Subdued Light Research Articles

‘In the Womb Yet Out‐of‐Doors’: Penumbra and the Spaces In‐Between

AbstractIn a wide‐ranging and erudite article that is historical, cultural and philosophical, Stephen Kite explores the experiential nature of the penumbra. He reminds us of some of architecture's most celebrated spaces where subdued light calms architectural vistas. Flooding us with notable examples, he examines the penumbra in terms of sublimity, prospect and refuge, and psychoanalysis.

Mother Schema, Obstetric Dilemma, and the Origin of Behavioral Modernity.

What triggered the emergence of uniquely human behaviors (language, religion, music) some 100,000 years ago? A non-circular, speculative theory based on the mother-infant relationship is presented. Infant “cuteness” evokes the infant schema and motivates nurturing; the analogous mother schema (MS) is a multimodal representation of the carer from the fetal/infant perspective, motivating fearless trust. Prenatal MS organizes auditory, proprioceptive, and biochemical stimuli (voice, heartbeat, footsteps, digestion, body movements, biochemicals) that depend on maternal physical/emotional state. In human evolution, bipedalism and encephalization led to earlier births and more fragile infants. Cognitively more advanced infants survived by better communicating with and motivating (manipulating) mothers and carers. The ability to link arbitrary sound patterns to complex meanings improved (proto-language). Later in life, MS and associated emotions were triggered in ritual settings by repetitive sounds and movements (early song, chant, rhythm, dance), subdued light, dull auditory timbre, psychoactive substances, unusual tastes/smells and postures, and/or a feeling of enclosure. Operant conditioning can explain why such actions were repeated. Reflective consciousness emerged as infant-mother dyads playfully explored intentionality (theory of mind, agent detection) and carers predicted and prevented fatal infant accidents (mental time travel). The theory is consistent with cross-cultural commonalities in altered states (out-of-body, possessing, floating, fusing), spiritual beings (large, moving, powerful, emotional, wise, loving), and reports of strong musical experiences and divine encounters. Evidence is circumstantial and cumulative; falsification is problematic.

Evaluation of the Major Steps in the Conventional Protocol for the Alkaline Comet Assay.

Single cell gel electrophoresis, also known as the comet assay, has become a widespread DNA damage assessment tool due to its sensitivity, adaptability, low cost, ease of use, and reliability. Despite these benefits, this assay has shortcomings, such as long assay running time, the manipulation of multiple slides, individually, through numerous process steps, the challenge of working in a darkened environment, and reportedly considerable inter- and intra-laboratory variation. All researchers typically perform the comet assay based upon a common core approach; however, it appears that some steps in this core have little proven basis, and may exist, partly, out of convenience, or dogma. The aim of this study was to critically re-evaluate key steps in the comet assay, using our laboratory’s protocol as a model, firstly to understand the scientific basis for why certain steps in the protocol are performed in a particular manner, and secondly to simplify the assay, and decrease the cost and run time. Here, the shelf life of the lysis and neutralization buffers, the effect of temperature and incubation period during the lysis step, the necessity for drying the slides between the electrophoresis and staining step, and the need to perform the sample workup and electrophoresis steps under subdued light were all evaluated.

Lux Perpetua: The Poetry of Seamus Heaney, from Door into the Dark to Electric Light

Light shines perpetually in Seamus Heaney's poetry. It has its first glimmerings in poems conceived in darkness and brought into luminous being. The interplay of darkness and light becomes the prevailing metaphor in a poetry of memory and perception, especially in the books published in the 1960s. In the 1970s and 1980s, writing in a subdued light becomes a necessary condition in a tense and sometimes turbulent political climate. A more equable light shines in the work composed in the 1990s, enabling a poetry of meditation and spiritual scrutiny, of quiet celebration and elegy. In later poems, light is cast on a host of theological and eschatological mysteries, and inspires a visionary apprehension of final things. All of these processes of light, from the glimmerings of poetic imagination in the moment of composition to the equation of light with political freedom and philosophical understanding, are validated for Heaney by their profound significance and appeal in Romantic poetry and poetics.

Metamorphosis of the invasive ascidian Ciona savignyi: environmental variables and chemical exposure.

