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Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.

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The evolution of metazoan body plans has involved changes to the Hox genes, which are involved in patterning the body axis and display striking evolutionary conservation of structure and expression. Invertebrates contain a single Hox cluster whereas tetrapods possess four clusters. The zebrafish has seven unlinked hox clusters, a finding that is difficult to reconcile with the notion that genomic complexity, reflected by Hox cluster number, and morphological complexity are causally linked, as the body plan of the zebrafish is not obviously more complex than that of the mouse or human. Why have the additional hox genes in zebrafish been conserved? To address the role of these additional zebrafish hox genes, we have examined the duplicate hoxB5 genes, hoxB5a, and hoxB5b. Conservation of gene duplicates can occur when one gene acquires a new function (neofunctionalization), or when the ancestral function is divided between the two duplicates (subfunctionalization). hoxB5a and hoxB5b are expressed in distinct domains, and their combined expression domain is strikingly similar to that of single Hoxb5 genes in other species. The biochemical functions encoded by the two genes were studied by overexpression, which resulted in identical developmental defects in the anterior hindbrain and cranial neural crest, suggesting strongly that hoxB5a and hoxB5b have equivalent biochemical properties with respect to early development. From these studies, we conclude that conservation of hoxB5a and hoxB5b is likely the result of division of the ancestral Hoxb5 function between the two genes, without significant changes in biochemical activity. These results suggest a resolution to the conundrum of the extra hox genes and clusters in the zebrafish, since if any of the additional hox genes in the zebrafish are similarly subfunctionalized, they are unlikely to supply novel genetic functions. Thus, the morphological complexity potentially conferred by the majority of additional zebrafish hox clusters may not be substantially greater than that conferred by the four tetrapod clusters.

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We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.

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The oxidative behavior of Auranofin, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato- S(triethylphosphine)gold(I), was investigated by using cyclic voltammetry (CV) in 0.1 M Bu(4)NPF(6)/CH(2)Cl(2) and 0.1 M Bu(4)NPF(4)/CH(2)Cl(2) solutions using Pt working and auxiliary electrodes and a Ag/AgCI reference. CV studies at scan rates from 50-2,000 mV/s and Auranofin concentrations between 1 and 4 mM, show two irreversible oxidation processes occurring at +1.1 V and +1.6 V vs. Ag/AgCl. Ph(3) (p-thiocresolate) was also investigated as a reference for comparison of the oxidation processes in Auranofin to that of other phosphine gold thiolate complexes previously reported. The electrochemical response appears to be sensitive to adsorption at the electrode as well as to the nature of the supporting electrolyte solution. Repeated cycling shows a build up of products at the electrode.

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Gold-thiolate/disulfide exchange reactions of (p-SC(6)H(4)Cl)(2) with Ph(3)PAu(SC(6)H(4)CH(3)), dppm(AuSC(6)H(4)CH(3))(2), and dppe(AuSC(6)H(4)CH(3)) (2) were investigated. The rate of reactivity of the gold-thiolate complexes with (p-SC(6)H(4)Cl)(2) is: dppm(AuSC(6)H(4)CH(3))(2)>> dppe(AuSC(6)H(4)CH(3))(2)>Ph(2)PAu (SC(6)H(4)CH(3)). This order correlates with conductivity measurements and two ionic mechanisms have been evaluated. (1)H NMR experiments demonstrate that in the reaction of dppm(AuSC(6)H(4)CH(3))(2) with (p-SC(6)H(4)Cl)(2), the mixed disulfide, ClC(6)H(4)SSC(6)H(4)CH(3), forms first, followed by the formation of (p-SC(6)H(4)CH(3))(2). The rate law is first order in (pp-SC(6)H (4)Cl)(2) and partial order in dppm(AuSC(6)H(4)CH(3))(2). Results from electrochemical and chemical reactivity studies suggest that free thiolate is not involved in the gold-thiolate/disulfide exchange reaction. A more likely source of ions is the dissociation of a proton from the methylene backbone of the dppm ligand which has been shown to exchange with D(2)O. The implications of this are discussed in terms of a possible mechanism for the gold-thiolate/disulfide exchange reaction.

