Identification of 3-Aryl-1-benzotriazole-1-yl-acrylonitrile as a Microtubule-Targeting Agent (MTA) in Solid Tumors.

Recently, a compound derived from recent scientific advances named 34 has emerged as the focus of this research, the aim of which is to explore its potential impact on solid tumor cell lines. Using a combination of bioinformatics and biological assays, this study conducted an in-depth investigation of the effects of 34. The results of this study have substantial implications for cancer research and treatment. 34 has shown remarkable efficacy in inhibiting the growth of several cancer cell lines, including those representing prostate carcinoma (PC3) and cervical carcinoma (HeLa). The high sensitivity of these cells, indicated by low IC50 values, underscores its potential as a promising chemotherapeutic agent. In addition, 34 has revealed the ability to induce cell cycle arrest, particularly in the G2/M phase, a phenomenon with critical implications for tumor initiation and growth. By interfering with DNA replication in cancer cells, 34 has shown the capacity to trigger cell death, offering a new avenue for cancer treatment. In addition, computational analyses have identified key genes affected by 34 treatment, suggesting potential therapeutic targets. These genes are involved in critical biological processes, including cell cycle regulation, DNA replication and microtubule dynamics, all of which are central to cancer development and progression. In conclusion, this study highlights the different mechanisms of 34 that inhibit cancer cell growth and alter the cell cycle. These promising results suggest the potential for more effective and less toxic anticancer therapies. Further in vivo validation and exploration of combination therapies are critical to improve cancer treatment outcomes.

Confocal investigation on colocalization between tubulin posttranslational modifications and associated proteins in rat C6 glioma cells.

Glioblastoma multiforme is the most lethal brain tumor. In the study of mechanisms underlying its development attention has been paid to the microtubular network of its cells, mainly on ßIII tubulin, considered as a marker of malignancy. In the present work, we chose to investigate the tubulin code in glioblastoma cells, analyzing the degree of interaction between tubulin post-translational modifications and different proteins associated with them. The pattern of diverse associated proteins such as EB-1, CLIP-170 and kinesin-1 and their degree of co-distribution with the most abundant post-translational tubulin modifications (tyrosination, acetylation and polyglutamylation) were evaluated. Through immunofluorescence we have shown that EB-1, CLIP-170 and kinesin-1 were well detectable in glioblastoma cells. The double fluorescence and colocalization index between the post-translational modifications of tubulin and associated proteins showed that tyrosinated α-tubulin has significantly high affinity with EB-1, CLIP-170 and kinesin-1, while for acetylated and polyglutamylated tubulin, the degree of interaction with the three associated proteins evaluated was less apparent. Data presented in this paper underline the importance of a thorough analysis of the microtubular mechanics in glioblastoma cells. This may suggest new experimental therapeutic approaches able to act more selectively on the microtubular network of cells in this type of cancer.

Correlation between wing bone microstructure and different flight styles: The case of the griffon vulture (gyps fulvus) and greater flamingo (phoenicopterus roseus).

Flying is the main means of locomotion for most avian species, and it requires a series of adaptations of the skeleton and of feather distribution on the wing. Flight type is directly associated with the mechanical constraints during flight, which condition both the morphology and microscopic structure of the bones. Three primary flight styles are adopted by avian species: flapping, gliding, and soaring, with different loads among the main wing bones. The purpose of this study was to evaluate the cross-sectional microstructure of the most important skeletal wing bones, humerus, radius, ulna, and carpometacarpus, in griffon vultures (Gyps fulvus) and greater flamingos (Phoenicopterus roseus). These two species show a flapping and soaring flight style, respectively. Densitometry, morphology, and laminarity index were assessed from the main bones of the wing of 10 griffon vultures and 10 flamingos. Regarding bone mineral content, griffon vultures generally displayed a higher mineral density than flamingos. Regarding the morphology of the crucial wing bones involved in flight, while a very slightly longer humerus was observed in the radius and ulna of flamingos, the ulna in griffons was clearly longer than other bones. The laminarity index was significantly higher in griffons. The results of the present study highlight how the mechanics of different types of flight may affect the biomechanical properties of the wing bones most engaged during flight.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model.

BACKGROUND: Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. METHODS: Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/ß tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. RESULTS: The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. CONCLUSIONS: Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Investigation of the effects of Lycium barbarum polysaccharides against cadmium induced damage in testis.

This study describes the effects of Lycium barbarum polysaccharides (LBP) on testicular damage induced by cadmium (Cd). Adult male rats were i.p. injected with CdCl2 (4mg/Kg, once) with or without LBP pretreatment (300mg/Kg orally, once a day, for 30days). Testis weight, morphological/histological structure and oxidative stress parameters were evaluated. Several adverse effects were observed after CdCl2 injection, with a significant decrease in body/testis weight ratio (P<0.05), gross morphological changes with hyperemia of the parenchyma, increased volume and alteration in the structure of the seminiferous tubules. Furthermore, Cd intoxication caused a significant decrease of glutathione (GSH) and Trolox equivalent antioxidant capacity (TEAC) in testis (P<0.05) together with a significant increase (P<0.01) of 3-nitro-l-tyrosine (3NT) while malondialdehyde (MDA) did not change. LBP pretreatment caused slight signs of improvement in the morphology of the seminiferous tubules. Our results confirm that Cd induces testicular damage and suggest the oxidative stress involvement. LBP could ameliorate Cd testicular damage but further investigations are needed.

Nitrosative-induced posttranslational α-tubulin changes on high-glucose-exposed Schwannoma cell line.

OBJECTIVE: One of the major complications during prolonged hyperglycemic condition is the onset of the so-called diabetic neuropathy, that can affect the peripheral nervous system. Evidence has reported that glucose-induced oxidative stress could be a key mediator in this process, impairing the cytoskeletal structures, such as microtubules. In general, much attention is paid to the possible nitrosative-induced changes of neurons during hyperglycemic conditions, while little is known of the Schwann cells. METHODS: Using morphological examination, immunofluorescence staining and western blot analysis, the possible hyperglycemic oxidative-induced microtubular changes in the RT4 Schwannoma cell line was investigated. RESULTS: After 72 hrs of 180 mM d-glucose exposure, a decrease of total, tyrosinated and detyrosinated α-tubulins was found, whereas a significant increase of the acetylated α-tubulin isotype and 3-nitro-l-tyrosine was present, both through western blot and immunofluorescence staining. Moreover a downregulation of catalase and deacetylase Sirt2 enzymes was detected. CONCLUSION: Our data underline the importance of nitrosative-induced microtubular alterations in the PNS, during hyperglycemic conditions, highlighting that Schwann cells may be directly involved in the pathogenesis of diabetic neuropathy, through the impairment of their microtubular network.

Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

Nitrosative stress induces proliferation and viability changes in high glucose-exposed rat Schwannoma cells.

OBJECTIVES: Schwann cells may be involved in the pathogenesis of several neuropathies, such as those linked to an excess of d-glucose. Indeed, hyperglicemic condition can often result in the production of high reactive/nitrosative oxygen species concentration and possible damage of several cell structures. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on RT4 Schwannoma cell lines. METHODS: Cells were cultured for 72hrs in the presence of 180 mM D-glucose. Morphology, growth rate, cell viability, catalase evaluation and Western blot were performed. RESULTS: In D-glucose-exposed cells, 3-Nitrotyrosine increase and subsequent modifications in cell morphology, growth rate, viability and catalase activity were found. CONCLUSION: Our findings suggested a possible primary role played by Schwann cells in the hyperglicemic neuropathy pathogenesis, through the excessive production of RNS and a decrease in antioxidant defense systems, bearing out the importance of the "nitrosative hypothesis" in the hyperglicemic-induced nervous system complications.

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Aromatase expression in cultured fetal sheep astrocytes after nitrosative/oxidative damage.

Aromatase, the enzyme converting androgens into estrogens, is involved in many brain processes such as neural differentiation and plasticity or the prevention of cell death. We have previously observed an increase in aromatase immunoreactivity in sheep neurons exposed in vitro to the oxidant 3-nitro-L: -tyrosine. However, little is known regarding the way that sheep astrocytes cope with nitrosative stress, a condition occurring in sheep in the pathogenesis of neurodegenerative disorders such as scrapie and Maedi-Visna. Our aim has been to evaluate the effects of 3-nitro-L-tyrosine on astrocyte primary cultures from 90-day-old fetal sheep brain. Living cells were observed and characterized by immunofluorescence with a GFAP antibody, which indicated that the majority of the cells were astrocytes. A viability assay was performed on both untreated and treated cells. Reverse transcription with the polymerase chain reaction was undertaken to monitor time- and dose-dependent variations in aromatase gene expression. Stressed astrocytes showed signs of deterioration, were reduced in number, and appeared round with few short processes; the cell death rate was ∼30%. Aromatase expression was detected starting from a 24-h exposure to 1 mM 3-nitro-L-tyrosine and reached the highest levels at 72 h. Thus, oxidative damage probably results in the local production of neuroprotective estradiol by reactive astrocytes via the aromatization of testosterone.

