Immunofluorescence Microscopic Examination Research Articles

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

The tongue of the red-eared slider (Trachemys scripta elegans): morphological characterization through gross, light, scanning electron, and immunofluorescence microscopic examination

The red-eared slider (Trachemys scripta elegans) is renowned for its remarkable adaptations, yet much of its complex biology remains unknown. In this pioneering study, we utilized a combination of gross anatomy, scanning electron microscopy (SEM), light microscopy, and immunofluorescence techniques to examine the tongue’s omnivorous adaptation in this species. This research bridges a critical knowledge gap, enhancing our understanding of this intriguing reptile. Gross examination revealed a unique arrowhead-shaped tongue with a median lingual fissure and puzzle-piece-shaped tongue papillae. SEM unveiled rectangular filiform, conical, and fungiform papillae, with taste pores predominantly on the dorsal surface and mucous cells on the lateral surface of the papillae. Histologically, the tongue’s apex featured short rectangular filiform and fungiform papillae, while the body exhibited varying filiform shapes and multiple taste buds on fungiform papillae. The tongue’s root contained lymphatic tissue with numerous lymphocytes surrounding the central crypt, alongside lingual skeletal musculature, blood and lymph vessels, and Raffin corpuscles in the submucosa. The lingual striated muscle bundles had different orientations, and the lingual hyaline cartilage displayed a bluish coloration of the ground substance, along with a characteristic isogenous group of chondrocytes. Our research represents the first comprehensive application of immunofluorescence techniques to investigate the cellular intricacies of the red-eared slider’s tongue by employing seven distinct antibodies, revealing a wide array of compelling and significant findings. Vimentin revealed the presence of taste bud cells, while synaptophysin provided insights into taste bud and nerve bundle characteristics. CD34 and PDGFRα illuminated lingual stromal cells, and SOX9 and PDGFRα shed light on chondrocytes within the tongue’s cartilage. CD20 mapped B-cell lymphocyte distribution in the lingual tonsil, while alpha smooth actin (α-SMA) exposed the intricate myofibroblast and smooth muscle network surrounding the lingual blood vessels and salivary glands. In conclusion, our comprehensive study advances our knowledge of the red-eared slider’s tongue anatomy and physiology, addressing a significant research gap. These findings not only contribute to the field of turtle biology but also deepen our appreciation for the species’ remarkable adaptations in their specific ecological niches.

Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl−/HCO3 − exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4’s C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl−/HCO3 − exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin’s interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.

Immunofluorescence Assay of Ablated Colorectal Liver Metastases: The Frozen Section of Image-Guided Tumor Ablation?

PurposeTo validate an immunofluorescence assay (IFA) detecting residual viable tumor (VT) as intraprocedural thermal ablation (TA) zone assessment and demonstrate its prognostic value for local tumor progression (LTP) after colorectal liver metastasis (CLM) TA. Materials and MethodsThis prospective study, approved by the institutional review board, included 99 patients with 155 CLMs ablated between November 2009 and January 2019. Tissue samples from the ablation zone (AZ) center and minimal margin underwent immunofluorescent microscopic examination interrogating cellular morphology and mitochondrial viability (IFA) within 30 minutes after ablation. The same tissue samples were subsequently evaluated with standard morphologic and immunohistochemical methods. The sensitivity, specificity, and overall accuracy of IFA versus standard morphologic and immunohistochemical examination were calculated. The LTP-free survival rates were evaluated for the 12-month follow-up period. ResultsOf the 311 tissue samples stained, 304 (98%) were deemed evaluable. Of these specimens, 27% (81/304) were considered positive for the presence of VT. The accuracy of IFA was 94% (286/304). The sensitivity and specificity were 100% (63/63) and 93% (223/241), respectively. The 18 false-positive IFA assessments corresponded to samples that included viable cholangiocytes. The 12-month LTP-free survival was 59% versus 78% for IFA positive versus negative for VT AZs, respectively (P < .001). There was no difference in LTP between margin positive only and central AZ–positive tumors (25% vs 31%, P = 1). ConclusionsThe IFA assessment of the AZ can be completed intraprocedurally and serve as a valid real-time biomarker of complete tumor eradication or detect residual VT after TA. This method could improve tumor control by TA.

