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Cell biology of antigen processing in vitro and in vivo
The conversion of exogenous and endogenous proteins into immunogenic peptides recognized by T lymphocytes involves a series of proteolytic and other enzymatic events culminating in the formation of peptides bound to MHC class I or class II molecules. Although the biochemistry of these events has been studied in detail, only in the past few years has similar information begun to emerge describing the cellular context in which these events take place. This review thus concentrates on the properties of antigen-presenting cells, especially those aspects of their overall organization, regulation, and intracellular transport that both facilitate and modulate the processing of protein antigens. Emphasis is placed on dendritic cells and the specializations that help account for their marked efficiency at antigen processing and presentation both in vitro and, importantly, in vivo. How dendritic cells handle antigens is likely to be as important a determinant of immunogenicity and tolerance as is the nature of the antigens themselves.
Two-Step Regulation of a Meristematic Cell Population Acting in Shoot Branching in Arabidopsis
Shoot branching requires the establishment of new meristems harboring stem cells this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.
Conflict of interest statement
The authors have declared that no competing interests exist.
Fig 1. Existence of a meristematic cell…
Fig 1. Existence of a meristematic cell population with a fixed developmental fate in leaf…
Fig 2. AM initiation requires STM -expressing…
Fig 2. AM initiation requires STM -expressing cells.
(A) Schematic representation of axillary bud formation in…
Fig 3. Low level STM expression is…
Fig 3. Low level STM expression is required for meristematic cell identity and axillary buds…
Fig 4. STM expression in rev-6 and…
Fig 4. STM expression in rev-6 and REV expression in the leaf axil.
Fig 5. Direct up-regulation of STM expression…
Fig 5. Direct up-regulation of STM expression by REV.
(A) RT-qPCR analysis of STM expression…
Fig 6. Epigenetic modification of the STM…
Fig 6. Epigenetic modification of the STM locus.
(A) RT-PCR with primers amplifying the REV…
Fig 7. Conceptual model showing meristematic cells…
Fig 7. Conceptual model showing meristematic cells maintenance and up-regulation during AM initiation.
Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.
Giardia lamblia, like most human intestinal parasitic protozoa, sustains fundamental morphological and biochemical changes to survive outside the small intestine of its mammalian host by differentiating into an infective cyst. However, the stimulus that triggers this differentiation remains totally undefined. In this work, we demonstrate the induction of cyst formation in vitro when trophozoites are starved for cholesterol. Expression of cyst wall proteins was detected within encystation-specific secretory vesicles 90 min after the cells were placed in lipoprotein-deficient TYI-S-33 medium. Four cloned lines derived from two independent Giardia isolates were tested, and all formed cysts similarly. Addition of cholesterol, low density or very low density lipoproteins to the lipoprotein-deficient culture medium, inhibited the expression of cyst wall proteins, the generation of encystation-specific vesicles, and cyst wall biogenesis. In contrast, high density lipoproteins, phospholipids, bile salts, or fatty acids had little or no effect. These results indicate that cholesterol starvation is necessary and sufficient for the stimulation of Giardia encystation in vitro and, likely, in the intestine of mammalian hosts.
How do non-differentiating bacteria adapt to starvation?
Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.
The signals for starvation response are transduced through elevated [Ca2+]i in Dictyostelium cells
The mechanism by which cells recognize starvation to allow subsequent cellular development was analyzed using Dictyostelium discoideum, with special emphasis on Ca2+ as a crucial signal transducer in intra- and intercellular communications. As was expected, the cytosolic Ca2+ concentration ([Ca2+]i) in aequorin-expressing cells (RHI76 derived from D. discoideum Ax-3) was temporarily increased, when 3-5 microM thapsigargin (Tg), a specific inhibitor of the Ca(2+)-ATPase, was added into the cells incubated in semistarvation medium (SS-medium: 1 vol of growth medium plus 7 vol either of 20 mM Na2/K-phosphate buffer (pH 6.2) or of Bonner's salt solution (BSS)). Essentially the same result was obtained by the application of 5 microM nigericin (Ng), an acid ionophore to cells under the semistarved condition. Here it is of interest to note that in the SS-medium Tg and Ng are capable of enhancing cell differentiation as exemplified well by the earlier acquisition of chemotactic response to cAMP, possibly inducing the starvation response through the [Ca2+]i increase. From Western blot analysis of phosphotyrosine (pTyr)-containing proteins using anti-pTyr antibody, it was found that the pTyr-phosphorylation levels of 97-, 80-, and 45-kDa proteins increase specifically in response to starvation. Interestingly, Tg and Ng induced such a change of the 80-kDa protein in the cells incubated in the SS-medium. Taken together these results strongly suggest that the temporal increase of [Ca2+]i may be a matter of importance for signal transduction coupled with starvation response.
Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells
Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.
Keywords: Dulbecco's modified Eagle's medium (DMEM) cellular protein expression opti-MEM serum starvation transfection.
