Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

Identification of a key amino acid of LuxS involved in AI-2 production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2(-) variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ～100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter.     

Examination of the dose distribution of volumetric modulated arc radiotherapy using a high-definition multi-leaf collimator for breast cancer patients with irradiated regional lymph nodes

BackgroundA high-definition multi-leaf collimator (HD-MLC) with 5- and 10-mm fine MLCs is useful for radiotherapy. However, it is difficult to irradiate the mammary gland and supraclavicular region using a HD-MLC because of the narrow field of volumetric modulated arc radiotherapy (VMAT). Therefore, we aimed to evaluate the dose distribution of the VMAT dose using a HD-MLC in 15 patients with left breast cancer undergoing postoperative irradiation of breast and regional lymph nodes, including the internal mammary node.Materials and methodsThe following four plans were generated: three-arc VMAT using HD-MLC (HD-VMAT), two tangential arcs and one-arc VMAT using HD-MLC (tHD-VMAT), three-dimensional conformal radiotherapy (3DCRT) using HD-MLC, and two-arc VMAT using the Millennium 120-leaf MLC (M-VMAT). We assessed the doses to the target volume and organs at risk.ResultsThe target dose distributions were higher for HD-VMAT than 3DCRT. There were no significant differences in the heart mean dose (D_mean) or lung volume receiving 20 Gy (V20 Gy) between HD-VMAT and 3DCRT. The heart D_mean and lung V20 Gy of tHD-VMAT were higher than those of HD-VMAT, and the heart D_mean of M-VMAT was higher than that of HD-VMAT. However, the target doses of tHD-VMAT, M-VMAT, and HD-VMAT were equivalent.ConclusionsIn cases of the mammary gland and regional lymph node irradiation, including the internal mammary node in patients with left breast cancer, HD-VMAT was not inferior to M-VMAT and provided a better dose distribution to the target volume and organs at risk compared with 3DCRT and tHD-VMAT.     

TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.     

Assembly of a mixture of isomeric BChl c from Chlorobium limicola as determined by intermolecular 13C-13C dipolar correlations: coexistence of dimer-based and pseudo-monomer-based stackings

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.     

Localization of mRNAs for phosphatidylinositol phosphate kinases in the mouse brain during development

The gene expression for seven phosphatidylinositol phosphate kinases (PIPKs)-types Ialpha, Ibeta, Igamma, types IIalpha, IIbeta, IIgamma, and type III-was examined using in situ hybridization histochemistry, in the mouse brain during normal development. In the embryonic mouse brain, positive expression signals were detected only for the genes encoding PIPK Igamma and PIPK IIbeta in both the cerebral ventricular and mantle zones, with weaker signals in the former zone. On the other hand, the genes encoding all PIPKs were essentially detected in the external granule cell layer which represents the germinal zone for the neuronal granule cells. In the postnatal brain, among the seven PIPKs, the expression for genes encoding PIPK Igamma and IIbeta is evident in most gray matter, while the expression for the other five types was weak in the cortical gray matter and negligible in most non-cortical gray matter such as the diencephalon and brain stem nuclei. While the expression for most PIPKs in the mature hippocampus was distinct, the expression in the CA3 and the dentate gyrus was less definite for the genes encoding PIPK Ialpha and IIgamma, respectively. The distinct expression for the gene encoding PIPK IIalpha was detected in the postnatal white matter such as the cerebellar medulla, the corpus callosum, the hippocampal fimbriae, and the internal capsule.     

Aging modulates susceptibility to mouse liver Mallory-Denk body formation

Mallory-Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions found in several liver diseases and consist primarily of the cytoskeletal proteins, keratins 8 and 18 (K8/K18). Recent evidence indicates that the extent of stress-induced protein misfolding, a K8>K18 overexpression state, and transglutaminase-2 activation promote MDB formation. In addition, the genetic background and gender play an important role in mouse MDB formation, but the effect of aging on this process is unknown. Given that oxidative stress increases with aging, the authors hypothesized that aging predisposes to MDB formation. They used an established mouse MDB model-namely, feeding non-transgenic male FVB/N mice (1, 3, and 8 months old) with 3,5 diethoxycarbonyl-1,4-dihydrocollidine for 2 months. MDB formation was assessed using immunofluorescence staining and biochemically by demonstrating keratin and ubiquitin-containing crosslinks generated by transglutaminase-2. Immunofluorescence staining showed that old mice had a significant increase in MDB formation compared with young mice. MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62. Old mouse livers had increased oxidative stress. In addition, 20S proteasome activity and autophagy were decreased, and endoplasmic reticulum stress was increased in older livers. Therefore, aging predisposes to experimental MDB formation, possibly by decreased activity of protein degradation machinery.     

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes.

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.     

Effects of synthetic and naturally occurring flavonoids on Na+,K+-ATPase: aspects of the structure-activity relationship and action mechanism

A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na+, K+-adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na+, K+-ATPase from dog kidney cortex when present at concentrations from 40-1000 microM. Flavones possessing cyclohexyl instead of the phenyl group (i.e., 2-cyclohexyl-benzopyran-4-one derivatives), were the most potent with IC50 at 257-320 microM. Structure-activity relationships were observed among the following mono-substituted flavones as: i) 2-cyclohexyl-benzopyran-4-one much greater than 2-phenyl-benzopyran-4-one; ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one greater than 2-cyclohexyl-6-hydroxybenzopyran-4-one greater than 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly any of the flavonoids were able to block [3H]ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na+, K+-ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme.