Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins,including nitrated proteins

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.     

Factors influencing immunity and resistance of Pediococcus acidilactici to the bacteriocin, pediocin AcH

In pediocin AcH producing Pediococcus acidilactici strains the genes for both the production of pediocin and immunity against it are encoded in an 8.9 kb plasmid pSMB74. Following loss of this plasmid, the variants lost the ability to produce pediocin AcH, but some retained the resistance against it. This resistance was a transient trait, acquired while nonproducing cells grew in the presence of pediocin AcH but lost when the cells were grown in the absence of it.     

Hearing Loss and Hair Cell Death in Mice Given the Cholesterol-Chelating Agent Hydroxypropyl-β-Cyclodextrin

Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, 2-hydroxypropyl-β-cyclodextrin (HPβCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPβCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPβCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology.     

Genetic transformation of somatic cells. VI. Transfer of the trait of the rate of loss of transformant phenotype by means of DNA from cells containing the thymidine kinase gene of the herpes simplex virus

Using dot-hybridization with thymidine kinase gene (tk gene) of Herpes simplex virus type 1 (HSV 1) of DNA preparations obtained from isolated metaphase chromosomes and lysate fractions of metaphase cells, which presumably contain smaller particles compared to metaphase chromosomes, it has been shown that the tk gene of HSV 1 is localized in chromosomes of cells of transformant clones unstable in TK+-phenotype. The DNA isolated from the metaphase chromosomes from cells of transformant clones is 1.5- or 2-fold more efficient in transforming TK-Chinese hamster cells than is the total high molecular weight DNA from the same cells. Upon transformation of TK- cells by the high molecular weight DNA from the tk gene of HSV 1-containing clones, varying in the rate of the loss of TK+-phenotypes, the character "rate of the loss of transformant phenotype" is transferred together with the tk gene of HSV 1 in 22% of cases. Cells of rerevertant clones, produced from TK- subclones of transformant clones, display the rate of the loss of transformant phenotype characteristic of cells of parental TK+-clones. A comparison of the results allows a conclusion that DNA sequences, determining the character "rate of the loss of transformant phenotype", are linked tightly with the transforming DNA proper containing the tk gene of HSV 1, but are not localized inside such a DNA.     

The influence of the genotype on the process of ageing of chick lens cells in vitro

We reported previously that changes in crystallin expression in differentiating long-term primary cultures of lens cells from five different chick genotypes are similar to those which occur in vivo between hatching and the 8-week-old adult. These changes followed a similar program in all genotypes but occurred more rapidly in cells from the fast-growing than from the slow-growing genotypes. The present study examines ageing changes in lens cell populations from the same five genotypes, over a 4-6 month period, using long-term serial subcultures. The capacity for lentoid differentiation was progressively lost, but the rate of loss was inversely related to the intrinsic growth rate of the cells of these genotypes, occurring at the first passage in the slowest-growing strain, while fifth passage cells of the fastest-growing strain still retained some lentoid-forming capacity. The rate of loss of crystallin expression was also inversely related to the genetic growth rate, but the sequence of changes appears to be nonrandom, since it was broadly similar in all genotypes, starting with a preferential loss of delta-crystallin, as occurs in vivo; although alpha- and beta-crystallins were undetectable in late dedifferentiated cultures, the capacity of the cells for their synthesis was still present. Cultures from both fast-growing genotypes eventually showed senescence, but those from all three slow-growing genotypes underwent transformation. The major cell component in late cultures of all genotypes was actin.     

Exploitation of the HIV-1 coat glycoprotein, gp120, in neurodegenerative studies in vivo

Neuronal loss has often been described at post-mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which lead neurones to death. In particular, the HIV-1 coat glycoprotein, gp120, has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein, and that this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV-associated dementia.     

Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential

Human plasma has been demonstrated to contain factors that induce the sequential expression of nonterminal and terminal adipocyte differentiation in 3T3 T mesenchymal stem cells. We now report the development of methods for the isolation of purified populations of nonterminally differentiated cells and terminally differentiated cells, and we show that it is possible to experimentally induce transition from the nonterminal to the terminal state of differentiation. With this model system it is therefore now possible to examine the biological and molecular processes associated with the terminal event in differentiation, i.e., the irreversible loss of proliferative potential. In this regard, we demonstrate that transition from the nonterminal to terminal state of differentiation is a complex metabolic process that consists of at least two steps and that this process can be triggered by pulse exposure to an inducer for approximately 12 h but that approximately 24-48 h is required for the process to be completed. The data also establish that induction of the terminal event in differentiation requires protein synthesis but not RNA and DNA synthesis. These and additional results suggest that loss of proliferative potential associated with the terminal event in cellular differentiation is a distinct regulatory process, and we suggest that defects in this regulatory process may be of etiological significance in the pathogenesis of specific human diseases, especially cancer.     

Neuroglia in the inferior olivary nucleus during normal aging and Alzheimer's disease

It is likely that neuronal loss occurs in certain brain regions in Alzheimer's Disease (AD) without any neurofibrillary pathology. In the human principle inferior olivary nucleus (PO), we have shown that neuronal loss is about 34% (Lasn et al. Journal of Alzheimer Disease, 2001; 3: 159-168), but the fate of the neuroglial cells is unknown. Since the unique network of neurons and neuroglial cells and their cohabitation are essential for normal functioning of CNS, we designed a study to estimate the total number of oligodendrocytes and astrocytes in normally aged and AD brains. The study is based on 10 control and 11 AD post-mortem human brains. An unbiased stereological fractionator method was used. We found significant oligodendroglial cell loss (46%) in AD as compared to control brains, while the total number of astrocytes showed a tendency to decrease. It is likely that the ratio of oligodendroglial cells to neurons remains unchanged even in degenerative states, indicating that oligodendroglial cells parallel neuronal loss. Astroglial cells did not increase in total number, but the ratio to neurons was significantly increased due to the neuronal loss. Using a novel unbiased quantitative method, we were able to describe significant oligodendroglial loss in the PO but the pathogenic mechanism behind remains unknown.     

p73 suppresses polyploidy and aneuploidy in the absence of functional p53

Previous studies showed that p53 plays a central role in G1 and DNA damage checkpoints, thus contributing to genomic stability. We show here that p73 also plays a role in genomic integrity but this mechanism is manifest only when p53 is lost. Isolated p73 loss in primary cells does not induce genomic instability. Instead, it results in impaired proliferation and premature senescence due to compensatory activation of p53. Combined loss of p73 and p53 rescues these defects, but at the expense of exacerbated genomic instability. This leads to rapid increase in polyploidy and aneuploidy, markedly exceeding that of p53 loss alone. Constitutive deregulation of cyclin-Cdk activities and excess failure of the G2/M DNA damage checkpoint appear to fuel increased ploidy abnormalities upon p53/p73 loss, while primary mitotic defects do not play a causal role. These data indicate that p73 is essential for suppressing polyploidy and aneuploidy when p53 is inactivated.     

The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl's effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia.     

Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum

The sexual development, macrocyst formation, of Dictyostelium discoideum is initiated by sexual fusion of cells. The sexual fusion is only taken place under the culture conditions of excess water and darkness. Under these conditions, cells acquire the fusion competence, but lose it when cell density is high. The loss of the fusion competence is caused by accumulation of ammonia excreted by cells in a culture. Ammonia suppresses the fusion competence of cells at a certain concentration, and consequently inhibits formation of macrocysts and induces fruiting-body formation. Thus, excess water induces the sexual development by diluting ammonia and lack of water induces the asexual development.     

A low-molecular-weight factor which stimulates loss of EDTA-resistant cell cohesion in Dictyostelium

Abstract. During dedifferentiation, mutant HI4 of Dictyostelium discoideum loses the capacity to release and respond chemotactically to cyclic adenosine monophosphate (cAMP) in a normal fashion, but it retains EDTA-resistant cohesiveness, the membrane glycoprotein gp80, and the capacity to reaggregate rapidly by non chemotactic means [21]. In wild-type cells, these last three characteristics are lost at prescribed times in the normal program of dedifferentiation. In the present study, we tested whether dedifferentiating wild-type cells or medium conditioned by dedifferentiating wild-type cells will stimulate the loss of EDTA-resistant cohesiveness in dedifferentiating mutant cells. Results are presented which demonstrate that wild-type cells release a factor into the medium which causes the loss of EDTA-resistant cohesiveness in mutant cells at the correct time in the dedifferentiation program. The factor is heat sensitive, resistant to lyophilization, and exhibits a molecular weight of less than 5,000 daltons. However, although the factor stimulates the loss of EDTA-resistant cohesiveness, it does not stimulate the loss of the glycoprotein gp80, the membrane molecule implicated in cohesion. Several explanations for these results are proposed.     

