from 1 to 12 transmembrane segments. By means of computer-based prediction, functional Cl⁻ channels have been synthesized artificially, revealing that many possible ion pores are hidden in channel, transporter or unidentified hydrophobic membrane proteins. Thus, novel Cl⁻-conducting pores may be occasionally discovered, and evidence from molecular biologic studies will clarify their physiologic and pathophysiologic roles. Received 28 July 2005; received after revision 25 August 2005; accepted 21 September 2005     

Enhanced susceptibility of T lymphocytes to oxidative stress in the absence of the cellular prion protein

The cellular prion glycoprotein (PrP^C) is ubiquitously expressed but its physiologic functions remain enigmatic, particularly in the immune system. Here, we demonstrate in vitro and in vivo that PrP^C is involved in T lymphocytes response to oxidative stress. By monitoring the intracellular level of reduced glutathione, we show that PrP^−/− thymocytes display a higher susceptibility to H₂O₂ exposure than PrP^+/+ cells. Furthermore, we find that in mice fed with a restricted diet, a regimen known to increase the intracellular level of ROS, PrP^−/− thymocytes are more sensitive to oxidative stress. PrP^C function appears to be specific for oxidative stress, since no significant differences are observed between PrP^−/− and PrP^+/+ mice exposed to other kinds of stress. We also show a marked evolution of the redox status of T cells throughout differentiation in the thymus. Taken together, our results clearly ascribe to PrP^C a protective function in thymocytes against oxidative stress.     

HCN2 channels in local inhibitory interneurons constrain LTP in the hippocampal direct perforant path

Neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to modulate spontaneous activity, resting membrane potential, input resistance, afterpotential, rebound activity, and dendritic integration. To evaluate the role of HCN2 for hippocampal synaptic plasticity, we recorded long-term potentiation (LTP) in the direct perforant path (PP) to CA1 pyramidal cells. LTP was enhanced in mice carrying a global deletion of the channel (HCN2^−/−) but not in a pyramidal neuron-restricted knockout. This precludes an influence of HCN2 located in postsynaptic pyramidal neurons. Additionally, the selective HCN blocker zatebradine reduced the activity of oriens-lacunosum moleculare interneurons in wild-type but not HCN2^−/− mice and decreased the frequency of spontaneous inhibitory currents in postsynaptic CA1 pyramidal cells. Finally, we found amplified LTP in the PP of mice carrying an interneuron-specific deletion of HCN2. We conclude that HCN2 channels in inhibitory interneurons modulate synaptic plasticity in the PP by facilitating the GABAergic output onto pyramidal neurons.     

Application de méthodes d'analyse biochimique à une étude taxonomique: les corégones du lac de Neuchâtel

Summary Two closely related forms ofCoregonus from Lake Neuchatel were examined cytologically and biochemically, in order to ascertain the chromosome number and the DNA content of haploid and diploid nuclei.Coregonus fera has 2N=78 ± 2 chromosomes, and a DNA content (diploid) of 5.8 × 10⁻⁹ mg;Coregonus macrophthalmus, 2N=78⁺ ± 3, DNA content of 6.1 × 10⁻⁹ mg. The difference between the two DNA constants is statistically significant. These results do not support the hypothesis which postulates that polyploidy may be a determining factor in the speciation of these fishes.      

Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line

Cancer stem cells (CSCs) play an important role in the development, invasion, and drug resistance of carcinoma, but the exact phenotype and characteristics of ovarian CSCs are still disputable. In this study, we identified cancer stem cell-like cells (CSC-LCs) and investigated their characteristics from the ovarian adenocarcinoma cell line 3AO. Our results showed that CSC-LCs were enriched in sphere-forming test and highly expressed CD44⁺CD24⁻. The spheres and CD24⁻ cells possessed strong tumorigenic ability by transplantation into nonobese diabetic/severe combined immunodeficient mice. CD44⁺CD24⁻ cells expressed stem cell markers and differentiated to CD44⁺CD24⁺ cells by immunofluorescence assay and fluorescence-activated cell-sorting analysis. In vitro experiments verified that CD44⁺CD24⁻ cells were markedly resistant to carboplatin and paclitaxol. In conclusion, our study identifies the CD44⁺CD24⁻ phenotype, self-renewal, high tumorigenicity, differentiation potential, and drug resistance of ovarian CSC-LCs. Our findings may provide the evidence needed to explore a new strategy in the treatment of ovarian cancer.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

