98 research outputs found
Thymosin Beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study
Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymeras
Cell starvation increases uptake of extracellular Thymosin β4 and its complexes with calcium
Cell metastasis is the main cause of cancer mortality. Inhibiting early events during cell metastasis and invasion could significantly improve cancer prognosis, but the initial mechanisms of cell transition and migration are barely known. Calcium regulates cell migration, whilst Thymosin β4 is a G-actin and iron binding peptide associated with tumor metastasis and ferroptosis. Under normal cell growth conditions, intracellular free calcium ions and Thymosin β4 concentrations are strictly regulated, and are not influenced by extracellular supplementation. However, cell starvation decreases intracellular Thymosin β4 and increases extracellular peptide uptake above the normal range. Unexpectedly, cell starvation significantly increases internalization of extracellular Ca2+/Thymosin β4 complexes. Elucidating the role of Ca2+/Thymosin β4 in the early events of metastasis will likely be important in the future to develop therapies targeting metastasis
Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes
In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs
Cellular trafficking of thymosin beta-4 in HEPG2 cells following serum starvation
Thymosin beta-4 (Tβ4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeabilit
Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae)
Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied
Thymosin beta-4 prenatal administration improves fetal development and halts side effects due to preterm delivery
Objective: Thymosin beta 4 (TB4) is the most abundant member of the beta-thymosin family in humans. The main physiological role of TB4 is the regulation of actin polymerization. TB4 is also involved in angiogenesis, cell survival, cell migration and fetal development. The aim of this study was to evaluate the activity of TB4 as a fetal growth promoter when administered during pregnancy.
Materials and methods: Our protocols have been carried out in full conformity with the rules and guidelines expected for this kind of trial. 10 pregnant mice received the same injection regimen. Only 6 of these 10 are part of this experiment because they were pregnant. At 10:00 a.m. on day E14 and E17 of gestation mice were weighed and treated with an intraperitoneal injection of TB4 (Regene RX, Rockville, MD, USA; 6 mg/kg in PBS).
Results: The mothers treated with TB4 for two days precisely E14 and E17, showed a higher cranio-caudal length when compared to control newborns. At histology, maternal TB4 treatment was associated with more advanced development of lungs, heart, kidney, cerebral cortex and notochord.
Conclusions: Our study shows that TB4 administration during gestation may act as a powerful fetal growth promoter, by accelerating the development of newborn organs and tissues
Exploring cell surface markers and cell-cell interactions of human breast milk stem cells
Background: Breakthrough studies have shown that pluripotent stem cells are present in human breast milk. The expression of pluripotency markers by breast milk cells is heterogeneous, relating to cellular hierarchy, from early-stage multi-lineage stem cells to fully differentiated mammary epithelial cells, as well as weeks of gestation and days of lactation. Design and methods: Here, we qualitatively analyze cell marker expression in freshly isolated human breast milk cells, without any manipulation that could influence protein expression. Moreover, we use electron microscopy to investigate cell-cell networks in breast milk for the first time, providing evidence of active intercellular communication between cells expressing different cellular markers. Results: The immunocytochemistry results of human breast milk cells showed positive staining in all samples for CD44, CD45, CD133, and Ki67 markers. Variable positivity was present with P63, Tβ4 and CK14 markers. No immunostaining was detected for Wt1, nestin, Nanog, OCT4, SOX2, CK5, and CD34 markers. Cells isolated from human breast milk form intercellular connections, which together create a cell-to-cell communication network. Conclusions: Cells freshly isolated form human breast milk, without particular manipulations, show heterogeneous expression of stemness markers. The studied milk staminal cells show "pluripotency" at different stages of differentiation, and are present as single cells or grouped cells. The adjacent cell interactions are evidenced by electron microscopy, which showed the formation of intercellular connections, numerous contact regions, and thin pseudopods
Human breast milk cells are positive for the pioneer transcription factor ISL1
Objective: ISL1 is a pioneer transcription factor that plays important roles in cell lineage specification and differentiation, by programming the epigenome and recruiting additional regulatory factors. The aim of this study is to determine whether the human breastmilk contains ISL1-positive stem cells, and, if so, to describe the subcellular localization of ISL1. Materials and methods: Breast milk was obtained from fourteen healthy females during the first 2-6 months of lactation. Cell morphology was examined in the breast milk with the automatic ThinPrep® processor (Hologic® Inc.) in commercial Cytological ThinPrep® solution (Hologic® Inc.), followed by standard immunohistochemical staining of ISL1. Results: ISL1 had a granular diffuse cytoplasmic localization, with varying intensity of staining in both single and grouped cells. Nuclear staining was also present, as was staining of intracellular and extracellular vesicles with ISL1 antibody. Conclusions: These preliminary results suggest that ISL1 could distinguish a readily available source of putative stem cells in human breast milk. These stem cells may complete the network created between the mother and the newborn during gestation, thereby improving the efficiency of programming and reprogramming postnatal events
Interindividual variability in L1CAM expression in the human kidney during development: are there implications for fetal programming of kidney diseases presenting in adulthood?
L1 cell adhesion molecule (L1CAM) is a member of the immunoglobulin superfamily of cell adhesion molecules. The present study investigated the expression of L1CAM during the development in the fetal human kidney at different gestational ages, to reach a better knowledge on the role of L1CAM in renal morphogenesis
Glomerular filtration rate decline in T2DM following diagnosis. The Verona newly diagnosed diabetes study-12
Aims: Nephropathy is a complication of type 2 diabetes, with increased albuminuria and reduced glomerular filtration rate (GFR) as biomarkers. Rates of progression to end-stage-renal disease are variable among patients. In this study we have examined the GFR decline in newly diagnosed T2DM. Methods: A cohort of 410 patients with newly diagnosed T2DM and with at least four serum creatinine during the follow-up period were recruited. A linear model was used to calculate the decline in eGFR. A multivariable logistic model was used to identify independent predictors of rapid eGFR decline. Results: Average follow-up was 12.4 years. The eGFR change was −0.80 ± 2.23 ml/min/1.73 m2 per year. Patients were arbitrarily stratified into rapid decliners (≤-3.0 ml/min/1.73 m2 per year), moderate decliners (-2.9/-1 ml/min/1.73 m2 per year) and slow/no decliners (>-1.0 ml/min/1.73 m2 per year). Subjects in the 3 categories were 11.4%, 27.3%, and 61.3%, respectively. Albuminuria was the stronger predictor of rapid eGFR decline. Conclusions: A rapid decline in eGFR occurs in approximately 1 out of 10 newly diagnosed subjects. This rapid decline can be predicted by widely accessible clinical features, such as albuminuria. Identification of rapid decliners may help to reduce progression toward advanced stages of nephropathy
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