Morpho-functional characterization of cultured pigment cells fromRana esculenta L. Liver

Summary A simple method to isolate and culture liver pigment cells fromRana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells fromRana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance. This research was partly supported by grant of Ministero della Pubblica Istruzione, Ricerca Scientifica.     

Fractional Analyses of Soybean Sterols in Four Classes

A well growing methane oxidizing bacterium was isolated in pure culture from a natural source through studying production of single cell protein. This bacterium was identified as a new species Methylomonas flagellata. It was shown to be a gram-negative rod, 1.0 to 1.2 by 2.0 to 3.0 μ, and motile by means of polar flagella. Formation of a type of “immature cysts” was observed.

This bacterium showed relatively high growth rate of μ=0.17 hr^?1 for a pure methane oxidizing bacterium.     

Transdifferentiated Mesenchymal Stem Cells as Alternative Therapy in Supporting Nerve Regeneration and Myelination

1. Aims: Demyelination plays a crucial role in neurodegenerative processes and traumatic disorders. One possibility to achieve remyelination and subsequent restoration of neuronal function is to provide an exogenous source of myelinating cells via transplantation. In this context, mesenchymal stem cells (MSCs) have attracted interest. They are multipotent stem cells that differentiate into cells of the mesodermal lineage like bone, cartilage, fat, and muscle. Although adult, their differentiation potential is remarkable, and they are able to transdifferentiate.2. Methods: We transformed cultivated rat MSCs into myelinating cells by using a cytokine cocktail. Transdifferentiated MSCs were characterized by an enhanced expression of LNGF-receptor, Krox20, and CD104, and a decreased expression of BMP receptor-1A as compared to untreated MSCs. The myelinating capacity was evaluated in vitro and in vivo. Therefore, PC12 cells, normally unmyelinated, were cocultivated with MSCs, transdifferentiated MSCs, and Schwann cells, or the respective cells were grafted into an autologous muscle conduit bridging a 2-cm gap in the rat sciatic nerve. Myelination of PC12 cells was demonstrated by electron microscopy. In vivo, after 3 and 6 weeks regeneration including myelination was monitored histologically and morphometrically. Autologous nerves and cell-free muscle grafts were used as control.3. Results: Schwann cells and transdifferentiated MSCs were able to myelinate PC12 cells after 14 days in vitro. In vivo, autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate myelination was noted in the Schwann cell groups and, albeit with restrictions, in the transdifferentiated MSC groups, while regeneration in the MSC groups and in the cell-free groups was impaired.4. Conclusion: Our findings demonstrate that it may be possible to differentiate MSCs into therapeutically useful cells for clinical applications in myelin defects.     

Solitary terminal cells of Aphanizomenon gracile (Cyanobacteria,Nostocales) can divide and renew trichomes

An attempt was made to find evidence that morphologically distinct terminal cells of filamentous cyanobacterium Aphanizomenon gracile strain CCALA 8 are capable of dividing and forming trichomes. Based on our current knowledge, the division of morphologically diversified terminal cells is possible in nostocalean cyanobacteria. However, this process has been observed only in a few species. Terminal cells of A. gracile differ morphologically from other vegetative cells of a trichome, as they are not hyaline and can sometimes be found as solitary cells in cultures. Hence, it was reasonable for us to suspect that these cells are capable of dividing and forming trichomes. We observed terminal cells under a light and transmission electron microscope. Microscopic observations revealed that the septum formed in both solitary terminal cells and in terminal cells attached to trichomes. Our study is the first to demonstrate division and renewal of trichomes in terminal cells of A. gracile. Previously, such mode of reproduction was described only for another nostocalean cyanobacterium Raphidiopsis mediterranea. Moreover, our findings further emphasize the variability among members that belong to the genus Aphanizomenon , in which a type species (A. flos‐aquae) has hyaline cells incapable of dividing and renewing trichomes, while A. gracile can additionally propagate by solitary terminal cells division. This additional feature distinguishing A. gracile from typical species of Aphanizomenon, such as A. flos‐aquae, might be valuable for resolving taxonomic position of the species considering ambiguous genetic relationship between A. gracile and A. flos‐aquae.     

The Temporal Relationship between Protein Phosphatase, Mitochondrial CytochromecRelease, and Caspase Activation in Apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochromec.Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochromecin apoptosis induced by the Fas receptor. We demonstrate that cytochromecis released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochromec.The protein phosphatase inhibitor calyculin A prevented cytochromecrelease and apoptosis induced by both agents, suggesting that release of cytochromecis required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochromec.The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochromecand apoptosis induced by both insults.     