δ-Protocadherins: unique structures and functions

δ-Protocadherins constitute a group of cadherins characterized by several conserved motifs in their cytoplasmic domains. We present a phylogenetic analysis that further divides this group into δ1-protocadherins (comprising protocadherin-1, −7, −9 and −11 or -X/Y) and δ2-protocadherins (comprising protocadherin-8, −10, −17, −18 and −19). The δ-protocadherin genes, which are located on different chromosomes in man and mouse, have a similar gene structure. They are expressed as multiple splice forms, differing mostly in their cytoplasmic domains. Some δ-protocadherins were reported to mediate weak cell-cell adhesion in vitro and cell sorting in vivo. In addition, individual δ-protocadherins might play important roles in signaling pathways, as they bind to proteins such as TAF1/Set, protein phosphatase-1α and the Frizzled 7 receptor. The spatiotemporally restricted expression of δ-protocadherins in different tissues and species and the results of their functional analysis, mainly in Xenopus, suggest that they play multiple, tightly regulated roles in vertebrate development. Received 18 July 2005; received after revision 26 August 2005; accepted 2 September 2005     

Dependence of 6β-acetoxy-7α-hydroxyroyleanone block of Kv1.2 channels on C-type inactivation

Voltage-gated K⁺ (Kv) channels exhibit slow or C-type inactivation during continuous depolarization. A selective pharmacological agent targeting C-type inactivation is hitherto lacking. Here, we report that 6β-acetoxy-7α-hydroxyroyleanone (AHR), a diterpenoid compound isolated from Taiwania cryptomerioides, can selectively modify C-type inactivation of Kv1.2 channels. Extracellular, but not intracellular, AHR (50 μM) dramatically accelerated the slow decay of Kv currents and left-shifted the steady-state inactivation curve. AHR blocked Kv currents with an IC₅₀ of 17.7 μM. AHR did not affect the kinetics and voltage-dependence of Kv1.2 channel activation. Channel block by AHR was independent of intracellular K⁺ concentration. In addition, effect of AHR was much attenuated in a Kv1.2 V370G mutant defective in C-type inactivation. Therefore, block of Kv1.2 channels by AHR did not appear to involve direct occlusion of the outer pore but depended on C-type inactivation. AHR could thus be a probe targeting Kv channel C-type inactivation gate.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana

Cellulose microfibrils containing crystalline β-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-β-glucanases. The mutants also accumulate a readily extractable β-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps. Received 5 January 2001; received after revision 25 April 2001; accepted 25 April 2001     

Genetically engineered avidins and streptavidins

Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, K_d≈10⁻¹⁴–10⁻¹⁶ M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology. Received 22 June 2006; received after revision 1 August 2006; accepted 21 September 2006     

Zeitbedarf der Längsteilung von miteinander verzwirnten Fadenmolekülen

Summary The time requirement is treated for a longitudinal fission by Brownian movement of a very long particle consisting of two or more filaments twisted a great number of times round each other to form a double spiral. It is shown that a comparatively swift disintegration is obtained by partial rotation or torsion round the axis of the spiral, resulting in a loosing of the spiral structure and subsequent separation of the constituents by translational Brownian movement. The time required to separate a double spiral consisting of about 900 turns of a height of 3.4 × 10⁻⁷ cm and a radius of 10⁻⁷ cm, thus having a length of 3 × 10⁻⁴ cm being realized approximately by deoxyribonucleic acid is found by this mechanism to be about 50 to 80 s. The time required to undo the same spiral by unwrapping it turn by turn would be about 150 days. The result of the considerations is related to observations published byAlexander andSteacy on deoxyribonucleic acid. An additional remark stresses the importance of stereochemical asymmetry for the practicability of the mechanism and therefore the importance of optical activity for the time requirement of such disintegrations or transformations of high polymer material occurring in living organisms.      

