Appearance Of Apoptotic Cells Research Articles

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development.

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

CHAC1 exacerbates arsenite cytotoxicity by lowering intracellular glutathione levels.

We here examined whether CHAC1 is implicated in arsenite (As(III))-induced cytotoxicity in HaCaT cells. We found that HaCaT cells in which the intracellular GSH levels were elevated by transfection with CHAC1 siRNA showed decreased sensitivity to As(III) compared to the control cells. Treatment with BSO (an inhibitor of GSH biosynthesis) abolished the decrease in sensitivity to As(III), suggesting that an increase in intracellular GSH levels was involved in the decrease in sensitivity to As(III) due to the decrease in the levels of CHAC1 expression. When we examined the expression of CHAC1 after exposure of HaCaT cells to As(III), the levels of CHAC1 were increased. Since CHAC1 is a proapoptotic factor, we examined appearance of apoptotic cells and cleavage of caspase-3 after exposure to As(III) to determine whether As(III)-induced CHAC1 up-regulation was involved in apoptosis induction. The results showed that induction of apoptosis by As(III) exposure was not detected in CHAC1 siRNA-transfected cells. Together, our findings indicate that CHAC1 is involved in the sensitivity of HaCaT cells to As(III) by regulating the intracellular GSH levels, and in particular, CHAC1 is involved in As(III)-induced apoptosis.

A mathematical model of wound healing in bovine corneal endothelium

We developed a mathematical model to describe healing processes in bovine corneal endothelial (BCE) cells in culture, triggered by mechanical wounds with parallel edges. Previous findings from our laboratory show that, in these cases, BCE monolayers exhibit an approximately constant healing velocity. Also, that caspase-dependent apoptosis occurs, with the fraction of apoptotic cells increasing with the distance traveled by the healing edge. In addition, in this study we report the novel findings that, for wound scratch assays performed preserving the basal extracellular matrix: i) the healing cells increase their en face surface area in a characteristic fashion, and ii) the average length of the segments of the cell columns actively participating in the healing process increases linearly with time. These latter observations preclude the utilization of standard traveling wave formalisms to model wound healing in BCE cells. Instead, we developed and studied a simple phenomenological model based on a plausible formula for the spreading dynamics of the individual healing cells, that incorporates original evidence about the process in BCE cells. The model can be simulated to: i) obtain an approximately constant healing velocity; ii) reproduce the profile of the healing cell areas, and iii) obtain approximately linear time dependences of the mean cell area and average length of the front active segments per column. In view of its accuracy to account for the experimental observations, the model can also be acceptably employed to quantify the appearance of apoptotic cells during BCE wound healing. The strategy utilized here could offer a novel formal framework to represent modifications undergone by some epithelial cell lines during wound healing.

Comparative Histological Study on the Effect of Tramadol Abuse on the Testis of Juvenile and Adult Male Albino Mice.

As a synthetic analog of codeine, tramadol is often prescribed to treat mild to moderate pains. This study was designed to estimate and compare the histological effect of tramadol on testes of both juvenile and adult male albino mice. A total number of 40 healthy male albino mice were classified into two main groups as follows: group I (juvenile group, includes 20 mice aged three weeks) subdivided equally into group Ia (control group received isotonic saline) and group Ib (tramadol-treated group received 40 mg/kg/d tramadol orally for 30 days); group II (adult group, includes 20 mice aged two months) subdivided equally into group IIa (control group received isotonic saline) and group IIb (tramadol-treated group). Juvenile and adult tramadol-treated groups showed numerous testicular changes, including blood vessels congestion, widening of intercellular spaces, vacuolization in interstitial tissues, luminal germ cells exfoliation, and increased expression of caspase-3 that indicated cellular apoptosis. In the ultrastructural examination, spermatogenic cells degenerated with the frequent appearance of apoptotic cells. Sertoli cells showed vacuolations, large lipid droplets, and disrupted intercellular cell junctions. These observed testicular changes were markedly observed in the juvenile group. Testicular abnormalities and apoptotic changes can be caused by tramadol administration. These abnormalities are more common in juvenile mice.