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

Seasonal Effect on Developmental Competence, Oxidative Status and Tubulin Assessment of Prepubertal Ovine Oocyte.

The reproductive seasonality of domestic animals is often manipulated in order to have more reproductive periods for commercial purposes related to the production of milk and meat. It is scientifically proven that such an alteration of the reproductive activity in sheep entails a deterioration in oocyte quality, leading to an inability to generate embryos. Since oocytes obtained from prepubertal ewes can be incorporated into an in vitro embryo production system and considering that their quality is crucial to the success of in vitro procedures, the aim of this work was to investigate the effect of seasons on the quality of prepubertal ovine oocytes collected in autumn and spring. Ovaries were collected from a local slaughterhouse from 30-40-day-old suckling lambs during both seasons. Following 24 h of in vitro maturation, oocytes developmental competence, reactive oxygen species (ROS) intracellular levels, and mitochondrial activity were evaluated, and a tubulin assessment was performed. The results on embryo production, as a percentage of first divisions and number of blastocysts obtained, were significantly higher in oocytes collected in the spring. Mitochondrial activity in oocytes was higher, and ROS production significantly lower, in spring than in autumn. Tubulin PTMs (tyrosinated and acetylated α-tubulin) showed a higher immunoreactivity in oocytes collected in spring compared with autumn sampling. Our data showed that seasons may affect the developmental competence, energetic status, and tubulin assessment of oocytes recovered from prepubertal ewes. Therefore, special care should be taken when choosing the period of the year for prepuberal ovine oocytes collection aimed at in vitro embryo reproduction programs.

Expression and distribution of P450-aromatase in the ovine hypothalamus at different stages of fetal development.

OBJECTIVES: An important step of sexual differentiation is the conversion of testosterone to estrogen by aromatase leading to masculinization and defeminization of the fetal brain areas crucial for normal sexual behavior and reproduction. Brain sexual differentiation occurs throughout a critical period starting from different prenatal stages depending on the species. Such period goes on from gestation day (GD) 30 to 100GD in the sheep. The fetal sheep brain is reported to aromatize androgens to estrogens at 64GD. The main goal of this work was to evaluate aromatase expression in sheep hypothalami during the whole period of sexual differentiation (35GD, 55GD, 80GD, 115GD) and whether differences may be observed depending on gestational stage and sex. METHODS: Sections at the hypothalamic level underwent immunoperoxidase technique employing anti-aromatase and anti-androgen receptor antibodies. Samples from 35GD and 55GD were also processed with in situ hybridization using aromatase cDNA probe. Blot analyses were performed to quantify possible aromatase immunoexpression differences between sexes. For sexing, samples at 35GD and 55GD underwent DNA extraction and SRY amplification. RESULTS: Our results revealed aromatase and androgen receptor immunoreactivity along the whole period of sexual differentiation. Both molecules were detected in many brain regions and markedly in the periventricular area. The highest aromatase and androgen receptor amounts were observed at 35GD and 55GD, when aromatase was more abundant in females than in males. CONCLUSIONS: In conclusion, the sheep can be included among the species where aromatase is highly expressed in the hypothalamus during the whole period of sexual differentiation.

Morphological features and microtubular changes in vitrified ovine oocytes.

Cryobanking of oocytes collected from prepubertal donors may supply a virtually unlimited number of female gametes for both basic research and commercial applications. Prepubertal oocytes show some structural and functional limitations compared to the adult ones that may impair their ability to recover damages from cryopreservation. In oocytes, the meiotic spindle is acutely sensitive to temperature deviation, but capable of regeneration following cryopreservation. In the present work, we studied the effects of vitrification and post-warming incubation on the microtubular cytoskeleton and the tubulin post-translational modifications (tyrosination and acetylation) in prepubertal and adult oocytes. Obtained results showed that prepubertal oocytes are more affected by vitrification-induced injuries than adult ones. In fact, prepubertal oocytes showed more severe alterations of the meiotic spindle conformation and a higher percentage of parthenogenetic activation compared to adult ones. Moreover, in the adult oocytes the equilibrium between tyrosinated and acetylated α-tubulin was restored after 4 h of post-warming incubation. Diversely, in prepubertal oocytes the imbalance between tyrosinated and acetylated α-tubulin was increased during post-warming incubation. Our study shows that prepubertal oocytes react differently to the insults provoked by vitrification compared to adult oocytes, showing an impaired ability to recover from vitrification-induced injuries. In the evaluation of oocyte ability to recover from vitrification-induced injuries, tubulin post-translational modifications represent an important indicator for assessing oocyte quality.

Different nitrosative-induced microtubular modifications and testosterone neuroprotective effects on high-D-glucose-exposed neuroblastoma and glioma cells.

