Fourth Day Of Culture Research Articles

Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy – achieving similar function using different components – and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs.

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

Application of the white-rot fungus Trametes sp. (C3) laccase in the removal of acetaminophen from aqueous solutions

Effluents containing drugs originating from hospitals, veterinary clinics, residences, and pharmaceutical industries, pass through the sewage treatment system and are discharged into the aquatic environment, causing water contamination. The methods used in sewage treatment are ineffective for these types of micropollutants. Laccase, an enzyme produced by white-rot fungi, has been used to successfully remove drugs in waters. The objective of this work was to evaluate the removal of acetaminophen from aqueous solutions by the fungus Trametes sp. (C3). Ethanol showed efficiency in inducing the production of the laccase enzyme and in vitro removal tests the amount of drug decreased by 76 %. The use of the 1-hydroxybenzotriazole (HBT) as mediator caused a lower removal rate than the assay without its presence, indicating that it acted as an inhibitor of degradation. In in vivo tests, there was a 79 % removal rate of acetaminophen on the fourth day of culture. Toxicity assays showed that after the action of the laccase there was toxicity to the larval stage of Artemia salina.

Biological effects of trans, trans-farnesol in Leishmania amazonensis.

Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite. The study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome. Exogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production. The findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections.

In-situ removal of aquaculture waste nutrient using floating permeable nutrient uptake system (FPNUS) under mixotrophic microalgal scheme

The current study investigated the in-situ aquaculture nutrient removal from fish tanks using mixotrophic Scenedesmus in a floating permeable nutrient uptake system (FPNUS) and compared with nutrient concentration in control, autotrophy, and bacterial nitrogen removal (BNR) treatments. In the first run, results were not as expected due to the missing PO4−-P as the mixotrophic growth in flasks with PO4−-P was 55.86% more than growth in aquaculture wastewater. With PO4−-P addition in FPNUS, average and maximum removal rates under mixotrophy reached 2.53 and 10.96 mg/(L·d), respectively. The average mixotrophic removal rate was 40.31 and 81.42% higher than removal rates under autotrophy and BNR. Daily nutrient loading and removal were matched only in mixotrophy after fourth day of culture. These results show the great potential for nutrient removal using mixotrophic microalgae-based FPNUS due to its high efficiency, capability of in-situ treatment and nutrient recycling through biomass utilization.

Fabrication and characterization of electrospun psyllium husk‐based nanofibers for tissue regeneration

AbstractThe present study reports for first time the blending of psyllium husk (PH) powder/gelatin (G) in the polymer‐rich composition of polyvinyl alcohol (PVA) to make an electrospinnable solution. The composite was prepared in 3 different ratios viz., 100% (wt/wt) (PVA + PH), 75% + 25% (PVA + 75PH + 25G) (wt/wt) and 50% + 50% (PVA + 50PH + 50G) (wt/wt) in 6% PVA solution. Optimum electrospinning parameters were evaluated for all the prepared blends. The fabricated nanofibers were characterized by scanning electron microscopy (SEM), attenuated total reflectance‐Fourier transform infrared, differential scanning calorimetry, porosity percentage, and fiber orientation using ImageJ software. A qualitative in vitro degradation study at room temperature is supported by SEM images. The cellular interactions were characterized by MTT assay of NIH‐3T3 fibroblast cells for 2 and 4 days with an optimum cell growth of >50% by fourth day of culture and long‐term cultivation of L929‐RFP cells was observed for 10 days. The nanofibers were formed in the range of 49–600 nm. PVA + 75PH + 25G when cultured with L929‐RFP cells exhibited highest fluorescence intensity and thus supported cellular proliferation significantly. Based on the results obtained from various analyses, we anticipate that fabricated psyllium‐based nanofiber can be used as a promising candidate for wound healing and other biomedical applications.

Long-Term Toxicity of ZnO Nanoparticles on Scenedesmus rubescens Cultivated in Semi-Batch Mode.