Sensitive-stage embryo irradiation affects embryonic neuroblasts and adult motor function

BackgroundCranial radiation therapy for treating childhood malignancies in the central nervous system or accidental radiation exposure may result in neurological side effects in surviving adults. As tissue homeostasis is maintained by stem cells, understanding the effect of radiation on neural stem cells will provide clues for managing the neurological effects. Drosophila embryos were used as a model system whose sensitivity to irradiation-induced cell death changes from the sensitive to resistant stage during development.ObjectiveDrosophila embryos at the radiation-sensitive stage were irradiated at various doses and the radiation sensitivity was tested regarding the appearance of apoptotic cells in the embryos and the embryonic lethality. Cell fates of the neural stem cells called neuroblasts (NBs) and adult motor function after irradiation were also investigated.ResultIrradiation of Drosophila embryos at the radiation-sensitive stage resulted in a dose-dependent increase in the number of embryos containing apoptotic cells 75 min after treatment starting at 3 Gy. Embryonic lethality assayed by hatch rate was induced by 1 Gy irradiation, which did not induce cell death. Notably, no apoptosis was detected in NBs up to 2 h after irradiation at doses as high as 40 Gy. At 3 h after irradiation, as low as 3 Gy, the number of NBs marked by Dpn and Klu was decreased by an unidentified mechanism regardless of the cell death status of the embryo. Furthermore, embryonic irradiation at 3 Gy, but not 1 Gy, resulted in locomotor defects in surviving adults.ConclusionEmbryonic NBs survived irradiation at doses as high as 40 Gy, while cells in other parts of the embryos underwent apoptosis at doses higher than 3 Gy within 2 h after treatment. Three hours after exposure to a minimum dose of 3 Gy, the number of NBs marked by Dpn and Klu decreased, and the surviving adults exhibited defects in locomotor ability.

Rational design of novel N‐alkyl amine analogues of noscapine, their chemical synthesis and cellular activity as potent anticancer agents

The scaffold structure of noscapine (an antitussive plant alkaloid) was modified by inducting N-aryl methyl pharmacophore at C-9 position of the isoquinoline ring to rationally design and screened three novel 9-(N-arylmethylamino) noscapinoids, 15-17 with robust binding affinity with tubulin. The selected 9-(N-arylmethylamino) noscapinoids revealed improved predicted binding energy of -6.694kcal/mol for 15, -7.118kcal/mol for 16 and -7.732kcal/mol for 17, respectively in comparison to the lead molecule (-5.135kcal/mol). These novel derivatives were chemically synthesized and validated their anticancer activity based on cellular studies using two human breast adenocarcinoma, MCF-7 and MDA-MB-231, as well as with a panel of primary breast tumor cells. These derivatives inhibited cellular proliferation in all the cancer cells that ranged between 3.2 and 32.2μM, which is 11.9 to 1.8 fold lower than that of noscapine. These novel derivatives effectively arrest the cell cycle in the G2/M phase followed by apoptosis and appearance of apoptotic cells. Thus, we conclude that 9-(N-arylmethyl amino) noscapinoids, 15-17 have a high probability to be a novel therapeutic agent for breast cancers.

Rational design, chemical synthesis and cellular evaluation of novel 1,3-diynyl derivatives of noscapine as potent tubulin binding anticancer agents

We present a new class of derivatives of noscapine, 1,3-diynyl-noscapinoids of an antitussive plant alkaloid, noscapine based on our in silico efforts that binds tubulin and displays anticancer activity against a panel of breast cancer cells. Structure-activity analyses pointed the C-9 position of the isoquinoline ring which was modified by coupling of 1,3-diynyl structural motifs to rationally design and screened a series of novel 1,3-diynyl-noscapinoids (20–22) with robust binding affinity with tubulin. The selected 1,3-diynyl-noscapinoids, 20–22 revealed improved predicted binding energy of −6.568 kcal/mol for 20, -7.367 kcal/mol for 21 and -7.922 kcal/mol for 22, respectively in comparison to the lead molecule (−5.246 kcal/mol). These novel derivatives were chemically synthesized and validated their anticancer activity based on cellular studies using two human breast adenocarcinoma, MCF-7 and MDAMB-231, as well as with a panel of primary breast cancer cells isolated from patients. Interestingly, all these derivatives inhibited cellular proliferation in all the cancer cells that ranged between 6.2 to 38.9 μM, which is 6.7 to 1.5 fold lower than that of noscapine. Unlike previously reported derivatives of noscapine that arrests cells in the S-phase, these novel derivatives effectively inhibit proliferation of cancer cells, arrests cell cycle in the G2/M-phase followed by apoptosis and appearance of apoptotic cells. Thus, we conclude that 1,3-diynyl-noscapinoids have great potential to be a novel therapeutic agent for breast cancers.

