We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.

Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.

Morphometric evaluation of adenomas of the colon to determine their degree of dysplasia is still controversial, since subjective factors influence the decision concerning benignancy or malignancy. Some indices have been proposed to assist in categorizing intermediate lesions into benign or malignant. However, these indices show a wide range of distribution. To evaluate the reliability of one such index, the index of structural atypism (ISA, area of glands/area of stroma), we studied polyps as a whole, and found great variability of index values within a single polyp.

We have examined the roles of pertinent extracellular matrix molecules in the formation of the neural crest cell migration patterns in the sclerotome of the mouse embryo. The present data indicate that permissiveness for migration is inversely correlated with chondroitin sulfate content. Experimental removal of chondroitin sulfate proteoglycans in the embryo causes neural crest cells to migrate even within the posterior half of the somite, which they do not invade ordinarily. Moreover, three different sclerotomal regions defined by the presence or absence of the ventromedial and/or ventrolateral pathways are present along the anteroposterior axis and undergo systematic temporal changes that affect migration patterns. The most anterior portion of the sclerotome is conducive to both ventromedial and ventrolateral migration (Anterior Region). The intermediate portion is conducive to ventromedial migration only (Intermediate Region). No neural crest cells are seen within the posterior portion of the sclerotome (Posterior Region). At this level, they are observed exclusively in the dorsolateral space adjacent to the roof of the neural tube. With advancing embryonic development, the rostrocaudal length of the Anterior Region decreases and is accompanied by a corresponding enlargement of the Intermediate Region. These results suggest that temporal and regional differences in the sclerotome contribute to the neural crest cell migration patterns in the mouse. To refine our understanding of the underlying mechanisms, regional differences and temporal changes in the distribution of extracellular matrix molecules have been examined during migration. In the sclerotome, chondroitin sulfate displays distinct distribution patterns that are closely correlated with the migration patterns of mouse neural crest cells. Furthermore, their migration patterns are altered in embryos treated with the inhibitors of chondroitin sulfate proteoglycan biosynthesis, sodium chlorate, and beta-D-xyloside. In inhibitor-treated embryos, neural crest cell migration occurs even in the posterior portion of the sclerotome. The metameric organization of dorsal root ganglia is disturbed in these embryos. Our observations provide novel evidence for the importance of sclerotomal chondroitin sulfate distribution patterns in mouse crest cell migration patterns. We conclude that systematic spatiotemporal changes in the distribution of chondroitin sulfate proteoglycans are a key requisite for the formation of migration patterns of mouse neural crest cells in the sclerotome.

We analyzed 47 primary sporadic human renal cell carcinomas (39 clear cell and 8 non-clear cell) for mutations of the von Hippel-Lindau (VHL) tumor suppressor gene using the polymerase chain reaction and single strand conformational polymorphism analysis of DNA. All of the positive cases in single strand conformational polymorphism analyses were further characterized by direct sequencing. Somatic mutations were detected in 22 (56%) of 39 clear cell renal carcinomas including 15 deletions, 3 insertions, 3 missense mutations, and 1 nonsense mutation. Nineteen of these mutations predicted to produce truncation of the VHL protein. These mutations mainly occurred in the last one-third region of exons 1, 2, and 3. In addition, loss of heterozygosity of the VHL gene was observed in 16 (84%) of 19 informative clear cell renal carcinomas. No somatic mutations were detected in 8 non-clear cell carcinomas. These results show that the VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinomas, especially in the clear cell subtype renal cell carcinoma. Clear cell carcinoma might be distinguished from other pathological types of renal cell carcinomas by molecular genetic techniques.

To determine whether long-term oral administration of UFT, a combination of 5-fluorouracil and uracil, in addition to conventional estrogen therapy improved the response and survival of the patients with advanced stage D2 prostate adenocarcinoma, a randomized prospective study was performed with either estrogen alone (Honvan 200 mg/day or presexol 1 mg/day: group A) or estrogen plus UFT (400 mg/day:group B). This study comprises 34 newly diagnosed patients with poorly differentiated prostatic adenocarcinoma (18 patients in group A and 16 in group B). Survival from all causes of death or cancer-specific death were compared using Kaplan-Meier actual methods among the patients separated by histological composition of tumors analyzed WHO histologic patterns, score of extent of disease (EOD), and with or without normalization of serum PSA or PAP levels after treatment. Although combination therapy with UFT against overall survival was effective without statistical significance, better survival in this group than the patients treatment with estrogen alone assessed among the patients whose tumor contained more than 70% of medullary and/or column-cord histological components. The survivals among the patients with EOD score 3 and whose serum PSA or PAP levels did not lead to decrease within normal levels after treatment were also better in group B than in group A. These findings suggest the validity of the combination therapy with UFT in addition to estrogen against highly advanced prostatic cancer patients whose tumor composed of abundant non-hormone-refractor histological components.

