Predictions of the somatic mutation and mortalization theories of cellular ageing are contrary to experimental observations

An accumulation of recessive lethal somatic mutations has often been proposed as a basis of cellular ageing. We have developed a mathematical model for the somatic mutation theory as applied to the finite in vitro lifespan of diploid fibroblast populations. Provided the mutation rate is sufficiently high, the model readily explains the cessation of proliferation of fibroblast cultures, but it predicts a much lower proportion of viable cells than is observed experimentally and also requires an unrealistically short cell division time. It is noted that the somatic mutation model is formally quite similar to the “mortalization” theory of Shall & Stein (1979), and that the mortalization theory is also incompatible with the same experimental data. We conclude that neither the somatic mutation theory nor the mortalization theory can explain the observed features of the growth of fibroblast populations in vitro. We discuss the possibility that deleterious mutations become important in the terminal stages of the lifespan, when they may accumulate as an indirect result of a general breakdown in information transfer between macromolecules.     

Targeting telomerase via its key RNA/DNA heteroduplex

Telomerase is a promising "universal" anticancer target. It has been demonstrated that inhibition of telomerase leads to mortalization and death of previously immortal cell lines. We are interested in targeting telomerase by binding to the RNA/DNA duplex that forms during its catalytic cycle. The RNA strand of this duplex is a component of telomerase and acts as a template to direct the synthesis of the single-stranded DNA telomere. We have hypothesized that molecules that bind to this duplex will inhibit the enzyme by either preventing strand dissociation or by sufficiently distorting the substrate, thereby causing a misalignment of key catalytic residues. To test this hypothesis we have examined the activity of telomerase in the presence of a range of intercalating molecules, known for their broad duplex binding properties. Of the nine compounds we examined, four show promising lead activity in the low micromolar range. A kinetic analysis of the telomeric products suggests that these compounds do not act by stabilizing G-quartets, thereby supporting the telomeric RNA/DNA heteroduplex as the site of action. We anticipate using these lead compounds as the basis for combinatorial variation to increase the affinity and specificity for the target telomerase.     

A bimolecular mechanism for the cell size control of the cell cycle

A molecular model for the control of cell size has been developed. It is based on two molecules, one (I) acts as an inhibitor of the entrance into S phase, and it is synthetised just after cell separation in a fixed amount per nucleus. The other (A) is an activator of the S phase, and it is synthetised at a ratio proportional to the overall protein accumulation. The activator reacts stoichiometrically with (I), and after all the (I) molecules have been titrated, (A) begins to accumulate. When it reaches a threshold value, it triggers the onset of DNA replication. This model was tested by simulation and when applied to the case of unequal division explains a number of features of an exponentially growing yeast cell population: (a) the lengths of TP (cycle time of parent cells) and TD (cycle time of daughter cells) verify the condition exp(- KTP ) + exp(- KTD ) = 1; (b) the changes of the average cell size of populations at different growth rates; (c) the frequency of parents and daughters at various growth rates; (d) the increase of cell size at bud initiation for cells of increasing genealogical age; (e) the existence of a TP - TB period (difference between the cycle time of parents and the length of budded phase) that depends linearly upon the doubling time of the population.     

Characterization of the cell of origin for small cell lung cancer

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.     

Characterization of the cell of origin for small cell lung cancer

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.Key words: Rb, p53, SCLC, cell of origin, cancer, lung, neuroendocrine     

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

Bacteria are surrounded by a complex cell envelope made up of one or two membranes supplemented with a layer of peptidoglycan (PG). The envelope is responsible for the protection of bacteria against lysis in their oft‐unpredictable environments and it contributes to cell integrity, morphology, signaling, nutrient/small‐molecule transport, and, in the case of pathogenic bacteria, host–pathogen interactions and virulence. The cell envelope requires considerable remodeling during cell division in order to produce genetically identical progeny. Several proteinaceous machines are responsible for the homeostasis of the cell envelope and their activities must be kept coordinated in order to ensure the remodeling of the envelope is temporally and spatially regulated correctly during multiple cycles of cell division and growth. This review aims to highlight the complexity of the components of the cell envelope, but focusses specifically on the molecular apparatuses involved in the synthesis of the PG wall, and the degree of cross talk necessary between the cell division and the cell wall remodeling machineries to coordinate PG remodeling during division. The current understanding of many of the proteins discussed here has relied on structural studies, and this review concentrates particularly on this structural work.     

Non-hormonal cell types in the pituitary candidating for stem cell

Hormone balances in the body are primarily governed by the hypothalamus-pituitary system. For its pivotal role, the pituitary gland relies on an assortment of different hormone-producing cell types, the proportions of which dynamically change in response to fluctuating endocrine demands. Mechanisms of pituitary cellular plasticity are at present far from understood, and may include proliferation and transdifferentiation of hormonal cells. Whether new cells also originate by recruitment from stem cells is unsettled, although this idea has frequently been proposed. Here, I will review these data by focusing on the non-hormonal cell types that have been advanced as candidates for the pituitary stem cell position.     