Protective effects of citicoline-containing eye drops against UVB-Induced corneal oxidative damage in a rat model

It has been reported that citicoline increases antioxidant activity in some tissues. However, the effect of citicoline on corneal wound-healing has not yet been demonstrated. The aim was to investigate the protective effects of citicoline on ultraviolet B (UVB) radiation–induced corneal oxidative damage in a rat model. Four groups (eight animals each) were investigated: controls; UVB only; UVB/citicoline; and citicoline only. Corneal oxidative damage was induced by exposure to UVB radiation at 560 μW/cm2 for five days in the UVB-exposed groups and 1% citicoline eye drops were applied (3xday) for eight days in the two citicoline groups. Corneal surface damage was evaluated by opacity and fluorescein staining. Corneal injury was assessed biochemically by measuring the concentrations of glutathione (GSH) and malondialdehyde (MDA) and the activity of corneal superoxide dismutase (SOD) and catalase. Matrix metalloproteinase (MMP) −2 and −9 and caspase-3 were evaluated by immunofluorescent staining and microscopic examination and by Western blot analysis. Corneal gene expression analysis was performed for vascular endothelial growth factor (VEGF), interleukin-1 beta (IL-1β) and transforming growth factor-beta (TGF-β). UVB radiation caused significant epithelial damage and evident opacity in the cornea, together with a local decrease in SOD, catalase and GSH activity. Corneal MDA concentrations increased with UVB exposure. The UVB/Citicoline group had significantly less corneal damage, greater SOD, catalase and GSH activity, and decreased MDA concentrations compared to the UVB only group (p < 0.05). Expression of TGF-β, IL-1β and VEGF was significantly lower in the citicoline/UVB group compared to the UVB group (p < 0.05). Interestingly, TGF-β expression was lower in the citicoline only group compared with controls. Immunfluorescent staining and Western blot analysis showed increased MMP-2, -9 and caspase-3 in the UVB only group compared with the UVB/citicoline group. It was shown that citicoline treatment may be effective in suppressing oxidative stress and controlling inflammation in UVB corneal injury.

Ascleposide, a natural cardenolide, induces anticancer signaling in human castration-resistant prostatic cancer through Na+ /K+ -ATPase internalization and tubulin acetylation.

Cardiac glycosides, which inhibit Na+ /K+ -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+ /K+ -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC). Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+ /K+ -ATPase was determined using confocal immunofluorescence microscopic examination. Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+ /K+ -ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na+ /K+ -ATPase and downregulated the protein expression of Na+ /K+ -ATPase α1 subunit. Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+ /K+ -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na+ /K+ -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development.

High Prevalence and Diversity of Zoonotic and Other Intestinal Parasites in Dogs from Eastern Spain.

The diversity and frequency of enteric parasites in dog populations in the Castellón province (Eastern Spain) were assessed using a prospective cross-sectional epidemiological survey. A total of 263 canine fecal samples were collected between July 2014 and July 2016. Detection of intestinal parasites was conducted by routine coprological methods. In addition, identification of Giardia duodenalis and Cryptosporidium spp. was carried out by direct immunofluorescence microscopy, whereas the presence of Strongyloides spp. was assessed by real-time PCR in a selected number of specimens. Based on conventional and/or immunofluorescence microscopy examination, 65.8% (95% confidence interval: 59.7-71.5) of the investigated dogs were found infected by at least one gastrointestinal parasite. G. duodenalis (35.4%) and members of the family Ancylostomatidae (27.0%) were the most prevalent protozoan and helminth parasites found, respectively. Other pathogens potentially infective to humans included Toxocara canis (8.0%), Cryptosporidium spp. (6.8%), and Strongyloides spp. (1.1%). Frequency of occurrence of helminthic, but not protozoan, enteroparasites was geographical origin dependent (p = 0.02), with dogs living in coastal areas presenting higher infection rates than those living in inland regions. Similarly, rural dogs were significantly more infected than urban dogs (p < 0.001). Our results revealed that zoonotic agents were common in dogs from the Castellón province. Animals from rural areas and sheltered dogs were particularly at risk of these infections.

Tubular Injury and Dendritic Cell Activation Are Integral Components of Light Chain-Associated Acute Tubulointerstitial Nephritis.

