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Germline mutations in the p53 tumor suppressor gene have been identified in patients with Li-Fraumeni syndrome (LFS) and patients with Li-Fraumeni-like syndrome (LFL). However, to date, germline p53 mutations in patients not fulfilling the criteria of LFS or LFL have been reported only very rarely. In our study, a novel germline c.584T>C (p.Ile195Thr) mutation of the p53 gene was found in a 21-year-old male with a glioblastoma and colon cancer. He had no family history of cancer within second-degree relatives, and loss of the wild-type p53 allele and overexpression of p53 protein were observed in both tumors. Functional analyses revealed transactivation and growth suppressive function activities of the Thr195-type p53 to be impaired. These results suggest germline p53 mutations to possibly be responsible for a subset of young adult patient with multiple malignant tumors, even those not meeting the clinical criteria for LFS or LFL.

PINK1 was recently found to be associated with PARK6 as the causative gene. We performed mutation analysis in eight inbred families whose haplotypes link to the PARK6 region. We identified six pathogenic mutations (R246X, H271Q, E417G, L347P, and Q239X/R492X) in six unrelated families. All sites of mutations were novel, suggesting that PINK1 may be the second most common causative gene next to parkin in parkinsonism with the recessive mode of inheritance.

AlphaB-crystallin is known as a small heat shock protein with a cytoprotective function. This study was undertaken to investigate the relationship between alphaB-crystallin and changes seen in Alzheimer's disease. The distribution and immunohistochemical characteristics of alphaB-crystallin positive neurones in the cerebral cortices of 4 patients with Alzheimer's disease were examined. AlphaB-crystallin positive neurones were mainly distributed in the limbic and paralimbic regions, namely parahippocampal gyrus, fusiform gyrus, cingulate cortex, middle and superior frontal gyrus, and insular cortex, which corresponded to commonly affected regions in Alzheimer's disease. Moreover, such neurones were present predominantly in the III or V layer of the cerebral cortex. The number of alphaB-crystallin positive neurones increased in parallel with the neuronal loss. Logistic regression analysis revealed a significant relation between the density of alphaB-crystallin positive neurones and that of extracellular neurofibrillary tangles (NFTs), with a correlation coefficient (r) of 0.57 and P < 0.0001 in 14 regions of the cerebral cortex. In contrast, the relation was not statistically significant between the density of alphaB-crystallin positive neurones and that of classical senile plaques, diffuse plaques or intracellular NFTs. Modified Gallyas-Braak (GB) staining on alphaB-crystallin positive neurone demonstrated several patterns of the structures: faint GB positive structures in the swollen perikaryon with straight neurites, fine granules compressed and contorted into fuzzy bundles, intensely GB positive filamentous structures together with fine granules and very intensely GB positive ring-like NFTs in a swollen perikaryon with curved neurites. In positive neurones, the density of ring-like NFTs correlated with that of atrophic perikaryon, or bent neurites and a decrease in the immunoreactivity of alphaB-crystallin. These data suggest that a close relationship exists between the appearance of alphaB-crystallin in neurones, extracellular NFTs, and neurofibrillary formation in alphaB-crystallin positive neurones in Alzheimer brain.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.

Research into familial Parkinson's disease (PD) remained at a virtual standstill in Europe and the US for several decades until a re-challenge by Japanese neurologists regarding an autosomal recessive form of PD. In 1965, our research group at Nagoya University examined familial cases of early-onset parkinsonism characterized by autosomal recessive inheritance, diurnal fluctuation of symptoms (alleviation after sleep), foot dystonia, good response to medication, and benign course without dementia. An inborn error of metabolism in some dopamine-related pathway was suspected. The clinical study of four families with the disease, named as "early-onset parkinsonism with diurnal fluctuation (EPDF)", was published in Neurology in 1973. The pathological study of a case in 1993 revealed neuronal loss without Lewy bodies in the substantia nigra. Based on these clinical and pathological evidences, EPDF was defined as a distinct disease entity. Screening for the EPDF gene was started in 1994 in collaboration with Juntendo University. With the discovery of parkin gene in 1998, EPDF was designated as PARK2. Of our 16 families examined for gene analysis, 15 proved to be PARK2, and the remaining one, PARK6.

