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PubMed1976

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: changes in oxidative phosphorylation and synthesis of mitochondrial phospholipids.

Changes in the capacity of mitochondria to carry out oxidative phosphorylation and in the rate of synthesis and incorporation of phospholipids into mitochondria were measured during the germination of conidiospores of Neurospora crassa. The competence of isolated mitochondria to carry out coupled respiration was very low during the first 3 h growth, but it increased rapidly, reaching maximal levels at 5 to 6 h growth. Changes in mitochondrial function were the same in cells grown in 2% sucrose- or 15% glucose-supplemented medium. The rate of synthesis of mitochondrial phospholipids was very low during the first 2 h growth and increased to maximal levels between 3 and 5 h. The rate of synthesis of mitochondrial phospholipids was approximately three times higher in cells grown in 15% glucose than in those grown in 2% sucrose. The maximal rate of synthesis of mitochondrial phospholipids occurred during spore germination and preceded attainment of full competence for oxidative phosphorylation. The lipid-rich condition of the mitochondrial resulting from the high rate of synthesis of phospholipids in glucose-grown cells is postulated to be related to the whorled inclusions observed in thin sections of Neurospora cells.

Antimycin A↗
PubMed1994

Transposon mutagenesis of Nostoc sp. strain ATCC 29133, a filamentous cyanobacterium with multiple cellular differentiation alternatives.

Nostoc sp. strain ATCC 29133 (PCC 73102; Nostoc 29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc 29133. By use of a Tn5 derivative, Tn5-1063 (Km(r)Bm(r)Sm(r)), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc 29133 were generated at a frequency of approximately 1 x 10(-6), 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc 29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc 29133, and the transposition site identified from a Nostoc 29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc 29133.

Conjugation, Genetic↗
PubMed1993

Spatial restriction of cellular differentiation.

Myxococcus xanthus cells differentiate into myxospores within a fruiting body, an aggregate of approximately 10(5) cells. Previous work had discerned an inner and outer domain within the fruiting body differentiated by cell density and cell alignment. To test whether the two domains might play different roles in spore differentiation, developmentally regulated gene fusions were screened for expression restricted to one domain or the other. Transcriptional lacZ fusions to 80 developmentally regulated genes were examined and eight fusions were found that restricted expression to the inner domain, while one fusion, omega 7621, showed initial expression in the outer domain. Initial omega 7621 expression coincided with patches of spore precursors evident in bright-field microscopy. Later in development, both omega 7621 expression and the patches expanded inward, eventually filling both the inner and outer domains. Previous work had also shown that high cell density and cell alignment are required for transmission of the C-signal, which is needed to initiate spore differentiation. Evidence is presented for a novel morphogenetic mechanism in which C-signaling in the outer (high density) domain initiates spore differentiation. It is proposed that spore precursors are passively transported from the outer to the inner domain by the movements of undifferentiated rod cells. Reconstruction experiments showed that developing rod cells move with sufficient force to displace spores. Spore precursors thus accumulate in the inner domain where they express spore-specific genes at high levels and account for inner domain specific expression.

Biological Transport↗
PubMed1994

The DCC gene product in cellular differentiation and colorectal tumorigenesis.

The analysis of human colorectal tumors has revealed frequent loss of heterozygosity (LOH) of the long arm of chromosome 18. A novel gene, DCC (deleted in colorectal cancer), located within the region of LOH on chromosome 18q was identified and has been implicated as a tumor suppressor gene. We have now shown that DCC encodes a membrane-bound protein of the immunoglobulin-CAM family, as demonstrated by cell-surface labeling, immunohistochemical analysis, and sequencing of cDNA clones. The DCC protein was found in axons of the central and peripheral nervous system and in differentiated cell types of the intestine. Colorectal tumors that lost their capacity to differentiate into mucus producing cells uniformly lacked DCC expression and loss of a chromosome 18q allele was often accompanied by loss of DCC expression in colon tumors. These results provide evidence that DCC encodes a cell surface-localized protein and emphasize the inverse relationship between differentiation and tumorigenesis.