The scope of this study was to investigate the toxic effects of zinc oxide (ZnO) nanoparticles (NPs) on freshwater microalgae, in long-term semi-batch feeding mode at two different hydraulic retention times (HRTs) (20 and 40 days). A freshwater microalgae, Scenedesmus rubescens, was employed and exposed to a semi-continuous supply of ZnO NPs at a low concentration of 0.081 mg/L for a period of 28 d. Experiments were conducted under controlled environmental conditions. Τhe impact of ZnO NPs on S. rubescens, which was assessed in terms of nutrient removal, biomass growth, and algal lipid content. Semi-batch mode cultures showed that low ZnO NP concentrations at an HRT of 40 d did not have any negative effect on microalgae growth after the fourth day of culture. In contrast, algal growth was inhibited up to 17.5% at an HRT of 20 d in the presence of ZnO NPs. This might be attributed to the higher flow rate applied and ZnO NPs load. A positive correlation between nutrient removal and microalgae growth was observed. The algal lipid content was, in most cases, higher in the presence of ZnO NPs at both HRTs, indicating that even low ZnO NPs concentration cause stress resulting in higher lipid content.

Sexually dimorphic gene expression and neurite sensitivity to estradiol in fetal arcuate Kiss1 cells.

Kiss1 neurons of the arcuate (ARC) nucleus form an interconnected network of cells that communicate via neurokinin B (encoded by Tac2) and its receptor (encoded by Tacr3) and play key roles in the control of the reproductive axis through sex hormone-regulated synthesis and release of kisspeptin peptides (Kp, encoded by Kiss1). The aim of this study was to determine whether the Kiss1 cell population of the ARC already displays sexually dimorphic features at embryonic age E16.5 in mice. At this time of development, Kiss1-GFP- and Kp-immunoreactive cell bodies were restricted to the ARC and not found in the pre-optic area (POA). The Kiss1-GFP cell population was identical in size between sexes but had significantly lower Kiss1, Tac2, and Tacr3 mRNA levels and lower Kp-ir fiber density in the POA in male compared to female fetuses. Receptors for androgen (Ar) and estrogen (Esr1, Esr2, Gpr30) and the Cyp19a1 gene (encoding the estradiol-producing enzyme aromatase) transcripts were also detected in fetal ARC Kiss1-GFP cells with significant sex differences for Ar (higher in males) and Esr1 (higher in females). Functional studies on primary cultures of sorted fetal Kiss1-GFP cells revealed a significant negative effect of estradiol treatment on neurite outgrowth on the fourth day of culture in the female group specifically. We conclude that the ARC Kiss1 cell population is already sexually differentiated at E16.5 and that its morphogenetic development may be particularly vulnerable to estradiol exposure at this early developmental time.

Pulsatile tubular cardiac tissues fabricated by wrapping human iPS cells-derived cardiomyocyte sheets.

The purpose of this study was to fabricate pulsatile tubular cardiac tissue using cell sheet based-tissue engineering. First, we fabricated human induced pluripotent stem cell (hiPSc)-derived cardiomyocyte sheets and normal human dermal fibroblast (NHDF) sheets which are harvested from temperature responsive culture dishes only by lowering the temperature. Then tubular cardiac tissues are formed by wrapping one hiPSc-derived cardiomyocyte sheet and three NHDF sheets around an octagonal column, and both ends of the tubular tissue were covered with fibrin and collagen gel. The octagonal column with the tubular tissue was connected to an in vitro circulation system in a culture box. After four-day culture, the cardiac tissue survived and pulsated spontaneously in the circulation system. Furthermore, the analysis with a Millar catheter inserted into the cardiac tubes revealed significant inner pressure changes generated by their beating. In addition, the tubular cardiac tissue pulsated in response to the electrical stimulation. Although histological analyses demonstrated that cardiac troponin T-positive cells stratified the inner surface of the tubular tissues, gene expression analyses showed an immature state of these cardiomyocytes. Thus, cell sheet-based tissue engineering realized human pulsatile tubular cardiac tissue fabrication and we believe that these tubular cardiac tissues should contribute to future drug screening and regenerative therapy for heart diseases.

The Dynamics of Water Quality During Culture of Snakehead Fish (Channa striata) in The Aquarium

