Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

BIDIRECTIONAL AC-DC CONVERTERS USING ONE CYCLE CONTROL (OCC) FOR ELECTRIC VEHICLES

The electric vehicle (EV) market has seen a rapid growth in the recent past. With an increase in the number of electric vehicles on road, there is an increase in the number of high capacity battery banks interfacing the grid. The battery bank of an EV, besides being the fuel tank, is also a huge energy storage unit. Presently, it is used only when the vehicle is being driven and remains idle for rest of the time, rendering it underutilized. Whereas on the other hand, there is a need of large energy storage units in the grid to filter out the fluctuations of supply and demand during a day. EVs can help bridge this gap. The EV battery bank can be used to store the excess energy from the grid to vehicle (G2V) or supply stored energy from the vehicle to grid (V2G ), when required. To let power flow happen, in both directions, a bidirectional AC-DC converter is required. This thesis concentrates on the bidirectional AC-DC converters which have a control on power flow in all four quadrants for the application of EV battery interfacing with the grid. This thesis presents a bidirectional interleaved full bridge converter topology. This helps in increasing the power processing and current handling capability of the converter which makes it suitable for the purpose of EVs. Further, the benefit of using the interleaved topology is that it increases the power density of the converter. This ensures optimization of space usage with the same power handling capacity. The proposed interleaved converter consists of two full bridges. The corresponding gate pulses of each switch, in one cell, are phase shifted by 180 degrees from those of the other cell. The proposed converter control is based on the one-cycle controller. To meet the challenge of new requirements of reactive power handling capabilities for grid connected converters, posed by the utilities, the controller is modified to make it suitable to process the reactive power. A fictitious current derived from the grid voltage is introduced in the controller, which controls the converter performance. The current references are generated using the second order generalized integrators (SOGI) and phase locked loop (PLL). A digital implementation of the proposed control ii scheme is developed and implemented using DSP hardware. The simulated and experimental results, based on the converter topology and control technique discussed here, are presented to show the performance of the proposed theory.

Exploration of the cell-cycle genes found within the RIKEN FANTOM2 data set

The cell cycle is one of the most fundamental processes within a cell. Phase-dependent expression and cell-cycle checkpoints require a high level of control. A large number of genes with varying functions and modes of action are responsible for this biology. In a targeted exploration of the FANTOM2-Variable Protein Set, a number of mouse homologs to known cell-cycle regulators as well as novel members of cell-cycle families were identified. Focusing on two prototype cell-cycle families, the cyclins and the NIMA-related kinases (NEKs), we believe we have identified all of the mouse members of these families, 24 cyclins and 10 NEKs, and mapped them to ENSEMBL transcripts. To attempt to globally identify all potential cell cycle-related genes within mouse, the MGI (Mouse Genome Database) assignments for the RIKEN Representative Set (RPS) and the results from two homology-based queries were merged. We identified 1415 genes with possible cell-cycle roles, and 1758 potential paralogs. We comment on the genes identified in this screen and evaluate the merits of each approach.

BIDIRECTIONAL AC-DC CONVERTERS USING ONE CYCLE CONTROL (OCC) FOR ELECTRIC VEHICLES

The electric vehicle (EV) market has seen a rapid growth in the recent past. With an increase in the number of electric vehicles on road, there is an increase in the number of high capacity battery banks interfacing the grid. The battery bank of an EV, besides being the fuel tank, is also a huge energy storage unit. Presently, it is used only when the vehicle is being driven and remains idle for rest of the time, rendering it underutilized. Whereas on the other hand, there is a need of large energy storage units in the grid to filter out the fluctuations of supply and demand during a day. EVs can help bridge this gap. The EV battery bank can be used to store the excess energy from the grid to vehicle (G2V) or supply stored energy from the vehicle to grid (V2G ), when required. To let power flow happen, in both directions, a bidirectional AC-DC converter is required. This thesis concentrates on the bidirectional AC-DC converters which have a control on power flow in all four quadrants for the application of EV battery interfacing with the grid. This thesis presents a bidirectional interleaved full bridge converter topology. This helps in increasing the power processing and current handling capability of the converter which makes it suitable for the purpose of EVs. Further, the benefit of using the interleaved topology is that it increases the power density of the converter. This ensures optimization of space usage with the same power handling capacity. The proposed interleaved converter consists of two full bridges. The corresponding gate pulses of each switch, in one cell, are phase shifted by 180 degrees from those of the other cell. The proposed converter control is based on the one-cycle controller. To meet the challenge of new requirements of reactive power handling capabilities for grid connected converters, posed by the utilities, the controller is modified to make it suitable to process the reactive power. A fictitious current derived from the grid voltage is introduced in the controller, which controls the converter performance. The current references are generated using the second order generalized integrators (SOGI) and phase locked loop (PLL). A digital implementation of the proposed control ii scheme is developed and implemented using DSP hardware. The simulated and experimental results, based on the converter topology and control technique discussed here, are presented to show the performance of the proposed theory.

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/- mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/- background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/- mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Cell death control : the interplay of apoptosis and autophagy in the pathogenicity of sclerotinia sclerotiorum

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways. © 2008 Elsevier Ltd. All rights reserved.

IFN-gamma inhibits growth of WISH cells in a cell cycle phase-specific manner

Treatment of WISH (human amnion) cells with interferon-gamma (IFN-gamma) inhibits their growth. Release of the cells from IFN-gamma-mediated growth inhibition led to a rapid and significant increase in DNA synthesis, followed by doubling of cell numbers. The DNA synthesis profile was strikingly similar to that shown by WISH cells released from growth arrest by the G(1)/S phase inhibitor, aphidicolin, This strongly suggested that IFN-gamma treatment leads to growth inhibition of WISH cells at the G(1)/S boundary of the cell cycle. In contrast, IFN-alpha blocked growth of these cells at the G(0)/G(1) boundary.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.