Light chain-associated acute tubulointerstitial nephritis (LC-ATIN) is a variant of light chain proximal tubulopathy (LCPT). It is characterized by interstitial inflammation with tubulitis and deposition of monoclonal light chains in the tubulointerstitium. LC-ATIN is a rather poorly recognized pattern of LCPT and not much is known about this entity. To determine the clinicopathologic features of patients with LC-ATIN and investigate the proximal tubular injury and mechanism of interstitial inflammation in LC-ATIN. A total of 38 cases of LC-ATIN were identified from the archives of 5043 renal biopsy specimens. In all cases, routine light microscopic examination, immunofluorescence, and electron microscopic examination were performed. In selected cases, immunofluorescent staining of dendritic cells and immunohistochemical staining for 4 tubular injury markers-KIM-1, p53, bcl-2, and Ki-67-were performed. A characteristic finding in LC-ATIN cases was immunofluorescence staining of monoclonal light chains along tubular basement membranes in linear fashion and inside proximal tubular cells with a granular pattern. No monoclonal light chains were present in glomerular or vascular compartments confirmed with immunofluorescence, electron microscopy, and ultrastructural gold labeling. Ten of 15 LC-ATIN cases (67%) were concurrently positive for the 4 tubular injury markers. Dendritic cells were identified within the tubulointerstitium in the renal biopsy specimens, interacting with surrounding tubules with light-chain deposits and inflammatory cells. Significant proximal tubular injury occurs associated with LC-ATIN, and the monoclonal light chains accumulated in proximal tubular cells contribute to the injury. Dendritic cells are involved in the pathogenesis of interstitial inflammation in LC-ATIN.

Clinicopathologic features of lipoprotein glomerulopathy: observation of 6 cases

Objective: To investigate the clinicopathologic features of lipoprotein glomerulopathy (LPG). Methods: A total of 6 cases (5 males and 1 female, with a mean age of 27.5 years and age range of 11-53 years) of lipoprotein glomerulopathy with complete clinicopathologic data were enrolled. Except for light microscope, immunofluorescence and electron microscopic examination, renal biopsy tissues were checked by oil red O staining. The gene map of apolipoprotein E (ApoE) of 2 cases were analyzed. Results: All 6 cases presented with heavy proteinuria or nephrotic syndrome, and high level of low-density lipoprotein (LDL), very low-density lipoprotein (vLDL), ApoE. Family history of LPG was found in 3 cases, and 2 patients progressed to uremia, or even to death. Pathologic features showed that lipoprotein deposited in glomerulus capillary lumen and renal tubular epithelial cells. Gene analysis demonstrated that 2 cases expressed abnormal ApoE gene (162G>C and 455G>C). Conclusions: Lipoprotein glomerulopathy is autosomal-recessive disease with mutation of ApoE. Common clinical manifestations of LPG are heavy proteinuria or nephrotic syndrome, with a poor prognosis. Renal biopsy pathologic diagnosis can confirm this kidney disease. Emboli of lipoprotein being observed in glomerulus capillary lumen is the pathological feature of LPG.

Os efeitos condrotóxicos e apoptóticos de levobupivacaína e bupivacaína na articulação do joelho de coelhos

BackgroundA single dose injection or continuous infusion of local anesthetics into the joint space is considered to be a well‐defined analgesia technique. The aim of this study was to investigate the chondrotoxic and apoptotic effects of single‐dose intra‐articular injection of levobupivacaine and bupivacaine on rabbit knee joint tissues. Materials and methodsThe animals were allocated into two groups each containing 20 rabbits. 0.5% levobupivacaine (Group L) and 0.5% bupivacaine (Group B) were applied intra‐articularly to the left posterior joints of rabbits. At the same time, normal saline was applied to the right posterior leg knee joints of rabbits in both groups and used as a control (Group S). At the end of the 7th and 28th days after the intraarticular injections, ten randomly chosen rabbits in each group were killed by applying intraperitoneal thiopental. Sections of cartilage tissue samples were stained for light microscopic examinations and the TUNEL method was used to investigate apoptotic cells. ResultsAs a result of immunofluorescence microscopic examination, the number of apoptotic cells in Group B at day 7 and day 28 were both significantly higher than Group L and S (p<0.05). Also, the number of apoptotic cells in Group L at day 7 and day 28 were both significantly higher than Group S (p<0.05). ConclusionsWe found that bupivacaine is more chondrotoxic than other anesthetic agent and increases the number of apoptotic cells. These results indicated that bupivacaine caused high chondrotoxic damage and it led to more apoptotic activation than levobupivacaine.