Intensive culture of snakehead fish used artificial feed with high protein content. The impact of the high use of artificial feed will cause waste that can cause poor water quality and cause fish mortality. The purpose of this research was to know the dynamics of water quality during culture of snakehead fish Channa striata in the aquarium. The test animals used were snakehead fish with a length of 6.90 ± 0.49 cm and weight of 2.57 ± 0.59 g. Fish was maintenanced with a density of 2 fish L-1, 4 fish L-1 and 6 fish L-1. Fish was cultured for four days in an aquarium with size of 60x40x40 cm3 and water volume of 20 L. Water quality were observed, i.e, temperature, pH, dissolved oxygen, TAN, nitrite, nitrate, orthophosphate, hardness and alkalinity. The results showed that pH, temperature, hardness, TAN, nitrite, nitrate and ortophosphate were not significantly different between treatments (P>0.05), while dissolved oxygen and alkalinity were showed different significantly (P<0.05). On the fourth day of culture with a density of 4 fish L-1 and 6 fish L-1 has led to water saturation in the culture media. Dynamic of water quality was started on the third day of culture, whereas nitrites could not be converted to nitrate maximally. High levels of nitrate in the snakehead culture indicated that that this culture could be combined with an integrated aquaculture with plants.

Transfection of bovine spermatogonial stem cells in vitro.

Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (P<0.05) in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups (P<0.05). In comparison with these three days, the rate of infected cells was higher when transfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture.

Enhanced production of nojirimycin via Streptomyces ficellus cultivation using marine broth and inhibitory activity of the culture for seeds of parasitic weeds.

Root parasitic weeds, such as Orobanche spp. and Striga spp., cause serious damage to crops. Recently, it was demonstrated that nojirimycin (NJ) selectively inhibits seed germination in these weeds. In this study, we modified the medium for Streptomyces ficellus to increase its production of NJ and evaluated the culture as an antiparasitic weed agent. We screened alternatives to Pharmamedia™, an additive in the original medium, and found that marine broth stimulated NJ production. Moreover, soluble starch-depleted medium could maintain S. ficellus NJ production. The NJ concentration reached 710 mg/L after four-day batch culture in starch-depleted marine broth medium, which was 17-fold higher than that in the Pharmamedia™ medium. The culture in the marine broth medium inhibited seed germination of Orobanche spp. and Striga spp. as effectively as a standard solution of NJ.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

MtTdp2α-overexpression boosts the growth phase of Medicago truncatula cell suspension and increases the expression of key genes involved in the antioxidant response and genome stability

Aside from the great importance of Leguminosae in food and agriculture industry, legume model systems like Medicago truncatula are also essential tools to dissect complex cellular pathways and retrieve valuable information to other crops. Here, we investigated the roles played by the tyrosyl-DNA phosphodiesterase 2α (MtTdp2α) gene in cell viability and proliferation using M. truncatula suspension cultures. Our research hypothesis is that the overexpression of MtTdp2α, implicated in the removal of transient topoisomerase/DNA covalent complexes, can impact on cell suspension viability. M. truncatula suspension cultures derived from leaf explants of MtTdp2α-overexpressing lines and a control line carrying the empty vector were used. Our results showed that the control line reached the stationary growth phase by the fourth day of culture while the transgenic lines presented an extended exponential growth, reaching the stationary phase at day six following culture. The MtTdp2α-overexpressing lines also showed increased viability as compared to the control line. The transcript levels of MtSOD, MtAPX, MtMT2, MtMRE11, MtNBS1, MtRad50, MtOGG1 and MtFPG were significantly enhanced in the transgenic lines as compared to control. Overall, our results show that the MtTdp2α overexpression impacts in a positive manner on cell viability and proliferation in suspension cultures. Additionally, our study provides an insight on the suitability of M. truncatula cell suspension cultures as a promising alternative to evaluate potential protective mechanisms, with results comparable to those obtained when using whole plants.

Cryopreservation of gametophytic thalli of Ulva prolifera (Ulvales, Chlorophyta) from Korea

Cryopreservation is needed to ensure long-term storage without any genetic drift and contamination. To conserve Ulva species, gametophytic thalli of Ulva prolifera (Chlorophyta) were first cryopreserved at −196 °C using the two-step cooling method with cooling rate of 1 °C min−1 to −40 °C. Three cryoprotectants (dimethyl sulfoxide (DMSO), glycerol, and proline) at five concentrations (5, 10, 15, 20, and 25 %) and mixed cryoprotectants were assessed to determine the optimal solution. The mixture of cryoprotectants with the highest viability was 10 % glycerol in combination with 5 % DMSO and 5 % proline, with a viability of 89.2 %. The cryoprotectant treated singly with the highest viability was 20 % glycerol with a viability of 91.6 %. However, there was no statistically significant difference between 20 % glycerol and the mixed treatment. An evaluation of long-term storage in liquid nitrogen showed very high viability of 91.2–92.1 % with no statistically significant effect from storage time up to 120 days. The specimens developed normally in culture after cryopreservation for 120 days with the rate of gametogenesis reaching 95.7 % on the fourth day of culture. The released gametes developed normally into gametothalli. In conclusion, the use of glycerol as a cryoprotectant was very effective for cryopreservation of the gametothalli of U. prolifera. With 20 % glycerol, viability was very high as 91.6 %. The results suggest that gametothalli of U. prolifera can be cryopreserved completely by a treatment with 15–25 % glycerol singly as well as by a mixture of cryoprotectants with usual efficiency as a cryoprotectant.

