p53 protein overexpression and K-rascodon 12 mutation in pancreatic ductal carcinoma: Correlation with histologic factors

The correlation of p53 protein overexpression and the K-ras codon 12 mutatlon wlth histologlc type, grade of cytologic atypla, depth of lnvasion and other histologlc prognostic factors was studied In paraffin sectlons from 43 ductectatic-and 70 solid-type pancreatic ductal carcinomas. Overexpression of p53 was found in 23.3% (10143) of ductectatic carcinomas (17.2% of intraductal and 35.7% of lnvaslve carclnomas) and in 61.4% (43/70) of solid carcinomas. In ductectatic cancers, p53 overexpression was detected In 14.8% (4/27) of carcinomas wlth lowgrade atypla (CAL), 50.0% (5110) of carcinomas wlth high-grade atypla (CAH) and in 16.7% (In) of mixed low- and hlgh-grade cancers. In the last group, expression was restricted to an area of CAH. In solld cancers, p53 overexpression did not dlffer by histologic type or grade. Overexpresslon of p53 and K-ras mutatlons did not correlate with histologlc prognostic factors (lymphatic, venous and perineural Invasion, and lymph node metastasls) in ductectatlc and solld cancers or depth of invasion of solld carclnomas. Our data suggest that p53 alteratlon occurs at an early intraductal stage of solld carcinoma, irrespectlve of cellular atypla, but Is low in ductectatic CAL and becomes hlgher In ductectatlc CAH. K-ras mutatlon, present In a high percentage of tumors of all groups and not correlating with the factors above, showed no changes In frequency with tumor progression.     

Topographical gradient in expression of R2D5 antigen in superior olivary nuclei and hippocampal dentate gyrus of the cat.

An immunohistochemical analysis of the cat central nervous system revealed that a monoclonal antibody which recognizes a soluble cytosolic protein, R2D5, bound two regions in a prominent spatial gradient. In the medial and lateral superior olivary nuclei of the brainstem, R2D5 immunoreactivity appeared as a gradient across a population of topographically ordered principal neurons. The spatial gradient corresponded to the tonotopic organization in the superior olivary nuclei: i.e., R2D5 immunoreactivity tended to occur more frequently and intensely in low-frequency neurons than in high-frequency neurons. Granule cells in the hippocampal dentate gyrus also had a pronounced spatial gradient in R2D5 immunoreactivity expression, and this gradient corresponded to the septotemporal axis of the hippocampus. Granule cells of the temporal (ventral) portions of the hippocampus were labeled intensely with R2D5 antibody, while those located in progressively more septal (dorsal) portions had gradually less immunoreactivity. These results suggest that in both the superior olivary nuclei and the hippocampal dentate gyrus, neurons differ in intrinsic properties by their position along specific axes. They suggest also that the hippocampus has an intrinsic functional organization related to the spatial gradient along its septotemporal axis.     

Studies of bovine enterovirus structure by ultraviolet resonance Raman spectroscopy

The structural comparison of bovine enterovirus MZ468 strain before and after the heat treatment was studied by ultraviolet resonance Raman (UVRR) spectra excited at both 235 and 251 nm. The difference between full, heated full and purified empty particles, which were expected as an in vitro model of uncoating, were demonstrated. At 235 nm excitation, the Raman bands of the capsid protein dominated in all the UVRR spectra. The UVRR spectra of the empty particles exhibited non-homogenious broadening for tryptophan W3 band and W7 Fermi doublet bands, which were characteristics of hydrophobic environment, when compared with those of the full particles. The results indicates that some Trp indole rings of the full particles were packaged inside the viral capsids and not strained by virion assembly. On the other hand, the Raman bands assigned to guanine residues of the single stranded-RNA genome were enhanced strongly in the 251-nm excited UVRR spectrum. The spectral differences between the packaged (full particles) and the unpackaged virions (heated full particles) indicates that some guanine residues had strong hydrogen bonds in the full particles.     

Lymphocyte subpopulations and T-cell subsets in human oral cancer tissues: immunohistologic analysis by monoclonal antibodies

A series of T-cell-specific monoclonal antibodies (Leu-1, Leu-2a, and Leu-3a) and B-cell-specific monoclonal antibody (HLB-1) were used to detect the localization and intensity of infiltration of lymphocyte subpopulations and T-cell subsets in frozen sections of 17 patients with the oral cancer. The vast majority of the lymphocyte infiltrates in the oral cancer tissues were reactive with Leu-1. In contrast, B cells were detectable with HLB-1 in only 2 of 17 cases. Leu-2a-positive cells were dominant in four cases, whereas Leu-3a positive cells were dominant in only three cases. In seven cases, both cells infiltrated to the same degree. Leu-2a positive cells tended to be dominant in the cases with earlier clinical stages.     

