Loss of wild-type Jak2 allele enhances myeloid cell expansion and accelerates myelofibrosis in Jak2V617F knock-in mice.

JAK2V617F is the most common mutation found in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p (9pLOH) involving the JAK2 locus has been observed in ∼30% of MPN patients. JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the roles of wild-type JAK2 (JAK2 WT) and JAK2V617F alleles in the development of MPNs, we have used conditional Jak2 knock-out and Jak2V617F knock-in mice and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like MPN in mice, loss of Jak2 WT allele in hemizygous or homozygous Jak2V617F mice results in markedly increased white blood cells, neutrophils, reticulocytes and platelets in the peripheral blood, and significantly larger spleen size compared with heterozygous Jak2V617F mice. Hemizygous or homozygous Jak2V617F mice also exhibit accelerated myelofibrosis compared with mice expressing heterozygous Jak2V617F. Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN. Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean.

Chalcone synthase (CHS), the key enzyme in the flavonoid biosynthesis pathway, is encoded by a multigene family, CHS1-CHS8 and dCHS1 in soybean. A tandem repeat of CHS1, CHS3 and CHS4, and dCHS1 that is believed to be located in the vicinity comprises the I locus that suppresses coloration of the seed coat. This study was conducted to determine the location of all CHS members by using PCR-based DNA markers. Primers were constructed based on varietal differences in either the nucleotide sequence of the 5'-upstream region or the first intron of two cultivars, Misuzudaizu, with a yellow seed coat (II), and Moshidou Gong 503, with a brown seed coat (ii). One hundred and fifty recombinant inbred lines that originated from a cross between these two cultivars were used for linkage mapping together with 360 markers. Linkage mapping confirmed that CHS1, CHS3, CHS4, dCHS1, and the I locus are located at the same position in molecular linkage group (MLG) A2. CHS5 was mapped at a distance of 0.3 cM from the gene cluster. CHS2 and CHS6 were located in the middle region of MLGs A1 and K, respectively, while CHS7 and CHS8 were found at the distal end of MLGs D1a and B1, respectively. Phylogenetic analysis indicated that CHS1, CHS3, CHS4, and CHS5 are closely related, suggesting that gene duplication may have occurred repeatedly to form the I locus. In addition, CHS7 and CHS8 located at the distal end and CHS2, CHS6, and CHS members around the I locus located around the middle of the MLG are also related. Ancient tetraploidization and repeated duplication may be responsible for the evolution of the complex genetic loci of the CHS multigene family in soybean.

Origin of cytoplasm substituted rice cultivars found in Japan.

Genetic variation of Japanese rice cultivars were examined. Five of 450 lowland cultivars and another five of 200 upland cultivars were determined as the indica type by using isozyme genotypes and the remainder were of the japonica type. The major characteristics of these indica cultivars, revealed a slender shape of grains, a short apiculus hair length, a positive allele for Ph reaction, and allele-3 for the Pgd1 locus. Three of these indica cultivars showed a non-deletion ORF100, which is essential to the japonica-type plastid. The plastid subtype identity (PS-ID) sequences of these plastids is 6C7A, which is also a japonica-specific repeat unit. Thus, these cultivars were concluded to be naturally generated cytoplasm substituted lines. These plastids were introduced into a indica genetic background from japonica cultivars grown elsewhere. The rest of the indica cultivars revealed a deletion-type ORF100 and plastid subtype 8C8A, both of which are indica-specific. These cultivars carried indica-type allelic constitutions for diagnostic isozyme loci. However, other characters were identical to the cytoplasm-substituted cultivars in Japan. In East and Southeast Asia, cultivars carrying a indica-type nuclear genotype with a japonica-type plastid are restricted to Aus cultivars in the Bengal region. Genetic and historical records suggest that Japanese indica cultivars and the Aus cultivars are closely related. The Aus cultivars acquire necessary genetic constitutions from both indica and japonica cultivars through naturally occurring out-crossing to adapt to a particular cultivation condition in the region. The wide adaptability enabled them to be introduced into a northern region like Japan.

Analysis of the duplicated CHS1 gene related to the suppression of the seed coat pigmentation in yellow soybeans.

