Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Stem cell niches: famished paneth cells, gluttonous stem cells

Adult tissue stem cells adjust to environmental changes. A new study in the?mouse intestine reveals that caloric restriction causes Paneth cells to repress mTORC1 signaling; this in turn stimulates proliferation of neighboring stem cells.     

Mucosubstance histochemistry of Brunner's glands, pyloric glands and duodenal goblet cells in the ferret

The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods. The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man. The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomucins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Mucosubstance histochemistry of Brunner's glands,pyloric glands and duodenal goblet cells in the ferret

Summary The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods.The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man.The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomycins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.     

Noise-driven stem cell and progenitor population dynamics

Background

The balance between maintenance of the stem cell state and terminal differentiation is influenced by the cellular environment. The switching between these states has long been understood as a transition between attractor states of a molecular network. Herein, stochastic fluctuations are either suppressed or can trigger the transition, but they do not actually determine the attractor states.

Methodology/Principal Findings

We present a novel mathematical concept in which stem cell and progenitor population dynamics are described as a probabilistic process that arises from cell proliferation and small fluctuations in the state of differentiation. These state fluctuations reflect random transitions between different activation patterns of the underlying regulatory network. Importantly, the associated noise amplitudes are state-dependent and set by the environment. Their variability determines the attractor states, and thus actually governs population dynamics. This model quantitatively reproduces the observed dynamics of differentiation and dedifferentiation in promyelocytic precursor cells.

Conclusions/Significance

Consequently, state-specific noise modulation by external signals can be instrumental in controlling stem cell and progenitor population dynamics. We propose follow-up experiments for quantifying the imprinting influence of the environment on cellular noise regulation.     

Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G(1)-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.     

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.     

The progenitor cells of the embryonic telencephalon and the neonatal anterior subventricular zone differentially regulate their cell cycle

For the last 10 years our laboratory has been studying the proliferation, migration and differentiation of neuronal progenitor cells located in the anterior part of the postnatal forebrain subventricular zone (SVZa). SVZa-derived cells possess a number of proliferative characteristics that distinguish them from the other progenitor cells in the central nervous system. This review summarizes our recent findings, in which we compared the pattern of cell cycle inhibitory proteins expressed by the neonatal SVZa to that of telencephalic ventricular zone cells.     

Solution structure of cryptdin-4, a mouse paneth cell alpha-defensin

Mammalian defensins are abundant antimicrobial peptides that contribute to host defense. They are characterized by several conserved amino acids, including six invariant cysteine residues which form three intramolecular disulfide bonds and stabilize the tertiary structure. Cryptdin-4 (Crp4), a mouse alpha-defensin with potent in vitro bactericidal activity, has a primary structure distinct from all known alpha-defensins in that its polypeptide backbone uniquely lacks three residues between Cys(IV) and Cys(V). NMR diffusion experiments showed that Crp4 is monomeric in solution, and its three-dimensional solution structure, determined by two-dimensional proton NMR, consists of a triple-stranded antiparallel beta-sheet with the beta-strands joined to each other by a series of tight turns and a beta-hairpin. However, the overall beta-sheet content in Crp4 is lower than that of other alpha-defensin structures, while the shape and orientation of the Crp4 beta-hairpin also differ from those of other alpha-defensin structures. These structural characteristics combined with the high overall cationicity of Crp4 may contribute to its broad bactericidal spectrum and membrane disruptive activity.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue

Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H³TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H³TdR exposure indicates that 55% of total CFC's in adult marrow are in G₀ or G₁ with a volume of 410 μ³, 42% are in S phase and of volume 450–950 μ³, and the remainder are in G₂ and mitosis with a volume of between 600–950 μ³. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G₂ component. H³TdR exposure confirmed the non-cycling status of these fetal progenitor cells.     

FGF10 maintains distal lung bud epithelium and excessive signaling leads to progenitor state arrest,distalization, and goblet cell metaplasia

Background  

Interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and Fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. The fibroblast growth factor 10 (Fgf10) null mouse is characterized by the absence of lung bud development. Previous studies have shown that this requirement for Fgf10 is due in part to its role as a chemotactic factor during branching morphogenesis. In other endodermal organs Fgf10 also plays a role in regulating differentiation.     

The cell cycle,cell death,and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11–12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.