The establishment and regeneration of a range ofCuphea germplasm t in vitro

The range of Cuphea species previously investigated in vitro has been extended, and the potential of a number of species for uptake into gene transfer systems evaluated. Eight species were successfully established in culture and assessments of relative subculturing potential rates after 4 weeks varied from 10.3 per initial shoot in C. wrightii to 2.3 in C. leptopoda. In tissue regeneration studies, callus formation was rapid and efficient from seven of the eight lines investigated and the species C. lutea, C. lanceolata, C. leptopoda, C. paucipetala and C. laminuligera (in addition to the previously reported C. wrightii) regenerated shootsin vitro from leaf explants after three to six weeks culture on a medium containing 8.8 μM BA, in a two-stage protocol. A high incidence of in vitro flowering was also recorded in C. lutea, C. leptopoda and C. paucipetala. Though initial growth rates were comparable, it was found that extended culture on a matrix of Phytagel rather than agar was detrimental to subsequent shoot development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Corticotropin-releasing hormone-immunopositive nerve elements in apposition to chicken gonadotropin-releasing hormone I-containing perikarya in Japanese quail (Coturnix coturnix japonica) brain

The neuroanatomic basis of how stress inhibits reproduction in birds is not understood. To address this question we used double-label immunofluorescence histochemistry to determine whether corticotropin-releasing hormone (CRH)-immunoreactive (ir) neuronal elements contact chicken gonadotropin-releasing hormone I (cGnRH I)-ir somata in brains of Japanese quail. The double-label system used a sheep anti-cGnRH I primary antibody with a secondary antibody conjugated to dichlorotriazinylaminofluorescein dihydrochloride for green fluorescence and a rabbit anti-CRH antibody with a secondary conjugated to Texas Red for red fluorescence. Immunohistochemical (IHC) distribution of both peptides resembled that in previous reports using single-label IHC. In four areas of the quail brain in which CRH nerve fibers and cGnRH I somata co-occurred (bed nucleus commissural pallii, nucleus preopticus medialis, nucleus septalis lateralis and nucleus accumbens), numerous instances were found of CRH-ir nerve fibers or terminals in apposition to cGnRH I cell bodies. These interactions provide a potential neuroanatomic route by which CRH may directly inhibit the activity of cGnRH-I-containing neurons, thereby inhibiting gonadotropin output and halting or slowing the progression of reproductive cycles. It remains to be demonstrated by electron microscopy whether these interactions, which appear abundant by IHC, represent instances of synaptic contact, as has been demonstrated to occur in analogous areas in mammalian species.     

5′-Anchored simple-sequence repeat primers are useful for analysing potato somatic hybrids

Somatic hybrids between Solanum tuberosum cv 'Brodick' and S. sanctae-rosae were selected for having elevated chromosome numbers, intermediate morphology and potato cyst nematode resistance (derived from S. sanctae-rosae). DNA was extracted from this material and subjected to inter-SSR PCR directed by oligonucleotide primers comprising 5′-anchored di- and tri-nucleotide repeat motifs. PCR products were separated by polyacrylamide gel electrophoresis and visualised by silver staining. An average of 30.31 bands per primer were generated in each parent and, on average, 8.19 of these bands were polymorphic. S. sanctae-rosae-specific bands were observed in the hybrid material as was evidence of deletions, substitutions and rearrangements. The relative merits of this technique for the analysis of somatic hybrid material are discussed. Received: 23 July 1996 / Revision received: 22 October 1996 / Accepted: 22 March 1999     

Oral estrogen masculinizes female zebra finch song system

It is well established that parenteral treatment of female zebra finch chicks with estradiol masculinizes their song control nuclei and that as adults they are capable of song. Concern over the widespread use of putative environmental estrogens caused us to ask whether oral exposure to estrogens (a natural route of exposure) could produce similar effects. We dosed chicks orally with estradiol benzoate (EB; 1, 10, 100, and 1000 nmol/g of body mass per day, days 5-11 posthatch), the non-ionic surfactant octylphenol (100 and 1000 nmol/g), or the pesticides methoxychlor (100 and 1000 nmol/g) and dicofol (100 nmol/g) and measured their song control nuclei as adults. EB treatment produced increases in song nuclei comparable to that induced by parenteral administration of estrogens. This is the first study of which we are aware to use an oral route of administration, which simulates the natural process of parent birds feeding their nestlings. We conclude that oral exposure to estradiol alters song control nuclei and we report in a related paper (Millam et al., 2001) that such exposure severely disrupts reproductive performance. Although we detected no influence of xenobiotics on induction of song control nuclei the possibility remains that oral exposure to xenoestrogens in high enough doses could affect development.     

