Stereological quantification of olfactory receptor neurons in mice.

The total number of olfactory receptor neurons (ORNs) in the mouse main olfactory epithelium (MOE) was estimated using stereological sampling. Noses and skulls of male and female 8-week-old C57BL/6J mice were de-calcified, embedded in paraffin, cut into 10-µm-thick sections serially at 100-µm intervals, and processed for immunohistochemistry for the olfactory marker protein (OMP), a specific marker for ORNs. The number of OMP (+) receptor neurons was measured using an optical fractionator with the Stereo-Investigator software. The mean values of the total number of OMP (+) receptor neurons in the unilateral MOE were 5,140,000±380,000 in males and 5,210,000±380,000 in females, with no significant differences between the sexes. We concluded that the total number of ORNs in the unilateral MOE is approximately 5×10(6) in mice.

Interleukin-10 gene polymorphism reflects the severity of chronic immune thrombocytopenia in Japanese patients.

INTRODUCTION: T-helper cell type 1 (Th1) polarization of the immune response has been documented in patients with chronic immune thrombocytopenia (ITP). Interleukin (IL)-10 is the most important factor regulating Th1 and T-helper type 2 cytokine synthesis. This study evaluated the impact of IL-10 polymorphisms on both susceptibility to, and severity of, chronic ITP. METHODS: We analyzed -1082(G/A), -812(C/T), and -592(C/A) IL-10 polymorphisms in 90 patients with adult chronic ITP and 202 race- and sex-matched healthy controls. RESULTS: No significant differences in the genotype or haplotype frequencies were observed between the patient with chronic ITP and the control group. However, more patients with the -592AA genotype showed a severe thrombocytopenic state (platelet count <10 x 109/l) than those with the -592CC/CA genotypes (44.1%vs. 19.6%, P = 0.01). Furthermore, more patients with the ATA/ATA haplotype showed a severe thrombocytopenic state than those without the ATA/ATA haplotype (44.1%vs. 19.6%, P = 0.01). CONCLUSION: According to our data, patients with low producer type of IL-10 polymorphisms have more severe thrombocytopenia, suggesting that IL-10 gene polymorphisms may reflect the severity of ITP.

Heirloom tomato gene bank: assessing genetic divergence based on morphological, agronomic and molecular data using a Ward-modified location model.

Accessions in gene banks need to be characterized and evaluated to determine their genetic diversity. We made a joint diversity analysis of the tomato gene bank of the Universidade Estadual do Norte Fluminense Darcy Ribeiro in Rio de Janeiro state, using the Ward-modified location model. Forty Solanum lycopersicum accessions were characterized and evaluated for 22 morphoagronomic descriptors and 131 random amplified polymorphic DNA markers. Based on the pseudo-F and pseudo-t(2) criteria, the optimal number of groups was established as five. Variability within groups was high for both continuous and discrete nominal data. The first two canonical variables explained about 90% of the inter-group variability. Care should be taken in using the Ward-modified location model technique to avoid incorporating excessive and unnecessary markers, which could favor molecular markers when compared with morphoagronomic variables. However, the minimum number of markers is germplasm- dependent and must be recalculated for each new divergence analysis.

Comparison of multivariate statistical algorithms to cluster tomato heirloom accessions.

Use of multivariate statistical algorithms is considered an important strategy to quantify genetic similarity. Local varieties and traditional (heirloom) seeds of genotypes are key sources of genetic variation. The Universidade Estadual do Norte Fluminense (UENF), Rio de Janeiro, Brazil, has a tomato gene bank with accessions that have been maintained for more than 40 years. We compared various algorithms to estimate genetic distances and quantify the genetic divergence of 40 tomato accessions of this collection, based on separate and joint analyses of discrete and continuous variables. Differences in continuous variables and discrete and joint analyses were calculated based on the Mahalanobis, Cole Rodgers and Gower distances. Although opinions differ regarding the validity of joint analysis of discrete and continuous data, we found that analyzing a larger number of variables together is viable and can help in the discrimination of accessions; the information that is generated is relevant and promising for both, the accessions conservation and the use of genetic resources in breeding programs.

Myelodysplastic syndrome with chromosome 5 abnormalities: a nationwide survey in Japan.

