Effect of gene flow on spatial genetic structure in the riparian canopy tree Cercidiphyllum japonicum revealed by microsatellite analysis.

Few studies have analyzed pollen and seed movements at local scale, and genetic differentiation among populations covering the geographic distribution range of a species. We carried out such a study on Cercidiphyllum japonicum; a dioecious broad-leaved tree of cool-temperate riparian forest in Japan. We made direct measurement of pollen and seed movements in a site, genetic structure at the local scale, and genetic differentiation between populations covering the Japanese Archipelago. Parentage analysis of seedlings within a 20-ha study site indicated that at least 28.8% of seedlings were fertilized by pollen from trees outside the study site. The average pollination distance within the study site was 129 m, with a maximum of 666 m. The genotypes of 30% of seedlings were incompatible with those of the nearest female tree, and the maximum seed dispersal distance within the study site was over 300 m. Thus, long-distance gene dispersal is common in this species. The correlation between genetic relatedness and spatial distance among adult trees within the population was not significant, indicating an absence of fine-scale genetic structure perhaps caused by high levels of pollen flow and overlapping seed shadows. Six populations sampled throughout the distribution of C. japonicum in Japan showed significant isolation-by-distance but low levels of genetic differentiation (F(ST) = 0.043), also indicating long-distance gene flow in C. japonicum. Long-distance gene flow had a strong influence on the genetic structure at different spatial scales, and contributes to the maintenance of genetic diversity in C. japonicum.

Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using a real-time RT-PCR assay.

Using a real-time RT-PCR method, we analyzed the expression of e1a2 BCR-ABL mRNA in bone marrow samples from 13 patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia (ALL) at different time points during chemotherapy and after bone marrow transplantation (BMT). The detection limit of the method, assessed using serial dilutions of ALL/MIK cells, was found to be 1:10(5), similar to what is observed for the conventional RT-nested PCR method. The e1a2 BCR-ABL values were normalized with respect to those of the housekeeping gene GAPDH. The decrease in the e1a2 BCR-ABL/GAPDH ratio after remission induction chemotherapy reflects well the response to chemotherapy and consequently correlates with the prognosis. Although molecular remission was achieved by chemotherapy alone, some patients relapsed, and the e1a2 BCR-ABL/GAPDH ratios in these cases progressively increased to the levels seen prior to hematological relapse. Long-term hematological complete remission (more than 30 months) could be achieved in cases in which a more than 4.0 log decrease in the e1a2 BCR-ABL/GAPDH ratio was obtained by chemotherapy alone, and BMT was then performed. In conclusion, real-time RT-PCR allows for an evaluation of the kinetics of e1a2 BCR-ABL/GAPDH expression during the various phases of chemotherapy or after BMT and may be effective for the indication and control of disease relapse in Ph-positive ALL patients.

Diagnosis and monitoring of human cytomegalovirus diseases in patients with human immunodeficiency virus infection by use of a real-time PCR assay.

We used a real-time PCR assay to measure human cytomegalovirus (HCMV) DNA load in whole blood and plasma of 70 patients who were infected with human immunodeficiency virus type 1. Break points of 3.0 x 10(3) copies/mL in whole blood and 1.0 x 10(3) copies/mL in plasma were well-correlated with the existence of definite HCMV disease (sensitivity, 93% and 86%; specificity, 89% and 85%; positive predictive value, 70% and 63%; and negative predictive value, 98% and 95%, respectively). In patients with < 50 cells/microL of CD4(+) T lymphocytes, positive predictive values increased to 78% and 71%, respectively. The viral loads of all patients who received anti-HCMV therapy declined to < or =2.0 x 10(2) copies/mL in parallel with the improvement of clinical symptoms. These findings show that the HCMV DNA load quantified with our method is a useful tool for diagnosis of HCMV diseases and for monitoring the disease activity in patients infected with HIV-1.

Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis.

We developed a new quantitative system for diagnosis of invasive pulmonary aspergillosis (IPA) using real-time automated polymerase chain reaction (PCR). Intra-assay and interassay precision rates for in vitro examination were 2.53% and 2.20%, respectively, and the linearity of this assay was obtained when there were >20 copies/well. We examined 323 samples taken from 122 patients with hematological malignancies, including 33 patients with IPA and 89 control patients. Blood samples were subjected to PCR antigen detection methods, using enzyme-linked immunosorbent assay (ELISA) and determination of plasma (1-->3)-beta-D-glucan (BDG) concentration. The sensitivities of PCR, ELISA, and BDG measurement for diagnosis of IPA were 79%, 58%, and 67%, respectively; the specificities were 92%, 97%, and 84%. Positive findings on PCR preceded those of computed tomography by -0.3+/-6.6 days, those of BDG measurement by 6.5+/-4.9 days, and those of ELISA by 2.8+/-4.1 days. Real-time PCR was sensitive for IPA diagnosis, and quantitation was accurate.