Adenocarcinoma, Mucinous↗
PubMed1969

Nucleic acid metabolism during cytokinin induced cellular differentiation.

Edstrom's microphoresis technique has been employed to determine the quantitative alterations in nucleic acid content and base composition of individual cells associated with the initiation of bud primordia in Funaria hygrometrica. The filamentous protonema of this moss initiates bud cells which through repeated divisions form the leafy gametophores. The cytokinin, 6-furfurylaminopurine (kinetin), was used to induce the differentiation of bud cells from protonematal cells. The total RNA content of kinetin-induced bud cells (22.0 mumug/cell) was nearly 15 times that of protonematal cells (1.6 mumug/cell). The same dramatic increase in total RNA was apparent in bud cells which developed spontaneously in older cultures. As would be predicted, the adenine (A) to guanine (G) ratio for DNA from bud and protonematal cells was identical (0.7). The A:G ratio for RNA from bud cells (1.0) was much lower than that from protonematal cells (1.7). Thus, kinetin-induced differentiation in this system involves a dramatic increase in total RNA, the base composition of which approaches that of DNA. The base composition (A:G ratio) of DNA remains constant.

Cell Differentiation↗
PubMed1988

Turnover of inositol phospholipids in cultured murine keratinocytes: possible involvement of inositol triphosphate in cellular differentiation.

The relationship between the turnover of inositol phospholipids (PtdIns) and the growth and differentiation of normal murine keratinocytes in culture was studied. Addition of myo-[U-14C]inositol to freshly plated cells resulted in a linear incorporation of radiolabel into the inositol phospholipids of the proliferating basal cells in culture during the initial 36 h, after which time the rate of radiolabel incorporation into the cells declined. The decrease in the incorporation of the radiolabel into the PtdIns, particularly the more highly phosphorylated PtdIns-4P and PtdIns4,5P2, correlated with the marked hydrolysis of these polyphosphoinositides and the rapid hydrolytic release of the inositol phosphates (InsP2 and InsP3). The transient accumulation of the InsPs correlated with the onset of differentiation of these cells. To ascertain whether the above observations of keratinocytes that were undergoing normal proliferative and differentiating phases in culture are consistent with the more synchronized populations of proliferative and differentiating cells, we investigated the turnover of PtdIns in a Ca2+-regulated system of homogenous populations of proliferating mouse keratinocytes in 0.09 mM Ca2+, and a differentiating population in 1.8 mM Ca2+. Our data from system revealed rapid hydrolysis of the PtdIns in the prelabeled low-Ca2+ proliferating cells immediately after a switch from the low to normal extracellular Ca2+ medium. Associated with this hydrolysis was the rapid and transient accumulation of the InsPs (maximum of 60 sec). The hydrolysis of the PtdIns and the accumulation of the InsP3 were not observed when the prelabeled proliferating cells were switched from a low to a low extracellular Ca2+ medium. These results suggest that the rapid hydrolysis of the PtdIns, particularly PtdIns4,5P2, which was accompanied by the hydrolytic release of InsP3, could be the initiating signal to program proliferating keratinocytes into differentiation.

Animals↗
PubMed1990

Cellular differentiation and histogenesis of rat glandular stomach cancers.