Achene morphology and slime structure in some taxa of Artemisia L. and Neopallasia L. (Asteraceae)

We have examined slime cell distribution on the surface of the achenes of some Artemisia and Neopallasia taxa, as well as slime composition, envelope formation during the hydration, and slime relation to different morphological features and environmental factors. The results of the studies show a characteristic pattern of slime cells distribution, which could differ between taxa. The slime in the taxa studied belongs to the cellulose type and consists of two components i.e., pectins and cellulose. Although all fruits contain slime cells, not all of them show the slime envelope formation. Plants occurring in dry habitats (such as A. barrelieri) or annual species (such as A. annua) are characterised by a large amount of slime and a fast process of slime envelope formation. Slime production has not been observed in some polyploid populations (A. campestris and A. campestris ssp. sericea) and in two species occurring in relatively fertile habitats (A. verlotiorum, A. vulgaris). A reason for this may be either the immaturity of polyploid fruits leading to the production of a scarce, not detectable slime amount or, alternatively, the occurrence of not functional slime cells. Slime facilitates and stimulates the germination, as well as the adherence of the fruits to the ground or to animals (for dispersal). The slime could play important role in the distribution and colonisation of new habitats in many Artemisia taxa.     

Isolation and identification of clara cells from rabbit lung

Summary A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion. Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy. This research was supported in part by the U.S. Environmental Protection Agency under an interagency agreement relating to the Federal Interagency Energy/Environmental Research and Development Program.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Studies on the Discrimination of SCP-related Yeast by Proton Magnetic Resonance Spectroscopy: Structural Changes in Cell Wall Mannan of Candida subtropicalis Grown in Different Media

Applicability of PMR spectroscopy to the discrimination of the SCP-related yeast was investigated because the yeast was inactivated by heat treatment. When Candida subtropicalis was cultured in a medium containing glucose as a sole source of carbon, its cell wall mannan (mannan A) showed a PMR spectral pattern characterized by three intense peaks at δ: 4.97, 5.10, 5.29 and two small peaks at δ: 4.90, 5.19. Mannan (mannan B) from the yeast cultured in a medium containing n-pentadecane and triton X–100 showed a different spectral pattern in which the signals were observed at δ: 4.97, 5,10, 5.29, the intensity ratios of the signals were also different from those of mannan A. Acetolysis mannan was analyzed to compare the difference between two specified structures by using a gel elution method, methylation analysis and PMR spectra. Mannan B contained a less amount of the a (1→3) linkage than mannan A did, and differed from mannan A in its distribution pattern of side chain units. Our previous results together with the present ones proved the PMR method to be effective for the discrimination of the yeast.     

Demonstration of Aminopeptidase Activities Secreted by Amsonia tabernaemontana Walt. Cells

Using synthetic substrates, an uncomplicated and sensitive procedure for the determination of extracellular aminopeptidase was developed. The studied enzyme produced by the tested plant material (calli, cell suspension culture and roots of Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrates β‐naphthylamides (βNA) and 4‐(phenylazo) phenylamides (PAP‐amide) of the amino acids to β‐naphthylamine and 4‐(phenylazo) aniline, respectively, and amino acid. The β‐naphthylamides of the amino acids were applied for the identification of extracellular aminopeptidase, whereas the 4‐(phenylazo) phenylamides of the amino acids were used for the determination of intra‐ and extracellular aminopeptidase activity. By simultaneous azocoupling of β‐naphthol with Fast Garnet GBC salt on agar plates a corresponding brown‐red hardly water‐soluble azo‐dye was produced. The evaluation of dyed zones allowed the extracellular aminopeptidase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat‐inactivated cells (10 min at 100 °C) or in medium inoculated without substrate. On the agar plates with substrate and sterile Amsonia seedlings, changes in coloration were observed indicating a release of aminopeptidase from the roots during germination. The results show a 91.0 % intracellular and 9.0 % extracellular distribution of aminopeptidase activity, when a cell suspension culture of A. tabernaemontana Walt. as the plant material was used. The agar plate method described permits the rapid, uncomplicated and specific detection of plant producers of extracellular aminopeptidase, which could be particularly useful in future inhibitory and/or biotechnological studies.     