Phycocyanin from Spirulina platensis Induces Cytotoxicity and Apoptosis in T47D Cells

Breast cancer cases in Indonesia have risen in alarming rate. Herbal medicines have been developed to complement conventional cancer therapy and to lessen the negative effects of the therapies. Phycocyanin, blue-green photosynthetic pigment from Spirulina platensis, becomes a potential candidate of herbal medicine for cancer therapy due to its capability to eliminate various cancer cell line on in vitro level. This research aimed to analyse the effectivity of phycocyanin to induce cytotoxic and apoptotic activity of T47D cells (breast cancer cell line). MTT assay was perform to measure IC50 value. Double-staining assay and flow cytometry were applied to visualize the appearance of apoptotic cells and to measure the percentage of apoptotic and necrotic cells. Result showed that the IC50 value of phycocyanin towards T47D cells is 1650 μg/mL. Phycocyanin was also proven to induce apoptosis at IC50 with the apoptosis percentage of 45.93%. This research presented different results from previous researches that used phycocyanin from saltwater Spirulina platensis. Based on the results of this study, phycocyanin has the potential to be developed as anticancer agent by inducing apoptosis.

High-Dose Vitamin C Preadministration Reduces Vancomycin-Associated Nephrotoxicity in Mice.

Vancomycin is recommended for treating severe infections caused by Gram-positive cocci, including methicillin-resistant Staphylococcus aureus. However, renal damage often occurs as a side effect because vancomycin is mainly excreted via the kidneys. The mechanism of vancomycin-associated nephrotoxicity is thought to involve the elevation of oxidative stress in the kidneys. Vitamin C (VC) has strong antioxidant properties; therefore, we evaluated the effect of high-dose VC preadministration on vancomycin-associated nephrotoxicity. Vancomycin was intraperitoneally injected into mice once daily for 7 d. Additionally, high-dose VC was intraperitoneally injected into mice at 30 min before vancomycin administration for 7 d. The plasma creatinine and urea nitrogen levels were increased by vancomycin treatment; however, high-dose VC preadministration suppressed the increase in these levels. Histological examination also revealed that high-dose VC preadministration reduced the characteristics of vancomycin-associated nephrotoxicity, such as dilated renal tubules with casts, the dilation of renal proximal tubules, and tubular epithelial desquamation. Furthermore, high-dose VC preadministration reduced the appearance of apoptotic cells presumably derived from the epithelial cells in the dilated proximal tubules. Thus, intraperitoneally injected high-dose VC preadministration reduced vancomycin-associated nephrotoxicity in mice. These novel findings may indicate that vancomycin-associated nephrotoxicity in humans may be reduced by high-dose VC preadministration.

Energetically significant unconventional π-π contacts involving fumarate in a novel coordination polymer of Zn(II): In-vitro anticancer evaluation and theoretical studies

A new fumarato bridged Zn(II) coordination polymer viz. catena[μ-fumaratobis(3,5-dimethylpyrazole)zinc(II)trihydrate],{[Zn2(μ-fum)2(Hdmpz)4]·3H2O}n (1) (where, fum = fumarate, Hdmpz = 3,5-dimethylpyrazole) has been synthesized in purely aqueous medium at room temperature. The compound is structurally characterized by single crystal X-ray diffraction, FT-IR, electronic spectroscopy, PXRD and TGA. The zig-zag polymeric chain of 1 self assembles into a 2D supramolecular network via CH⋯π, CH⋯C, NH⋯O and CH⋯O interactions. Particularly relevant in 1, since in addition to the aforementioned non-covalent interactions, the electron rich π-system of fumarate is involved in energetically significant unconventional π–π contacts with the electron poor π-cloud of Hdmpz, which have been analyzed by DFT calculations and characterized using the non-covalent interaction (NCI) plot index. The strong H-bond agrees well with the MEP analysis since it is established between the H-bond donor involving NH group of Hdmpz and the H-bond acceptor involving O-atom of carboxylate moiety. The compound has been investigated for anticancer activities considering cell cytotoxicity, apoptosis and molecular docking as parameters in Dalton’s lymphoma (DL) malignant cancer cell line and results were compared with cisplatin (reference drug) under the same experimental conditions. Interestingly, negligible cytotoxic effect was observed against normal cells (PBMC) in comparison to the cancer cells (DL). The treatment of the compound caused appearance of apoptotic cells as evident by fluorescence based nuclear morphology study. The in silico study reveals that the compound interacts with the antiapoptotic proteins such as bcl-2, bcl-XL and Mcl-1 which may lead to apoptotic cell death.