Development and assessment of enzyme immunoassay for platelet-derived microparticles.

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 microm filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Establishment of a follicular lymphoma cell line (FLK-1) dependent on follicular dendritic cell-like cell line HK.

A novel cell line, FLK-1, was established from bone marrow cells of a patient with follicular lymphoma by means of co-culture with follicular dendritic cell (FDC)-like cell line HK. Immunophenotypic analysis showed that FLK-1 expressed CD10, CD19, CD20, CD38, IgG and HLA-DR, which is a typical feature of germinal center B cells. Cytogenetic analysis of FLK-1 demonstrated t(14;18)(q32;q21) translocation involving BCL2 and immunoglobulin heavy chain genes. Especially noteworthy is that the growth of FLK-1 was found to be dependent on a FDC line, HK. When HK cells were removed from the culture, FLK-1 cells stopped growing and eventually died. An apoptotic mechanism appeared to be involved as indicated by the presence of chromosome condensation and DNA ladder formation. The culture experiment using micropore membranes showed that soluble factor(s) of HK cells supported the growth, while direct cell-to-cell contact appeared to be necessary for longterm cell proliferation. These findings suggest the importance of the micro-environment for follicular lymphoma cells to grow. The FLK-1 cell line may thus prove to be useful for studying the growth mechanism of follicular lymphoma and provide new insights into the pathogenesis of follicular lymphoma.

Treatment of Relapsing Ph+ Acute Lymphoblastic Leukemia with Donor Leukocyte Infusion Followed by Quantitative Monitoring of Residual Disease.

The case of a 34-year-old man with relapsing Ph+ acute lymphoblastic leukemia (ALL), which occurred five months after allogeneic bone marrow transplantation, is described. He was originally treated with aggressive chemotherapy, which put him in hematological remission, and he subsequently received donor leukocyte infusion (DLI) form the original donor. To assess the efficacy of this adoptive immunotherapy, we monitored minor-BCR/ABL (m-BCR/ABL) mRNA levels using the recently established real-time quantitative RT-PCR (RQ-PCR) assay. The results were compared with those obtained using conventional qualitative RT-PCR assays run in parallel. RQ-PCR, but not RT-PCR-based, minimum residual disease (MRD) detection showed a good correlation with the rapid changes documented during the post-DLI clinical course. Currently, six months after DLI, the patient continues to be in remission, which is consistent with the undetectable levels of m-BCR/ABL mRNA in the leukemic clone using RQ-PCR found in this study. Thus, monitoring of m-bcr/abl transcripts using RQ-PCR provides more useful information on a clinical assessment of MRD.

Hepatic undifferentiated (embryonal) sarcoma in an adult: a case report and review of the literature.

Undifferentiated (embryonal) sarcoma of the liver (USL) is a rare malignant tumour with a poor prognosis. The absence of specific symptoms, the rapid tumour growth, the normality of the common tumour markers, and the consequential delay in the diagnosis often result in significant enlargement of the tumour. To our knowledge, there have been only 42 reported cases of USL in adults worldwide during the 40 years since the clinicopathological entity of USL was defined. We report here a 27-year-old male with USL who has been treated successfully with surgical resection and adjuvant chemotherapy using ifosfamide, adriamycin and cisplatin. Although the prognosis of the disease remains generally poor, long-term survival has been achieved recently in patients with a combination of surgery and multi-agent chemotherapy.

Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation.

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10(7) copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.

Evaluation of nucleotide derivatives and sugar nucleotide analogs as inhibitors of glycosyltransferases.

The inhibition of glycosyltransferases was studied using uridine monophosphate derivatives and uridine diphosphate sugar analogs. Modification in the nucleoside portion caused selective inhibition of glycosyltransferases.

Bernard-Soulier syndrome Kagoshima: Ser 444-->stop mutation of glycoprotein (GP) Ib alpha resulting in circulating truncated GPIb alpha and surface expression of GPIb beta and GPIX.

The platelet membrane glycoprotein (GP)Ib/IX complex is composed of three polypeptides, GPIb alpha, GPIb beta, and GPIX, and functions as a platelet receptor for von Willebrand factor. All three subunits are reported to be requisite for efficient surface expression of the complex. The absence of the GPIb/IX complex on platelet membrane is the hallmark of a congenital qualitative platelet disorder, termed the Bernard-Soulier syndrome (BSS). We describe here the molecular basis of a novel variant phenotype of BSS in a female patient, designated as BSS Kagoshima. Her platelets completely lacked the surface expression of GPIb alpha, but expressed a residual amount of GPIb beta and GPIX. Unexpectedly, her platelets and plasma contained a truncated GPIb alpha polypeptide with an apparent molecular weight of 116 kD (under nonreducing conditions). The amounts of truncated protein were 23% and 60% of the normal values in platelets and plasma, respectively. The abnormal protein contained a normal amount of sialic acid as demonstrated by digestion with neuraminidase. DNA sequencing analysis showed a homozygous single nucleotide substitution from the serine codon (TCA) to a nonsense codon (TAA) at residue 444 in the GPIb alpha gene. The mutant gene generated a truncated GPIb alpha molecule lacking the transmembrane region and cytoplasmic tail. Her parents were heterozygotes for the mutation. These findings suggest that this type of truncated GPIb alpha was produced, normally glycosylated, and subsequently secreted into the plasma. Furthermore, the truncated GPIb alpha might be associated with the process of the surface expression of incomplete GPIb/IX complex, GPIb beta and GPIX.