© 2018 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
Cell Differentiation in Dictyostelium discoideum
We have shown previously that amoebae of D. discoideum strain V12 M2 starved at low density in the presence of cyclic AMP fail to form either stalk cells or prespore cells a low molecular weight factor released by cells at high density promotes stalk formation under these conditions but formation of prespore cells requires ‘cell contact’. Here we summarise evidence that:
Elevated intracellular cyclic AMP levels are required for all developmental gene expression beyond the preaggregative phase, and ammonia antagonises this expression in some way. However, the action of ammonia is not pathway specific.
‘Cell contact’ is a specific requirement for entry into the prespore pathway of gene expression since isolated cells provided with cyclic AMP synthesise much reduced amounts of the presporespecific enzyme uridine diphosphate (UDP) galactose polysaccharide transferase but normal amounts of the pathway-indifferent enzyme glycogen phosphorylase.
The ‘cell contact’ mechanism is uniquely sensitive to low concentrations of pronase. This protease selectively inhibits transferase synthesis and blocks in vitro spore differentiation (in a spore-forming mutant). It does not prevent chemotactic aggregation, stream formation, or stalk cell formation in the presence of cyclic AMP.
Differential regulation of NM23-H1 under hypoxic and serum starvation conditions in metastatic cancer cells and its implication in EMT
Multiple stresses are prevalent inside the tumor microenvironment rendering tumor growth, neighboring invasion and metastasis of the cancer cells to distant organs. NM23-H1 is the first metastasis suppressor gene identified and known to be implicated as an important regulator of stress-induced metastasis. Herein, we demonstrated that prototypical NM23-H1 expression diminished during hypoxia and serum starvation in Panc-1/MDA-MB-231 cells, but converse invasion patterns were obtained in these two diverse stresses. Supportingly, a compelling discrete difference in mRNA and protein levels of NM23-H1 was achieved in hypoxia as well as serum starvation. Knockdown of NM23-H1 activates EMT whereas the similar effects are subdued in serum starvation where NM23-H1 down-modulation prompted E-cadherin upregulation. Stable NM23-H1 expression augmented E-cadherin levels along with retardation in invadopodea formation and invasion. In hypoxia/serum starvation excess NM23-H1 effectively modulated the Twist1 promoter activity. Thus, differential regulation of NM23-H1 may corroborate/abrogate EMT depending on the nature of stress, tumor microenvironment and cellular context.
Keywords: E-cadherin EMT Hypoxia NM23-H1 Panc-1 Serum-starvation TME.
(ROS). A reactive intermediate oxygen species that usually contains an unpaired electron in its outermost shell of electrons. Examples include: superoxide anion (O2 •− ), hydroxyl radical (OH • ), hydroxyl ion (OH − ), hydrogen peroxide (H2O2) and hypochlorite ion (OCl − ).
The electron-transport chain comprises complexes I–IV and an ATPase, which couples the transfer of an electron from NADH or FADH to molecular oxygen (O2). This process pumps protons (H + ) across the mitochondrial membrane, which generates a proton gradient, the energy of which drives ATP synthesis by the ATPase.
An enzyme and chromatin-remodelling factor that deacetylates histone proteins. Because deacetylation is energetically favoured, the class-I and -II deacetylases do not require any cofactors, such as ATP or NAD. However, the Sir2 class-III histone deacetylase uses NAD as a co-substrate.
One of the cleavage products of nicotinamide adenine dinucleotide (NAD) that is produced in each deacetylation cycle (along with acetyl-ADP-ribose). It is also a non-competitive inhibitor of Sir2 deacetylases.
A protein that has sequence homology to the conserved enzymatic domain of yeast Sir2. In mammals, there are seven sirtuins, SIRT1–7.
FORKHEAD TRANSCRIPTION FACTOR
A member of a conserved family of transcription factors. Forkhead transcription factors belong to the winged-helix class of DNA-binding proteins. They function as key regulators of stress resistance and metabolism in mammals and of lifespan in C. elegans.
(WAT). A depot of specialized cells that function as the main storage site for fat in the form of triglycerides. WAT has different functions, such as a heat insulator, a mechanical cushion and, most importantly, a source of energy. Also, WAT has been found to produce hormones, the levels of which are regulated by the amount of stored fat.
A state of abnormally high levels of insulin in the blood. One cause of hyper-insulinaemia is high insulin production by pancreatic β-cells to compensate for insulin resistance in peripheral tissues.
A state of high levels of glucose in the blood. Hyperglycaemia is usually an early sign of insulin resistance, which can lead to diabetes mellitus.
A strain of mice that carries a spontaneous dominant mutation in the Wld locus that stabilizes the axons of peripheral neurons from degeneration after neuronal damage. The Wld locus is at a translocation that fuses the gene encoding ubiquitin fusion degradation protein 2a (UFD2a), a ubiquitin-chain-assembly factor, to the complete sequence of the nicotinamide mononucleotide adenyl-transferase-1 (Nmnat1) gene. The resulting increase in NMNAT1 expression causes the phenotype.
(UCPs). A family of mitochondrial-inner-membrane proteins. They allow the leakage of protons down the electrochemical gradient that is generated by respiration, thereby uncoupling oxygen consumption and ATP synthesis from the production of chemical energy.
A depot of fat cells that is used for thermogenesis in rodents, brown adipose tissue derives its colour from rich vascularization and densely packed mitochondria these properties reflect its high metabolism of fat in response to cold exposure.