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.

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.

Methylation status of c-myc oncogene in leukemic cells: hypomethylation in acute leukemia derived from myelodysplastic syndromes.

DNA methylation plays an important role in gene regulation. We have analyzed the methylation status of CCGG sites in and around the human proto-oncogene c-myc in blood cells from patients with acute and chronic leukemias and with myelodysplastic syndromes using restriction endonucleases. The 5' region of c-myc was unequivocally hypomethylated in all the 58 specimens studied, including 10 from normal bone marrow and 1 from human placenta. In contrast, the 3' region was hypermethylated in a great majority of cases. However, this region was hypomethylated in 1 of 12 patients with de novo acute myeloid leukemia, 1 of 6 patients with chronic myeloid leukemia, and 4 of 5 patients with acute myeloid leukemia preceded by a documented stage of myelodysplastic syndromes. One possible mechanism for the 3' region of c-myc to have remained hypomethylated may be a "delayed methylation" during transforming events toward a more aggressive stage of the disease, but the precise mechanism is unknown.

Localization of metallothionein in hair follicles of normal skin and the basal cell layer of hyperplastic epidermis: possible association with cell proliferation.

Metallothionein is a low-molecular-weight metal-binding protein. Although it is inducible by a variety of agents and ubiquitously present in many tissues, its physiologic functions are still not clear. The present study was undertaken to determine the possible functions of metallothionein in both the proliferation and differentiation of epidermal keratinocytes. Metallothionein was detected immunohistochemically in hair matrix cells of the bulb and cells of the outer root sheath of anagen hair follicles, but not in dermal papillae in normal skin in the back of mice. In hyperplastic epidermal tissue, induced by either a phorbol ester tumor promoter or cholera toxin, the basal cells of the interfollicular epidermis stained strongly for metallothionein. Elevated expression of mRNA of the metallothionein gene was also demonstrated when the skin was stimulated by agents that induced hyperplasia. Papillomas produced by two-stage carcinogenesis protocols also stained for metallothionein. These observations suggest that metallothionein is involved in the proliferation of epidermal keratinocytes.

ALL- and CML-type BCR/ABL mRNA transcripts in chronic myelogenous leukemia and related disorders.

Using the reverse transcription polymerase chain reaction, we investigated acute lymphoid leukemia (ALL)-type, and chronic myelogenous leukemia (CML)-type BCR/ABL mRNA expression in a total of 66 patients with chronic myeloproliferative disorder (CMPD). Thirty-six of 37 patients with CML were positive for CML-type mRNA. Thirteen of the 25 CML had ALL-type mRNA expression. The patients with ET, PV, MF, and CMML did not have any detectable BCR/ABL expression. The most remarkable finding was that two patients, a Ph1-positive CML patient and a patient with a presumptive diagnosis of essential thrombocythemia (ET), showed only ALL-type chimeric mRNA expression.

Detection of c-myc oncogene amplification in a CML blastic phase patient with double minute chromosomes.

Double minute chromosomes (dmin) are relatively rare in leukemias. Cytogenetic analysis of blood cells from a woman with blastic phase chronic myelogenous leukemia (BC-CML) showed numerous dmin chromosomes and complex abnormalities including a Philadelphia (ph(1))-chromosome. Oncogene amplification in hematopoietic malignancies is also rare. Using PCR, we retrospectively investigated the extent of c-myc gene amplification in DNA extracted from stored blood smears from the patient. To qualify the PCR products, the beta-globin gene was used as the internal reference gene and it was co-amplified with the c-myc gene. The extent of amplified c-myc was about 6.8-fold. This finding suggests that the c-myc gene was amplified in dmin and that the gene amplification contributes to the progression to acute leukemia or rapid growth of leukemic cells.

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.

Hair dye use and occupational exposure to organic solvents as risk factors for myelodysplastic syndrome.

To investigate the relationships of personal hair dye use and environmental factors to myelodysplastic syndromes (MDS), we conducted a case-control study in Japan. A total of 111 MDS cases and 830 controls randomly selected from the residents in the same prefecture of cases using telephone directories responded to a health questionnaire. The odds ratio (OR) for ever having used hair dye was 1.99 (95% confidence interval (CI) 1.17-3.38) and there were statistically significant trends in risk with increasing duration and number of hair dye use. Occupational exposure to organic solvents was marginally associated with the risk of MDS (OR = 1.99; 95% CI 0.97-4.10).

