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

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

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

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney

Background: A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching. Design and methods: Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination. Results: Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching. Conclusions: The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud

Chronic complications in patients with newly diagnosed type 2 diabetes: prevalence and related metabolic and clinical features: the Verona Newly Diagnosed Type 2 Diabetes Study (VNDS) 9

INTRODUCTION: We explored the presence of chronic complications in subjects with newly diagnosed type 2 diabetes referred to the Verona Diabetes Clinic. Metabolic (insulin secretion and sensitivity) and clinical features associated with complications were also investigated.RESEARCH DESIGN AND METHODS: The comprehensive assessment of microvascular and macrovascular complications included detailed medical history, resting ECG, ultrasonography of carotid and lower limb arteries, quantitative neurological evaluation, cardiovascular autonomic tests, ophthalmoscopy, kidney function tests. Insulin sensitivity and beta-cell function were assessed by state-of-the-art techniques (insulin clamp and mathematical modeling of glucose/C-peptide curves during oral glucose tolerance test).RESULTS: We examined 806 patients (median age years, two-thirds males), of whom prior clinical cardiovascular disease (CVD) was revealed in 11.2% and preclinical CVD in 7.7%. Somatic neuropathy was found in 21.2% and cardiovascular autonomic neuropathy in 18.6%. Retinopathy was observed in 4.9% (background 4.2%, proliferative 0.7%). Chronic kidney disease (estimated glomerular filtration rate <60mL/min/1.73 m2) was found in 8.8% and excessive albuminuria in 13.2% (microalbuminuria 11.9%, macroalbuminuria 1.3%).Isolated microvascular disease occurred in 30.8%, isolated macrovascular disease in 9.3%, a combination of both in 9.1%, any complication in 49.2% and no complications in 50.8%.Gender, age, body mass index, smoking, hemoglobin A1c and/or hypertension were independently associated with one or more complications. Insulin resistance and beta-cell dysfunction were associated with macrovascular but not microvascular disease.CONCLUSIONS: Despite a generally earlier diagnosis for an increased awareness of the disease, as many as ~50% of patients with newly diagnosed type 2 diabetes had clinical or preclinical manifestations of microvascular and/or macrovascular disease. Insulin resistance might play an independent role in macrovascular disease.TRIAL REGISTRATION NUMBER: NCT01526720

Cytogenetical studies in five Atlantic Anguilliformes fishes

The order Anguilliformes comprises 15 families, 141 genera and 791 fish species. Eight families had at least one karyotyped species, with a prevalence of 2n = 38 chromosomes and high fundamental numbers (FN). The only exception to this pattern is the family Muraenidae, in which the eight species analyzed presented 2n = 42 chromosomes. Despite of the large number of Anguilliformes species, karyotypic reports are available for only a few representatives. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n = 38; 8m+14sm+10st+6a; FN = 70) and four species of Muraenidae, Enchelycore nigricans (2n = 42; 6m+8sm+12st+16a; FN = 68), Gymnothorax miliaris (2n = 42; 14m+18sm+10st; FN = 84), G. vicinus (2n = 42; 8m+6sm+28a; FN = 56) and Muraena pavonina (2n = 42; 6m+4sm+32a; FN = 52), collected along the Northeastern coast of Brazil and around the St Peter and St Paul Archipelago were analyzed. Typical large metacentric chromosomes were observed in all species. Conspicuous polymorphic heterochromatic regions were observed at the centromeres of most chromosomes and at single ribosomal sites. The data obtained for Ophichthidae corroborate the hypothesis of a karyotypic diversification mainly due to pericentric inversions and Robertsonian rearrangements, while the identification of constant chromosome numbers in Muraenidae (2n = 42) suggests a karyotype diversification through pericentric inversions and heterochromatin processes

Iperuricemia e rischio cardiovascolare. [Review]

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