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca2+]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca2+-ATPase and Na+/K+-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.

Paclitaxel production is enhanced in suspension‐cultured hazel (Corylus avellana L.) cells by using a combination of sugar, precursor, and elicitor

Hazel (Corylus avellana L.) has recently been drawing attention as an alternative source of taxol. In the present study, the effects of sugar type, and different concentrations of phenylalanine (Phe) and vanadyl sulfate (V) on the production of taxol in C. avellana were investigated. A factorial experiment was used to optimize the concentrations of the precursor and elicitor. The cells were treated with Phe and V on the fourth day of culture and were harvested every 2 days until the 10th day. By increasing the Phe and V supply, taxol production increased during the culture period and the maximum level of 4.2 μg/g (dry weight) was obtained at day 10 by combining 3 μM of Phe and 0.05 and 0.1 mM of V in media supplemented with fructose (3%). The time course study on taxol production suggested that the appropriate time for using Phe is day 4 of culture, and day 8 for V. Overall, taxol production in C. avellana cell suspension culture was improved by the use of the combined strategy.

Cell proliferation in in vivo‐like three‐dimensional cell culture is regulated by sequestration of ERK1/2 to lipid rafts

Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25β1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions. Four-day post-confluent culture of GD25β1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules. GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels. These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.

Probing the microenvironmental conditions for induction of superficial zone protein expression

To determine the in vitro conditions which promote expression of superficial zone protein (SZP). Chondrocytes from 6-month-old calves were expanded in monolayer culture and the expression of SZP in alginate bead and monolayer culture was quantified with quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining. The effect of oxygen tension on SZP expression was determined by qRT-PRC analysis of cells cultured in two dimension (2D) and three dimension (3D) under hypoxic (1% pO2) or normoxic (21% pO2) conditions. Finally, to examine the effect of cyclic tensile strain on expression of SZP in 2D and 3D cultures, chondrocytes encapsulated in alginate beams or seeded on type I collagen coated polydimethylsiloxane (PDMS) chambers were subjected to 5% strain at 1 Hz, 2 h/day for 4 days or 2 h at the fourth day of culture and mRNA levels were quantified. Bovine chondrocytes in monolayer showed a drastic decrease in SZP expression, similar in trend to the commonly reported downregulation of type II collagen (Col2). Chondrocytes embedded in alginate beads for 4 days re-expressed SZP but not Col2. SZP expression was higher under normoxic conditions whereas Col2 was upregulated only in alginate beads under hypoxic conditions. Cyclic mechanical strain showed a tendency to upregulate mRNA levels of SZP. A microenvironment encompassing a soft encapsulation material and 21% oxygen is sufficient for fibroblastic chondrocytes to re-express SZP. These results serve as a guideline for the design of stratified engineered articular cartilage and suggest that microenvironmental cues (oxygen tension level) strongly influence the pattern of SZP expression in vivo.

Identification of monogenic dominant male sterility and its suppressor gene from an induced mutation using a broccoli (Brassica oleracea var. italica) microspore culture

Male sterility (MS) has been studied intensively in Brassica crops to ensure high purity and the protection of F1 hybrids. In the preceding trial, broccoli microspores were treated with a mutagen, n-nitroso-n-methyl urethane (NMU), during the cultural process to produce MS. Many MS plants were derived from the 0.01 μM and 0.1 μM treatments just before and at the fourth day of culture, respectively. In this study, most MS plants inherited sterility from a single recessive gene. Only one plant, 9-4C-106, which originated from the 0.1 μM treatment on the fourth day, was controlled by a single dominant gene. Another dominant mutant gene suppressing the expression of this dominant MS gene was discovered in a plant treated with 0.01 μM NMU. A hybrid breeding system using this MS structure is discussed.