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.

Fulminant, CMV-associated, haemophagocytic syndrome following unrelated bone marrow transplantation.

We report a case of haemophagocytic syndrome (HPS) occurring after allogeneic bone marrow transplantation (BMT) for acute promyelocytic leukaemia (APL) in a patient in fourth complete remission (CR). Anti-cytomegalovirus (CMV) antibody (Ab) was negative in this patient before BMT. BMT was performed from an HLA-identical unrelated donor who was positive for CMV Ab. After bone marrow engraftment and haematological recovery, severe acute graft-versus-host disease (GVHD) developed. This patient was treated with methylprednisolone in addition to cyclosporin A (CsA). Acute GVHD showed partial improvement, but CMV antigenaemia was observed. Despite administration of gancyclovir and immunoglobulin, CMV antigenaemia showed no improvement and HPS developed. As no other infections or malignancies were observed, we suspect that CMV infection was the trigger for development of HPS.

Metallothionein induction as a potent means of radiation protection in mice.

A striking resistance to lethal damage from a single dose of 6-8 Gy of X rays has been found in mice which had received various pretreatments to induce metallothionein (MT) synthesis in the liver prior to irradiation. Mice were injected with manganese (10 mg Mn/kg) or cadmium (3 mg Cd/kg) salt subcutaneously, or a patch of dorsal skin (2 X 2 cm2) was excised 1 or 2 days prior to irradiation. The increased tolerance of these mice to radiation was established by a marked decrease of mortality rate, an increase of mean survival time, a reduction of weight loss, and a smaller decrease in the number of leukocytes as compared with the control group. The LD50/30 for control mice was 6.3 Gy, while the corresponding values for the groups pretreated with Mn, Cd, and skin excision were 7.5, 7.7, and 7.9 Gy, respectively. The normal level of MT in mouse liver was approximately 25 micrograms/g tissue. This level increased 2.5- to 3-fold 24 h after 6.3 Gy irradiation. The MT levels of mice pretreated with Cd, Mn, and skin excision were increased 8-, 5-, and 7-fold, respectively, prior to irradiation as compared with the preirradiation control. These results indicate that the induction of MT in mouse liver is a significant factor in the mechanism of protection against radiation.

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.

Effect of granulocyte-colony stimulating factor on empiric therapy with flomoxef sodium and tobramycin in febrile neutropenic patients with hematological malignancies. Kan-etsu Hematological Disease and Infection Study Group.

The clinical effects of concomitant use of granulocyte-colony stimulating factor (G-CSF) on empiric antibiotic therapy in febrile neutropenic patients were evaluated in a randomized fashion. Two hundred and fourteen neutropenic febrile episodes (neutrophil counts < 1.0 x 10(9)/l) were treated with flomoxef sodium and tobramycin with or without G-CSF. The resolution of fever at day 4 (excellent response) or at day 7 (good response) was deemed effective. Among 157 evaluable episodes, the observed excellent responses were 31 (38.8%) and the good responses were 20 (25.0%) in the G-CSF group; those in the control group were 26 (33.8%) and 25 (32.5%), respectively. The overall efficacy rate was 63.8% (51/80) in the G-CSF group and 66.2% (51/77) in the control group (not significant). The initial neutrophil count was 0.186 +/- 0.249 x 10(9)/l in the G-CSF group and 0.235 +/- 0.290 x 10(9)/l in the control group, and rose to 2.889 +/- 4.198 x 10(9)/l and 0.522 +/- 0.844 x 10(9)/l, respectively, at day 7. These results indicate that G-CSF does not affect the rate of response to empiric antibiotic therapy in febrile neutropenic patients, although a significant effect of G-CSF was observed on neutrophil recovery.

99Tcm-labelled chimeric human/mouse anti-granulocyte antibody bone marrow scintigraphy: a preliminary clinical study.