Sphingosine-1-Phosphate Receptor 1, Expressed in Myeloid Cells, Slows Diet-Induced Atherosclerosis and Protects against Macrophage Apoptosis in Ldlr KO Mice.

We generated myeloid specific sphingosine-1-phosphate receptor 1 (S1pr1) deficient mice by crossing mice that had myeloid specific expression of Cre recombinase (lyzMCre) with mice having the S1pr1 gene flanked by loxP recombination sites. We transplanted bone marrow from these mice and control lyzMCre mice with intact macrophage S1pr1 gene expression into low-density lipoprotein (LDL) receptor gene (Ldlr) deficient mice. The resulting chimeras were fed a high fat atherogenic diet for nine or twelve weeks and evaluated for atherosclerosis development in the aortic sinus. Selective S1pr1 deficiency in bone marrow-derived myeloid cells resulted in accelerated development of atherosclerosis, necrotic core formation and the appearance of apoptotic cells within atherosclerotic plaques of Ldlr knockout mice in response to a high fat diet. Examination of macrophages in culture revealed that the sphingosine-1-phosphate receptor 1 selective agonist, SEW2871 or high density lipoprotein (HDL), protected macrophages against apoptosis induced by endoplasmic reticulum (ER) stress or oxidized LDL, through activation of phosphatidylinositol-3-kinase/Akt signaling. Targeted S1pr1-deletion prevented Akt activation and protection against apoptosis by either SEW2871 or HDL. Our data suggests that sphingosine-1-phosphate receptor 1 in macrophages plays an important role in protecting them against apoptosis in vitro and in atherosclerotic plaques in vivo, and delays diet induced atherosclerosis development in Ldlr deficient mice.

Salidroside attenuates hypoxia/reoxygenation-induced human brain vascular smooth muscle cell injury by activating the SIRT1/FOXO3α pathway.

It has been reported that salidroside (SAL), a natural dietary isothiocyanate, exhibits neuroprotective roles in cerebral ischemia-reperfusion injury. However, to the best of our knowledge, its underlying protective mechanism remains unknown. Sirtuin 1 (SIRT1) is a class III histone deacetylase involved in a variety of cellular functions. SIRT1 has been identified as a mediator of cerebral ischemia and may induce neuroprotection by activating various intracellular downstream targets, such as forkhead box protein O3α (FOXO3α). Therefore, the present study aimed to investigate whether SAL protects human brain vascular smooth muscle cells (HBVSMC) against hypoxia/reoxygenation (H/R) injury, which is a cell model of cerebral ischemia-reperfusion injury, through regulating the SIRT1-activited signaling pathway. The present study revealed that H/R treatment significantly reduced the expression of SIRT1 protein in HBVSMCs. Additionally, pretreatment with SAL reversed the H/R-induced decrease in cellular viability, increased caspase-3 activity, the appearance of apoptotic cells and the apoptosis rate in HBVSMCs. SAL attenuated the H/R-induced decrease in the expression of SIRT1 and phosphorylated FOXO3α protein in HBVSMCs, suggesting that the protective role of SAL in H/R injury occurs via the SIRT1/FOXO3α pathway. Furthermore, sirtinol, a SIRT1-specific inhibitor, suppressed the inhibitory effects of SAL on H/R-induced cytotoxicity and apoptosis as indicated by the downregulation of cell viability and upregulation of caspase-3 activity and apoptosis rate induced by sirtinol treatment in HBVSMCs. The reversal effects of SAL on H/R-induced alternation of B-cell lymphoma (Bcl-2) and Bcl-2 associated X protein expression were also attenuated by sirtinol. These results suggest that SAL exhibits neuroprotective effects against H/R injury by activating the SIRT1/FOXO3α pathway, which may become a novel potential therapeutic target for the treatment of cerebral ischemic disease.

Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0μM) for 48h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24h of 0.25μM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48h of 0.25μM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

8-Farnesyloxycoumarin induces apoptosis in PC-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity.

Prostate cancer is the second most common cancer in men worldwide. Overexpression of 15-lipoxygenase-1 (15-LOX-1) has been reported in prostate cancer patients. This study aimed to investigate the cytotoxic and anticancer effects of 8-farnesyloxycoumarin (8f), a prenylated coumarin, by inhibition of 15-LOX-1 activity, in prostate cancer cells. The activity of 15-LOX-1 and the inhibitory effects of 8f on this enzyme were first assessed in PC-3 and DU145 prostate cancer cells. The MTT assay was used to examine the cytotoxicity effects of 8f on PC-3 cells following 15-LOX-1 inhibition. To determine the type of cell death, chromatin condensation and DNA damage were examined by DAPI staining and comet assay, respectively. Furthermore, the effects of 8f on the cell cycle were evaluated by PI staining and flow cytometry. The activity of 15-LOX-1 was determined to be higher in PC-3 compared with DU145 cells; thus, this cell line was selected for further experiments. 8f induced cell death in PC-3 cells in a dose-dependent and time-dependent manner, with IC50 values similar to cisplatin, which was used as a control. However, 8f did not significantly affect the viability of HFF3, human foreskin fibroblast cells, under identical conditions. The appearance of apoptotic cells after 8f treatment was confirmed by the presence of PC-3 cells containing condensed chromatin as shown by DAPI staining. The comet assay indicated the induction of DNA damage in cancerous cells compared with normal cells. In addition, 8f induced a potent G1 cell-cycle arrest in PC-3 cells. Our results showed that the antitumor effects of 8f on PC-3 cells were promoted by apoptosis induction, probably via inhibition of 15-LOX-1 activity, thus suggesting that 8f may have therapeutic value in prostate cancer treatment.

Protective effects of melatonin and selenium against apoptosis of olfactory sensory neurons: A rat model study.

Selenium plays a role in the prevention of oxidative damage and has been linked to regulatory functions in cell growth, apoptosis, cell survival, and cytotoxicity. Melatonin has an antioxidant effect, which protects against a number of free radical species. Given its antioxidant properties, melatonin has been widely known to inhibit neuronal apoptosis. We examined the cytoprotective effects of melatonin and selenium in rat olfactory sensory neurons after rhinosinusitis by immunohistochemical evaluation of olfactory bulb mucosa. Rhinosinusitis was induced bilaterally in 24 animals. Twenty-four rats were randomly divided into three equal groups. The melatonin group was treated with intraperitoneal (i.p.) melatonin and ampicillin-sulbactam, the selenium group was treated with i.p. selenium and ampicillin-sulbactam, the antibiotic group was treated with i.p. ampicillin-sulbactam; all three groups were treated for 10 days. After a period of 10 days of treatment, the animals were killed for immunohistochemical analyses. All olfactory bulb mucosae were removed immediately. No histochemical differences were found in the three groups. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells were detected in each group. In the antibiotic group, the appearance of apoptotic cells was higher, whereas the number of apoptotic cells significantly decreased in the melatonin group. When compared with the selenium group, fewer terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells were observed in the melatonin group, which was not significant. In the antibiotic group, the cytoplasmic active caspase-3 and Bax immunostaining in the olfactory epithelium and glandular cells of stroma were higher when compared with the immunostaining in melatonin and selenium groups. Active caspase-3 and Bax immunostaining in the subepithelial stroma was dramatically reduced in the melatonin group. In contrast, the staining intensity and the number of Bcl-2 immunopositive cells were significantly increased in the melatonin group. In the selenium group, Bax and active caspase-3 were moderately immunopositive in the epithelium and subepithelial stroma. However, Bcl-2 immunostaining was more pronounced in the olfactory epithelium and some stromal cells. Our results indicated the possibility that the supplementation of melatonin and selenium, two antioxidant agents for the treatments in the rhinosinusitis rat model, might be reduced or prevent anosmia.

