Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine

During inhalation of allergens and experimental sepsis, formation of brominated tyrosine has been reported. In this study, we first examined the immunogenicity of brominated protein prepared by treatment of N-bromosuccinimide (NBS). The immunized serum obtained reacted with brominated bovine serum albumin (BSA). The NBS dose-dependent formation of immunoreactivity, which was estimated by enzyme-linked immunosorbent assay, was observed, and the increase coincided with 3,5-dibromotyrosine (DiBrY) formation in the modified BSA, which was chemically determined by liquid chromatography/quadrupole tandem mass spectrometry (LC/MS/MS). Second, by use of immunized mice, monoclonal antibodies to the brominated one were prepared. The two established novel monoclonal antibodies obtained from the immunized mice reacted with DiBrY, 3,5-dichlorotyrosine (DiClY), and 3,5-diiodotyrosine (DiIY). Moreover, 3,5-dihalo-4-hydroxybenzoic acids (3,5-dichloro-4-hydroxybenzoic acid and 3,5-dibromo-4-hydroxybenzoic acid) were recognized by these antibodies. These results suggest that dihalogenated tyrosines (DiBrY, DiClY, and DiIY) are the epitopes. Lastly, we used the antibody in an immunohistochemical study. Lipopolysaccharide (LPS) was intraperitoneally administered to mice, and livers were removed. Positive staining of LPS-treated mouse liver tissues by both the anti-dihalotyrosine antibody and anti-myeloperoxidase antibody was estimated, suggesting that inflammatory tissue damage induces the formation of dihalotyrosine in vivo.     

Direction of action potential propagation influences calcium increases in distal dendrites of the cricket giant interneurons

To understand the relationship between the propagation direction of action potentials and dendritic Ca(2+) elevation, simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and intradendritic membrane potential were performed in the wind-sensitive giant interneurons of the cricket. The dendritic Ca(2+) transients induced by synaptically-evoked action potentials had larger amplitudes than those induced by backpropagating spikes evoked by antidromic stimulation. The amplitude of the [Ca(2+)](i) changes induced by antidromic stimuli combined with subthreshold synaptic stimulation was not different from that of the Ca(2+) increases evoked by the backpropagating spikes alone. This result means that the synaptically activated Ca(2+) release from intracellular stores does not contribute to enhancement of Ca(2+) elevation induced by backpropagating spikes. On the other hand, the synaptically evoked action potentials were also increased at distal dendrites in which the Ca(2+) elevation was enhanced. When the dendritic and axonal spikes were simultaneously recorded, the delay between dendritic spike and ascending axonal spike depended upon which side of the cercal nerves was stimulated. Further, dual intracellular recording at different dendritic branches illustrated that the dendritic spike at the branch arborizing on the stimulated side preceded the spike recorded at the other side of the dendrite. These results suggest that the spike-initiation site shifts depending on the location of the activated postsynaptic site. It is proposed that the difference of spike propagation manner could change the action potential waveform at the distal dendrite, and could produce synaptic activity-dependent Ca(2+) dynamics in the giant interneurons.     

RobA-lnduced Multiple Antibiotic Resistance Largely Depends on the Activation of the AcrAB Efflux

RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a ΔompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1^? mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.     

Long-Term Colonization by blaCTX-M-Harboring Escherichia coli in Healthy Japanese People Engaged in Food Handling

The actual state of intestinal long-term colonization by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 μg/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla_CTX-M were repeatedly recovered from 11 of the 13 carriers of bla_CTX-M-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla_CTX-M-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla_CTX-M-15 or bla_CTX-M-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.     

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography-mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP-TLC blotting) and (ii) gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). CEOOH was applied to DPPP-TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC-EI-MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC-EI-MS analysis combined with DPPP-TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.     

Highly sensitive histamine-sensitization test for residual activity of pertussis toxin in acellular pertussis vaccine

Histamine-sensitization test method based on histamine-sensitizing death is widely used for controlling residual activity of pertussis toxin in acellular pertussis vaccines. The test method evaluates the residual activity according to the death of mice injected with a test vaccine after histamine challenge and the test result, therefore, depends on the sensitivity of mice. A highly sensitive test method based on change in rectal temperature of mice has been used in Japan for many years but has limited feasibility in other countries. We examined the possibility of a test method using dermal temperature measured by infrared thermometer to reduce animal suffering instead of rectal temperature. The dermal temperature method was shown to be as sensitive as the rectal temperature method. Furthermore, the dermal as well as rectal temperature methods can evaluate the activity of a test vaccine in relative to a reference preparation so as to allow direct comparison of the test results among different laboratories. The activity by means of the dermal temperature method was also found to be well consistent with that by the rectal temperature method.     

The influence of natural mineral water on aquaporin water permeability and human natural killer cell activity

Aquaporins are the intrinsic membrane proteins functioning as water channel to transport water and/or mineral nutrients across the biological membrane systems. In this research, we aimed to clarify if the selected mineral water can affect aquaporin functions in vitro and the assumption of the mineral water can modify aquaporin expression and activate natural killer cell activity in human body. First, we expressed six human and eight plant aquaporin genes in oocytes and compared the effect of different kinds of natural mineral water on aquaporin activity. The oocyte assay data show that Hita tenryosui water could promote water permeability of almost all human and plant aquaporins in varying degrees, and freeze-dry and organic solvent extraction could reduce AQP2 activity but pH change and boiling could not. Second, each volunteer in two groups (10 in one group) received an oral Hita tenryosui or tap water load of 1000 ml/day for total four weeks. We found that these two kinds of water did not directly affect the relative expression levels of AQP1 and AQP9 in the blood cells, but intriguingly, the natural killer cell activities of the volunteers drinking Hita tenryosui water were significantly improved, suggesting that Hita tenryosui water has obvious health function, which opens a new and interesting field of investigation related to the link between mineral water consumption and human health and the therapies for some chronic diseases.     

Structural insights into the novel diadenosine 5',5‴-P¹,P⁴-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

Rv2613c is a diadenosine 5′,5?-P¹,P⁴-tetraphosphate (Ap₄A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (Ap_nA) hydrolases and Ap₄A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family Ap_nA hydrolases than to that of typical Ap₄A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with Ap_nA phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family Ap_nA hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap₄A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis.     

Transpiration from shoots triggers diurnal changes in root aquaporin expression

Root hydraulic conductivity (Lp(r)) and aquaporin amounts change diurnally. Previously, these changes were considered to be spontaneously driven by a circadian rhythm. Here, we evaluated the new hypothesis that diurnal changes could be triggered and enhanced by transpirational demand from shoots. When rice plants were grown under a 12h light/12h dark regime, Lp(r) was low in the dark and high in the light period. Root aquaporin mRNA levels also changed diurnally, but the amplitudes differed among aquaporin isoforms. Aquaporins, such as OsPIP2;1, showed moderate changes, whereas root-specific aquaporins, such as OsPIP2;5, showed temporal and dramatic induction around 2h after light initiation. When darkness was extended for 12h after the usual dark period, no such induction was observed. Furthermore, plants under 100% relative humidity (RH) showed no induction even in the presence of light. These results suggest that transpirational demand triggers a dramatic increase in gene expressions such as OsPIP2;5. Immunocytochemistry showed that OsPIP2;5 accumulated on the proximal end of the endodermis and of the cell surface around xylem. The strong induction by transpirational demand and the polar localization suggest that OsPIP2;5 contributes to fine adjustment of radial water transport in roots to sustain high Lp(r) during the day.