Utility of fluorescence in situ hybridization for ploidy and p57 immunostaining in discriminating hydatidiform moles

Discrimination between complete moles (CMs), partial moles (PMs), and hydropic abortions (HAs) is important as the risk of persistent gestational trophoblastic disease (GTD) differs for each condition. We evaluated whether ancillary fluorescence in situ hybridization (FISH) with a set of chromosome enumeration probes (CEP) for chromosomes X, Y, and 17 and p57 immunostaining could improve the clinical diagnosis. Forty-one products of conception (POC) were reclassified according to clinical performance, morphology, p57 immunostaining results, and FISH results. The accuracy of histological examination alone was 85% for the original diagnosis. FISH analysis showed diploidy in 19 of 20 CMs and triploidy in 4 of 6 PMs. The concordance rate was 92.5% on using the CEP probes. p57 Staining was negative in all CMs and positive in all PMs and HAs. Chromosomal abnormality was detected in 3 cases of HA by using FISH. In conclusion, combined p57 immunostaining and FISH with a set of 3 CEP probes for chromosomes X, Y, and 17 could be useful in the classification of hydatidiform moles.     

Purple nonsulfur bacteria diversity in activated sludge and its potential phosphorus-accumulating ability under different cultivation conditions

This study investigates the diversity and the potential phosphorus-accumulating ability among the purple nonsulfur (PNS) bacteria. Traditional methods and molecular biotechniques were applied. Microscopic visualization using 4′,6-diamidino-2-phenylindole staining as well as chemical analysis demonstrated that most of the isolated PNS bacteria presented different levels of phosphorus accumulation. Four of the pure cultures, denoted as Rhodopseudomonas palustris CC1, CC7, G11, and GE1, based on their differences in the PNS’s pufM gene, exhibited higher internal phosphorus content compared to other isolated strains in this study. In addition, substantial polyphosphate accumulation was observed after the bacteria entered their stationary growth phase. Among them, the isolated R. palustris G11 could accumulate internal phosphorus up to 13%–15% of its cell dry weight under anaerobic illuminated incubation conditions. When the incubation status was switched from anaerobic to aerobic, the bacterial phosphorus content had a tendency to decrease slightly or remain about the same throughout the whole aerobic stage. The growth rate and biomass were higher when the PNS bacteria grew under photoheterotrophic conditions rather than the chemoheterotrophic ones. Furthermore, the environmental pH value could affect the contents of internal bacterial phosphate. Results of this study demonstrated that PNS bacteria are a group of the polyphosphate-accumulating organisms, of which this ability had never been properly studied. The conditions that PNS bacteria accumulating polyphosphate presented from this study were unique and showed characteristics that were different from the well-known enhanced biological phosphorus removal model.     

Mitochondrial DNA phylogeography of Glyptothorax fokiensis and Glyptothorax hainanensis in Asia

Whole mitochondrial DNA cytochrome b sequences in 62 fish from 13 locations in Southeast China identified two major clades corresponding to two allopatric taxa, Glyptothorax fokiensis fokiensis and Glyptothorax fokiensis hainanensis . Reciprocal monophyly and a molecular clock separation between these two taxa of 2·3 million years indicate these taxa should be elevated to species. Mismatch distributions and Fu's F _S statistic suggest that both G. fokiensis and G. hainanensis have experienced recent population expansions. Analysis of molecular variance indicates that most of the genetic variation resides among populations within both species, with Φ _ST= 0·645 for G. fokiensis and 0·801 for G. hainanensis , suggesting restricted gene flow among populations. Significant correlations between the geographic and the genetic distances provide support for the importance of geographic isolations between populations. Nested clade analysis also confirms low levels of genetic exchanges between the two major groups and between populations within each group. The phylogeographical pattern among populations of Glyptothorax in East Asia can be attributed to historical fragmentations, demographic expansions and occasional long-distance dispersals stimulated by tectonic activity and Ice Age climate changes.     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

Activation of Calcitonin Receptor and Calcitonin Receptor-like Receptor by Membrane-anchored Ligands

G protein-coupled receptors (GPCRs) are the most important pharmaceutical targets, and more than 40% of drugs in use today modulate GPCR signaling. A major hurdle in the development of therapies targeting GPCRs is the drug candidate''s nonselective actions in multiple tissues. The ability to spatially control GPCR signaling would provide a venue for developing therapies that require targeted GPCR signaling. Here, we show that the fusion of a RAMP1 co-receptor with the calcitonin gene-related peptide (CGRP), or calcitonin, transforms the RAMP1 from a co-receptor to bona fide membrane-anchored ligands (CGRP-RAMP1 and CAL-RAMP1). The CAL-RAMP1 selectively activates the calcitonin receptor (CR), whereas, the CGRP-RAMP1 activates both the calcitonin receptor-like receptor (CLR) and CR. Unlike a free peptide, which moves freely in the extracellular space and differentiates targets based on molecular affinity, the anchored CGRP-RAMP1 and CAL-RAMP1 ligands confine their activities to individual cells. In addition, our study showed that a CGRP8–37-RAMP1 chimera, but not RAMP1, functions as an antagonist for CGRP-RAMP1-mediated signaling, suggesting that the activation of CLR by CGRP-RAMP1 shares similar molecular mechanisms with the CGRP-mediated activation of CLR/RAMP1 receptor complexes. Taken together, our finding thus provides a novel class of ligands that activate CR and CLR exclusively in an autocrine manner and a proof-of-concept demonstration for future development of targeted therapies aimed at these receptors in specific cell populations.     

Folate Deficiency Triggers an Oxidative-Nitrosative Stress-Mediated Apoptotic Cell Death and Impedes Insulin Biosynthesis in RINm5F Pancreatic Islet β–Cells: Relevant to the Pathogenesis of Diabetes

It has been postulated that folic acid (folate) deficiency (FD) may be a risk factor for the pathogenesis of a variety of oxidative stress-triggered chronic degenerative diseases including diabetes, however, the direct evidence to lend support to this hypothesis is scanty. For this reason, we set out to study if FD can trigger the apoptotic events in an insulin-producing pancreatic RINm5F islet β cells. When these cells were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature proceeding through a mitochondria-dependent pathway. In addition to evoke oxidative stress, FD condition could also trigger nitrosative stress through a NF-κB-dependent iNOS-mediated overproduction of nitric oxide (NO). The latter compound could then trigger depletion of endoplasmic reticulum (ER) calcium (Ca²⁺) store leading to cytosolic Ca²⁺ overload and caused ER stress as evidence by the activation of CHOP expression. Furthermore, FD-induced apoptosis of RINm5F cells was found to be correlated with a time-dependent depletion of intracellular gluthathione (GSH) and a severe down-regulation of Bcl-2 expression. Along the same vein, we also demonstrated that FD could severely impede RINm5F cells to synthesize insulin and their abilities to secret insulin in response to glucose stimulation were appreciably hampered. Even more importantly, we found that folate replenishment could not restore the ability of RINm5F cells to resynthesize insulin. Taken together, our data provide strong evidence to support the hypothesis that FD is a legitimate risk factor for the pathogenesis of diabetes.     

Interaction between obesity and genetic polymorphisms in the apolipoprotein CIII gene and lipoprotein lipase gene on the risk of hypertriglyceridemia in Chinese

To understand the effects of the interaction between genetic polymorphisms and obesity on the risk of hypertriglyceridemia (HTG), two polymorphisms, an SstI polymorphism on the apolipoprotein CIII gene and a HindIII polymorphism on the lipoprotein lipase gene, were analyzed in 339 Chinese subjects with (82 cases in the HTG group) or without HTG (257 cases in the control group). Our data revealed that the frequencies of obesity, the SstI minor allele (S2), and the HindIII major allele (H+) in the HTG group were significantly higher than in the control group. Subgroup analysis revealed that the association between these two polymorphisms and HTG occurred predominantly in nonobese subjects and in subjects with the less hypertriglyceridemic genotype of another polymorphism. Multivariate logistic regression analysis showed that all three risk factors (obesity, S2-containing chromosome, and H+ homozygosity) were associated with HTG, and an interaction was found between obesity and H+ homozygosity for the occurrence of HTG. The risk of HTG increased significantly with combinations of risk factors. Subjects can be divided into low or high risk groups for HTG using such combinations. These results provide evidence of interaction between obesity and the HindIII polymorphism of the lipoprotein lipase gene on the risk of HTG. Received: 27 November 1996 / Accepted: 28 March 1997     

Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13-mers precisely spaced relative to the 9-mers

The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs. Three 13-mers tandemly repeated at one end of the origin and four 9-mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters. Mutant origins were constructed with altered spacing of the 9-mers relative to the 13-mers. Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro. Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation. These results suggest that interaction with the 9-mers positions at least one subunit of DnaA to recognize directly the nearest 13-mer for DNA melting.