Growth hormone reverses excitotoxic damage induced by kainic acid in the green iguana neuroretina

It is known that growth hormone (GH) is expressed in extrapituitary tissues, including the nervous system and ocular tissues, where it is involved in autocrine/paracrine actions related to cell survival and anti-apoptosis in several vertebrates. Little is known, however, in reptiles, so we analyzed the expression and distribution of GH in the eye of green iguana and its potential neuroprotective role in retinas that were damaged by the intraocular administration of kainic acid (KA). It was found, by Western blotting, that GH-immunoreactivity (GH-IR) was expressed as two isoforms (15 and 26kDa, under reducing conditions) in cornea, vitreous, retina, crystalline, iris and sclera, in varying proportions. Also, two bands for the growth hormone receptor (GHR)-IR were observed (70 and 44kDa, respectively) in the same tissues. By immunofluorescence, GH-IR was found in neurons present in several layers of the neuroretina (inner nuclear [INL], outer nuclear [ONL] and ganglion cell [GCL] layers) as determined by its co-existence with NeuN, but not in glial cells. In addition, GH and GHR co-expression was found in the same cells, suggesting paracrine/autocrine interactions. KA administration induced retinal excitotoxic damage, as determined by a significant reduction of the cell density and an increase in the appearance of apoptotic cells in the INL and GCL. In response to KA injury, both endogenous GH and Insulin-like Growth Factor I (IGF-I) expression were increased by 70±1.8% and 33.3±16%, respectively. The addition of exogenous GH significantly prevented the retinal damage produced by the loss of cytoarchitecture and cell density in the GCL (from 4.9±0.79 in the control, to 1.45±0.2 with KA, to 6.35±0.49cell/mm2 with KA+GH) and in the INL (19.12±1.6, 10.05±1.9, 21.0±0.8cell/mm2, respectively) generated by the long-term effect of 1mM KA intraocular administration. The co-incubation with a specific anti-GH antibody, however, blocked the protective effect of GH in GCL (1.4±0.23cell/mm2) and INL (11.35±1.06), respectively. Furthermore, added GH induced an increase of 90±14% in the retinal IGF-I concentration and the anti-GH antibody also blocked this effect. These results indicate that GH and GHR are expressed in the iguana eye and may be able to exert, either directly of mediated by IGF-I, a protective mechanism in neuroretinas that suffered damage by the administration of kainic acid.

Conditioned umbilical cord tissue provides a natural three-dimensional storage compartment as in vitro stem cell niche for human mesenchymal stroma/stem cells

BackgroundThe use of large amounts of human multipotent mesenchymal stroma/stem cells (MSC) for cell therapies represents a desirable property in tissue engineering and banking in the field of regenerative medicine.Methods and resultsWhereas cryo-storage of umbilical cord (UC) tissue pieces in liquid nitrogen without ingredients was associated with predominant appearance of apoptotic cells after thawing and re-culture, progressive growth of MSC was observed following use of cryo-medium. Moreover, conditioning of UC tissue pieces by initial explant culture and subsequent cryo-storage with cryo-medium accelerated a further MSC culture after thawing. These findings suggested that conditioning of UC tissue pieces provides an in vitro stem cell niche by maintenance of a 3-dimensional natural microenvironment for continuous MSC outgrowth and expansion. Indeed, culture of GFP-labeled UC tissue pieces was accompanied by increased outgrowth of GFP-labeled cells which was accelerated in conditioned UC tissue after cryo-storage. Moreover, cryopreserved conditioned UC tissue pieces in cryo-medium after thawing and explant culture could be cryopreserved again demonstrating renewed MSC outgrowth after repeated thawing with similar population doublings compared to the initial explant culture. Flow cytometry analysis of outgrowing cells revealed expression of the typical MSC markers CD73, CD90, and CD105. Furthermore, these cells demonstrated little if any senescence and cultures revealed stem cell-like characteristics by differentiation along the adipogenic, chondrogenic and osteogenic lineages.ConclusionsExpression of MSC markers was maintained for at least 10 freeze/thaw/explant culture cycles demonstrating that repeated cryopreservation of conditioned UC tissue pieces provided a reproducible and enriched stem cell source.