In this study, the effects of environmental variables on larval metamorphosis of the solitary ascidian Ciona savignyi were investigated in a laboratory setting. The progression of metamorphic changes were tracked under various temperature, photoperiod, substrate, larval density, and vessel size regimes. Metamorphosis was maximised at 18 °C, 12:12 h subdued light:dark, smooth polystyrene substrate, and 10 larvae mL−1 in a twelve-well tissue culture plate. Eliminating the air-water interface by filling culture vessels to capacity further increased the proportion of metamorphosed larvae; 87 ± 5% of larvae completed metamorphosis within 5 days compared to 45 ± 5% in control wells. The effects of the reference antifouling compounds polygodial, portimine, oroidin, chlorothalonil, and tolylfluanid on C. savignyi were subsequently determined, highlighting (1) the sensitivity of C. savignyi metamorphosis to chemical exposure and (2) the potential to use C. savignyi larvae to screen for bioactivity in an optimised laboratory setting. The compounds were bioactive in the low ng mL−1 to high µg mL−1 range. Polygodial was chosen for additional investigations, where it was shown that mean reductions in the proportions of larvae reaching stage E were highly repeatable both within (repeatability = 14 ± 9%) and between (intermediate precision = 17 ± 3%) independent experiments. An environmental extract had no effect on the larvae but exposing larvae to both the extract and polygodial reduced potency relative to polygodial alone. This change in potency stresses the need for caution when working with complex samples, as is routinely implemented when isolating natural compounds from their biological source. Overall, the outcomes of this study highlight the sensitivity of C. savignyi metamorphosis to environmental variations and chemical exposure.

Protocol optimization and evaluation of rice varieties response to <i>in vitro</i> regeneration

Understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. The key factors for enhancing successful regeneration are genotypes, tissue source of explants, combination and concentration of growth regulators, and culture conditions. In the present study different methods of regeneration were tested in a set of 12 rice accession representing indica, japonica, aromatic and wild groups. The highest frequency of shoot regeneration was achieved using mesocotyls derived from in vitro grown seeds cultured on Murashige and Skoogs, basal medium without growth regulators, when cultured on medium supplemented with Benzyl Adenine (BA) 0.5 mg/l under subdued light at 25°C ± 2°C. Under these conditions mesocotyls (Plurality of meristems) produced 3 to 4 tiller shoots in primary culture. One seed/One single mesocotyls segment produced over 5 to 9 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Through callusing phase different rice cultivars produced different percentage of callusing however Basmati 370 gave high percentage in response to callussing, organogenesis and root formation. In between these two methods, the direct shoot regeneration gave 80% - 100% result besides the wild species in respect with their genotype whereas the indirect organogenesis gave only 10% - 30% of plantlets. So the direct multiplication from mesocotyls is an efficient method for plantlet regeneration of rice cultivars through in vitro culture. Tissue culture derived plants when evaluated with rice markers no variability is found. For regeneration genotypes and its trigger ing factors like medium composition, culture conditions and type of explants are playing a combined role.

Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines

Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC(10) drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.

Highly efficient plant regeneration and in vitro polyploid induction using hypocotyl explants from diploid mulberry (Morus multicaulis 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.

Work flow with digital intraoral radiography: A systematic review

Objective. This systematic review evaluates the six most frequently emphasized advantages of working with digital radiography: less working time, lower radiation dose to the patient, fewer retakes and errors, wider dynamic range, easier access to patient information and easier image storage and communication. Moreover, some clinical aspects and possible disadvantages of digital imaging that were not foreseen at the beginning of the digital era, such as patient discomfort, damage to the receptor, degradation of the image, cross-contamination and viewing conditions, were assessed. Material and methods. The literature search used the PubMed database with no limits and was performed during June to August 2009. Search strategies are described in the text for each of the mentioned tasks. A hand search of task-specific journals supplemented the search strategies. Results. Time seems to be saved when switching from film to digital imaging in dental practice, a dose reduction may not be obtained, retakes and errors may be increased, the dynamic range may be wider with photostimulable storage phosphor (PSP) plates but not with sensors, the effect on patient information has not been well studied and storage and communication create new challenges with regard to handling large files and image compression. In addition, patient discomfort seems to be pronounced with sensors compared with PSP plates and film, the PSP plate may be scratched in clinical use and a two-layer barrier seems to be needed to prevent contamination of the receptor. The type of monitor may not be of major concern if the image is viewed in a room with subdued light. Conclusions. Not all of the predicted advantages with digital compared to film-based radiography hold true in daily clinical work. Of particular interest is the relationship between number of images, retakes and the dose given to the patient.

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K(D) values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.

Characterization of Fluorescent Sterol Binding to Purified Human NPC1

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (<1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.

Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.

The Effect of Wavelet and Discrete Cosine Transform Compression of Digital Radiographs on the Detection of Subtle Proximal Caries

