In vitro plantlet formation by organogenesis in E. camaldulensis and by somatic embryogenesis in Eucalyptus citriodora

Adventitious shoots were formed through callus on leaf explants of Eucalyptus camaldulensis Dehnh. (River red gum) taken from shoot cultures of mature trees. Callus formed in dark on a medium containing 1 g/l casein hydrolysate, 3 mg/l 1-naphthaleneacetic acid, 0.1 mg/l 6-benzyladenine and 50 g/l sucrose. Shoot initiation occurred in 4 weeks on calli shifted to light on a regeneration medium containing 10% coconut milk, 0.5 mg/l 6-benzyladenine and 20 g/l sucrose. Rooting occured in dark on a liquid medium containing 4 mg/l 1-naphthaleneacetic acid. Zygotic embryos of Eucalyptus citriodora Hook f. (Lemon scented gum) cultured in dark on a medium containing 3 mg/l 1-naphthaleneacetic acid and 50 g/l sucrose formed somatic embryoids which grew to normal plantlets on the same regeneration medium used for organogenesis.Abbreviations BAP 6-benzyladenine - CH Casein hydrolysate - CM Coconut Milk - NAA 1-naphthaleneacetic acid NCL Communication no. 4162     

Some conditions apply: Systems for studying Plasmodium falciparum protein function

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.     

Thermoluminescence and photoluminescence studies on γ‐ray‐irradiated Ce3+,Tb3+‐doped potassium chloride single crystals

Single crystals of KCl doped with Ce³⁺,Tb³⁺ were grown using the Bridgeman–Stockbarger technique. Thermoluminescence (TL), optical absorption, photoluminescence (PL), photo‐stimulated luminescence (PSL), and thermal‐stimulated luminescence (TSL) properties were studied after γ‐ray irradiation at room temperature. The glow curve of the γ‐ray‐irradiated crystal exhibits three peaks at 420, 470 and 525 K. F‐Light bleaching (560 nm) leads to a drastic change in the TL glow curve. The optical absorption measurements indicate that F‐ and V‐centres are formed in the crystal during γ‐ray irradiation. It was attempted to incorporate a broad band of cerium activator into the narrow band of terbium in the KCl host without a reduction in the emission intensity. Cerium co‐doped KCl:Tb crystals showed broad band emission due to the d–f transition of cerium and a reduction in the intensity of the emission peak due to ⁵D₃–⁷F_j (j = 3, 4) transition of terbium, when excited at 330 nm. These results support that energy transfer occurs from cerium to terbium in the KCl host. Co‐doping Ce³⁺ ions greatly intensified the excitation peak at 339 nm for the emission at 400 nm of Tb³⁺. The emission due to Tb³⁺ ions was confirmed by PSL and TSL spectra. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical sensing of DNT by engineered olfactory yeast strain

With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1alpha, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene-responsive receptor.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

Artificial chromosome vectors and expression of complex proteins in transgenic animals

Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.     

Effect of thermal annealing on the structural and optical properties of tris‐(8‐hydroxyquinoline)aluminum(III) (Alq3) films

Tris‐(8‐hydroxyquionoline)aluminum (Alq₃) was synthesized and coated on to a glass substrate using the dip coating method. The structural and optical properties of the Alq₃ film after thermal annealing from 50°C to 300°C in 50° steps was studied. The films have been prepared with 2 to 16 layers (42–324 nm). The thickness and thermal annealing of Alq₃ films were optimized for maximum luminescence yield. The Fourier transform infrared spectrum confirms the formation of quinoline with absorption in the region 700 ? 500/cm. Partial sublimation and decomposition of quinoline ion was observed with the Alq₃ films annealed at 300°C. The X‐ray diffraction pattern of the Alq₃ film annealed at 50°C to 150°C reveals the amorphous nature of the films. The Alq₃ film annealed above 150°C were crystalline nature. Film annealed at 150°C exhibits a photoluminescence intensity maximum at 512 nm when excited at 390 nm. The Alq₃ thin film deposited with 10 layers (220 nm) at 150°C exhibited maximum luminescence yield. Copyright © 2014 John Wiley & Sons, Ltd.     

Transgenic animal production and animal biotechnology

Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.