Effects of modulators of protein kinase C on human sperm capacitation.

OBJECTIVE: To examine the effects of stimulators or inhibitors of protein kinase C on capacitation and protein phosphorylation in human sperm. DESIGN: Capacitated sperm treated with or without modulators of protein kinase C were monitored by the chlortetracycline fluorescence assay. Capacitation was confirmed by the ability of sperm to undergo the acrosomal reaction in response to mouse zonae pellucidae. 32P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effect of protein kinase C stimulator, 12-O-tetradecanoyl-phorbol-13-acetate, on protein phosphorylation. RESULTS: The treatment of sperm with protein kinase C stimulators resulted in the following: [1] the rapid appearance of the clear perimeter pattern, featuring distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece; [2] an accelerated ability to undergo the acrosomal reaction; and [3] an enhanced phosphorylation of 57.5-kd sperm phosphoprotein. Furthermore, these stimulatory effects were inhibited by protein kinase C inhibitors. CONCLUSION: Protein phosphorylation mediated by protein kinase C may be involved in the regulation of human sperm capacitation.

Calyculin A, protein phosphatase inhibitor, enhances capacitation of human sperm.

OBJECTIVE: To examine the effects of protein phosphatase inhibitors on capacitation and protein phosphorylation in human sperm. DESIGN: The chlortetracycline (CTC) fluorescence assay was used to monitor capacitated sperm treated with or without protein phosphatase inhibitors. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to Ca++ ionophore A23187 or mouse zonae pellucidae. 32P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effects of protein phosphatase inhibitors on protein phosphorylation. RESULTS: The treatment of sperm with calyculin A resulted in the following: [1] the rapid appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece, in a dose-dependent manner in the 1 to 100 nM range; [2] an accelerated ability to undergo the acrosome reaction; and [3] an enhanced phosphorylation of sperm phosphoproteins in a dose-related fashion in the 1 to 100 nM range. A similar stimulatory effect was observed only with a 100-fold higher concentrations of okadaic acid, another protein phosphatase inhibitor. CONCLUSION: Our results strongly suggest that protein phosphorylation and dephosphorylation may be involved in the regulation of human sperm capacitation.

Antibody dependent cell mediated cytotoxicity on human cervical carcinoma cell line, ME-180, with human monoclonal antibody.

A human monoclonal antibody (MCA), CLN-IgG, showed cytotoxic effect in vitro against the cervical carcinoma cell line, ME-180, by antibody dependent cell-mediated cytotoxicity (ADCC). To determine which fractions of cells in peripheral blood lymphocyte (PBL) mediate ADCC, PBL were separated with nylon wool column and sheep red blood cells (SRBC). Both adherent cells (monocyte) and non-T, non-B cells showed cytotoxicity by ADCC. Human non-T, non-B cells showed higher cytotoxic activity against ME-180 cells than monocytes. Furthermore murine effector cells were less effective in ADCC than human effector cells with human MCA.

Synthesis of 2'-deoxy and 2',3'-dideoxynucleoside derivatives from thioglycosides.

The synthesis of both 2'-deoxy and 2',3'-dideoxynucleoside derivatives by the reaction of thioglycosides with nucleoside bases was examined. The stereochemical outcome at the anomeric position was found to depend on the protecting groups and the C-3 configuration in the sugar moiety, the kind of activator, and the reaction temperature. Based on these findings, 2'-deoxy-D-xylo nucleoside and 2',3'-dideoxynucleoside derivatives have been synthesized in beta-selective manner.

Deltamycins, new macrolide antibiotics. III. Chemical structures.

The structures of deltamycins A1,A2,A3 and A4 belonging to the basic macrolide family of antibiotics were determined mainly from their spectral properties. Deltamycin A4 was identified as carbomycin A having an isovaleryl group on the mycarose moiety of the molecule. Deltamycin A1,A2 and A3 possess similarities to the structure of deltamycin A4, but they have acetyl, propionyl and n-butyryl group, respectively, in the place of isovaleryl group of deltamycin A4. These structures were confirmed by chemical synthesis from deltamycin X (4''-O-deacyldeltamycin) and the corresponding acyl chlorides.