Seed coat color in soybean is controlled by the classically defined I ( Inhibitor) locus. The seeds of most commercial soybean varieties are yellow due to the presence of a dominant allele of the I locus ( I: yellow seed coat, or i(i) : pigmented hilum and yellow seed coat), which inhibits seed coat pigmentation. Analysis of spontaneous mutations from I (yellow seed coat) to i (pigmented seed coat) has shown that these mutations are correlated with the deletion of a duplicated chalcone synthase gene-1 ( CHS1) region. In the current study, we isolated the duplicated CHS1 region from a soybean cultivar with a I/I genotype (cv Miyagi shirome) and determined its structure. The results showed that the duplicated CHS1 contained intact regulatory and coding regions. We designated the duplicated CHS1 as ICHS1. In the hypocotyls of Miyagi shirome, the cDNA derived from ICHS1 mRNA was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, whereas in the immature seed coats it was suggested that the amount of transcripts from ICHS1 and/or another type of CHS1 ( CHS1.1) was very low. Interestingly, in the Miyagi shirome genome with a I/I genotype, ICHS1 was closely linked to the truncated CHS3, and sequence comparison showed that this cluster probably arose from the CHS1-CHS3 cluster by a 1.8-kb deletion event.

Absence of estrogen receptor-alpha expression in human ovarian clear cell adenocarcinoma compared with ovarian serous, endometrioid, and mucinous adenocarcinoma.

The mechanism that regulates growth in ovarian clear cell adenocarcinoma (CCA) is not well understood. A high incidence of concurrent endometriosis with CCA may indicate that estrogen is a growth promotor in CCA. To determine estrogen as a growth promotor, the authors investigated the presence or absence of estrogen receptor-alpha (ER-alpha), ER-beta, progesterone receptor, and dioxin receptor (i.e., aromatic hydrocarbon receptor) in clinically resected ovarian CCA, serous adenocarcinoma (SAC), endometrioid adenocarcinoma (EAC), and mucinous adenocarcinoma (MAC) specimens using an immunohistochemical method. Expression of ER-alpha and ER-beta messenger ribonucleic acid was examined by reverse transcription-polymerase chain reaction in three established CCA cell lines: KK, RMG-1, and HAC-II. None of the surgically resected CCA and CCA cell lines showed positive staining for ER-alpha. Conversely, 97.2% of SACs, 100% of EACs, and 70% of MACs showed positive nuclear staining for ER-alpha (p < 0.001). Conversely, positive ER-beta staining for CCA (39.3%) was similar to that of SAC (41.7%) and MAC (30.0%). EAC (75%) showed a higher expression of ER-beta (p < 0.02). Progesterone receptor was detected in only 10.7% of CCA, compared with SAC and EAC (SAC, 86.1%; EAC, 91.7%; p < 0.01). Aromatic hydrocarbon receptor was detected in all histologic types at an incidence of approximately 50% to 60%. Messenger ribonucleic acid of ER-alpha and ER-beta was not detected in the three CCA cell lines. These findings indicate biologic characteristics that distinguish CCA from other types of ovarian epithelial cancer.

Enhanced expression and differential inducibility of soybean chalcone synthase genes by supplemental UV-B in dark-grown seedlings.

By developing gene-specific RT-PCR and using filters to allow transmission down to 290 nm (UV-B+) or blocking all radiation below 320 nm (UV-B(-)), the effect of UV-B+ and UV-B- light on expression of each of the presently known seven members of soybean chalcone synthase (CHS) gene family in dark-grown seedlings was analyzed. Dark expression was detectable already in 18 h dark-germinating embryos, with progressive increases on successive days, suggesting that chs belongs to a class of genes expressed very early during germination, and that the expression at this stage is either constitutive or induced by non-light-dependent factors present in the seed or made available following imbibition. Exposure of 18 h dark-germinating embryos to UV-B- or to UV-B+ light did not lead to an increase in chs signal. However, the 24 h dark-germinating embryos showed a distinct effect of UV-B+, interestingly coinciding with the stage when the head of seedlings was in the process of being pushed up above ground by stem elongation, suggesting the possibility of a developmental switch modulating the appearance of UV-B response. The response to UV-B- was most prominent in chs1 and almost silent in chs2, while the up-regulation by UV-B+ was most prominent in chs5 and chs6 and much less so in chs2. Interestingly, chs2 was noted to be the only member of the Gmchs gene family devoid of H-box, raising the possibility that the H-box may be a good indicator of the photo-inducibility of a chs gene.

An allele of the ripening-specific 1-aminocyclopropane-1-carboxylic acid synthase gene (ACS1) in apple fruit with a long storage life.

An allele of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (Md-ACS1), the transcript and translated product of which have been identified in ripening apples (Malus domestica), was isolated from a genomic library of the apple cultivar, Golden Delicious. The predicted coding region of this allele (ACS1-2) showed that seven nucleotide substitutions in the corresponding region of ACS1-1 resulted in just one amino acid transition. A 162-bp sequence characterized as a short interspersed repetitive element retrotransposon was inserted in the 5'-flanking region of ACS1-2 corresponding to position -781 in ACS1-1. The XhoI site located near the 3' end of the predicted coding region of ACS1-2 was absent from the reverse transcriptase-polymerase chain reaction product, revealing that exclusive transcription from ACS1-1 occurs during ripening of cv Golden Delicious fruit. DNA gel-blot and polymerase chain reaction analyses of genomic DNAs showed clearly that apple cultivars were either heterozygous for ACS1-1 and ACS1-2 or homozygous for each type. RNA gel-blot analysis of the ACS1-2 homozygous Fuji apple, which produces little ethylene and has a long storage life, demonstrated that the level of transcription from ACS1-2 during the ripening stage was very low.

