Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live andin situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

High frequency plant regeneration from thin cell layer explants of Brassica napus

Explants composed of the epidermis and 4–9 layers of subepidermal cells were excised from internodes of Brassica napus L. ssp. oleifera cv. Westar and cultured on modified Murashige and Skoog (MS) medium. The three or four terminal internodes excised from plants at an early stage (before any flower buds had opened) were shown to be the best explant source. Both cytokinin and auxin were required for induction of shoot organogenesis. Of six auxins tested, only naphthaleneacetic acid (NAA) was effective in shoot bud initiation. All four cytokinins tested (when associated with 0.5 mgl^-1 NAA) promoted organogenesis, but at differing frequencies. The highest shoot induction frequency was obtained at 10–15 mgl^-1 benzyladenine (BA). The organogenic response was strongly affected by the nitrogen content of the medium. The best response was observed when NO₃ ^- was the sole nitrogen source (supplied as KNO₃) in the range 30–90 mM. Sucrose and glucose were equally supportive in shoot regeneration with the optimal levels at 0.12 M and 0.15 M, respectively. Shoots were rooted on medium free of growth regulators and mature plants were grown in the greenhouse. Plants were also recovered from leafy structures which differed morphologically and histologically from shoot buds.     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

大蒜愈伤组织诱导条件初报

以白皮大蒜和大头蒜为材料,报道了大蒜品系、外植体的低温前处理、外植体大小及取材种类不同对愈伤组织诱导的影响。结果表明：（１）无论是４℃低温保藏或室温保存前处理,大头蒜的消毒效果均优于白皮大蒜,４℃低温前处理可降低外植体出愈污染;（２）只有带表皮的外植体才可诱导愈伤组织,外植体较大（０．５ｃｍ×０．３ｃｍ×０．２ｃｍ）,继代期愈伤组织产生量较多;外植体偏小（０．２ｃｍ×０．１ｃｍ×０．１ｃｍ）,极少产生愈伤组织;（３）４℃冷藏１８ｄ对大头蒜愈伤组织诱导有一定影响,但对白皮大蒜影响不大。冷藏可推迟出愈１～２ｄ。     

Direct plant regeneration from leaf explants of Plumbago species and its genetic fidelity through RAPD markers

In vitro plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige & Skoog (1962) medium supplemented with 1.5 mg litre^?1 6‐benzylaminopurine, 0.25 mg litre^?1 indole‐3‐acetic acid, 50 mg litre^?1 adenine sulfate and 3% (w/v) sucrose. The shoot initials developed within 2–3 wk on the leaf margin as well as from the wounds of the leaf. High frequency shoot‐bud regeneration was achieved on similar medium in subsequent subcultures. The semi‐mature leaves produced more shoot‐buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi‐mature explants produced shoot‐buds per leaf explant within 4 wk of culture. Shoots rooted easily on medium having half‐strength basal Murashige & Skoog (1962) medium supplemented with 0.25 mg litre^?1 indole‐3‐butyric acid and 2% (w/v) sucrose; 84–92% of the in vitro rooted plantlets survived in the greenhouse. The regenerated plantlets appeared morphologically similar to the mother plants. No variation was detected among the regenerated plants by the use of Randomly Amplified Polymorphic DNA (RAPD) markers. This method might be useful for assessing plant improvement programmes.     

High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells

Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial–mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway^TM recombination into the Bac-to-Bac^TM system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft⁺) was 5–8 mg/L culture. rHTuft⁺ was characterized by SDS–PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft⁺ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft⁺, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.     

Transformation of Nicotiana tabacum,N. debneyi,and N. rustica: Inheritance and protoplast expression of antibiotic resistance

Agrobacterium tumefaciens strains harbouring plasmid vectors pBCAT1, pVU1011 or pMON806 were used to transform leaf explants of Nicotiana tabacum cultivars Delgold and Candel, N. debneyi, and N. rustica var. NRT. Transgenic plants resistant to the selective agents kanamycin, hygromycin or methotrexate were regenerated and used as sources of leaf mesophyll protoplasts. Protoplasts divided and regenerated plants in the presence of selective agents at levels inhibitory to protoplasts of non-transformed plants. Cross-resistance of protoplasts to more than one selective agent was not observed in this study which suggests that this approach may lead to an efficient interspecific somatic hybrid selection system.     

不同因子对山药愈伤组织诱导的影响

研究了基因型、外植体、植物激素和光暗等因子对山药愈伤组织诱导的影响。结果表明 :(1)不同基因型山药均能诱导形成愈伤组织 ,但出愈率不同 ,其排列顺序是 :“4 7号”山药 >铁棍山药 >太谷山药。 (2 )不同外植体在同一种培养基上的诱导率存在差异。同一外植体在不同植物激素浓度配比中 ,诱导率也不同。叶片、茎段、零余子的最佳激素配比均为 6 - BA2 (mg/ L,以下单位同 ) +NAA2 ,诱导率分别为 53.3%、 6 5.6 %、 10 0 % ,茎尖的最佳激素配比为 6 - BA2 +NAA0 .2 ,诱导率为 93.3%。 (3)植物激素在愈伤组织诱导过程中起关键的作用。细胞分裂素与生长素组合使用优于单一激素。二者的浓度配比不同 ,出愈率也不同。 (4 )光暗条件对不同外植体愈伤组织诱导的影响不同。暗培养有利于零余子的诱导 ,而光培养则有利于叶片的诱导。