The chondrotoxic and apoptotic effects of levobupivacaine and bupivacaine on the rabbit knee joint

BackgroundA single dose injection or continuous infusion of local anesthetics into the joint space is considered to be a well-defined analgesia technique. The aim of this study was to investigate the chondrotoxic and apoptotic effects of single-dose intra-articular injection of levobupivacaine and bupivacaine on rabbit knee joint tissues. Materials and methodsThe animals were allocated into two groups each containing 20 rabbits. 0.5% levobupivacaine (Group L) and 0.5% bupivacaine (Group B) were applied intra-articularly to the left posterior joints of rabbits. At the same time, normal saline was applied to the right posterior leg knee joints of rabbits in both groups and used as a control (Group S). At the end of the 7th and 28th days after the intraarticular injections, ten randomly chosen rabbits in each group were killed by applying intraperitoneal thiopental. Sections of cartilage tissue samples were stained for light microscopic examinations and the TUNEL method was used to investigate apoptotic cells. ResultsAs a result of immunofluorescence microscopic examination, the number of apoptotic cells in Group B at day 7 and day 28 were both significantly higher than Group L and S (p<0.05). Also, the number of apoptotic cells in Group L at day 7 and day 28 were both significantly higher than Group S (p<0.05). ConclusionsWe found that bupivacaine is more chondrotoxic than other anesthetic agent and increases the number of apoptotic cells. These results indicated that bupivacaine caused high chondrotoxic damage and it led to more apoptotic activation than levobupivacaine.

Patogeneza zapaleń naczyń związanych z przeciwciałami przeciwko cytoplazmie neutrofilów

ABSTRAKTZapalenia naczyń związane z przeciwciałami przeciwko cytoplazmie neutrofilów (antineutrophil cytoplasmic antibodies-associated vasculitis – AAV) to grupa chorób zajmujących małe naczynia i charakteryzujących się ich martwiczym zapaleniem oraz obecnością przeciwciał przeciwko cytoplazmie neutrofilów (antineutrophil cytoplasmic antibodies – ANCA) przy braku lub obecności jedynie skąpej ilości immunoglobulin w ścianie naczyń. Przeciwciała przeciwko cytoplazmie neutrofilów to przeciwciała klasy G skierowane przeciwko składnikom ziarnistości granulocytów obojętnochłonnych. Dowody na udział ANCA w patogenezie AAV pochodzą zarówno z obserwacji klinicznych, badań in vitro, jak i badań na zwierzętach. Przyczyny pojawiania się ANCA są cały czas wnikliwie badane, gdyż istnieje wiele nie w pełni zrozumiałych ścieżek patogenetycznych prowadzących do rozwoju choroby. Wciąż pojawiają się też nowe doniesienia pozwalające lepiej zrozumieć sposób powstawania patologicznych zmian wykrywanych u pacjentów z AAV.W niniejszej pracy przedstawiono najbardziej prawdopodobne mechanizmy odpowiadające za powstawanie ANCA, jak również możliwą rolę przeciwciał w patogenezie rozwoju zapalenia naczyń.

Histopathological patterns of renal diseases in egyptian children: A single-center experience.

The present cross-sectional, retrospective study was aimed to determine the histopathological spectrum of renal diseases in Egyptian children and to evaluate the indications, safety, and efficacy of percutaneous renal biopsy (PRB) in a large tertiary center in Egypt. PRBs performed at the Department of Pediatrics, Tanta University Hospital over a period of nine years (from January 2007 to December 2015) were included. Light microscopic (LM) examination was performed in all cases while immunofluorescence and electron microscopic examination were performed in selected cases. Two hundred and thirty renal biopsies were performed during the study duration. Nine biopsies were excluded from the study due to insufficient sample (<7 glomeruli per specimen) giving a PRB efficacy rate of 96.1%. Results of 221 renal biopsies performed on 210 patients from native kidneys were described. Ninety-seven patients were male (46.19%) and 113 were female (53.81%) with age ranging from three months to 18 years (mean 10.51 ± 3.81 years). The main indications of renal biopsy were nephrotic syndrome (NS) (43.89%), lupus nephritis (23.53%), and recurrent or persistent hematuria (10.41%). The most common finding on LM examination of renal biopsies from children with NS was minimal change disease (22.17%). Secondary nephropathies were mostly due to lupus (23.53%). IgA nephropathy was found in eight patients (3.62%). Local pain at the site of biopsy was the most common minor complication seen postbiopsy (60.58%). Transient gross hematuria was seen in 13 patients (5.88%) without urinary retention. Major complications that required surgical intervention or blood transfusion did not occur. NS was the main indication and minimal change disease was the most common histological finding of renal biopsies from Egyptian children. Complications and efficacy of renal biopsy procedure were comparable to that reported from developed countries.

