Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture

Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O₂ (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm² flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O₂ in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O₂, respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O₂ and 500 μl (1,480-μm depth) at 5% O₂ in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.     

Use of Acadian marine plant extract powder from Ascophyllum nodosum in tissue culture of Kappaphycus varieties

Three varieties of Kappaphycus alvarezii (Kapilaran, KAP), Tambalang purple (PUR), Adik-adik (AA), and one variety of Kappaphycus striatum var. sacol (green sacol (GS) were used to determine the efficiency of Acadian marine plant extract powder (AMPEP) as a culture medium at different concentrations, for the regeneration of young plants of Kappaphycus varieties, using tissue culture techniques for the production of seed stock for nursery and outplanting purposes for the commercial cultivation of carrageenophytes. A shorter duration for shoot formation was observed when the explant was treated with AMPEP + Plant Growth Regulator (PGR = PAA + zeatin at 1 mg L⁻¹) compared to AMPEP when used singly. However, four explants responded differently to the number of days required for shoot formation. The KAP variety took 46 days to form shoots at 3–4 mg L⁻¹ AMPEP + PGR; while PUR required 21 days at 3–5 mg L⁻¹ AMPEP and 3–4 mg L⁻¹ AMPEP + PGR. AA required 17 days at 3–5 mg L⁻¹ AMPEP and AMPEP + PGR; and GS 25 days at 1 mg L⁻¹ AMPEP + PGR. It was observed that among the four explants used, PUR and AA initiated shoot formation with the use of AMPEP only at higher concentrations (3–5 mg L⁻¹) after a shorter period. Only PUR responded positively to ESS/2 for shoot initiation. The use of AMPEP alone and/or in combination with PGR as a culture medium in the propagation of microplantlets using tissue culture technique is highly encouraging.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine

Diazinon and malathion are commonly used organophosphate insecticides in agriculture, industry, and in veterinary medicine as an ectoparasiticide. The importance to carry out in vitro reproductive toxicology assays lies on the need of knowing the alterations these insecticides may cause at cellular level, since they are endocrine disruptors that interfere with reproductive functions. The aim of this study was to evaluate in vitro oocyte viability, fertilization, and embryo development with different concentrations of diazinon and malathion. For in vitro fertilization (IVF), porcine oocytes and sperm were co-incubated for 7 h with increasing concentrations (50, 100, and 500 μM) of diazinon and malathion. For embryo development, fertilized oocytes were cultured in medium containing the same insecticide concentrations during 96 h for embryo development and 144 h for morulae formation. Diazinon did not affect oocyte viability and embryo divisions but decreased IVF (fertilization inhibition₅₀ = 502 μM) and morulae formation (morulae inhibition₅₀ = 344 μM). Malathion affected all the studied parameters: lethal concentration₅₀ = 1 mM, fertilization inhibition₅₀ = 443 μM, development inhibition₅₀ = 375 μM, and morulae inhibition₅₀ = 216 μM. The results of this study indicate that diazinon and malathion used in commercial formulation could be toxic, producing impairment in in vitro fertilization and embryo development. This is an approach for further investigations to find out cell damage mechanisms produced by these widely used insecticides.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Micropropagation and slow growth conservation of cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton)

Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about 70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of 20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation was detected among the in vitro-conserved plants compared with the mother plants.     

Ascitic fluid and serum from rats with acute pancreatitis injure rat pancreatic tissues and alter the expression of heat shock protein 60

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-α levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-α in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.     

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D _121 °C value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D _121 °C = 2.9 ± 0.5 min and Usk = 12.7 ± 2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D _121 °C value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Precision-cut dog renal cortical slices in dynamic organ culture for the study of cisplatin nephrotoxicity

The dog is the non-rodent species the most often used in preclinical drug safety evaluation. In this study, we established a new system of precision-cut dog renal cortical slices, evaluated their biochemical, functional, and morphological integrity, and determined the effects of cisplatin (cis-diamminedichloroplatinum (II), CDDP), a very potent nephrotoxic antineoplastic agent used to treat a variety of solid tumors, on the viability and histopathology of slices. Precision-cut renal cortical slices were made perpendicular to the cortical-papillary axis. Slices were incubated in DMEM/Ham's F12 culture medium containing 1 g/L glucose, 2 mmol/L glutamine, and 2 mmol/L heptanoic acid at 37°C in an atmosphere of 5% CO₂-70% O₂-25% N₂ in dynamic organ culture. Our results showed that slices maintained ATP and GSH content, protein synthesis, Na⁺-dependent uptake of glucose inhibited by phlorizin, PAH (p-aminohippuric acid) uptake inhibited by probenecid, and TEA (tetraethylammonium) uptake inhibited by mepiperphenidol for at least 6 h of culture, and morphological integrity up to 24 h. After 6 h of exposure, CDDP induced vacuolation and cell necrosis in the epithelial tubular cells of slices with a concentration-related increase in extension but not in severity. The development of the lesions started in the proximal tubules and extended to the distal tubules. The location and the extension of the lesions confirmed the observations in dog kidneys after in vivo treatment with CDDP by the intravenous route. The concentration-related decrease in slice viability after 6 h exposure to CDDP was in keeping with the extension of the histopathological lesions in the renal parenchyma. The slice viability was unaffected up to 0.63 mmol/L CDDP. At 1.25 and 2.5 mmol/L CDDP, slice viability fell by 35% and 75%, respectively. These results suggest that precision-cut dog renal cortical slices in culture may be suitable for addressing the specific nephrotoxicity issues encountered in this species.Abbreviations CDDP cis-diamminedichloroplatinum (II) - FIS freshly isolated slices - GSH glutathione - GSSG glutathione disulfide - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MGP methyl--D-glucopyranoside - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - PAH p-aminohippuric acid - PBS phosphate-buffered saline - TEA tetraethylammonium     

Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: Effects of oxygen tension

We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37°C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO₂–95% air (WPCS/air or ROCS/air) or 5% CO₂–70% O₂–25% N₂ (WPCS/ O₂ or ROCS/ O₂). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/ O₂ were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/ O₂ for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/ O₂ than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/ O₂. ROCS/ O₂ emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.