Bone marrow scintigraphy using 99Tcm-labelled chimeric anti-granulocyte antibody (anti-NCA-95) was performed in 17 patients with haematological disorders and skeletal metastases. Chimeric anti-NCA-95 antibody (chNCA95 Ab, 0.2 mg) labelled with 444 MBq 99Tcm was administered to obtain bone marrow images 4 h post-injection. One week later, an 111In-chloride bone marrow scan was performed on nine patients with haematological disorders. Lumbar bone marrow-to-background (L/B) and ilium-to-background (I/B) uptake ratios were calculated for each scan. In six patients with suspected skeletal metastases, 99Tcm-HMDP bone scans were performed. No patient had any adverse reaction or any immune reaction over 20 weeks. In the patients with haematological disorders, the L/B and I/B ratios of the 99Tcm-chNCA95 Ab scan were 3.41 +/- 0.90 and 1.23 +/- 0.31, whereas those of the 111In-chloride scan were 1.58 +/- 0.32 and 1.00 +/- 0.32, respectively. In assessing findings of irregular central bone marrow uptake and peripheral expansion of the bone marrow, the 99Tcm-chNCA95 Ab scan was much better than the 111In-chloride scan. In the six patients with suspected skeletal bone metastases, three true-positive and two true-negative results were observed. This preliminary study has revealed that 99Tcm-chNCA95 Ab scanning is safe and useful in the diagnosis of haematological disorders and skeletal metastases.

Acute myeloid leukemia accompanied by multiple thrombophlebitis.

A 66-year-old woman suffering from fever and thrombophlebitis was referred to our hospital. A peripheral blood examination revealed hyperleukocytosis with 96% blast cells and thrombocytopenia. The patient was diagnosed as having acute myeloid leukemia (AML) accompanied by disseminated intravascular coagulation (DIC). A marked decrease in protein C (PC) antigen and activity were observed. In this case, PC levels were lower than those observed in AML with DIC. Induction therapy for leukemia and treatment of DIC were started on the first day of hospitalization. The patient achieved complete remission, with PC antigen and activity levels normalized.

Fourteen-day oral combination dose toxicity study of CGS 16949 A (aromatase inhibitor) with 5-fluorouracil or tamoxifen in rats.

CGS 16949A, an aromatase inhibitor, was administered orally to female rats at doses of 1 and 10 mg/kg/day alone and in combination with tamoxifen (0.5 or 5 mg/kg/day) or 5-fluorouracil (20 mg/kg/day) for 14 days. CGS 16949A and tamoxifen combination: Increased food intake and body weight noted after CGS 16949A treatment were also observed following combination treatment, though to a lesser degree. Most of the clinical pathological features noted following combination treatment were similar to those induced by single compound treatment. Gross pathological and histopathological changes ascribed to the antiestrogenic action of CGS 16949A, such as increased ovarian weight, decreased uterine weight, cystic follicles and atrophied uterus and vaginal epithelium, were alleviated by combination treatment, and were comparable in severity to those caused by tamoxifen alone. No severe toxic changes were induced by combination treatment. CGS 16949A and 5-fluorouracil combination: Increased body weight noted after CGS 16949A treatment was also observed following combination treatment, though to a lesser degree. Most of the changes caused by single compound treatment, including the aforementioned effects of CGS 16949A on the genital organs, were also noted following combination treatment. There was no evidence of enhancement of the effects by combination treatment.

Growth and DNA synthesis of folate- and methionine-depleted L1210 mouse leukemia cells in culture.

The growth and DNA synthesis of L1210 mouse leukemia cells were examined under folate- and methionine-deficient conditions. Cell proliferation was dependent on methionine supplementation rather than on folate concentration. The UdR suppression value was abnormally high in the folate-deficient condition. However, it was also high when the methionine was low, despite folate supplementation. In accordance with this, UdR incorporation was significantly improved with various folates by cells grown in low-methionine conditions. Methionine depletion resulted in marked impairment of UdR incorporation regardless of folate concentration. These findings indicate close metabolic interrelations between folate and methionine, which may be relevant to the pathological biochemistry of human megaloblastic anemia.

Toward an assessment of social identity: the structure of group identification and its effects on in-group evaluations.

Two studies were conducted to explore relations among different aspects of group identification and their effects on in-group evaluations. Two aspects of identification were differentiated, namely, identification with the group membership (IDgroup) and with other group members (IDmember). The first of these was assumed to be further divided into its cognitive and affective subcomponents. An identification scale was developed and administered to students of a Japanese vocational school. Factor analyses in Studies 1 and 2 distinguished IDgroup and IDmember, but the cognitive and affective components of the former were not separated. Experimental studies concurrently undertaken confirmed many of the predictions and contentions by social identity theorists. Of particular importance was the result from Study 1 that members with low IDgroup deprecated the in-group when their negative social identity became salient, whereas those with high IDgroup (but not IDmember) did not. Both theoretical and applied implications are discussed.