The study compared diagnostic performances of 2 different image compression methods: JPEG (discrete cosine transform; Joint Photographic Experts Group compression standard) versus JPEG2000 (discrete wavelet transform), both at a compression ratio of 12:1, from the original uncompressed TIFF radiograph with respect to the detection of non-cavitated carious lesions. Therefore, 100 approximal surfaces of 50 tooth pairs were evaluated on the radiographs by 10 experienced observers using a 5-point confidence scale. Observations were carried out on a standardized viewing monitor under subdued light conditions. The proportion of diseased surfaces was balanced to approximately 50% to avoid bias. True caries status was assessed by serial ground sectioning and microscopic evaluation. A non-parametric receiver operating characteristic analysis revealed non-significant differences between the 3 image modalities, as computed from the critical ratios z not exceeding ±2 (JPEG/JPEG2000, z = –0.0339; TIFF/JPEG2000, z = 0.251;TIFF/JPEG, z = 0.914). The mean area beneath the curve was highest for TIFF (0.604) followed by JPEG2000 (0.593) and JPEG (0.591). Both intra-rater and inter-rater agreement were significantly higher for TIFF (ĸ_intra = 0.52; ĸ_inter = 0.40) and JPEG2000 images (ĸ_intra = 0.49; ĸ_inter = 0.38) than for JPEG images (ĸ_intra = 0.33; ĸ_inter = 0.35). Our results indicate that image compression with typical compression algorithms at rates yielding storage sizes of around 50 kB is sufficient even for the challenging task of radiographic detection of non-cavitated carious approximal lesions.

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Comparison of observer performance on soft-copy reading of digital chest radiographs: High resolution liquid-crystal display monitors versus cathode-ray tube monitors

The purpose of this study is to compare observer performance for detection of abnormalities on chest radiographs with 5-megapixel resolution liquid-crystal displays (LCD) and 5-megapixel resolution cathode-ray tube (CRT) monitors under bright and subdued ambient light conditions. Six radiologists reviewed a total of 254 digital chest radiographs under four different conditions with a combination of two types of monitors (a 5-megapixel resolution LCD and a 5-megapixel resolution CRT monitor) and with two types of ambient light (460 and 50 lux). The abnormalities analyzed were nodules, pneumothorax and interstitial lung disease. For each reader, the detection performance using 5-megapixel LCD and 5-megapixel CRT monitors under bright and subdued ambient light conditions were compared using multi-case and multi-modality ROC analysis. For each type of ambient light, the average detection performance with the two types of monitors was also compared. For each reader, the observer performance of 5-megapixel LCD and 5-megapixel CRT monitors, under both bright and subdued ambient light conditions, showed no significant statistical differences for detecting nodules, pneumothorax and interstitial lung disease. In addition, there was no significant statistical difference in the average performance when the two monitor displays, under both bright and subdued ambient light conditions, were compared.

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

Fluorescence-Guided Minimally Invasive Parathyroidectomy: A Novel Surgical Therapy for Secondary Hyperparathyroidism

Secondary hyperparathyroidism (SHPT) is a severe and frequent complication in patients with advanced chronic kidney disease, characterized by hyperplasia of all parathyroid glands and elevated serum parathyroid hormone levels. When surgery is required to prevent cardiovascular consequences, bone pain, osteoporosis, or even soft-tissue calcifications, detection of the enlarged glands often can be difficult because of their variability in number and location. A novel surgical technique, fluorescence-guided minimally invasive parathyroidectomy, may facilitate intraoperative localization of parathyroid glands. A 52-year-old woman with SHPT underwent minimally invasive videoscopic-assisted parathyroidectomy after photosensitization with aminolevulinic acid (ALA): Under special fluorescence illumination by D-Light (Karl Storz Co, Tuttlingen, Germany), bilateral neck exploration was performed. All enlarged parathyroid glands were identified because of their ALA-induced intense red fluorescence. Such surrounding structures as thyroid, lymph nodes, and soft tissue remained nonfluorescent and could be distinguished easily from parathyroid glands. Total parathyroidectomy with autotransplantation into the sternocleidoid muscle was performed. In patients with SHPT, exploration of all parathyroid glands during surgery is mandatory. However, to date, there is no convincing technical aid for the surgeon to facilitate this procedure. The ALA-induced fluorescence technique represents an innovative visual detection method for intraoperative identification of parathyroid glands. The technique serves as an additional tool requiring only moderate technical and clinical expenditure.

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25+/-2 degrees C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.

Plasma appearance of labeled β-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, beta-carotene, and retinol. Ingested doses of labeled carotenoids were 34 micromol for beta-carotene and 33 micromol for lutein. Peak plasma concentrations, areas under the plasma concentration-time curves (AUCs), and percentages of dose recovered at peak plasma concentrations were calculated. Average peak plasma concentrations were 0.38, 0.068, and 0.079 microM for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Average AUC values (over 28 days) were 42.8, 13.6, 13.2 microM h for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Percentages of dose recovered at peak plasma concentrations were 3.6, 0.7, and 0.7% for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. A positive relationship was observed between baseline plasma retinol levels and [13C]retinol plasma response. It is possible that this relationship was mediated either through some aspect of beta-carotene absorption or via the common pathways of metabolism for postdose and endogenous retinoid.

Key Role of Conserved Histidines in Recombinant Mouse β-Carotene 15,15′-Monooxygenase-1 Activity

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.