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21.
Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan. 相似文献
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Hypopigmenting agents: an updated review on biological, chemical and clinical aspects 总被引:4,自引:0,他引:4
Solano F Briganti S Picardo M Ghanem G 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2006,19(6):550-571
An overview of agents causing hypopigmentation in human skin is presented. The review is organized to put forward groups of biological and chemical agents. Their mechanisms of action cover (i) tyrosinase inhibition, maturation and enhancement of its degradation; (ii) Mitf inhibition; (iii) downregulation of MC1R activity; (iv) interference with melanosome maturation and transfer; (v) melanocyte loss, desquamation and chemical peeling. Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as this enzyme catalyses the rate-limiting step of pigmentation. Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce effects in clinical trials. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their efficacy and safety. Successful treatments need the combination of two or more agents acting on different mechanisms to achieve a synergistic effect. In addition to tyrosinase inhibition, other parameters related to cytotoxicity, solubility, cutaneous absorption, penetration and stability of the agents should be considered. The screening test system is also very important as keratinocytes play an active role in modulating melanogenesis within melanocytes. Mammalian skin or at least keratinocytes/melanocytes co-cultures should be preferred rather than pure melanocyte cultures or soluble tyrosinase. 相似文献
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Elsa N. Bou Ghanem Grant S. Jones Tanya Myers-Morales Pooja D. Patil Achmad N. Hidayatullah Sarah E. F. D'Orazio 《PLoS pathogens》2012,8(11)
Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlAm) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlAm), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlAm significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa. 相似文献
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It has been found that with mutation of two surface residues (Lys22 → Glu and His104 → Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5′…ImmG:N4′ and H257:Nδ…ImmG:O5′ consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4′…ImmG:O5′…H257:Nδ became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP. 相似文献
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Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early SN1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while pre-steady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1'-3H], [1'-14C], [2'-3H], [5'-3H], and [9-15N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1'-14C] and [9-15N] KIEs indicate a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1'-14C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state. 相似文献
28.
Ghanem M Saen-oon S Zhadin N Wing C Cahill SM Schwartz SD Callender R Schramm VL 《Biochemistry》2008,47(10):3202-3215
Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes. 相似文献
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Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX. 相似文献