Treatment of DBA/2J mice bearing a T1699 syngeneic mammary adenocarcinoma (Ts) with a single intraperitoneal (i.p.) injection of 100 ug melphalan produced complete tumor regression in about 65% of the animals treated; however, tumors recurred in about 85% of these regressors after 15-25 days' remission. The drug regimen was ineffective against Ts tumors growing in immunosuppressed or immunodeficient animals. Stimulation of immunologically intact Ts tumor-bearers with bacterial lipopolysaccharide (LPS) or with phytohemagglutinin (PHA) 3 days prior to melphalan therapy, on the other hand, produced not only higher rates of tumor regression but also significant increases in the number of permanent cures. A tumor induced by T1699 subline TR2 was resistant to the same regimen, although Ts and TR2 cells were equally susceptible to the cytotoxic and growth-inhibiting activities of the drug in vitro. In contrast, the combination of specifically armed monocytes and melphalan in vitro produced enhanced killing of Ts cells but not of TR2 cells. Analysis of the collaborative cytotoxicity between immune effector cells and melphalan indicated that exposure of tumor cells to killer cells increased the drug susceptibility of the tumor cells, but not the reverse. These results suggest a possible mechanism for in vivo resistance of tumors to chemotherapeutic agents that is not directly associated with the drug resistance of the tumor cells in vitro.

Properties of Ts, a long-term tissue culture line of T1699 mammary adenocarcinoma of DBA/2 mice, and two of its sublines - TR2 and TLI-1-were comparatively studied. Ts tumors produced no spontaneous metastases, nor did i.v. injection of up to 1 X 10(6) Ts cells produce a lung tumor nodule. Ts cells were susceptible to macrophage cytotoxicity in vitro and i.v. injected cells were rapidly destroyed in the lungs. TLI-1 tumors spontaneously metastasized to the lungs, and i.v. injection of 1 X 10(3) TLI-1 cells produced approximately 15 lung nodules per animal. TLI-1 cells were least susceptible to both macrophage-mediated cytolysis in vitro and in vivo host antitumor mechanisms. TR2 cells were intermediate with respect to all these properties. Differences in their susceptibility to macrophage cytotoxicity were recognized not only by normal peritoneal macrophages but also by murine macrophage lines. On the other hand, all the subpopulations were uniformly resistant to NK activity in vitro. These findings suggest that resistance of tumor cells in vitro to macrophage cytotoxicity corresponds with their in vivo metastatic potential.

Sera from DBA/2 mice bearing a syngeneic T1699 mammary adenocarcinoma not only induced specific antibody-dependent cellular cytotoxicity (ADCC) mediated by monocytes (and/or macrophages) but also armed (activated) such effector cells into specific killer cells in vitro. Removal of specific anti-tumor antibody (IgG2a subclass) by absorption with T1699 cells abolished the ADCC activity at the level of target cell coating but did not alter the level of effector-arming activity. Fractionation on Sephadex G-20 indicated more than one factor capable of inducing specific monocyte-mediated lysis of target cells in vitro, including immune complexes, free IgG2a 7S antibody (ADCC), and another factor(s) with a molecular weight very close to that of 7S immunoglobulins.

Intravenously (i.v.) or subcutaneously (s.c.) injected T1699 tumor cell subpopulations (Ts, and TLI-1) were destroyed more rapidly in BALB/c nu/nu athymic mice than in syngeneic DBA/2J mice. The number of artificial pulmonary metastases of TLI-1 tumors was significantly lower, and the growth rate of s.c. Ts, and TLI-1 tumors was correspondingly slower in the nude mice. No macroscopic outgrowth of pulmonary metastases of Ts tumors was detected in either group of s.c. tumor-bearers, even though the s.c. tumors progressed and killed the hosts. Unexpectedly, however, s.c. implantation in normal DBA/2J mice of lung homogenates not only from DBA/2J but also from s.c. Ts tumor-bearing BALB/c nu/nu mice produced tumors, suggesting that significant numbers of clonogenic Ts cells were present in the lungs of animals without spontaneous outgrowth of pulmonary metastases. Perturbations of s.c. Ts tumor-bearing DBA/2J or BALB/c nu/nu mice with immunosuppressive drugs or with anti-mouse thymocyte serum, respectively, induced rapid outgrowth of pulmonary metastases.

The nature of host-derived, specifically cytotoxic cells infiltrating solid murine mammary adenocarcinomas (line T1699) was investigated. Cell suspensions obtained from enzymatically dispersed tumors were separated by sedimentation velocity. The host cell fraction was heterogeneous and contained T-lymphocytes, non-phagocytic cells bearing Fc receptors, eosinophils and monocytes. Host cells equivalent in size to small lymphocytes and bearing Fc receptors were found to be the predominant cell type responsible for colony inhibition. These colony-inhibiting cells were insensitive to lysis with either anti-theta or anti-IgG serum and complement; and they were specific both in their induction and in their mediation and in their mediation of cytotoxicity. Host cells made up the predominant population in spontaneously regressive tumors but only a minor component in progressive tumors, and they were totally absent from progressive tumors in X-irradiated animals.