Restorative cell therapy for Parkinson's disease: a quest for the perfect cell

The development of a cell therapy for the neurodegenerative disorder Parkinson's disease is a realistic ambition. It is pursued by researchers and companies alike, and spans different donor tissue types of embryonic, fetal and adult origins. In this review, we briefly outline the past and current status of research and clinical trials with cell transplantation in Parkinson's disease. We discuss studies on donor tissue derived from embryonic ventral mesencephalon and assess the current research on various forms of stem cells of both embryonic and adult origins in the quest to develop a cell-based therapy for this debilitating movement disorder.     

Circuit maps for the cell

An assessment of the interactions between different receptor-mediated cellular signaling pathways may have revealed an unexpected degree of underlying compartmentalization and even simplicity in the cross-talk between these networks.     

Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism

The production of testosterone occurs within the Leydig cells of the testes. When production fails at this level from either congenital, acquired, or systemic disorders,the result is primary hypogonadism. While numerous testosterone formulations have been developed, none are yet fully capable of replicating the physiological patterns of testosterone secretion. Multiple stem cell therapies to restore androgenic function of the testes are under investigation. Leydig cells derived from bone marrow, adipose tissue, umbilical cord, and the testes have shown promise for future therapy for primary hypogonadism. In particular, the discovery and utilization of a group of progenitor stem cells within the testes, known as stem Leydig cells(SLCs), has led not only to a better understanding of testicular development, but of treatment as well. When combining this with an understanding of the mechanisms that lead to Leydig cell dysfunction, researchers and physicians will be able to develop stem cell therapies that target the specific step in the steroidogenic process that is deficient. The current preclinical studies highlight the complex nature of regenerating this steroidogenic process and the problems remain unresolved. In summary, there appears to be two current directions for stem cell therapy in male primary hypogonadism. The first method involves differentiating adult Leydig cells from stem cells of various origins from bone marrow, adipose, or embryonic sources. The second method involves isolating, identifying, and transplanting stem Leydig cells into testicular tissue. Theoretically, in-vivo re-activation of SLCs in men with primary hypogonadism due to age would be another alternative method to treat hypogonadism while eliminating the need for transplantation.     

Using cell engineering and omic tools for the improvement of cell culture processes

Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the ‘omic’ technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.     

The cell wall: a carbohydrate armour for the fungal cell

The cell wall is composed of a polysaccharide-based three-dimensional network. Considered for a long time as an inert exoskeleton, the cell wall is now seen as a dynamic structure that is continuously changing as a result of the modification of culture conditions and environmental stresses. Although the cell wall composition varies among fungal species, chemogenomic comparative analysis have led to a better understanding of the genes and mechanisms involved in the construction of the common central core composed of branched beta1,3 glucan-chitin. Because of its essential biological role, unique biochemistry and structural organization and the absence in mammalian cells of most of its constitutive components, the cell wall is an attractive target for the development of new antifungal agents. Genomic as well as drug studies have shown that the death of the fungus can result from inhibition of cell wall polysaccharide synthases. To date, only beta1,3 glucan synthase inhibitors have been launched clinically and many more targets remain to be explored.     

Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures

Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34(+) cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (q(gluc), in micromoles per cell per hour) and lactate generation rates (q(lac)) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum q(gluc) and q(lac) in cultures with different seeding densities and cytokine combinations. A two-population model (Q(lac) = alpha[CFC] + beta([TC] - [CFC ]), where [TC] is total cell concentration; Q(lac) is volumetric lactate production rate in micromoles per milliliter per hour; alpha is q(lac) for an average CFC; and beta is q(lac) for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34(+) cells. The values for alpha and beta that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34(+) cell content have similar values for beta and beta. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.     

Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth

Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.     

干细胞治疗糖尿病的研究进展

糖尿病是严重危害人类健康的疾病,近年来,随着干细胞理论及技术的发展,很多科学家都致力于应用干细胞治疗糖尿病,并取得了显著成效。干细胞移植治疗糖尿病的关键是干细胞的来源和定向诱导分化的手段,该文简要综述了目前在这些方面取得了诸多成果以及存在的问题。     

Haptoglobin for the treatment of sickle cell disease

There are three haptoglobin phenotypes in humans designated: Hp1–1, Hp2–1, and Hp2–2. The Hp1–1 phenotype has been shown to be protective against certain diseases, and this has been suggested to be the result of better anti-inflammatory and antioxidative properties compared to haptoglobin polymers of the other phenotypes when clearing cell-free haemoglobin. We propose the use of haptoglobin for the treatment of sickle cell disease, where an oxidative state exists caused by a high level of cell-free haemoglobin. A significant number of sickle cell disease patients are severely affected and experience regular acute painful episodes resulting in hospitalisation.Therapeutic treatments for sickle cell disease are limited and therefore haptoglobin could represent a vital alternative therapy. A method has been developed as part of the commercial fractionation of plasma for preparing haptoglobin enriched for dimers. This is significant as it uses a mixture of plasma of all haptoglobin phenotypes, and allows annual production of hundreds of kilograms quantities of haptoglobin that may be required to allow treatment of thousands of sickle cell disease patients worldwide.     

Thousands of rab GTPases for the cell biologist

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.