Thresholds for sweet, salt, and sour taste stimuli in cockatiels (Nymphicus hollandicus)

Little is known about avian taste perception and how taste affects food choice. We designed a study to determine the concentrations of aqueous solutions of common chemical taste stimuli that result in altered consumption patterns. Using two-choice taste-preference tests, we studied the taste thresholds of caged cockatiels (Nymphicus hollandicus) for aqueous solutions of potassium chloride, a phosphate buffer (to test pH), fructose, and glucose. First, the preferred and nonpreferred bottle locations were determined for each bird. Then, depending on the compound, the test solutions were placed in bottles in either the preferred or the nonpreferred locations and water was placed in the opposite bottle. Four parameters were measured at the end of 3-day test periods (total consumption, consumption from water side, consumption from test solution side, and proportion of nonpreferred side consumption to total consumption). Experiments were repeated with increasing concentrations of test flavors until intake variables were significantly affected (P < 0.05). Cockatiels distinguished (P < 0.05) between purified water and 0.16 mol L(-1) potassium chloride, 0.40 mol L(-1) fructose, or 0.16 mol L(-1) glucose. The test birds did not distinguish between water and 0.05 mol L(-1) mono- and dibasic sodium phosphate buffer solution at any tested pH within the range of 4.9-7.7. When these findings are compared to previous experiments with the same birds, it becomes clear that the gustatory reactions of cockatiels for two different stimuli (e.g., NaCl and KCl) from the same general taste category (salt, sweet, sour) can vary widely. This variation in the responses to related stimuli could be the result of a number of factors including anion effects (for salts and acids) as well as nongustatory physiological processes (e.g., as renal control of blood osmolarity). Zoo Biol 20:1-13, 2001. Copyright 2001 Wiley-Liss, Inc.     

Ranking and compacting binding segments of protein families using aligned pattern clusters

Background

Discovering sequence patterns with variation can unveil functions of a protein family that are important for drug discovery. Exploring protein families using existing methods such as multiple sequence alignment is computationally expensive, thus pattern search, called motif finding in Bioinformatics, is used. However, at present, combinatorial algorithms result in large sets of solutions, and probabilistic models require a richer representation of the amino acid associations. To overcome these shortcomings, we present a method for ranking and compacting these solutions in a new representation referred to as Aligned Pattern Clusters (APCs). To tackle the problem of a large solution set, our method reveals a reduced set of candidate solutions without losing any information. To address the problem of representation, our method captures the amino acid associations and conservations of the aligned patterns. Our algorithm renders a set of APCs in which a set of patterns is discovered, pruned, aligned, and synthesized from the input sequences of a protein family.

Results

Our algorithm identifies the binding or other functional segments and their embedded residues which are important drug targets from the cytochrome c and the ubiquitin protein families taken from Unitprot. The results are independently confirmed by pFam's multiple sequence alignment. For cytochrome c protein the number of resulting patterns with variations are reduced by 76.62% from the number of original patterns without variations. Furthermore, all of the top four candidate APCs correspond to the binding segments with one of each of their conserved amino acid as the binding residue. The discovered proximal APCs agree with pFam and PROSITE results. Surprisingly, the distal binding site discovered by our algorithm is not discovered by pFam nor PROSITE, but confirmed by the three-dimensional cytochrome c structure. When applied to the ubiquitin protein family, our results agree with pFam and reveals six of the seven Lysine binding residues as conserved aligned columns with entropy redundancy measure of 1.0.

Conclusion

The discovery, ranking, reduction, and representation of a set of patterns is important to avert time-consuming and expensive simulations and experimentations during proteomic study and drug discovery.

     

Inhibitory effects of biochanin A on titanium particle‐induced osteoclast activation and inflammatory bone resorption via NF‐κB and MAPK pathways

Revision operations have become a new issue after successful artificial joint replacements, and periprosthetic osteolysis leading to prosthetic loosening is the main cause of why the overactivation of osteoclasts (OCs) plays an important role. The effect of biochanin A (BCA) has been examined in osteoporosis, but no study on the role of BCA in prosthetic loosening osteolysis has been conducted yet. In this study, we utilised enzyme‐linked immunosorbent assay, computed tomography imaging, and histological analysis. Results showed that BCA downregulated the secretion levels of tumor necrosis factor‐α, interleukin‐1α (IL‐1α), and IL‐1β to suppress inflammatory responses. The secretion levels of receptor‐activated nuclear factor‐κB ligand, CTX‐1, and osteoclast‐associated receptor as well as Ti‐induced osteolysis were also reduced. BCA effectively inhibited osteoclastogenesis and suppressed hydroxyapatite resorption by downregulating OC‐related genes in vitro. Analysis of mechanisms indicated that BCA inhibited the signalling pathways of mitogen‐activated protein kinase (P38, extracellular signal‐regulated kinase, and c‐JUN N‐terminal kinase) and nuclear factor‐κB (inhibitor κB‐α and P65), thereby downregulating the expression of nuclear factor of activated T cell 1 and c‐Fos. In conclusion, BCA may be an alternative choice for the prevention of prosthetic loosening caused by OCs.     

Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.