Dissecting the contribution of p16(INK4A) and the Rb family to the Ras transformed phenotype

Although oncogenic Ras commonly contributes to the development of cancer, in normal primary cells it induces cell cycle arrest rather than transformation. Here we analyze the additional genetic changes required for Ras to promote cell cycle progression rather than arrest. We show that loss of p53 is sufficient for oncogenic Ras to stimulate proliferation in the absence of extrinsic mitogens in attached cells. However, surprisingly, we find that p53 loss is not sufficient for Ras to overcome anchorage dependence or contact inhibition. In contrast, expression of simian virus 40 (SV40) large T antigen (LT) allows Ras to overcome these additional cell cycle controls. Mutational analysis of SV40 LT shows that this action of SV40 LT depends on its ability to inactivate the retinoblastoma (Rb) family of proteins, in concert with the loss of p53. Importantly, we show that inactivation of the Rb family of proteins can be mimicked by loss of the cyclin-dependent kinase inhibitor p16(INK4A). p16(INK4A) is commonly lost in human tumors, but its contribution to the transformed phenotype is unknown. We demonstrate here a role for p16(INK4A) in the loss of cell cycle controls required for tumorigenesis and show how accumulating genetic changes cooperate and contribute to the transformed phenotype.     

Notch Signaling Mediates the Age-Associated Decrease in Adhesion of Germline Stem Cells to the Niche

Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.     

Maintenance of peritoneal B-1a lymphocytes in the absence of the spleen

Positive selection by autoantigens is believed to play an important role in the generation/maintenance of B-1a cells. Recently, it has been described that splenectomy results in the loss of an already established B-1a cell pool. To elucidate whether the spleen influences the peritoneal B-1a repertoire, we have analyzed the consequences of splenectomy in the recently established IgL-transgenic L2 mouse model. L2 mice are characterized by a severe block of B-2 development and predominance of B-1a cells, which exhibit a pronounced IgH oligoclonality, presumably due to positive selection by autoantigens. In this study, we show that, in striking contrast to splenectomized normal mice, L2 mice exhibit unchanged frequencies of peritoneal B-1a cells. The IgH repertoire of these B-1a cells, however, was severely perturbed in that the previously described predominant B-1a H chains were no longer present. The repertoire changes were partial since phosphatidylcholine-specific B-1a cells were present in similar numbers before and after splenectomy. Thus, splenic Ags appear to act as "survival factors" for major subsets of peritoneal B cells. The loss of B-1a cells in the absence of such factors is compensated by repertoire changes among B-1a cells in B cell lymphopenic L2 but not normal mice.     

Resistance of Erythrocytes of Hibernating Mammals to Loss of Potassium during Hibernation and during Cold Storage

In two species of hibernators, hamsters and ground squirrels, erythrocytes were collected by heart puncture and the K content of the cells of hibernating individuals was compared with that of awake individuals. The K concentration of hamsters did not decline significantly during each bout of hibernation (maximum period of 5 days) but in long-term bouts in ground squirrels (i.e. more than 5 days) the K concentration of cells dropped significantly. When ground squirrels were allowed to rewarm the K content of cells rose toward normal values within a few hours. Erythrocytes of both hamsters and ground squirrels lose K more slowly than those of guinea pigs (nonhibernators) when stored in vitro for up to 10 days at 5°C. In ground squirrels the rate of loss of K during storage is the same as in vivo during hibernation, and stored cells taken from hibernating ground squirrels also lose K at the same rate. The rate of loss of K from guinea pig cells corresponded with that predicted from passive diffusion unopposed by transport. The actual rate of loss of K from ground squirrel cells was slower than such a predicted rate but corresponded with it when glucose was omitted from the storage medium or ouabain was added to it. Despite the slight loss of K that may occur in hibernation, therefore, the cells of hibernators are more cold adapted than those of a nonhibernating mammal, and this adaptation depends in part upon active transport.     

