In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

Interaction between trehalose and alkaline-earth metal ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.     

Mumps virus V protein antagonizes interferon without the complete degradation of STAT1

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.     

Evaluation of extra capsular lymph node involvement in patients with extra-hepatic bile duct cancer

ABSTRACT: BACKGROUND: Lymph node metastasis (LNM) is one of the most important prognostic factors for extra-hepatic bile duct carcinoma (ExHBDC). Extra capsular lymph node involvement (ExCLNI) is the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The prognostic impact of ExCLNI has been shown to be mainly significant in head and neck malignancies. Recently, the prognostic impacts of ExCLNI have been evaluated in gastrointestinal malignancies. However, no data are available regarding the incidence and prognostic significance of extra-capsular lymph node involvement (ExCLNI) in resectable ExHBDCs. The aim of the present study is first to evaluate the incidence of ExCLNI in surgically-treated ExHBDCs and, second, to determine the prognostic impact of ExCLNI in patients with surgically-treated ExHBDCs. METHODS: A total of 228 patients, (110 cases of hilar cholangiocarcinoma and 118 cases of distal cholangiocarcinoma), with surgically-treated ExHBDCs were included in this retrospective study. ExCLNI was defined as the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The existence of ExCLNI and its prognostic value were analyzed as a subgroup of lymph node metastasis. RESULTS: ExCLNI was detected in only 22% of patients with lymph node metastasis of surgically-treated ExHBDC. The presence of ExCLNI correlated with distal cholangiocarcinoma (P = 0.002). On univariate analysis for survival, perineural invasion, vascular invasion, histological grade, and lymph node metastasis were statistically significant factors. On multivariate analysis, only lymph node metastasis was identified as a significant independent prognostic factor in patients with resectable ExHBDC. Subgroups of lymph node metastasis including the presence of ExCLNI, location of lymph node metastasis, and the number of lymph node metastasis had no statistically significant impact on survival. CONCLUSION: ExCLNI was present in only 22% of the LNM (7% of overall patients) in patients with surgically-treated ExHBDCs. And ExCLNI would have no impact on the survival of patients with surgically-treated ExHBDCs.     

Relationship among coronary plaque compliance, coronary risk factors and tissue characteristics evaluated by integrated backscatter intravascular ultrasound

ABSTRACT: BACKGROUND: The purpose of the present study was to evaluate the mechanical properties of coronary plaques and plaque behavior, and to elucidate the relationship among tissue characteristics of coronary plaques, mechanical properties and coronary risk factors using integrated backscatter intravascular ultrasound (IB-IVUS). Methods: Non-targeted plaques with moderate stenosis (plaque burden at the minimal lumen site: 50-70%) located proximal to the site of the percutaneous coronary intervention target lesions were evaluated by IB-IVUS. Thirty-six plaques (less calcified group: an arc of calcification [less than or equal to]10) in 36 patients and 22 plaques (moderately calcified group: 10< an arc of calcification [less than or equal to]60) in 22 patients were evaluated. External elastic membrane volume (EEMV) compliance, lumen volume (LV) compliance, plaque volume (PV) response (difference between PV in systole and diastole), EEM area stiffness index were measured at the minimal lumen site. Relative lipid volume (lipid volume/internal elastic membrane volume) was calculated by IB-IVUS. Results: In the less calcified group, there was a significant correlation between EEMV compliance and the relative lipid volume (r=0.456, p=0.005). There was a significant inverse correlation between EEM area stiffness index and the relative lipid volume (p=0.032, r =-0.358). The LV compliance and EEM area stiffness index were significantly different in the diabetes mellitus (DM) group than in the non-DM group (1.32 +/- 1.49 vs. 2.47 +/- 1.79 %/10 mmHg, p =0.014 and 28.3 +/- 26.0 vs. 15.7 +/- 17.2, p =0.020). The EEMV compliance and EEM area stiffness index were significantly different in the hypertension (HTN) group than in the non-HTN group (0.77 +/- 0.68 vs. 1.57 +/- 0.95 %/10 mmHg, p =0.012 and 26.5 +/- 24.3 vs. 13.0 +/- 16.7, p =0.020). These relationships were not seen in the moderately calcified group. Conclusion: The present study provided new findings that there was a significant correlation between mechanical properties and tissue characteristics of coronary arteries. In addition, our results suggested that the EEMV compliance and the LV compliance were independent and the compliance was significantly impaired in the patients with DM and/or HTN. Assessment of coronary mechanical properties during PCI may provide us with useful information regarding the risk stratification of patients with coronary heart disease.     

Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex

A key feature of the mammalian brain is its capacity to adapt in response to experience, in part by remodeling of synaptic connections between neurons. Excitatory synapse rearrangements have been monitored in vivo by observation of dendritic spine dynamics, but lack of a vital marker for inhibitory synapses has precluded their observation. Here, we simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbor of pyramidal neurons in the adult mammalian cortex using large-volume, high-resolution dual-color two-photon microscopy. We find that inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience. Further, we find inhibitory synapse and dendritic spine remodeling to be spatially clustered and that clustering is influenced by sensory input. Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.     

Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysis

Yeast is widely used to determine the tertiary structure of eukaryotic proteins, because of its ability to undergo post-translational modifications such as glycosylation. A mutant lacking S -adenosylmethionine synthesis has been reported as a suitable host for producing selenomethionine derivatives, which can help solve phase problems in protein crystallography. However, the mutant required external addition of S -adenosylmethionine for cell proliferation. Here, a selenomethionine-resistant Pichia pastoris mutant that showed S -adenosylmethionine autotrophy was isolated. Human lysozyme expressed by the mutant under the control of constitutive promoter contained selenomethionine at 65% occupancy, sufficient for use as a selenomethionine derivative for single-wavelength anomalous dispersion phasing.