A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity

Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression. These cells are cross-resistant to several structurally and functionally dissimilar drugs. Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics. In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines. Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells. This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene. The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM). We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations. To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin. These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells. In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells. Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.     

A mechanism for molecular asymmetry

Summary The origin of the molecular asymmetry of biological systems has been speculated upon extensively, and has been the object of numerous inconclusive experimental studies. That circularly polarized light (CPL) might have been the cause of this asymmetry was suggested in 1874 (van't Hoff 1897; Le Bel 1874). During the daylight morning (AM) there is a significant component of left (L) CPL in skylight, which reverses to right (R) CPL in the afternoon (PM) (Wolstencroft 1985). The rates or photochemical reactions of LCPL and RCPL are different for the R (right) and S (left) forms of chiral molecules (Flores et al. 1977). At sunset the ambient temperature at the surface of the earth is approximately 10°C higher than at sunrise. Most chemical reactions proceed faster at higher temperatures and for each 10°C rise in temperature chemical reaction rates increase by a factor of 1.8–4.1 (Taylor 1925). It is proposed that the combination of these four factors, LCPL in the AM compared to the RCPL in the PM, the different rates of photochemical reaction of the R and the S forms of an R-S racemic mixture with RCPL (and LCPL), the higher PM temperature, and the faster reaction rates in the PM could lead to a substantial deviation from equality in the degradation and formation of R and S enantiomeric forms of chiral molecules.     

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.     

A molecular basis for human hypersensitivity to aminoglycoside antibiotics.

We have investigated the distribution of mitochondrial DNA polymorphisms in a rare maternally transmitted genetic trait that causes hypersensitivity to aminoglycoside antibiotics, in the hope that a characterization of its molecular basis might provide a molecular and cellular understanding of aminoglycoside-induced deafness (AGD). Here we report that the frequency of a particular mitochondrial DNA polymorphism, 1555G, is associated nonrandomly with aminoglycoside-induced deafness in two Japanese pedigrees, bringing the frequency of this polymorphism to 5 occurrences in 5 pedigrees of AGD, and in 4 of 78 sporadic cases in which deafness was thought to be the result of aminoglycoside exposure; both frequencies are significantly different from the occurrence of this mutation in the hearing population, which was 0 in 414 individuals surveyed. The 1555G polymorphism occurred in none of 34 aminoglycoside-resistant individuals. We propose a specific molecular mechanism for aminoglycoside hypersensitivity in individuals carrying the 1555G polymorphism, based on the three-dimensional structure of the ribosome, in which the 1555G polymorphism favors aminoglycoside binding sterically, by increasing access to the the ribosome cleft.     

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MutT-related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8-oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K_m of 0.77 µM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT⁻ cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.     

A possible molecular mechanism for mammalian aging

The following model of aging is proposed: 1) defective proteins with anomalous primary structures are synthesized sometimes; 2) these defective proteins are precipitated in cells and intercellular spaces; 3) the precipitated proteins block them up under the influence of radicals; 4) a decrease of cell functional ability below some level results in the destruction of the organism regulation function. A formula is concluded connecting the life span (Tlife) with DNA-repair velocity (Vrep) and time of protein exchange (Tex): Tlife = (1/3).K.(Vper/Vdum).(Tfix + Tex), where K-admitted share of fixated proteins (fixated/native), Vdam-damage velocity, Tfix-fixation time of defective proteins. This analytical dependence was probed on literature data for man, elephant, cow, rabbit, guinea pig, golden hamster, rat, mouse and shrew. Tfix is shown to equal 5 divided by 10 days. A good agreement between the theoretical dependence Tlife(Tex) and literature data was obtained with the exception of the data for man.     

Endogenous retroviruses as potential hazards for vaccines

Retroviruses are classified as exogenous or endogenous according to their mode of transmission. Generally, endogenous retroviruses (ERVs) are not pathogenic in their original hosts; however, some ERVs induce diseases. In humans, a novel gammaretrovirus was discovered in patients with prostate cancer or chronic fatigue syndrome. This virus was closely related to xenotropic murine leukemia virus (X-MLV) and designated as xenotropic murine leukemia virus-related virus (XMRV). The origin and transmission route of XMRV are still unknown at present; however, XMRV may be derived from ERVs of rodents because X-MLVs are ERVs of inbred and wild mice. Many live attenuated vaccines for animals are manufactured by using cell lines from animals, which are known to produce infectious ERVs; however, the risks of infection by ERVs from xenospecies through vaccination have been ignored. This brief review gives an overview of ERVs in cats, the potential risks of ERV infection by vaccination, the biological characteristics of RD-114 virus (a feline ERV), which possibly contaminates vaccines for companion animals, and the methods for detection of infectious RD-114 virus.     