Disulfanyl peptide decreases melanin synthesis via receptor-mediated ERK activation and the subsequent downregulation of MITF and tyrosinase.

Bioactive peptides are commonly used in cosmeceutical purpose. This study was performed to search for an effective and short hypopigmenting peptide using normal human melanocytes as a screening model. A peptide that exhibits multitarget activities will be a promising peptide. Depigmenting effects were tested in normal human melanocytes. One peptide was selected, and signalling mechanism was investigated by Western blotting and immunofluorescent microscopic examination. A novel hypopigmenting peptide (dSHP) has been found to inhibit the production of melanin. This peptide significantly decreases tyrosinase activity but was not effective in a direct in vitro assay. It also induces the prolonged activation of ERK, and subsequently downregulates the levels of MITF. PD98059 abolished the dSHP-induced downregulation of MITF. These findings indicate that the dSHP-induced activation of ERK contributes to a reduced melanin synthesis via the downregulation of MITF. Fluorescent microscopic studies were consistent with such findings. Pertussis toxin reverses the downregulation of MITF, which means that the receptor-mediated ERK activation is involved. Moreover, it was also found that downregulation of MITF was clearly inhibited by lysosomal inhibitor (chloroquine). Novel tetrapeptide dSHP reduces the melanin synthesis by a receptor-mediated pathway. Furthermore, dSHP works by ERK activation and key transcription factor MITF degradation. Thus, it may be a good candidate as an effective hypopigmenting cosmetic agent.

Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells.

Autophagic activity reflects cellular response to drug treatment and can be regulated by STAT3 signaling. Resveratrol inhibits STAT3 activation and causes remarkable growth arrest and cell death of ovarian cancer (OC) cells. However, the autophagic status and its relevance with resveratrol’s anti-OC effects remain unclear. We analyzed the states of autophagic activities, the nature of autophagosomes and the levels of autophagy-related proteins (LC-3, Beclin 1 and STAT3) in resveratrol-treated CAOV-3 and OVCAR-3 OC cells using multiple approaches. We elucidated the correlation of STAT3 inhibition with autophagic activity by treating OC cells with an upstream inhibitor of STAT proteins, AG490. Resveratrol efficiently suppressed growth, induced apoptosis and inactivated STAT3 signaling of the two OC cell lines. We found enhanced autophagic activity accompanied with Beclin-1 upregulation and LC3 enzymatic cleavage in resveratrol-treated OC cells. Immunofluorescent (IF) microscopic and IF-based confocal examinations demonstrated the accumulation of cytoplasmic granules co-labeled with LC3 and cytochrome C in resveratrol- or AG490-treated OC cells. Using electron microscopy, we confirmed an increase in autophagosomes and mitochondrial spheroids in either resveratrol- or AG490-treated OC cells. This study demonstrates the abilities of resveratrol to enhance apoptotic and autophagic activities in OC cells, presumably via inactivating STAT3 signaling. Resveratrol or the selective JAK2 inhibitor also leads to mitochondrial turnover, which would be unfavorable for OC cell survival and sensitize OC cells to resveratrol.

Intracellular Delivery of Fluorescently Labeled Polysaccharide Nanoparticles to Cultured Breast Cancer Cells.

Nanoparticle delivery is becoming an increasingly more valuable technique in cancer drug treatments. The use of fluorescent probes, in particular, can provide noninvasive strategies to interrogate the internalization mechanisms of cancer cells and aid in drug design. Here we describe the delivery of fluorescently labeled polysaccharide-based nanoparticles to breast cancer cells in vitro and their subsequent immunofluorescence microscopy examination. The description of the synthesis, preparation, and delivery of the nanoparticles can be widely applicable to other in vitro drug delivery studies.

Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.

Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC). Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect. Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis. The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.

A Case of Focal Segmental Glomerulosclerosis in Turner Syndrome

Turner syndrome is usually accompanied with various anomalies. Congenital urological and renal abnormalities are often associated with this syndrome. The occurrence of glomerulonephritis is uncommon. An 18-year-old woman showed fatigue and profound proteinuria. She had been diagnosed with Turner syndrome in her age of 15. The kidney biopsy specimen examined by light microscopy, immunofluorescence and electron microscopic examination revealed focal segmental glomerulosclerosis. This is the first case report of focal segmental glomerulosclerosis in turner syndrome in South Korea.

Autophagy regulates colistin-induced apoptosis in PC-12 cells.

Colistin is a cyclic cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. Our recent study demonstrated that colistin induces apoptosis in primary chick cortex neurons and PC-12 cells. Although apoptosis and autophagy have different impacts on cell fate, there is a complex interaction between them. Autophagy plays an important role as a homeostasis regulator by removing excessive or unnecessary proteins and damaged organelles. The aim of the present study was to investigate the modulation of autophagy and apoptosis regulation in PC-12 cells in response to colistin treatment. PC-12 cells were exposed to colistin (125 to 250 μg/ml), and autophagy was detected by visualization of monodansylcadaverine (MDC)-labeled vacuoles, LC3 (microtubule-associated protein 1 light chain 3) immunofluorescence microscopic examination, and Western blotting. Apoptosis was measured by flow cytometry, Hoechst 33258 staining, and Western blotting. Autophagosomes were observed after treatment with colistin for 12 h, and the levels of LC3-II gene expression were determined; observation and protein levels both indicated that colistin induced a high level of autophagy. Colistin treatment also led to apoptosis in PC-12 cells, and the level of caspase-3 expression increased over the 24-h period. Pretreatment of cells with 3-methyladenine (3-MA) increased colistin toxicity in PC-12 cells remarkably. However, rapamycin treatment significantly increased the expression levels of LC3-II and beclin 1 and decreased the rate of apoptosis of PC-12 cells. Our results demonstrate that colistin induced autophagy and apoptosis in PC-12 cells and that the latter was affected by the regulation of autophagy. It is very likely that autophagy plays a protective role in the reduction of colistin-induced cytotoxicity in neurons.

Pauci-immune crescentic glomerulonephritis: A series of 21 cases

Background: Pauci-immune crescentic glomerulonephritis is the most common cause of crescentic glomerulonephritis (CrGN). Patients usually present with rapidly progressive glomerulonephritis (RPGN) with hematuria, proteinuria, and elevated serum creatinine levels. The characteristic feature of pauci-immune CrGN is focal necrotizing CrGN associated with little or no glomerular staining for immunoglobulin (Ig) by immunofluorescence microscopic examination. Most patients (80-85%) with pauci-immune CrGN, including the patients with and without systemic vasculitis, have antineutrophil cytoplasmic autoantibodies (ANCAs). Aims and Objectives: To study and compare the histological and laboratory features of ANCA-positive and ANCA-negative pauci-immune CrGN. Materials and Methods: Patients who were diagnosed with pauci-immune CrGN from January 2012 to May 2015, at BABINA Diagnostics, were included in this retrospective study. The terms pauci-immune and crescentic glomerulonephritis were defined according to standard guidelines. The demographic and laboratory data were extracted from the Laboratory Information System (LIS) for analysis. ANCA tests were performed by both indirect immunofluorescence (IIF) assay (EUROIMMUN, Lubeck, Germany) and antigen-specific enzyme-linked immunosorbent assay (ELISA) (ORGENTEC Diagnostika GmbH, Germany). Renal specimens were evaluated using direct immunofluorescence (for Ig and complement components) and light microscopy using routine and special stains. Results: Four (19%) out of the 21 cases of pauci-immune CrGN were ANCA negative. Acute features on histology were seen more than chronic features than ANCA-negative cases. Conclusion: Among the patients with pauci-immune CrGN, ANCA-negative patients were not rare. Compared with ANCA-positive patients, ANCA-negative patients had a lower percentage of normal glomeruli, more of chronic features, and a higher level of proteinuria.