Chromosome 5 abnormalities, deletion of the long arm of chromosome 5 (del(5q)) or monosomy 5 (-5), arise in about 10% of myelodysplastic syndromes (MDS), either as the sole cytogenetic abnormality or as part of complicated karyotype, and has distinct clinical implications for MDS. However, the prognostic factors of MDS patients with chromosome 5 abnormalities are not determined yet. In this study, 183 Japanese MDS patients with chromosome 5 abnormalities were analyzed. Estimated incidence of del(5q) and 5q- syndrome among MDS patients was 8.4 and 1.3%, respectively. Significant shorter overall survival (OS) and leukemia-free survival (LFS) were observed in -5 patients than del(5q) patients. Among del(5q) patients, addition of monosomy 7 or complex karyotype with more than three abnormalities were significantly related to shorter OS. LFS of del(5q) patients was divided into two risk groups by international prognostic scoring system (IPSS): low/intermediate (Int)-1 and Int-2/high groups. LFS sorted by World Health Organization classification-based prognostic scoring system (WPSS) was also divided into two groups: very low/low/Int and high/very high, and WPSS was able to predict the outcome of del(5q) patients more clearly than IPSS. Together with additional cytogenetic data, WPSS might be useful for clinical decision making in MDS patients with del(5q).

Expression of CD55 and CD59 on peripheral blood cells in patients with lymphoproliferative disease of granular lymphocytes.

Lymphoproliferative disease of granular lymphocytes (LDGL) is a disorder characterized by the clonal expansion of granular lymphocytes. It has recently been shown that the clonal expansion of granular lymphocytes occurs in patients with paroxysmal nocturnal hemoglobinuria (PNH) in a subclinical fashion. To test the possibility that LDGL patients share a PNH phenotype, we obtained peripheral blood cells from 20 patients with LDGL and examined the expression of the glycosylphosphatidyl inositol (GPI)-anchored proteins, CD55 and CD59. Compared with normal controls, however, a defective expression of CD55/59 was not observed on either granulocytes or erythrocytes from LDGL patients. An unexpected finding was the significantly lower CD55/59 expression on granular lymphocytes from patients with CD16(+)CD56(-) phenotype LDGL than from patients with CD16(+)CD56(+) phenotype LDGL, or natural killer (NK) and NK/T lymphocytes from healthy individuals. The positive correlation between the expression of CD56 and CD55/59 might have some relevance to the functional properties of the CD56(+) subset of large granular lymphocytes.

Variation in the loss of seed dormancy during after-ripening of wild and cultivated rice species.

BACKGROUND AND AIMS: The aim of this paper was to verify the variation in the loss of seed dormancy during after-ripening and the interspecific and interpopulation variability in the degree of dormancy of seven wild and two cultivated rice species comprising 21 populations and two cultivars. METHODS: Four wild rice species from South America, Oryza glumaepatula, O. latifolia, O. grandiglumis and O. alta, and two O. sativa cultivars were tested in one experiment. In a second experiment, five wild species, O. punctata, O. eichingeri, O.rufipogon, O. latifolia and O. glumaepatula, and one cultivated species (O. glaberrima) were evaluated. Initial germination tests were performed soon after the seeds were harvested and subsequently at 2-month intervals, for a total of six storage periods in the first experiment and three in the second. All tests were conducted in the dark at a temperature of 27 degrees C. KEY RESULTS: Different patterns of after-ripening among populations within and between species were observed. CONCLUSIONS: The cultivated species (O. sativa and O. glaberrima) and, amongst the wild species, the tetraploids O. latifolia, O. grandiglumis and the diploids O. eichingeri and O. punctata, had weak dormancy, losing it completely 2 months after harvest, while O. rufipogon and O. glumaepatula exhibited pronounced dormancy. The latter showed different patterns of after-ripening between populations indigenous to the Amazon region and those originating in the Paraguay River system. Seeds of Solimoes (Amazon) and Japura origin showed weak dormancy whereas those of Paraguay origin showed deep dormancy. Ecological differences among natural habitats may be involved in such differentiation.

Durable remission induced by rituximab-containing chemotherapy in a patient with primary refractory Burkitt's lymphoma.

We describe a 65-year-old man diagnosed with Burkitt's lymphoma arising from the intestine. The tumor cells had a mature B-cell immunophenotype and rearrangement of the c-myc gene. The patient was treated with intensive multiagent chemotherapy. After four courses of chemotherapy, an ileus developed due to a residual abdominal disease. We administered rituximab in combination with the same chemotherapy regimen. A dramatic clinical improvement was observed and abnormal uptake by 18F-fluorodeoxyglucose positron emission tomography disappeared. The patient experienced complete remission for 1 year. This encouraging result indicates that rituximab might be an important treatment choice in management of Burkitt's lymphoma.

X chromosome methylation-based chimerism assay for sex-mismatched hematopoietic stem cell transplantation.

Analysis of hematopoietic chimerism is important for monitoring engraftment, graft failure, and disease recurrence. Although several techniques are now available, their sensitivity is unsatisfactory. In sex-mismatched stem cell transplantation (SCT) with a female donor, Y chromosome-specific sequences have proven the most sensitive marker. However, in the case of a male donor, no such reliable marker has been available to date. In this study, we report a novel method we developed to detect microchimerism in female recipients who receive SCT from male donors. The X-linked human androgen receptor gene (HUMARA) contains a highly polymorphic CAG trinucleotide repeat. Near this polymorphic site are methyl-sensitive HpaII restriction enzyme sites. After HpaII digestion, unmethylated male HUMARA sequences are completely digested, while methylated female ones remain intact among the male origin cells. This allows a highly efficient detection of a small number of female cells. Combined with the nested PCR technique, the X chromosome methylation-based chimerism assay could attain a 10(-4) level of sensitivity, which is 1000-fold higher than that of conventional assays. The applicability of the method was confirmed in two transplant cases. This highly sensitive method can also be applied to detect minimal residual disease or microchimerism in conditions other than hematopoietic SCT.

[A Japanese perception of self and others: self-critical and other-enhancing biases].

Previous research has found that Japanese people make relatively critical appraisals of themselves while their appraisals of others are relatively flattering. In order to find the conditions under which these two evaluation biases occur, 144 Japanese undergraduates were first asked to list ten attributes of theirs and then to rate the desirability of possessing each of them (self judgment). Next, someone else in the same class rated the desirability of each (other judgment). Subsequently, all the attributes generated in the study were shown to a separate group of students, who rated general desirability of possessing each of the attributes (consensual judgment). Results showed that relative to consensual judgment, self-judgment was lower (self-criticism), but other judgment was no different for those attributes that were consensually negative. For those consensually positive, however, self judgment was no different from the consensual, but other judgment was higher (other-enhancement). Based on these findings, it was suggested that Japanese self-criticism operated in a relational self-improvement process whereby individuals sought to find and correct their shortcomings so as to meet socially shared standards of excellence.

Analysis of the distribution of CAG repeats and X-chromosome inactivation status of HUMARA gene in healthy female subjects using improved fluorescence-based assay.

We investigated the polymorphic CAG-repeat distribution and the X-inactivation status of the human androgen receptor (HUMARA) gene in 58 female Japanese volunteers. Polymerase chain reaction amplification was performed using a fluorescent-dye-labeled primer under conditions specific for GC-rich targets, and fragments were analyzed. To estimate the length of these fragments, FAM-labeled (blue fluorescent) products were simultaneously compared with ROM-labeled size markers (red) that were created by sequencing various HUMARA fragments. The number of polymorphic CAG repeats of HUMARA in 116 alleles from 58 female subjects ranged from 15 to 28. Of the 58 volunteers, 51 (88.0%) were heterozygous. In 96% of the heterozygous female subjects, the allelic differences were no greater than 6 repeats. X-chromosome inactivation was calculated as the ratio of the area of the smaller peak to the sum of the areas of the smaller and larger peaks. The average ratio was 0.38 (range, 0.09-0.50). Preferential use of 1 allele, by more than 75% (ratio. <0.25). was observed in 5 volunteers (10.9%). The clonal nature of a patient with chronic myelogenous leukemia was easily identified. This method is sensitive enough to discriminate a difference of 1 triplet repeat.

Risk factors for early death in patients undergoing treatment for multiple myeloma.

The survival time of myeloma patients improved from a few months to many years after treatment with melphalan. Perhaps chemotherapy more intensive than melphalan-prednisolone should be administered to patients at risk of early death. Therefore, early death must be accurately predicted. We analyzed 93 patients with recently diagnosed myeloma and found that 13 (14%) died within 6 months (early death). The most common cause of death was bacterial and fungal pneumonia when myeloma became uncontrollable. The response to conventional chemotherapy was poorer in patients at high risk of early death than the control group. Multivariate analysis showed that the serum level of beta-2 microglobulin was the only value that predicted early death.

Molecular detection of tumor cells at diagnosis invading the bone marrow and peripheral blood of patients with aggressive or indolent lymphomas.

We studied tumor cell invasions of bone marrow and peripheral blood in patients with various types of advanced non-Hodgkin's lymphoma by amplifying complementarity determining region III using the polymerase chain reaction (PCR) method and developing patient-specific probes. After molecular engineering, we could detect tumor cells in bone marrow from seven of 11 cases and in peripheral blood from six of 11 cases, despite negative results in four cases studied morphologically. Indolent cases were more likely to yield positive results than aggressive cases. The reason may be different biological behaviors among the histological types.