The gastric and intestinal phenotypic expressions of tumor cells in 18 adenomatous hyperplasias, 33 well-differentiated adenocarcinomas, and 16 undifferentiated adenocarcinomas (4 poorly differentiated adenocarcinomas, 10 signet-ring cell carcinomas and 2 mucinous adenocarcinomas) induced by N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide in the rat glandular stomach were studied by histochemical stainings for mucin and immunohistochemical staining for pepsinogen isozyme 1 (Pg 1). By histochemical staining for mucin [by the paradoxical concanavalin A method, the modified method with labeled peanut lectin, the galactose oxidase-Schiff (GOS) reaction, and the sialidase-GOS reaction] and immunohistochemical staining of Pg 1, gastric cancer cells of each histological group could be clearly classified into a gastric type, including mucous neck cell pyloric gland cell, and surface mucous cell subtypes, and an intestinal type, including goblet-cell, and intestinal absorptive cell subtypes. All tumors examined in this work consisted mainly of gastric-type cells but intestinal-type tumor cells were occasionally found among the gastric-type tumor cells. The incidences of intestinal-type cells in adenomatous hyperplasias (11.1%) and small well-differentiated adenocarcinomas (28.6%) were significantly less (P less than 0.05) than that in large well-differentiated adenocarcinomas (68.4%). The incidence of intestinal-type cells in small undifferentiated adenocarcinomas (25.0%) was also less than that in large ones (58.3%). The present results suggest the occurrence of change of phenotypic expression of tumor cells from the gastric type to the intestinal type during growth of tumors.

Adenocarcinoma↗
PubMed2005

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus.

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.

Amino Acid Sequence↗
PubMed1979

Regeneration of vitamin A deficient rat tracheal epithelium after mild mechanical injury. Growth kinetics and cellular differentiation.

Mild mechanical abrasion of tracheal epithelium of Vitamin A deficient rats removed the superficial cells and spared basal cells which divided to repopulate the damaged area. The proliferative cells passed through a period of DNA synthesis with the greatest numbers of thymidine incorporating cells in samples labelled 22 h after injury. A peak of cell division occurred at 32 h and there was no further DNA synthesis or cell division. The area of wounding exhibited squamous metaplasia while normal pseudostratified muco-ciliary structure was retained by adjacent epithelium which had not been injured. The data indicates that squamous metaplasia in the respiratory epithelium in longstanding Vitamin A deficiency is due to redirected differentiation of basal cells and is seen only after mitotic activity has occurred.

Animals↗
PubMed1979

The effect of vitamin A on cellular differentiation and mucous glycoprotein synthesis in long-term rat tracheal organ cultures.

Rat tracheal explants maintained as organ cultures exhibited a normal mucocillary epithelium for at least 46 days in the presence of retinyl acetate. In the absence of vitamin A the explant epithelium became quiescent or underwent a metaplastic change to a keratinizing squamous epithelium. This process was accelerated if explants were derived from vitamin A-deficient animals. Autoradiographic examination showed that [3H]glucosamine label accumulated in various cell types in the explant, but especially in the epithelium. It was found that the explants secreted mucous glycoproteins into the medium and that the production and biochemical characteristics of a specific mucin fraction were dependent upon the vitamin A status of the explant.

Animals↗
PubMed1993

Murine erythroleukemia cells contain two distinct GATA-binding proteins that have different patterns of expression during cellular differentiation.

GATA-1 is a major transcription factor of the erythroid lineage that has been implicated in the induced expression of a variety of red cell-specific genes during terminal differentiation of murine erythroleukemia cells. Although the GATA-1 protein is present at nearly equal levels before and after differentiation of murine erythroleukemia cells, in this study it was found that in the early commitment stages of the differentiation program there is a transient decrease in the GATA-1 mRNA and DNA binding activity levels due to a temporary block in transcription of the gene. Moreover, using a whole cell extraction procedure it was discovered that murine erythroleukemia cells contain a second GATA binding activity (denoted GATA-rel) which appears to be distinct from the GATA-1 factor based on its non-reactivity to two GATA-1 antisera. This protein has a limited tissue specificity, as it could not be detected in extracts from CHO, NIH 3T3, or COS cells. Similarly to the GATA-1 DNA-binding activity, the GATA-rel activity decreased during the early stages of differentiation. However, unlike GATA-1, GATA-rel activity did not return to pre-induced levels at later times. These results suggest that changes in gene expression during erythroid terminal differentiation may involve an interplay on levels of different GATA-binding factors.

Animals↗
PubMed2006

Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection.

Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.

Cell Line, Tumor↗