OBJECTIVES: Diabetic complications can often affect the central nervous system since the chronic exposure to hyperglycemia can result in the production of high concentration of reactive oxygen species with subsequent damage of several cell structures such as the cytoskeleton. In order to antagonize the oxidative status many substances have been tested as antioxidants. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on microtubular network of neuroblastoma and glioma mortalized cell lines, testing the possible neuroprotective effect of testosterone. METHODS: Neuroblastoma (C1300) and glioma (C6) cell lines were cultured in the presence of 300 mM (C1300) or 310 mM (C6) D-glucose, with or without 50 nM testosterone. After 72 hrs, morphology, growth rate, cell viability and catalase activity were evaluated. In addition, with the aim to detect any changes in the amount of tubulin isoforms, Western blot analysis was performed. RESULTS: In D-glucose-exposed cells, it was found a down-regulation of tubulin isoforms and an increase in 3-nitro-L-tyrosine and subsequent modifications in cell morphology, growth rate, viability and catalase activity. All these changes were more severe in neuroblastoma than in glioma cell line. When testosterone was added to the medium, all the parameters were very similar to controls. This neuroprotective action was well-detectable in C1300 cells, whereas testosterone was not able to recover significantly in C6 cells. CONCLUSION: Our results displayed: i) a selective action of high glucose on microtubules; ii) a different sensitivity to oxidative stress in neuronal and glial cells; iii) a different neuroprotective action of testosterone on neuronal and glial cells.

Tubulin post-translational modifications in developing dog primary neurons obtained with methods according to the 3Rs principles.

Microtubules play a crucial role during neuronal morphogenesis regulating many functions. In the study of these phenomena in vitro cellular models have been employed, mainly resorting to housed experimental animals. Among alternative models in neurobiological study, recently dog caught particular attention. In fact, the complexity of the canine brain, the life long span and the neurodegenerative pathologies render the dog a species more close to humans than rodents. Lately, growing interest in the limitation of the use of experimental animals, has stimulated the search for alternative experimental protocols. Starting from fetal dog brain, obtained by alternative way of sampling, we set neuronal primary cultures. Through immunofluorescence, we examined the presence and the cellular distribution of tubulin post-translational modifications as tyrosinated and acetylated α-tubulin, as markers of dynamic and stable microtubule respectively. In addition, we evaluated the pattern of two associated proteins which may slide on these two tubulin modifications, i.e. CLIP-170 and Kinesin-1. A clear positivity for tyrosinated and acetylated α-tubulin, was found. As far as the motor proteins are concerned, we detected a prevalence of CLIP-170 compared to kinesin-1 with a better overlapping between tyrosinated α-tubulin and CLIP-170. Our findings highlighted some original data about the role of the microtubular network during early phases of canine neuronal morphogenesis. In addition, the experimental protocol underlined the utility of this alternative model that allows to bypass both the scarcity of commercial canine neuronal cell lines and the need to resort to experimental dogs, respecting the 3Rs principles (reduction, refinement, and replacement).

Morphological and quantitative analysis on α-tubulin modifications in glioblastoma cells.

Glioblastoma is one of the primary tumors of the brain with high invasiveness and lethality. In the study of its pathophysiology in recent years much attention has been paid to the microtubular network, but exclusively to ß-III tubulin, whose presence in glioblastoma cells is associated with the degree of malignancy and diffusion. As is well known, the microtubular network performs its many functions thanks to various post-translational modifications, most of which affect the α-tubulin subunit. These modifications, able to coexist in the same microtubule, bind certain driving and cargo proteins, deeply influencing cellular functions. Since there are no data in the literature about the diverse post-translational modifications of tubulin in glioblastoma cells, this work aims to fill this gap. In the present work, through immunofluorescence, morphological analysis and Western blot, we studied the pattern of tyrosinated, detyrosinated, delta 2 (Δ2), acetylated and polyglutamylated tubulin. We detected good immunopositivity in fluorescence for almost all the modifications examined. Only Δ2 showed a very low signal. Western blot displayed that the most abundant tubulin modifications in glioblastoma cells were tyrosinated, acetylated and polyglutamylated. Morphological evaluation revealed that these modifications were more present along the cytoplasmic extensions, less evident around the cell body and always strongly evident in the mitotic spindles of the dividing cells. For the first time, the most abundant post-translational modifications and their cellular compartmentalization in glioblastoma cells have been highlighted, suggesting a novel approach in the study of their microtubular network and in the search of new experimental therapeutic strategies.

A recovery time after warming restores mitochondrial function and improves developmental competence of vitrified ovine oocytes.