Physiological and morphological properties of large non-pyramidal cells immunoreactive for cholecystokinin, parvalbumin or somatostatin were investigated in vitro in the frontal cortex of 18-22-day-old rats. These three peptides were expressed in separate populations including large cells. Cholecystokinin cells and parvalbumin cells made boutons apposed to other cell bodies, but differed in their firing patterns in response to depolarizing current pulses. Parvalbumin cells belonged to fast-spiking cells. Parvalbumin fast-spiking cells also included chandelier cells. In contrast, cholecystokinin cells were found to be regular-spiking non-pyramidal cells or burst-spiking non-pyramidal cells with bursting activity from hyperpolarized potentials (two or more spikes on slow depolarizing humps). Large somatostatin cells belonged to the regular-spiking non-pyramidal category and featured wide or ascending axonal arbors (wide arbor cells and Martinotti cells) which did not seem to be apposed to the somata so frequently as large cholecystokinin and parvalbumin cells. For electron microscopic observations, another population of eight immunohistochemically-uncharacterized non-pyramidal cells were selected: (i) five fast spiking cells including one chandelier cell which are supposed to contain parvalbumin, and (ii) three large regular-spiking non-pyramidal cells with terminals apposed to somata, which are not considered to include somatostatin cells, but some of which may belong to cholecystokinin cells. The fast-spiking cells other than a chandelier cell and the large regular-spiking non-pyramidal cells made GABA-positive synapses on somata (4% and 12% of the synapses in two small to medium fast-spiking cells, 22% and 35% of the synapses in two large fast-spiking cells, and 10%, 18% and 37% of the synapses in three large regular-spiking non-pyramidal cells). A few terminals of the fast-spiking and regular-spiking non-pyramidal cells innervated GABAergic cells. About 30% of the fast-spiking cell terminals innervated spines, but few of the regular-spiking non-pyramidal cell terminals did. A fast-spiking chandelier cell made GABA-positive synapses on GABA-negative axon initial segments. These results suggest that large GABAergic cells are heterogeneous in neuroactive substances, firing patterns and synaptic connections, and that cortical cells receive heterogeneous GABAergic somatic inputs.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Speleothem CaCO3 δ18O is a commonly employed paleomonsoon proxy. However, inferring local rainfall amount from speleothem δ18O can be complicated due to changing source water δ18O, temperature effects, and rainout over the moisture transport path. These complications are addressed using δ18O of planktonic foraminiferal CaCO3, offshore from the Yangtze River Valley (YRV). The advantage is that the effects of global seawater δ18O and local temperature changes can be quantitatively removed, yielding a record of local seawater δ18O, a proxy that responds primarily to dilution by local precipitation and runoff. Whereas YRV speleothem δ18O is dominated by precession-band (23 ky) cyclicity, local seawater δ18O is dominated by eccentricity (100 ky) and obliquity (41 ky) cycles, with almost no precession-scale variance. These results, consistent with records outside the YRV, suggest that East Asian monsoon rainfall is more sensitive to greenhouse gas and high-latitude ice sheet forcing than to direct insolation forcing.

Sphingosine 1-phosphate (S1P) is a serum-borne naturally occurring sphingolipid, specifically enriched in high-density lipoprotein (HDL) fractions. S1P binds to G-protein-coupled S1P1 receptors to activate endothelial NO synthase (eNOS) in vascular endothelial cells. We explored whether and how statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, modulate expression of S1P1 receptors and endothelial responses for subsequent stimulation with S1P or with HDL.

A long-standing question in nuclear physics is whether chargeless nuclear systems can exist. To our knowledge, only neutron stars represent near-pure neutron systems, where neutrons are squeezed together by the gravitational force to very high densities. The experimental search for isolated multi-neutron systems has been an ongoing quest for several decades1, with a particular focus on the four-neutron system called the tetraneutron, resulting in only a few indications of its existence so far2-4, leaving the tetraneutron an elusive nuclear system for six decades. Here we report on the observation of a resonance-like structure near threshold in the four-neutron system that is consistent with a quasi-bound tetraneutron state existing for a very short time. The measured energy and width of this state provide a key benchmark for our understanding of the nuclear force. The use of an experimental approach based on a knockout reaction at large momentum transfer with a radioactive high-energy 8He beam was key.