耐亚胺培南鲍曼不动杆菌医院内感染流行的分子机制研究

目的研究耐亚胺培南鲍曼不动杆菌的耐药谱特征及其医院内感染流行和耐药性产生的分子机制,为临床防治提供依据.方法4株耐亚胺培南鲍曼不动杆菌分离自2002年10月至2003年1月外科重症监护病房的感染患者,采用纸片扩散法及E-test进行药物敏感性检测及MIC值测定,肠杆菌科基因组内重复一致序列聚集合酶链反应(ERIC-PCR)进行克隆株的DNA分型,耐药质粒转移及消除试验、等电聚焦电泳、PCR扩增β-内酰胺酶基因及其克隆测序以识别其耐药基因和进行质粒定位.结果4株菌除对头孢哌酮/舒巴坦复合制剂的MIC值较低外,对头孢菌素类、氨基糖甙类和氟喹喏酮类等抗生素均显示出了较高水平的多重耐药性;DNA分型证实为同一克隆株;产OXA-23型碳青霉烯酶和PER-1型超广谱β-内酰胺酶(ESBLs);OXA-23定位在质粒上,PER-1定位在染色体上.结论本组耐亚胺培南鲍曼不动杆菌为多重耐药株,同一克隆株在不同感染个体间的相互传播导致了本次医院内感染的流行,产OXA-23和PER-1型β-内酰胺酶是其耐药性产生的重要原因.     

A molecular piston mechanism of pumping protons by bacteriorhodopsin

Summary In this review the proton-pumping mechanism proposed recently for bacteriorhodopsin [Chou, K. C. (1993) Journal of Protein Chemistry, 12: 337–350] is illustrated in terms of a phenomenological model. According to the model, the-ionone of the retinal chromophore in bacteriorhodopsin can be phenomenologically imagined as a molecular piston. The photon capture by bacteriorhodopsin would pull it up while the spontaneous decrease in potential energy would push it down so that it would be up and down alternately during the photocycle process. When it is pulled up, the gate of pore is open and the water channel for the proton translocation is through; when it is pushed down, the gate of pore is closed and the water channel is shut up. Such a model not only is quite consistent with experimental observations, but also provides useful insights and a different view to elucidate the protonpumping mechanism of bacteriorhodopsin. The essence of the model might be useful in investigating the mechanism of ion-channels of other membrane proteins.Abbreviations bR bacteriorhodopsin - All-trans bR bacteriorhodopsin with all-trans retinal chromophore - 13-cis bR bacteriorhodopsin with 13-cis retinal chromophore - All-trans bundle the 7-helix bundle in the all-trans bR - 13-cis bundle the 7-helix bundle in the 13-cis bR - rms root-mean-square     

A molecular mechanism for proton-dependent gating in KcsA

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. [1] identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.     

A non-receptor-mediated mechanism for internalization of molecular chaperones

The evolving realization that stress proteins, which have for many years been considered to be exclusively intracellular molecules under normal conditions, can be released from viable cells via a number of potential routes/pathways has prompted interest into their extracellular biology and intercellular signaling properties. That the stress proteins Hsp60, Hsp70 and gp96 can elicit both pro- and anti-inflammatory effects suggests that these molecules play a key role in the maintenance of immunological homeostasis, and a better understanding of the immunobiology of extracellular stress proteins might reveal new and more effective approaches for controlling and managing infectious disease, inflammatory disease and cancer. A number of cell surface receptors for stress proteins have been identified, and the intracellular consequences of these cell surface receptor-ligand interactions have been characterized. To date, studies into the intercellular signaling properties of stress proteins and their interactions with antigen presenting cells have focused on specific receptor-mediated uptake, and have not considered the fact that such cells can also take up proteins via non-specific endocytosis/pinocytosis. Herein we present a methodological approach for assessing receptor-mediated and non-receptor-mediated uptake of gp96 by rat bone marrow-derived dendritic cells.     

A molecular mechanism for bacterial susceptibility to zinc

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.     

Synaptic Plasticity: A molecular mechanism for metaplasticity

The results of expressing a constitutive form of a prominent synaptic kinase in transgenic mice suggest how there can be a sliding threshold for synapse modification, an important element in some learning theories.     

A new mechanism in plant engineering: The potential roles of microRNAs in molecular breeding for crop improvement

MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs that negatively modulate the expression of genes by inhibiting translation or by promoting the degradation of target mRNAs. miRNAs are now known to have greatly expanded roles in a variety of plant developmental processes, in signal transduction, and in the response to environmental stress and pathogen invasion. Because of their ability to inactivate either specific genes or entire gene families, artificial miRNAs function as dominant suppressors of gene activity when brought into a plant. Consequently, miRNA-based manipulations have emerged as promising new approaches for the improvement of crops. This includes the development of breeding strategies and the genetic modification of agronomic traits. Herein, we highlight new findings regarding the roles of miRNAs in plant traits, and describe the current miRNA-based plant engineering approaches. Finally, we consider the feasibility of modulating current approaches to address future challenges such as breeding programs to increase crop yield.