When a theory of aging ages badly

According to the widely acknowledged mitochondrial free radical theory of aging (MFRTA), the macromolecular damage that results from the production of toxic reactive oxygen species (ROS) during cellular respiration is the cause of aging. However, although it is clear that oxidative damage increases during aging, the fundamental question regarding whether mitochondrial oxidative stress is in any way causal to the aging process remains unresolved. An increasing number of studies on long-lived vertebrate species, mutants and transgenic animals have seriously challenged the pervasive MFRTA. Here, we describe some of these new results, including those pertaining to the phenotype of the long-lived Mclk1 ^+/− mice, which appear irreconcilable with the MFRTA. Thus, we believe that it is reasonable to now consider the MFRTA as refuted and that it is time to use the insight gained by many years of testing this theory to develop new views as to the physiological causes of aging.     

The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region

The human α₂-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn³⁹⁸-Lys⁴⁵²), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys⁴²², Lys⁴²⁹, Lys⁴³⁶, Lys⁴⁵²) are completely and two (Lys⁴⁰⁶, Lys⁴¹⁵) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys⁴⁵² as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys⁴⁵² and internal Lys⁴³⁶ of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr⁴⁴⁵ in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.     

Neuromuscular transmission inElectrophorus electricus (L.)

Résumé Le système neuromusculaire dansElectrophorus electricus est étudié avec des électrodes intracellulaires. La transmission neuromusculaire est supprimée avec D-tubocurarine (5 · 10^–7 p/v) ou avec un excès des ions Mg⁺⁺ (12 mM) dans la solution.     

Indole-3-carbinol enhances ultraviolet B-induced apoptosis by sensitizing human melanoma cells

Indole-3-carbinol (I3C) has been found to act against several types of cancer, while ultraviolet B (UVB) is known to induce the apoptosis of human melanoma cells. Here, we investigated whether I3C can sensitize G361 human melanoma cells to UVB-induced apoptosis. We examined the effects of combined I3C and UVB (I3C/UVB) at various dosages. I3C (200 μM)/UVB (50 mJ/cm²) synergistically reduced melanoma cell viability, whereas I3C (200 μM) or UVB (50 mJ/cm²), separately, had little effect on cell viability. DNA fragmentation assays indicated that I3C/UVB induced apoptosis. Further results show that I3C/UVB activates caspase-8, −3, and Bid and causes the cleavage of poly(ADP-ribose) polymerase. Moreover, I3C decreased the expression of the anti-apoptotic protein, Bcl-2, whereas UVB increased the translocation of Bax to mitochondria. Thus, an increased Bax/Bcl-2 ratio by I3C/UVB may result in melanoma apoptosis. In conclusion, our study demonstrated that I3C sensitizes human melanoma cells by down-regulating Bcl-2. Received 5 July 2006; received after revision 25 August 2006; accepted 11 September 2006     

Purinergic systems in microglia

Accumulating findings indicate that nucleotides play an important role in microglia through P2 purinoceptors. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X₁ – P2X₇) contain intrinsic pores that open by binding with ATP. P2Y receptors (8 types; P2Y_{1, 2, 4, 6, 11, 12, 13 and 14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. Microglia express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as ‘warning molecules’ especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain, chemotaxis and phagocytosis through nucleotide-evoked activation of P2X₄, P2Y₁₂ and P2Y₆ receptors, respectively. These findings indicate that extracellular nucleotides are important players in the central stage of microglial function. Received 19 April 2008; received after revision 20 May 2008; accepted 23 May 2008