Suppressor activity in the supernatant of T cell clones derived from chimeric mouse spleen cells

The supernatant from cultures of T cell clones derived from (BALB/c----C3H/He) chimeras suppresses BALB/c anti-C3H/He or BALB/c anti-C57BL/6 MLRs. When we studied the alloantigen specificity of the suppressor activity in culture supernatant, we observed three types of the suppressor activity (i.e., the suppressor activity against BALB/c anti-C3H/He MLR, against BALB/c anti-C57BL/6 MLR, and against both MLRs) on day 3 after stimulation of the T cell clones with 20% crude IL2 and feeder cells. Since the alloantigen specificity fluctuated somewhat with time, we considered that a time-course study was needed to determine it correctly. We thought it unlikely that any IFN-gamma or PGE2 in the culture supernatant of the T cell clones would have mediated the suppression. Our results suggest that alloantigen specific and non-specific suppressor T cells exist in bone marrow chimeras. The former appears to play an important role in inducing and maintaining transplantation tolerance, while the latter seems to have a rather harmful effect upon chimeras.     

Immunoexpression of the α subunit of a guanine nucleotide-binding protein (Go) in pulmonary neuroendocrine cells and neoplasms

The α subunit of a GTP-blndlng protein, Go, was investigated in pulmonary neuroendocrine neoplasms and fetal tissues of the lung by an immunohistochemlcal method. Positive immunostaining for the α subunit of Go (Goα) was found predominantly on the cell membrane and found occasionally in the cytoplasm. Typical carcinoids were all positively stained (9/9), and small cell carcinoma showed weaker and less frequent staining (5 positive cases in 10). Atypical carcinoids were variously stained (3/4). The tendency for obvious neuroendocrine differentiation to be immunohistochemically determined in typical carcinoids and not in small cell carcinoma is also true of staining for neuron specific enolase (NSE), chromogranin A (CG-A) and synaptophysin. In the lung, Goα-immunostaining was positive not only in nerve tissues but also in the airway epithelium. In the fetal lung, serial sections immunostained for NSE, CG-A and Goα confirmed that Goα-immunoreactive cells belong to the neuroendocrine cell population. The biological significance of Goα is unclear in normal and neoplastic lung tissues, but Goα is a useful marker of neuroendocrine cells and neoplasms of the lung.     

The roles of soluble osteopontin using osteopontin-transgenic mice in vivo: proliferation of CD4+ T lymphocytes and the enhancement of cell-mediated immune responses.

We generated transgenic mice expressing osteopontin (OPN) under the control of the alpha(1)-antitrypsin promoter. These mice (OPN-T mice) expressed OPN mRNA in liver and kidney, and released a large amount of plasma OPN, which increased after stimulation with turpentine oil. Before sensitization, the number of CD4+ T cells in lymph nodes was significantly higher in OPN-T than nontransgenic mice, and that in spleen was slightly higher, whereas that of CD8+ T cells was no different between OPN-T and nontransgenic mice. After sensitization, the CD4+ T cell numbers in spleen increased significantly, while there were almost no changes in the CD8+ T cells in lymph nodes and spleen. The intensity of contact hypersensitivity responses to 2,4-dinitrofluorobenzene (DNFB) was obviously enhanced in OPN-T mice. In the delayed-type hypersensitivity (DTH) model elicited by DNFB, the number of CD8+ T cells among DNFB-2,4,6-trinitrobenzenesulfonic acid (TNBS)-peritoneal exudate cells was significantly higher in OPN-T than nontransgenic mice, while there was almost no difference in that of CD4+ T cells. Adoptive transfer experiments revealed that the enhanced reactivity is carried by CD4+ and CD8+ T cells, respectively, although the ability of transferring DTH was significantly lower in CD8+ than in CD4+ T cells. The enhancement of CD8+ T cell migration was observed in OPN-T mice. These results suggest that OPN induces a proliferation of effector CD4+ and CD8+ cells in cell-mediated reactions and plays a role in the migration of CD8+ T cells.     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.