miR‐495 sensitizes MDR cancer cells to the combination of doxorubicin and taxol by inhibiting MDR1 expression

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR‐495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR‐495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol‐doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre‐treatment with miR‐495 before chemotherapy could improve the curative effect on MDR1‐based MDR cancer.     

Bacterial Contribution to Dissolved Organic Matter in Eutrophic Lake Kasumigaura,Japan

Incubation experiments using filtered waters from Lake Kasumigaura were conducted to examine bacterial contribution to a dissolved organic carbon (DOC) pool. Bacterial abundance, bacterial production, concentrations of DOC, total dissolved amino acids (TDAA), and total dissolved neutral sugars (TDNS) were monitored during the experiments. Bacterial production during the first few days was very high (20 to 35 μg C liter⁻¹ day⁻¹), accounting for 40 to 70% of primary production. The total bacterial production accounted for 34 to 55% of the DOC loss during the experiment, indicating high bacterial activities in Lake Kasumigaura. The DOC degradation was only 12 to 15%, whereas the degradation of TDAA and TDNS ranged from 30 to 50%, suggesting the preferential usage of TDAA and TDNS. The contribution of bacterially derived carbon to a DOC pool in Lake Kasumigaura was estimated using d-amino acids as bacterial biomarkers and accounted for 30 to 50% of the lake DOC. These values were much higher than those estimated for the open ocean (20 to 30%). The ratio of bacterially derived carbon to bulk carbon increased slightly with time, suggesting that the bacterially derived carbon is more resistant to microbial degradation than bulk carbon. This is the first study to estimate the bacterial contribution to a DOC pool in freshwater environments. These results indicate that bacteria play even more important roles in carbon cycles in freshwater environments than in open oceans and also suggests that recent increases in recalcitrant DOC in various lakes could be attributed to bacterially derived carbon. The potential differences in bacterial contributions to dissolved organic matter (DOM) between freshwater and marine environments are discussed.     

Setup accuracy and dose attenuation of a wooden immobilization system for lung stereotactic body radiotherapy

BackgroundWe evaluated the setup error and dose absorption of an immobilization system with a shell and wooden baseplate (SW) for lung stereotactic body radiotherapy (SBRT).Materials and methodsSetup errors in 109 patients immobilized with an SW or BodyFix system (BF) were compared. Dose attenuation rates of materials for baseplates were measured with an ion-chamber. Ionization measurements were performed from 90° to 180° gantry angle in 10° increments, with the ball water equivalent phantom placed at the center of the wood and carbon baseplates whose effects on dose distribution were compared using an electron portal imaging device.ResultsThe ratio for the anterior-posterior, cranial-caudal, and right-left of the cases within 3-mm registered shifts in interfractional setup error were 90.9%, 89.2%, and 97.4% for the SW, and 93.2%, 91.6%, and 98.0% for the BF, respectively. For intrafractional setup error, 98.3%, 97.4%, and 99.1% for the SW and 96.6%, 95.8%, and 98.7% for the BF were within 3-mm registered shifts, respectively. In the center position, the average (minimum/maximum) dose attenuation rates from 90° to 180° for the wooden and carbon baseplates were 0.5 (0.1/2.8)% and 1.0 (–0.1/10.1)% with 6 MV, respectively. The gamma passing rates of 2%/2 mm for the wooden and carbon baseplates were 99.7% and 98.3% (p < 0.01).ConclusionsThe immobilization system with an SW is effective for lung SBRT since it is comparable to the BF in setup accuracy. Moreover, the wooden baseplate had lower radiation attenuation rates and affected the dose distribution less than the carbon baseplate.     

Upregulation of LRRC8A by m5C modification-mediated mRNA stability suppresses apoptosis and facilitates tumorigenesis in cervical cancer

Cervical cancer (CC) is one of the most common gynecological malignancies with poor prognosis for advanced CC patients. LRRC8A is a volume-regulated anion channel protein involved in cellular homeostasis, but its role in CC remains largely unknown. In this study, we found that LRRC8A is elevated in CC and associated with poor prognosis. LRRC8A maintains cell survivals under the hypotonic condition, and promotes tumorigenesis through apoptosis suppression in vitro and in vivo. Notably, LRRC8A is upregulated by NSUN2-mediated m⁵C modification. m⁵C modified-LRRC8A mRNA is bound by the RNA binding protein YBX1 followed by the increased RNA stability. Moreover, loss of NSUN2 suppresses the proliferation and metastasis of CC cells, and NSUN2 expression is positively correlated with LRRC8A expression in CC. Altogether, our study demonstrates that the NSUN2-m⁵C-LRRC8A axis is crucial and would be a potential therapeutic target for CC.     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

Accumulation of hydroxyapatite in the kidney of streptozotocin-induced diabetic rat fed a low-zinc diet

Calcification occurred in the kidney of streptozotocin (STZ)-induced diabetic rats fed a low-zinc diet. The deposits were stained by the von Kossa method and were detected intracellularly in the tubular cells, mainly in the cortico-medullar region. The deposits were found to be a heterogenous substance on electron microscopy. There were various sizes of deposits, and the surfactant was very much distorted. Many deposits grew up to bind small particles, and the core-like substance was observed in the center of the deposit. The component of the deposit was analyzed by an X-ray microanalyzer, and was found to be calcium and phosphorus. The ratio of calcium to phosphorus was 2.159, which coincided with the ratio of standard hydroxyapatite. From these observations, the deposit is believed to be hydroxyapatite. It is thought that the core is formed at first, many particles are then bound to the core, and deposits grow up.