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Cyclic voltammetry (CV) experiments on LL(AuSR *)(2) complexes [LL = diphenylphosphinomethane (dppm), diphenylphosphinopentane (dpppn); R(*) = p-SC(6)H(4)CH(3)] show anodic sweeps that broaden by about 25 mV on going from the longer (dpppn) to the shorter (dppm) bidentate phosphine ligand. Changing concentrations had no effect on the shape of the waveform. The result suggests a weak intramolecular metal-metal interaction in dppm(AuSR *)(2) that correlates well with rate acceleration occurring in the reaction of dppm(AuSR *)(2) with organic disulfides. Quantum yields for cis-dppee(AuX)(2) [dppee = 1,2-bis(diphenylphosphino)ethylene; X = Cl, Br, I] complexes, (disappearance) Phi , are significantly higher in complexes with a softer X ligand, a trend that correlates well with aurophilicity. This result also suggests that electronic perturbation caused by Au(I)-Au(I) interactions is important in explaining the reactivity of some dinuclear gold(I) complexes. The crystal structure for cis-dppee(Aul)(2) shows short intramolecular Au(I)-Au(I) interactions of 2.9526 (6) A degrees , while the structure of trans-dppee(AuI)(2) , shows intermolecular Au(I)-Au(I) interactions of 3.2292 (9) A degrees . The substitution of .As for P results in a ligand, cis-diphenylarsinoethylene (cis-dpaee), that is photochemically active, in contrast to the cis-dppee ligand. The complexes, cis-dpaee(AuX)(2), are also photochemically active but with lower quantum yields than the cis-dppee(AuX)(2) complexes.

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The electrochemical deposition and stripping of mercury on gold surfaces was investigated to assess whether gold electrodes would return to mercury-free states after stripping analyses. X-ray photoelectron spectroscopy studies demonstrate the presence of mercury on gold foil electrodes that have undergone controlled-potential deposition procedures in Hg(2+) solutions (10 nM-0.1 mM) followed by stripping and cleaning in mercury-free electrolyte. Results show that mercury is not completely removed electrochemically from the gold electrodes, even when the oxidizing potential is +2.5 V vs Ag/AgCl. Bulk electrolyses deposition and stripping procedures coupled with cold vapor atomic absorption spectroscopic analyses of solutions after deposition and stripping are also reported. Results suggest that the nature of the gold electrode is fundamentally altered by irreversible adsorption of mercury; that is, mercury is adsorbed during deposition and some of the mercury is retained even after stripping and cleaning. The implications and strategies for using stripping analysis and gold electrodes for the measurement of mercury under the experimental conditions employed in this study are discussed.

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The orthodenticle orthologue Lox22-Otx was isolated from an annelid worm, the leech Helobdella triserialis. In situ hybridization reveals that embryonic expression of Lox22-Otx RNA is primarily restricted to an unsegmented head domain, including tissues in the foregut, surface ectoderm, and the head ganglion of the central nervous system. The patterns of head expression form concentric rings about the stomadeum and mark tissue domains that exhibit discrete behaviors during later morphogenesis and differentiation. Expression was also observed in one to two bilateral pairs of neurons in each segmental ganglion or neuromere of the body trunk. The largely head-specific expression of Lox22-Otx in this annelid species supports data from two other bilaterian phyla in suggesting the existence of a genetically defined head/trunk distinction. We suggest here that this head/trunk distinction is a synapomorphy of the Bilateria as a whole, and that it reflects the body plan of an early bilaterian ancestor. In addition, we discuss the possibility that the radial organization of gene expression and cell lineages in the leech's head domain may reflect the symmetry properties of a prebilaterian ancestor that had a radially symmetric body plan.

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The homeobox gene Lox2, a member of the HOM/Hox gene class, is expressed in a restricted domain along the anteroposterior (A-P) body axis of the leech Helobdella. The segmental tissues of the leech embryo arise from the parallel merger of five distinct and bilaterally paired cell lineages generated by embryonic stem cells or teloblasts. Injection of cell lineage tracers coupled with anti-LOX2 immunochemistry reveals that all five teloblast lineages generate central nervous system neurons that express the LOX2 protein, and that each lineage expresses LOX2 within a similar domain of body segments. Some lineally identified neurons display anti-LOX2 immunoreactivity over the entire expression domain, but the OM7 neuron has a distinctively high level of LOX2 expression, which is restricted to the seventh midbody ganglion. To ascertain the role of positional information in the axial patterning of LOX2 expression, we performed focal cell ablations that displaced one or another of the teloblast lineages out of segmental register with the other axial tissues. Such displacements brought about a corresponding shift in the LOX2 expression of the perturbed lineage, and had little or no effect on the LOX2 expression of the other, unperturbed lineages. This result indicates that the axial domain of LOX2 expression is not specified by positional cues acting coordinately across the various teloblast lineages, nor would it seem that the expression domain is imprinted from one lineage to the others. Rather, the different teloblast lineages acquire their axial patterns independently, and secondarily bring these patterns into alignment along the A-P axis through a process of morphogenetic assembly.