[Drug distribution of pelvic intraarterial infusion chemotherapy by bilateral internal iliac arterial catheter placement with coil occlusion of superior, inferior gluteal and obturator arteries].

The anticancer drug distribution in pelvic arterial infusion chemotherapy with bilateral internal iliac arterial catheter placement with coil occlusion of the superior, inferior and obturator arteries was indirectly evaluated by serially performed DSA, CTA and RI on 18 patients with pelvic malignancies. The distribution was judged to be Excellent (E), defined as uniform distribution in the entire lesion alone, in 9 of 18 patients (50%) evaluated prior to the start of the arterial infusion (AI), and as Good (G), defined as uniform distribution in the entire lesion and partly on an unaffected portion, in 9 (50%). Of the 11 patients evaluated after 7 times AI, E was found in 3 patients (27%), G in 7 (64%) and Fair (F), defined as distribution with a defect in the lesion, in one patient (9%). Of the 11 patients evaluated after 14 AI, E was found in 5 (45%), G in 4 (36%) and F in 2 (18%). Of the 7 patients evaluated after 21 AI, E was found in 2 (29%), G in 4 (57%) and F in one (14%). G was found in 2 of 2 patients (100%) evaluated after 28 AI. These results suggest that this method can maintain a favorable anticancer drug distribution even after multiple sessions of AI.

Organization of soybean chalcone synthase gene clusters and characterization of a new member of the family.

Chalcone synthase (CHS; EC 2.3.1.74), the first committed enzyme of the multibranched pathway of flavonoid/isoflavonoid biosynthesis is encoded by a multigene family in soybean, (Glycine max L. Merrill). Our results suggest that this gene family comprises at least seven members, some of which are clustered. We have identified four chs clusters in the allo-tetraploid G. max genome and chs5, a newly characterized member of the chs gene family is present in two of them. We describe the complete nucleotide sequence of chs5, the identification of its immediate neighbors and the organization of the four hitherto identified chs clusters in the Gm genome.

Platelet aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles--the role of alkaline phosphatase.

Human placental chorioepithelial brush border membrane, which is in direct contact with maternal blood flow, has platelet aggregation inhibiting activity. In the present study, the mechanism of this action has been examined in relation to ADP (adenosine diphosphate) degrading activity and alkaline phosphatase activity of brush border membrane vesicles (BBMV). BBMV prepared from human early and term placental villi, inhibited platelet aggregation induced by ADP. BBMV had potent ADP degrading (ADPase) activity. ADP was quickly degraded by BBMV. ADP degrading activity of BBMV was not so different between early and term placenta. Alkaline phosphatase activity of late placental BBMV was about three times greater than that of early placental BBMV. On the other hand, ADP degrading activity of late placental BBMV was almost the same as that of early placental BBMV. Inhibiting activity of platelet aggregation induced by ADP and ADP degrading activity of BBMV, were not inhibited by levamisole (alkaline phosphatase inhibitor).

Studies on placental inhibition of platelet aggregation: a comparison of human syncytiotrophoblast brush border and basal plasma membranes.

We compared the platelet aggregation inhibiting activity of human placental syncytiotrophoblast brush border membrane vesicles (BBMV) and basal plasma membrane vesicles (BpMV), and obtained the following results. Strong platelet aggregation inhibiting activity is found in placental BBMV. BBMV inhibited platelet aggregation induced by ADP (adenosine diphosphate) and arachidonic acid in a way which depended on the protein concentration of BBMV added. In contrast, BpMV showed no detectable platelet aggregation inhibiting activity. Quite high ADP degrading activity (ADPase activity) was present in the placental BBMV. ADP was quickly degraded by BBMV. In contrast, BpMV did not degrade ADP so quickly. Platelet TXB2 production was almost completely abolished at the protein concentration of 40 micrograms/ml of BBMV. In contrast, BpMV did not significantly inhibit platelet TXA2 (TXB2) production. These results show that syncytiotrophoblast brush border and basal plasma membranes of the human placenta have markedly different properties with respect to platelet aggregation inhibiting activity.

Properties of ADP-degrading activity of human placental syncytiotrophoblast brush border membrane vesicles.