Organic aerosol formation during the atmospheric degradation of toluene.

Organic aerosol formation during the atmospheric oxidation of toluene was investigated using smog chamber systems. Toluene oxidation was initiated by the UV irradiation of either toluene/air/NOx or toluene/air/CH3ONO/NO mixtures. Aerosol formation was monitored using scanning mobility particle sizers and toluene loss was monitored by in-situ FTIR spectroscopy or GC-FID techniques. The experimental results show that the reaction of OH radicals, NO3 radicals and/or ozone with the first generation products of toluene oxidation are sources of organic aerosol during the atmospheric oxidation of toluene. The aerosol results fall into two groups, aerosol formed in the absence and presence of ozone. An analytical expression for aerosol formation is developed and values are obtained for the yield of the aerosol species. In the absence of ozone the aerosol yield, defined as aerosol formed per unit toluene consumed once a threshold for aerosol formation has been exceeded, is 0.075 +/- 0.004. In the presence of ozone the aerosol yield is 0.108 +/- 0.004. This work provides experimental evidence and a simple theory confirming the formation of aerosol from secondary reactions.

Effect of cytokines on growth and differentiation of leukaemic cells with translocation t(6;9)(p23;q34).

The translocation t(6;9)(p23;q34) is detected infrequently in subtypes of haematological malignancies including acute myelogenous leukaemia (AML) and myelodysplastic syndrome (MDS). Although the t(6;9) leukaemia is commonly associated with bone marrow basophilia, the cytological characteristics of leukaemic cells are unclear. In the current study, we examined the in vitro effects of several cytokines on growth and differentiation of t(6;9) leukaemic cells. Isolated bone marrow mononuclear cells from four patients with t(6;9) (two MDS and two AML) were cultured for 14 d in the presence or absence of each cytokine. At the end of culture, viable cells were counted, and their histology was examined. Bone marrow cells obtained from 22 patients (10 AML, six AML from MDS, six MDS) lacking t(6;9) were used as controls. Compared with control cultures, significantly higher numbers of blasts appeared in the culture of bone marrow cells from t(6;9)-positive patients in response to stimulation with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or interleukin 3 (IL-3). Stem cell factor (SCF) had little effect. Neutrophil counts were also significantly increased in the presence of G-CSF or IL-3. SCF and IL-3 were potent in increasing basophil counts from t(6;9)-positive cultures. These findings suggest that bone marrow cells obtained from t(6;9) patients are highly sensitive to growth- and/or differentiation-promoting cytokines. Special attention should be paid to the use of "therapeutic" cytokines in these patients.

Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity.

We report a case of gammadelta T-cell-type large granular lymphocyte (LGL) leukemia (CD3 +,CD8 +, CD57 +,TCR gammadelta+), which was accompanied by pure red cell aplasia, neutropenia and thrombocytosis. Southern blotting analysis of the T-cell receptor beta gene showed the germline configuration, but clonal TCR J gamma rearrangements were identified. These granular lymphocytes demonstrated non-major histocompatibility complex-restricted cytotoxicitity. The serum-soluble FasL (sFasL) concentration of this patient was very high, whereas the serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 beta (IL-1beta), interleukin-2 (IL-2) and thrombopoietin were normal. After treatment with cyclosporin A, anemia and thrombocytosis were improved, and LGL and the elevated sFasL concentration decreased. These observations suggested that FasL may have played a role in the establishment of the clinical symptoms of this patient and could be useful as an indicator of disease activity.

An induction gene trap for identifying a homeoprotein-regulated locus.

An important issue in developmental biology is the identification of homeoprotein target genes. We have developed a strategy based on the internalization and nuclear addressing of exogenous homeodomains, using an engrailed homeodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. Eight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentially regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal cord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can internalize proteins, peptides, phosphopeptides, and antisense oligonucleotides, this strategy should be applicable to other intracellular targets for characterizing genetic networks involved in a large number of physiopathological states.

Two cases of chronic graft-versus-host disease with elevated levels of soluble Fas ligand in serum.

It has recently been shown that the Fas-Fas ligand (FasL) system may be one of the pathogeneses for acute graft-versus-host disease (GVHD), and it has been reported that serum soluble Fas ligand (sFasL) increases with the presence of acute GVHD. However, there is no report on a correlation between the Fas-FasL system and chronic GVHD. We present two cases of chronic GVHD with elevated levels of serum sFasL. Its level in each case was high at the onset of chronic GVHD, but it decreased with steroid therapy. Liver dysfunction also improved as the level of serum sFasL decreased. It appears in these cases that the Fas-FasL system was related to the pathogenesis of liver damage.