Isolation of the feline alpha1,3-galactosyltransferase gene, expression in transfected human cells and its phylogenetic analysis

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

The Induction of Intercellular Adhesion Molecule-1 on Human Umbilical Vein Endothelial Cells by a Heat-Stable Component of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Live Porphyromonas gingivalis enhanced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) in a bacterial dose-dependent manner. Inactivation of P. gingivalis by ultraviolet (UV), heat (56°C, 30 min), or sonication did not alter its stimulatory activity. ICAM-1 expression began to increase at 4 h after stimulation, reached a maximum at 12 h, and remained at the maximum for at least the next 8 h. This time course was similar to that of expression by Escherichia coli LPS. Furthermore, the effect of UV-inactivated P. gingivalis was not inhibited by boiling or polymyxin B treatment. In addition, the effect of P. gingivalis strain W83 on ICAM-1 expression was stronger than that of strain ATCC 33277. Our results suggested that some unidentified, heat-stable proteins, polysaccharides, or lipids may be the stimulatory factor(s), although the participation of LPS could not be completely ruled out. The ability of P. gingivalis to stimulate ICAM-1 expression on endothelial cells may play an important role in the pathogenesis of periodontal disease.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Arterial anatomical features of the upper palpebra

The arterial anatomical features of the upper palpebra were examined in both sides of seven fresh cadavers that had been systemically injected with a lead oxide/gelatin mixture. All specimens were stereoscopically radiographed for analysis of the three-dimensional structure of the arteries and were macroscopically dissected for observation of the relationships between the arteries and the other tissues. Cross-sections were prepared from one specimen and examined histologically. In all cases, there were four arterial arcades in the upper palpebra, namely, the marginal, peripheral, superficial orbital, and deep orbital arcades. Each arcade provided small vertical branches. The vertical branches coursed on both sides of the orbicularis oculi muscle and on both sides of the tarsal plate. From these small vertical branches, fine vessels branched off to the skin, muscle, and tarsal plate. These findings are important for avoiding complications such as bleeding and are useful for designing local flaps, such as switch flaps, for reconstructive surgical procedures.     

Vascular anatomy of the anterolateral thigh flap

Arterial and venous anatomy and their relation to the anterolateral thigh flap were examined in 10 specimens of six fresh cadavers in which radiopaque materials were injected into both the arterial and venous systems. Territories and positions of individual perforating arteries were measured, and the venous drainage pathway of the flap was analyzed. All specimens were radiographed stereoscopically to observe the three-dimensional structure of the arteries and veins. The territory of each perforating artery was smaller than expected. Most of the venous blood that had perfused the dermis was considered to pool in a polygonal venous network located in the skin layer and to enter the descending branch of the lateral circumflex femoral artery through large descending veins. The venous territories were considered different from the arterial territories. The findings in this study suggest that the design of the anterolateral thigh flap should be based on the venous architecture rather than on the arterial architecture and that the flap survival rate might be improved if thinning is performed appropriately.     

Amphotericin B covalent dimers bearing a tartarate linkage

Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.     

Low-temperature-induced structural changes in human lysozyme elucidated by three-dimensional NMR spectroscopy

The three-dimensional solution structures of human lysozyme were determined at 35 and 4 degrees C using the heteronuclear multidimensional NMR spectroscopy, which were compared with each other to clarify the structural response of this enzyme to lowering of the temperature. Together with the data of the temperature dependence experiments of the lytic activity against Micrococcus luteus, we consider the implication of the observed structural change for the low-temperature-induced reduction of the activity of human lysozyme. The structures of human lysozyme determined at the two temperatures are found to be similar, both of which comprise four alpha-helices (A- to D-helices) and three antiparallel beta-strands (beta(1)-beta(3)), leading to the constructions of the alpha- and beta-domains as previously identified in the X-ray crystal structure. A significant structural change was observed for the "active site lobe" comprising the loop region connecting C- and D-helices and the following D-helix, which moves toward the active site cleft located between the alpha- and beta-domains so as to obstruct the cleft according to the temperature lowering. It further appeared that the total volume as well as the accessible surface area of human lysozyme decreases with lowering of the temperature, suggesting that the internal cavity of this enzyme shrinks under low temperature environment. Because in human lysozyme the region comprising the active site lobe is responsible for turnover of the enzymatic reaction against the substrate, the low-temperature-induced structural change of the active site lobe presumably controls the efficiency of the lytic activity under low temperatures.