The enzymatic properties of ADP (adenosine diphosphate) degradation in human placental syncytiotrophoblast brush border membrane vesicles (BBMV) were explored and the following results were obtained. BBMV had high ADP degrading activity compared to homogenate of placental villi. ADP degrading activity of BBMV; 1.05 +/- 0.05 mumol/mg protein/min placental villi was 21 times higher than that of homogenate of placental villi. Hydrolysis of ADP by BBMV follows Michaelis-Menten saturation kinetics with an apparent Km of 10.9 +/- 0.8 microM and Vmax of 2.10 +/- 0.17 mumol/mg protein/min. The enzyme has a divalent cation requirement. EDTA (2 mM) was found to abolish ADP degrading activity but this could be restored by the addition of either magnesium or calcium ions. Maximum enzyme activity of ADP degradation in BBMV was observed at a pH close to 8.0. The enzyme was insensitive to vanadate, levamisole, oligomycin, ouabain and N-ethylmaleimide (NEM), omeprazole and adenosine (5') pentaphospho (5') adenosine.

Platelet aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles.

We investigated the platelet aggregation inhibiting activity of human placental brush border membrane vesicles (BBMV) and obtained the following results. A strong platelet aggregation inhibiting activity existed in placental BBMV. The BBMV inhibited the platelet aggregation induced by ADP, arachidonic acid, collagen and ristocetin in a dose-dependent manner. The protein concentration of BBMV giving 50 per cent inhibition was 52 +/- 6 micrograms/ml for ADP-induced platelet aggregation, 21 +/- 2 micrograms/ml for arachidonic acid-induced platelet aggregation, 19 +/- 2 micrograms/ml for collagen-induced platelet aggregation and 107 +/- 9 micrograms/ml for ristocetin-induced platelet aggregation. There was a high level of ADP degrading activity (ADPase activity) in the placental BBMV. ADP degrading activity of the BBMV: 10.5 +/- 0.5 mumol/mg protein/min was 21 times greater than that of homogenate of the placental villi. The placental BBMV inhibited platelet TXA2 production. In the 40 micrograms/ml protein concentration of placental BBMV, platelet TXA2 production was almost completely inhibited.

Characterization of human placental activity for transport of taurocholate, using brush border (microvillous) membrane vesicles.

The uptake of taurocholate into brush border membrane vesicles prepared from human full term placenta was studied using a rapid filtration technique. The taurocholate uptake into brush border membrane vesicles was sensitive to extravesicular osmolarity, and pre-incubation of the brush border membrane vesicles with the taurocholate increased the uptake of taurocholate into the brush border membrane vesicles. These findings indicate that the uptake of taurocholate by brush border membrane vesicles represents transport into vesicles. The uptake of taurocholate into vesicles was not dependent on Na+ electrochemical gradient (extravesicular > intravesicular). But this uptake was markedly increased when the intravesicular space was rendered electrically more positive by the use of lowly permeant anions or valinomycin-induced K+ diffusion membrane potentials. These findings indicate that the taurocholate transport into brush border membrane vesicles was dependent on membrane potential. The initial rate of taurocholate transport into brush border membrane vesicles exhibited saturation kinetics with respect to the taurocholate concentration, an apparent Km of 67 microM and Vmax of 0.30 nmol/mg protein/20 sec were calculated.

[The platelet aggregation inhibiting activity in human placenta--study on the ADP degrading activity of human placental villi].

We studied the platelet aggregation inhibiting activity and ADP degrading activity of human placental villi (tissue culture supernatant) and brush border membrane vesicles (BBMV) and obtained the following results. 1. There existed a platelet aggregation inhibiting activity in tissue culture supernatant of villi (S-villi) but not in that of decidua or amnion. The S-villi inhibited the platelet aggregation induced by ADP, but not that induced by collagen, arachidonic acid or ristocetin. And, there was also ADP degrading activity (ADPase activity) in the S-villi. ADP was quickly degraded by S-villi. When ADP was preincubated with S-villi, the platelet aggregation induced by ADP was completely lost. 2. There was very strong platelet aggregation inhibiting activity in placental BBMV. The BBMV almost completely inhibited the platelet aggregation induced by ADP, collagen, arachidonic acid and ristocetin. And there was very strong ADP degrading activity in the placental BBMV. ADP was quickly degraded by BBMV. When ADP was preincubated with BBMV, the platelet aggregation induced by ADP was completely lost. 3. The enzymatic character (heat stability, enzymatic kinetics, Ca++ dependency and pH dependency) of ADP degrading activity in BBMV was very similar to that in S-villi. 4. The ADP degrading activity of both S-villi and solubilized BBMV were fractionated by anion exchange column chromatography and gel filtration column chromatography in similar patterns, and it was shown that ADP degrading substance of both S-villi and solubilized BBMV had a molecular weight of about 60K.