A high ratio of G1 to G0 phase cells and an accumulation of G1 phase cells before S phase progression after injurious stimuli in the proximal tubule.

Proximal tubule (PT) cells can proliferate explosively after injurious stimuli. To investigate this proliferative capacity, we examined cell cycle status and the expression of cyclin‐dependent kinase inhibitor p27, a G1 phase mediator, in PT cells after a proliferative or injurious stimulus. Rats were treated with lead acetate (proliferative stimulus) or uranyl acetate (UA; injurious stimulus). Isolated tubular cells were separated into PT and distal tubule (DT) cells by density‐gradient centrifugation. Cell cycle status was analyzed with flow cytometry by using the Hoechst 33342/pyronin Y method. Most PT and DT cells from control rats were in G0/G1 phase, with a higher percentage of PT cells than DT cells in G1 phase. Lead acetate and UA administration promoted the G0‐G1 transition and the accumulation of G1 phase cells before S phase progression. In PT cells from rats treated with lead acetate or a subnephrotoxic dose of UA, p27 levels increased or did not change, possibly reflecting G1 arrest. In contrast, p27 became undetectable before the appearance of apoptotic cells in rats treated with a nephrotoxic dose of UA. The decrease in p27 might facilitate rapid cell cycling. The decreased number of p27‐positive cells was associated with PT cell proliferation in renal tissues after a proliferative or injurious stimulus. The findings suggest that a high ratio of G1 to G0 phase cells and a rapid accumulation of G1 phase cells before S phase progression in the PT is a biological strategy for safe, timely, and explosive cell proliferation in response to injurious stimuli.

P.5.a.006 Flavopiridol, an inhibitor of cyclin-dependent kinase 4, reverses cognitive deficits induced by β-amyloid peptide 1–42

It is known that growth hormone (GH) is expressed in extrapituitary tissues, including the nervous system and ocular tissues, where it is involved in autocrine/paracrine actions related to cell survival and anti-apoptosis in several vertebrates. Little is known, however, in reptiles, so we analyzed the expression and distribution of GH in the eye of green iguana and its potential neuroprotective role in retinas that were damaged by the intraocular administration of kainic acid (KA). It was found, by Western blotting, that GH-immunoreactivity (GH-IR) was expressed as two isoforms (15 and 26 kDa, under reducing conditions) in cornea, vitreous, retina, crystalline, iris and sclera, in varying proportions. Also, two bands for the growth hormone receptor (GHR)-IR were observed (70 and 44 kDa, respectively) in the same tissues. By immunofluorescence, GH-IR was found in neurons present in several layers of the neuroretina (inner nuclear [INL], outer nuclear [ONL] and ganglion cell [GCL] layers) as determined by its co-existence with NeuN, but not in glial cells. In addition, GH and GHR co-expression was found in the same cells, suggesting paracrine/autocrine interactions. KA administration induced retinal excitotoxic damage, as determined by a significant reduction of the cell density and an increase in the appearance of apoptotic cells in the INL and GCL. In response to KA injury, both endogenous GH and Insulin-like Growth Factor I (IGF-I) expression were increased by 70 ± 1.8% and 33.3 ± 16%, respectively. The addition of exogenous GH significantly prevented the retinal damage produced by the loss of cytoarchitecture and cell density in the GCL (from 4.9 ± 0.79 in the control, to 1.45 ± 0.2 with KA, to 6.35 ± 0.49 cell/mm2 with KA + GH) and in the INL (19.12 ± 1.6, 10.05 ± 1.9, 21.0 ± 0.8 cell/mm2, respectively) generated by the long-term effect of 1 mM KA intraocular administration. The co-incubation with a specific anti-GH antibody, however, blocked the protective effect of GH in GCL (1.4 ± 0.23 cell/mm2) and INL (11.35 ± 1.06), respectively. Furthermore, added GH induced an increase of 90 ± 14% in the retinal IGF-I concentration and the anti-GH antibody also blocked this effect. These results indicate that GH and GHR are expressed in the iguana eye and may be able to exert, either directly of mediated by IGF-I, a protective mechanism in neuroretinas that suffered damage by the administration of kainic acid.