Induction of apoptosis mediated by fas receptor and activation of caspase-3 in MRL-+/+ or MRL-lpr/lpr murine oocytes

The molecular mechanisms leading to ovarian follicular atresia in the typical pathways of programmed cell death remain to be clarified. Here we have demonstrated that the apoptotic signalling pathway in MRL-+/+ (MRL/+) murine oocytes is through the Fas receptor followed by the activation of caspase-3. In contrast, we found that the aberrant expression and dysfunction of the mutant Fas receptor in MRL-lpr/lpr (MRL/lpr) murine oocytes caused by insertion of the early transposable element (ETn) into the Fas gene were associated with an inability to activate the caspase cascade (especially caspase-3) and to induce nuclear DNA fragmentation. These findings indicate that the induction of apoptosis in MRL/lpr murine oocytes did not occur in the presence of a defective Fas receptor lacking the death domain to trigger the caspase cascade, suggesting a failure to induce ovarian follicular atresia.     

Production of hydroxyl radicals and alpha-dicarbonyl compounds associated with Amadori compound-Cu2+ complex degradation

The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1-n-butylamino-D-fructose (DBF) and N(alpha)-formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu2+ (but no other transition metal ions) to form a DBF-Cu2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu+ during incubation at 37 degrees C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and alpha-dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and a-dicarbonyl compounds from Amadori adducts in the presence of Cu2+.     

MEG responses during rhythmic finger tapping in humans to phasic stimulation and their interpretation based on neural mechanisms

 The phase-resetting experiment was applied to human periodic finger tapping to understand how its rhythm is controlled by the internal neural clock that is assumed to exist. In the experiment, the right periodic tapping movement was disturbed transiently by a series of left finger taps in response to impulsive auditory cues presented randomly at various phases within the tapping cycle. After each left finger tap, the original periodic tapping was reestablished within several tapping cycles. Influences of the disturbance on the periodic right finger tapping varied depending on the phase of the periodic right finger tapping at which each left finger tap was made. It was confirmed that the periodic tapping was disturbed not by the auditory cues but by the left finger taps. Based on this fact, in this paper each single left tap was considered as the stimulus, and the phase of the periodic tapping of the right index finger when the left tap was executed as the phase of the stimulus. Responses of the neural activities (magnetoencephalography, MEG), the tapping movement, and the corresponding muscle activities (electromyography) were simultaneously measured. Phase-resetting curves (PRCs) representing the degree of phase reset as a function of the phase of the stimulus were obtained both for the left sensorimotor cortex MEG response and for the right index finger tapping response. The shapes of both PRCs were similar, suggesting that the phase reset of the left sensorimotor cortex activities and that of the finger tapping rhythm were the same. Four out of eight subjects showed type-0 reset in Winfree's definition, and the others showed type-1 reset. For general limit-cycle oscillators, type-0 reset is obtained for relatively strong perturbations and type 1 for weak perturbations. It was shown that the transient response of MEG to the single left tap stimuli in type-0 subjects, where the phase was progressively reset, were different from those in type-1 subjects. Based on detailed analysis of the differences, a neural network model for the phase reset of the tapping rhythm is proposed. Received: 10 February 2000 / Accepted in revised form: 15 January 2002     

Frequent detection of anti-recoverin cytotoxic T-lymphocyte precursors in peripheral blood of cancer patients by using an HLA-A24-recoverin tetramer

Recoverin is a photoreceptor-specific calcium binding protein that is only expressed in the retina in normal tissues. Aberrant expression of recoverin, however, has been observed in several cancer tissues and may cause a very rare autoimmune disease, cancer-associated retinopathy (CAR). It has been suggested that CAR-positive cancer patients have a more favorable prognosis than CAR-negative cancer patients. To estimate the status of recoverin-specific T cells in such cancer patients, we generated an HLA-A24-recoverin peptide tetramer. By use of the tetramer, we could directly assess the frequency of CTL precursors (CTLp) of 20 HLA-A24(+) cancer patients with ten colon, six stomach and four breast cancers, and seven healthy individuals. Four cancer patients showed a CTLp frequency higher than 0.9%. Seven cancer patients including the former four patients and two healthy individuals showed specific anti-recoverin cytotoxic responses in an HLA-A24-restricted manner after in vitro stimulation with the recoverin peptide. Moreover, five cancer patients analyzed in an independent experiment using different peripheral blood mononuclear cells (PBMC) samples showed similar CTLp and cytotoxic T lymphocyte (CTL) frequencies and cytotoxic responses, suggesting that the CTLp frequency analyzed by the tetramer and the cytotoxic response may have a good correlation. Thus, we hypothesize that anti-recoverin CTLp may exist in some cancer patients, and that anti-recoverin CTL may be readily induced.     

Effect of the dose and composition of an autologous hapten-modified melanoma vaccine on the development of delayed-type hypersensitivity responses

We have reported that treatment of melanoma patients with a vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP) and preceded by low-dose cyclophosphamide induces delayed-type hypersensitivity (DTH) to autologous, unmodified tumor cells and that this response is a significant predictor of survival. We analyzed the vaccines prepared for 284 patients who were treated following resection of regional or distant metastases to find out whether the dose and composition determined the immunological response. A positive DTH response (> or =5 mm induration) to unmodified autologous tumor cells was induced in 57% of the patients (median: 5 mm; range: 0-22 mm). Regression analysis showed no significant association between the magnitude of DTH and the number of live (trypan blue exclusion) melanoma cells per dose over a dosage range of 0.5-25.0 x 10(6). Surprisingly, there was a small but significant positive relationship between the mean number of dead cells in the vaccines of a given patient and that patient's maximum DTH to unmodified melanoma cells. Only 37% of patients whose vaccines contained >50% live cells developed DTH, as compared with 69% and 65% of patients whose vaccines contained 26% to 50% or < or =25% live cells, respectively. Thus, it appears that dead tumor cells contribute to the immunogenicity of the DNP vaccine, but other factors such as the administration schedule may be more important determinants of immunological and clinical outcome.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.     

Effects of targeted overexpression of pleiotrophin on postnatal bone development

Pleiotrophin (PTN) is an extracellular matrix-associated growth/differentiation factor that, in post-natal life, is found mainly in bone and brain. Bone development was investigated in ptn-overexpressing mice between 1 and 30 weeks. In transgenics and controls, PTN (and its receptor syndecan-3) was synthesized by osteoblasts and was present in striated muscle. ptn over-expression enhanced intramembranous bone formation and had multiple effects on long-term bone growth. The pubertal growth spurt did not take place in transgenic mice, in which the growth trajectory was steady and continuous until 25 weeks. By 30 weeks, transgenic and control mice were of the same size, but the calcium content/mg bone was approximately 10% higher in the transgenics. PTN was also localized in growth plate and articular chondrocytes, but only in transgenic mice. In these, synthesis of type I collagen by articular chondrocytes was observed, as well as an encroachment of subchondral bone into the articular cartilage. The results suggest that PTN has multiple roles during in vivo bone formation and remodeling, probably acting as a co-factor or accessory protein that modulates the effects of primary signaling molecules.