The aim of the present study was to assess the ability of vitrified/warmed oocyte to recover from vitrification-induced damages after warming. In vitro matured, vitrified/warmed ovine oocytes were assessed for developmental competence, mitochondrial activity and distribution, ATP, ROS and catalase levels during 6 h of in vitro culture using fresh oocytes as control. ATP content in vitrified oocytes was lower than control during 4 h of post warming culture (p < .01). Vitrified oocytes were able to fill this gap only after 6 h of post-warming incubation. Moreover, mitochondrial activity was significantly lower (p < 0.01) in vitrified oocytes compared to controls, and this difference was maintained up to 2 h of incubation. Then the activity increased and at 4 h it was higher compared to controls (p < 0.01). These oocytes showed an increasing rate of clustered distribution of mitochondria which was lower than controls during the first 4 h of post warming culture (p < 0.01). ROS level was significantly higher at 0 h in vitrified compared to control oocytes and this difference was maintained also at 2 h and 6 h of incubation (p < 0.01). Catalase level was higher in vitrified oocytes than controls (p < 0.01) during the entire culture period. Cleavage and blastocyst rates were lower in vitrified oocytes compared to control ones during the two first time point of incubation period (p < .01), indeed they increased significantly from 0 to 4 h of incubation post warming (p < 0.01). The study demonstrated that vitrified/warmed oocytes need an extra time to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, time of damage recovery after vitrification could be used to standardize the vitrification protocols and to improve the developmental competence of vitrified/warmed oocytes.

Possible antioxidant effect of Lycium barbarum polysaccharides on hepatic cadmium-induced oxidative stress in rats.

The aim of this study was to investigate the potential protective effect of Lycium barbarum polysaccharides (LBP) pretreatment against cadmium (Cd)-induced hepatotoxicity in rats. Wistar rats were divided into control group, LBP group (300 mg/kg orally, once a day, for 30 days), Cd group (CdCl2 4 mg/kg i.p. once), and LBP + Cd group (LBP 300 mg/kg orally, once a day, for 30 days + CdCl2 4 mg/kg i.p. 24 h after the last treatment). Cd liver injury was examined by morphological/histological changes, transaminases, total protein concentration, and oxidative stress evaluated by MDA, 3NT, GSH, SOD, and TEAC activities. Cd intoxication caused gross morphological changes with hyperemia of the parenchyma, increased volume, and disappearance of the anatomical limits of the lobes associated with an increase of ALT, GSH, and TEAC in plasma and a decrease of MDA, GSH, and TEAC in liver, SOD, and total proteins in plasma. LBP pretreatment caused a slight improvement in the histological architecture and in the 3NT amount together with a significant improvement of hematic parameters. On the basis of the obtained results, we can affirm that LBP pretreatment can ameliorate liver conditions, but further studies are needed to better evaluate the protective antioxidant effects of LBP against Cd-induced toxicity.

Effects of testosterone on differentiation and oxidative stress resistance in C1300 neuroblastoma cells.

OBJECTIVES: Using undifferentiated mouse neuroblastoma cells (C1300), we have previously observed that testosterone (T) exerts a neuroprotective action against oxidative stress. Nitrogen intermediates induce the production of 3-nitro-L-tyrosine (3NT), an amino acid analogue involved in many neurodegenerative disorders. The aim of our work is to investigate T capability on C1300 cell differentiation. It is also evaluated whether differentiation could mitigate the nitrosative effects of 3NT. METHODS: The effects of both T and 3NT were studied on an undifferentiated cell line of neural origin (C1300). For this purpose, cell cultures underwent morphometric investigation, blot analyses and catalase activity assay. All data obtained were expressed as mean+/-SD and tested by one-way ANOVA or Student's t test. RESULTS: The results were compared with those gathered by means of N6,2'-O-dibutyryl-adenosine-3',5'-cyclic-mono-phosphate (db-cAMP), a well-known differentiating agent. T-exposed cells showed an irregular shape and exhibited long branching cytoplasmic extensions, which were longer than in db-cAMP cells. Moreover, T-exposure induced an increase in the expression of tyrosinated and acetylated alpha-tubulin while 3NT-incorporation into tubulin was markedly reduced. The action of antioxidant defence systems, namely catalase activity, was enhanced in cells exposed to T. CONCLUSION: This work highlighted the effects of db-cAMP on differentiation and neuroprotection, but even indicated that T exposure induced differentiation in C1300 cells and this process matches a significant neuroprotective effect. This action seemed to be more effective than in db-cAMP-treated cells. T is suggested, like other substances having antioxidant properties, to be of potential interest in the experimental therapy of neuropathological conditions.