Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic "birthdate." All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of approximately 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.
Endothelin-1 (ET-1), which is secreted from vascular endothelial cells, is not only a potent vasoconstrictor but also a vascular smooth muscle cell growth factor. The direct effect of ET-1 on vascular smooth muscle cells, mediated via its specific receptor may therefore play an important role in hypertension and atherosclerosis. Our previous studies indicated that ET-1 secretion from cultured aortic endothelial cells from spontaneously hypertensive rats (SHRs) at the prehypertensive stage (4 weeks old) was not significantly different from that of cells from age-matched Wistar-Kyoto (WKY) rats. In this study, the binding of ET-1 to cultured aortic smooth muscle cells from SHRs and WKY rats was studied. Using tissue explant techniques, rat aortic smooth muscle cells from SHRs and age-matched WKY rats of different ages (4 and 24 weeks old) were successfully cultured in vitro. The maximum binding capacity (Bmax) and binding affinity (Kd) of ET-1 to cultured aortic smooth muscle cells were evaluated by a receptor-binding assay. The data revealed that the affinity of ET-1 binding to smooth muscle cells was similar in all 4 groups of experimental rats. However, the Bmax of cultured smooth muscle cells from 24-week-old SHRs Was 2.5 times higher than of smooth muscle cells from age-matched WKY rats (8.64 ±0.72 vs 3.69±0.10 fmol/106 cells) and 1.5 times higher than in aortic smooth muscle cells from 4-week-old SHRs (8.64±0.72 vs 5.36±0.36 fmol/10 6 cells). These results suggest that hypertension in SHRs may be related to a high density of ET-1 receptors on arterial smooth muscle cells. (Jpn Circ J 1997; 61: 145 - 150)
Objective
To analyze the pediatric residents' competencies aiming at providing references for improving the quality of pediatric resident training and remodeling post-graduate education.
Methods
One hundred and eighty-four pediatric residents from Shanghai Jiao Tong University School of Medicine Affiliated Hospital participated in this cross-section investigation from December 2018 to February 2019. Using the cognitive questionnaire of pediatric residents' post competency to investigate the resident.
Results
Up to 45.7%(84/184)of respondents failed to define the competency. Within the top 11 competencies pediatric residents ranked, there was only one item effective use of communication in communication ability, and its importance score (4.67 ± 0.63), and there was no entry in the medical humanities literacy ranking in the top 11.
Conclusions
The awareness rate of resident positions is not high. In the standardized training of residents, the cultivation of medical humanities and communication skills were weak. Staged and hierarchical pediatric resident competency evaluation criteria should be developed.
Key words:
Pediatrics; Resident physician; Competency; Cognition
Abstract Introduction Approximately 50% of patients with sepsis‐induced acute lung injury and acute respiratory distress syndrome require mechanical ventilation. Patients with extended mechanical ventilator use routinely develop reinfections, which increases hospital stay, mortality, and health care cost. Some studies have pointed out inflammatory factors concentrations can affect ventilator weaning, but do not indicate changed inflammatory factors related to ventilator weaning during using ventilators. Objectives This study aimed to investigate during period of septic patients using ventilators, the inflammatory cytokines concentrations related with weaning rate. Methods Blood was collected from 35 septic patients before and during ventilator use on days 1, 7, 14, and 21 (or weaning). Results 58.3% (N = 20) of septic patients with mechanical ventilators were weaned successfully within 21 days (ventilator weaned group, VW), 16.7% (N = 6) did not wean within 21 days (ventilator dependent group, VD), and 25% died (death group) in hospital. Before ventilator use, higher C‐reactive protein (CRP), IL‐6, and IL‐8 levels were measured in the death group than in all other groups ( P < .05). During ventilator use, CRP, IL‐6, and IL‐8 concentrations declined significantly in VW and VD patients ( P < .05). In addition, IL‐6 concentrations in the VW group were significantly lower than in the VD group at 14 and 21 days ( P < .05). Conclusion The factors of ventilators weaning successfully such as disease control, nutritional status, and so on. The declined levels of serum inflammatory cytokines, especially IL‐6, improved inflammation status might be one factor of successfully weaning during septic patients on ventilators.