Coumarin derivatives protection against ROS production in cellular models of Aβ toxicities

The role of oxidative stress and free radicals in the development of Alzheimer's disease (AD) has been the focus of many recent studies. The role of hydrogen peroxide (H₂O₂) in AD is thought to be associated with Aβ (amyloid – β) damage in cells. A number of coumarin derivatives were previously found to be potent anti-inflammatory and antioxidant agents. Herein, these coumarin derivatives were tested as H₂O₂ scavengers with the DCF assay using two types of neuronal cells: (a) wild type (N2a) neuroblastoma cells and (b) APP/PS1 transgenic cell line expressing Aβ. Their scavenging activity was varied between the types of cell cultures and it was found to be concentration and time dependent in the mutant cells. Their protective role against cell death further supports this notion. These results suggest that these compounds could be used as a template in the design of new molecules with a possible role in AD.     

Identification of the Organism and Some Conditions of Peptidoglutaminase Formation

A peptidoglutaminase activity in microorganisms was detected using carbobenzoxy-l-glutamine or tertiary-amyloxycarbonyl-l-glutaminyl-l-proline as substrate. By screening, an organism which produces a relatively large amount of peptidoglutaminase was isolated from soil. The organism was identified as Bacillus circulans. The highest enzyme formation by the bacterium occurred during stationary growth phase in the basal medium containing lactose (0.5%) and polypepton (1%).     

The effects of myocyte enhancer factor 2A gene on the proliferation, migration and phenotype of vascular smooth muscle cells

The genetic basis for the phenotypic switching of vascular smooth muscle cells (VSMCs) is unclear in atherosclerosis. Recent studies showed that the 21‐base pair deletion mutation (Δ21) in myocyte enhancer factor 2A (MEF2A) gene could be an inherited marker for coronary artery disease. MEF2A mutation may affect the phenotypic switching of VSMCs. Human aortic VSMCs were used. Four groups of VSMCs transfected with green fluorescent protein plasmid (control group), MEF2A wild‐type (WT) plasmid (WT group), MEF2A Δ21 plasmid (Δ21 group) or MEF2A siRNA (siRNA group) were studied. The proliferation of VSMCs was determined by methylthiazolyldiphenyl‐tetrazolium bromide, and the migration of VSMCs was measured by Millicell chamber. The protein expressions of MEF2A, smooth muscle α‐actin, SM22α, osteopontin and p38 mitogen‐activated protein kinase signaling pathway were detected by Western blotting. MEF2A protein expression was knockdown by siRNA transfection. MEF2A protein was overexpressed in WT and Δ21 groups. Δ21 and siRNA groups obviously showed more proliferation (methylthiazolyldiphenyl‐tetrazolium bromide, 0.63 vs 0.66 vs 0.31, P < 0.01) and migration (52.6 vs 58.0 vs 21.2, P < 0.01) of VSMCs as compared with the WT group. In addition, the transfection of Δ21 and siRNA could induce the down‐regulation of smooth muscle α‐actin and SM22α (P < 0.01) and the up‐regulation of osteopontin (P < 0.01) in VSMCs. The phosphorylated p38 signaling pathway expression was significantly enhanced in the Δ21 and siRNA groups as compared with that of the WT group (P < 0.01). These results suggest that MEF2A dominant negative mutation and RNA silence could induce the phenotypic switching of VSMCs, leading to its increased proliferation and migration, and p38 mitogen‐activated protein kinase signaling pathway may participate in it. Copyright © 2011 John Wiley & Sons, Ltd.     

Nerve conduit based on HAP/PDLLA/PRGD for peripheral nerve regeneration with sustained release of valproic acid

The nerve conduits have been developed for nerve defect repair. However, no artificial conduits have obtained comparable results to autografts to bridge the large gaps. A possible reason for this poor performance may be a lack of sustainable neurotrophic support for axonal regrowth. Previous studies suggested nanocomposite conduits can be used as a carrier for valproic acid (VPA), a common drug that can produce effects similar to the neurotrophic factors. Here, we developed the novel bioabsorbable conduits based on hydroxyapatite/poly d -l -lactic acid (PDLLA)/poly{(lactic acid)-co-[(glycolic acid)-alt-(l -lysine)]} with sustained release of VPA. Firstly, the sustained release of VPA in this conduit was examined by high-performance liquid chromatography. Then Schwann cells were treated with the conduit extracts. The cell metabolic activity and proliferation were assayed by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide and bromodeoxyuridine staining. A 10-mm segment of rat sciatic nerve was resected and then repaired, respectively, using the VPA conduit (Group A), the PDLLA conduit (Group B), or the autografts (Group C). Nerve conduction velocities (NCVs), compound muscle action potentials (CMAPs), and histological staining were assayed following the surgery. The cell metabolic activity and proliferation were significantly increased (p < .05) by the extracts from VPA-conduit extract compared to others. NCVs and CMAPs were significantly higher in Groups A and C than Group B (p < .05). The nerve density of Groups A and C was higher than Group B. There was no significant difference between Groups A and C. Taken together, this study suggested the sustained-release VPA conduit promoted peripheral nerve regeneration that was comparable to the autografts. It holds potential for future use in nerve regeneration.     