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The unusual yellow color of Au(2)(dppm)(SR)(2) (R = 4-tolyl; dppm = diphenylphosphinomethane) is attributed to a red-shift in the S-->Au charge transfer caused by destabilization of the sulfur highest occupied molecular orbital (HOMO). Variable temperature experiments show two broad bands at -80 degrees C in the (31)P{(1)H} NMR spectrum of Au(2)(dppm)(SR)(2) and the activation energy for interconversion is 10 kcal/mol. Only one sharp band is observed down to -80 degrees C in the spectrum of the white complex, Au(2)(dppe)(SR)(2) (dppe = diphenylphosphinoethane). Molecular mechanics calculations on Au(2)(dppm)(SR)(2) and Au(2)(dppe)(SR)(2) reveal that, for Au(2)(dppe)(SR)(2), a series of maxima and minima, separated by 2.5 kcal/mol, occur every 120 degrees which is consistent with rotation around an unhindered carbon-phosphorus single bond. The Au atoms are not within bonding distance in any conformation. Computational results for Au(2)(dppm)(SR)(2) indicate one minimum energy structure in which the Au-P bonds are anti. There is a high energy conformation (9 kcal/mol above the global minimum) where overlap between golds is maximized. The implications of gold-gold bonding in this complex are discussed. The steric influence of the thiolate ligand has been examined by synthesizing a series of dinuclear gold(I) complexes in which the steric properties of the thiolate are varied: Au(2)(dppm)(SR)(2) (R = 2,6-dichlorophenyl; 2,6-dimethylphenyl; 3,5-dimethylphenyl). The 2,6-disubstituted complexes are white, while the 3,5-dimethyl complex is yellow. These results, along with VT-NMR experiments, are consistent with the conclusion that the more sterically-bulky thiolates hinder the close approach of the golds in the dinuclear complexes.

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The redox chemistry of mononuclear and dinuclear gold(I) phosphine arylthiolate complexes was recently investigated by using electrochemical, chemical, and photochemical techniques. We now report the redox chemistry of dinuclear gold(I) phosphine complexes containing aliphatic dithiolate ligands. These molecules differ from previously studied gold(I) phosphine thiolate complexes in that they are cyclic and contain aliphatic thiolates. Cyclic voltammetry experiments of Au(2) (LL)(pdt) [pdt = propanedithiol; LL = 1,2-bis(diphenylphosphino)-ethane (dppe), 1,3-bis(diphenylphosphino)propane (dppp), 1,4-bis(diphenylphosphino)butane (dppb), 1,5-bis(diphenylphosphino)pentane (dpppn)] in 0.1 M TBAH/CH(3)CN or CH(2)Cl(2) solutions at 50 to 500 mV/sec using glassy carbon or platinum electrodes, show two irreversible anodic processes at ca. +0.6 and +1.1 V (vs. SCE). Bulk electrolyses at +0.9 V and +1.4 V result in n values of 0.95 and 3.7, respectively. Chemical oxidation of Au(2)(dppp)(pdt) using one equivalent of Br(2) (2 oxidizing equivalents) yields 1,2-dithiolane and Au(2)(dppp)Br(2). The reactivity seen upon mild oxidation /= +1.3 V) is consistent with oxidation of gold(I) to gold(III). Structural and electrochemical differences between gold(I) aromatic and aliphatic thiolate oxidation processes are discussed.

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Between Dec 8, 1982, and Jan 29, 1983, four cases of primary bacteremia with Ewingella americana occurred in the intensive care unit of a community hospital. The patients developed high fever and leukocytosis, which gradually resolved after institution of antibiotic therapy. All infected patients had undergone either cardiovascular or peripheral vascular surgery. Epidemiologic investigation revealed that the probable source for the Ewingella outbreak was contamination of the ice bath used to cool syringes for cardiac output determinations. The nosocomial outbreak was terminated by the introduction of a closed cardiac output injectate delivery system.

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