During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.
It has been reported that the emergence of coronavirus disease 2019 (COVID-19) has changed the epidemiological characteristics of many pathogens, but the epidemiological characteristics of Mycoplasma (MP) infection in hospitalized children with community-acquired pneumonia (CAP) are not clear. The aim of this study was to answer this question. Children with CAP in three tertiary hospitals (hospitals A, B and C) from 2018 to 2023 were selected. Data on gender, age, number and date of MP infection were obtained from the medical record. The intensity of the epidemic was measured as a percentage of the number of CAP. In hospitals A and B, before the pandemic (in 2018 and 2019), the number of hospitalized children with MP pneumonia and the proportion of total pneumonia had shown a significant upward trend, but the control measures led to a slight decline. In hospital C, the number and percentage of hospitalized children with MP pneumonia were low before and during the COVID-19 period. After the epidemic control was lifted, the number and percentage of children with MP pneumonia in the three hospitals increased sharply, and the proportion of children aged more than 7 years old increased significantly in 2022 and 2023. From 2018 to 2019, there was already an epidemic trend of MP in the study hospital. From 2020 to 2022, after the outbreak of COVID-19, the incidence of MP pneumonia stabilized, but after the epidemic control was lifted, it broke out. This may be due to the severe restrictive measures taken early during the COVID-19 pandemic that effectively controlled the spread of MP, pausing its pandemic.
According to previous studies, endothelin-1 (ET-1) is the most potent growth factor in the regulation of vascular smooth muscle cell (VSMC) proliferation in spontaneously hypertensive rats (SHR). To evaluate if the dominant effect of ET-1-induced VSMC proliferation is achieved by autocrine regulation, aortic smooth muscle cells from four-week-old SHR and WKY (Wistar-Kyoto) rats were cultured in 24-well dishes, and the effects of ET-1 on VSMC proliferation were determined by (a) 3H-thymidine incorporation assays with different ET-1 blocking treatments, including a specific anti-ET-1 antibody; BQ-123, an ETA receptor blocker; and BQ-788, an ETB receptor blocker; and (b) examining the ET-1 blockade on the effects of treatment with other growth factors, including thrombin and angiotension II (AT-II). These results demonstrated that the anti-ET-1 antibody, BQ-123, BQ-788, and BQ-123 plus BQ-788 all caused dose-dependent inhibition of proliferation. A 90% inhibitory effect was observed at the maximum doses used except for BQ-123. The ET-1 receptor blockers inhibited thrombin-induced VSMC growth; however, they did not efficiently inhibit AT-II-induced VSMC growth. These results indicate that the autocrine effects of ET-1 play a predominant role in the proliferation of VSMCs from SHR and WKY rats. They also suggest that thrombin-induced VSMC growth is mediated by the autocrine effects of ET-1, and angiotensin II-induced VSMC growth is mediated by other signal pathways.
The pathogenesis of hypertension is not fully understood; endothelin 1 (EDN1) is involved in developing essential hypertension. EDN1 can promote vascular smooth muscle cell (VSMC) proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are 'dedifferentiated' cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the EDN1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced EDN1 expression in SHR VSMCs. We identified POU2F2 and CEBPB as the main molecules that enhance EDN1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the Edn1 promoter in male SHR VSMCs was from −1309 to −1279 bp. POU2F2 and CEBPB exhibited an additive role in the enhancer region of the EdnET1 promoter. POU2F2 or CEBPB overexpression sufficiently increased EDN1 expression, and co-transfection with the CEBPB and POU2F2 expression plasmids had additive effects on the activity of the Edn1 promoter and EDN1 secretion level of male WKY VSMCs. In addition, the knockdown of POU2F2 also revealed that POU2F2 is necessary to enhance EDN1 expression in SHR VSMCs. The enhancer region of the Edn1 promoter is highly conserved in rats, mice, and humans. POU2F2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2F2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.
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