Preparing normal tissue cells for space flight experiments

Deterioration of health is a problem in modern space flight business. In order to develop countermeasures, research has been done on human bodies and also on single cells. Relevant experiments on human cells in vitro are feasible when microgravity is simulated by devices such as the Random Positioning Machine or generated for a short time during parabolic flights. However, they become difficult in regard to performance and interpretation when long-term experiments are designed that need a prolonged stay on the International Space Station (ISS). One huge problem is the transport of living cells from a laboratory on Earth to the ISS. For this reason, mainly rapidly growing, rather robust human cells such as cancer cells, embryonic cells, or progenitor cells have been investigated on the ISS up to now. Moreover, better knowledge on the behavior of normal mature cells, which mimic the in vivo situation, is strongly desirable. One solution to the problem could be the use of redifferentiable cells, which grow rapidly and behave like cancer cells in plain medium, but are reprogrammed to normal cells when substances like retinoic acid are added. A list of cells capable of redifferentiation is provided, together with names of suitable drugs, in this review.     

莪术醇诱导人肝癌HepG2细胞衰老及其机制研究

为进一步探讨莪术醇的诱导细胞衰老的机制,该研究采用荧光定量PCR技术对莪术醇处理后细胞中81个细胞衰老相关基因差异表达谱进行分析,结果发现TP53及其下游基因p16Ink4a、p21Waf1/Cip1和p27Kip1等的表达水平显著升高,伴随ABL1、ALDH1A3、CHEK2、HRAS、PTEN等多个衰老信号通路启动与效应关联基因的转录显著增强,而CyclinA2、IGFBP3、SIRT1以及TERT等细胞周期进程与衰老信号通路的负性调控基因的表达水平则显著降低。Western印迹检测结果显示,p53及其下游周期素依赖性蛋白激酶抑制物(CKI)分子p21WAF1和p16INK4水平升高,CyclinA2水平降低,与PCR结果一致,并伴野生型p53-诱导的蛋白磷酸酶1(Wip1)水平显著增高,提示莪术醇可能通过激活p53信号通路诱导HepG2细胞衰老。该研究进一步发现莪术醇能够诱导HepG2细胞发生衰老表型改变,伴G0/G1期周期阻滞。     

Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group

 The lozenge locus is genetically complex, containing two functionally distinct units, cistrons A and B, that influence the structure of the compound eye. Extreme mutations of either cistron produce adult phenotypes that share similarities and that have striking differences. We have analyzed the expression of several developmentally important eye genes including boss, scabrous, rhomboid, seven-up, and Bar in lozenge mutant backgrounds representing both cistrons. This analysis follows the progressive recruitment of photoreceptor neurons during eye development and has confirmed that the initial development of photoreceptors is normal up to the five cell precluster stage (R8, R2/5 and R3/4). However, when lozenge is mutant, further eye development is perturbed. As cells R1, R6 and R7 are recruited, patterns of gene expression for seven-up and Bar become abnormal. We have also characterized the expression of two different enhancer trap alleles of lozenge. The lozenge product(s) appear to be first expressed in the eye disc in undifferentiated cells shortly after the five cell precluster forms. Then, as distinct cells are recruited to a fate, lozenge expression persists and is refined in those cells. Our data suggests that lozenge functions in cone cells and pigment cells as well as in specific glia. With respect to photoreceptor neurons, lozenge biases the developmental potential of cells R1, R6 and R7, by directly influencing the expression of genes important for establishing cell fate. Received: 26 July 1996 / Accepted: 6 January 1997     

Isolation,in vitro study,and stem cell markers for type A spermatogonia in a Characiformes species

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti‐Vasa, anti‐GFRα1, and anti‐OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.