PHLPP1 regulates contact inhibition by dephosphorylating Mst1 at the inhibitory site

Contact inhibition has been largely elusive despite that a loss of contact inhibition is a critical event for cancer development and progression. Here, we report that PHLPP1 is a binding protein for Mst1 and it modulates the Hippo pathway by dephosphorylating Mst1 at the inhibitory Thr³⁸⁷ of Mst1. Yap1 was localized predominantly in the nucleus but marginally in the cytoplasm in HeLa cells under sparse conditions, whereas the functional protein was more directed to sequestration in the cytoplasm under dense environments. Furthermore, loss of PHLPP1 resulted in a failure of the apoptotic control. It is interesting that down-regulated expression of PHLPP1 appears to mimic the loss of contact inhibition, a hallmark of cancer.     

WRN Loss Induces Switching of Telomerase-Independent Mechanisms of Telomere Elongation

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.     

Stabilization of the polarity axis in the zygotes of some Fucaceae

Summary The existence of a period of latent but stable polarity, i.e. a period in which the polarity axis has been irreversibly established but no morphological asymmetry can be detected, was studied in germinating populations of zygotes of Pelvetia fastigiata. We found that the time course of the loss of sensitivity to a single polarity-axis-determining light stimulus coincided with the time course of germination (rhizoid outgrowth), up to the germination of about one-third the population, showing that the cells remained responsive to the light stimulus until or almost until the appearance of visible asymmetry. In the later part of the germination period, some of the zygotes may have lost their light sensitivity somewhat before rhizoid outgrowth, suggesting that at least some of the ungerminated cells may at this time possess, for a brief period, a latent but stable polarity axis.The loss of responsiveness with time to an antagonistic, second light stimulus followed the same time course as the loss of sensitivity to a single light stimulus. There was no suggestion of the existence of a latent but stable polarity axis in any members of the population in this experiment.An analysis of data of Jaffe (1968) on polarity-axis determination and germination in P. fastigiata following a single light stimulus yields essentially the same conclusions as our own single-light-stimulus experiments.In contrast, analysis of data of Whitaker and Lowrance (1936) on Fucus furcatus indicated that in these zygotes latent but stable determination of the polarity axes had taken place 3–4 hours before germination. A similar situation emerges from the analysis of another experiment of Jaffe (personal communication) with P. fastigiata zygotes in which the loss of sensitivity to an orienting light stimulus appeared accelerated and germination may have been delayed in comparison with his 1968 data.We conclude, therefore, that populations of Fucaceae zygotes may vary with regard to the existence of a latent but stable polarity axis. However, when existence of such a latent, stable polarity axis can be inferred, its duration usually is brief, and it seems to be in most cases limited to a small fraction of the individuals of the total population at any particular time. In order to infer rigorously the existence of latent but stable polarity axes in populations of germinating zygotes and similar cells, it is essential to obtain the time courses for axis stabilization and for the development of visible asymmetry simultaneously.     

Not quite dead enough: on bacterial life, culturability, senescence, and death

A number of regulatory networks are functionally integrated in starving cells of Escherichia coli to reduce oxidation of target macromolecules and to enhance the cell's ability to withstand environmental insults. However, despite the fact that starving wild-type E. coli cells enhance their capacity to manage oxidative stress, the proteins of these cells become increasingly oxidized and the cells gradually lose their ability to reproduce. Indeed, it has been argued that starved and growth-arrested bacterial cells show the same signs of senescence as aging cells of higher organisms and that free radicals may be involved in the gradual loss of bacterial culturability observed in a stationary phase culture. Another model suggests that the apparent loss of viability of starved cells is a programmed and adaptive response in which the cells enter a reversible non-culturable state; the theory of the formation of viable but non-culturable cells. Recent data concerning the physiology and biochemistry of starved E. coli cells favor the model that starvation-induced loss of culturability is the result of stochastic deterioration rather than a programmed and adaptive phenomenon, and these data will be reviewed here.