Decrease in bacterial production over the past three decades in the north basin of Lake Biwa,Japan

Limnology - In Lake Biwa, the largest lake in Japan, external pollutant loads have decreased since the 1980s, leading to improved water quality, such as reduction in biochemical oxygen demand (BOD)...     

Involvement of intracellular caveolin‐1 distribution in the suppression of antigen‐induced mast cell activation by cationic liposomes

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross‐linking of high‐affinity immunoglobulin E receptor in a time‐ and dose‐dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the inhibition of store‐operated Ca²⁺ entry. Further, we revealed that the mechanism underlying an impaired [Ca²⁺]_i increase is the inhibition of the activation of the phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Yet, how cationic liposomes inhibit the PI3K‐Akt pathway is still unclear. Here, we focused on caveolin‐1, a major component of caveolae, which is reported to be involved in the activation of the PI3K‐Akt pathway in various cell lines. In this study, we showed that caveolin‐1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin‐1 to the plasma membrane and the colocalization of caveolin‐1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

Structural requirement of C‐terminal region of chicken lysozyme signal peptide

Abstract

The mechanisms of signal peptide cleavage has not been fully elucidated yet. In previous investigation, we have examined the effect of chicken lysozyme signal peptide mutations on the secretion of human lysozyme. During this study, we determined that the hydrophobic bulky amino acid Val at position ‐1 inhibited the function of signal peptide. To determine why the ‐1Val suppressed the function of signal peptide, turn‐promoting amino acids Pro and Gly were introduced after ‐lVal to prevent the signal peptide from forming α‐helix and induce β‐turn around the cleavage site. This mutation resulted in no processing of signal peptide and no secretion of human lysozyme. However, the replacement of ‐1Val with Ala permitted a functional signal. Based on these results, three dimensional models around the cleavage site of each signal peptide were made, which show that bulky side chain at ‐1 residue of signal peptide limits the reaction space for signal peptidase and suppresses cleavage by steric hindrance. We suggest that the bulky side chain at ‐1 residue suppresses the signal peptide cleavage by its local steric hindrance and not by a change in whole structure around the cleavage site. On the other hand, introduction of Pro at position +1 did not inhibit signal cleavage completely resulting in poor secretion and processing efficiency although Pro in position +1 has been recently reported to block cleavage of the prokaryotic signal peptide. The mechanism of cleavage of prokaryotic signal may be different than that of eukaryotic signal.     

Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.     

Vertical Distribution and Estimated Doses from Artificial Radionuclides in Soil Samples around the Chernobyl Nuclear Power Plant and the Semipalatinsk Nuclear Testing Site

For the current on-site evaluation of the environmental contamination and contributory external exposure after the accident at the Chernobyl Nuclear Power Plant (CNPP) and the nuclear tests at the Semipalatinsk Nuclear Testing Site (SNTS), the concentrations of artificial radionuclides in soil samples from each area were analyzed by gamma spectrometry. Four artificial radionuclides (²⁴¹Am, ¹³⁴Cs, ¹³⁷Cs, and ⁶⁰Co) were detected in surface soil around CNPP, whereas seven artificial radionuclides (²⁴¹Am, ⁵⁷Co, ¹³⁷Cs, ⁹⁵Zr, ⁹⁵Nb, ⁵⁸Co, and ⁶⁰Co) were detected in surface soil around SNTS. Effective doses around CNPP were over the public dose limit of 1 mSv/y (International Commission on Radiological Protection, 1991). These levels in a contaminated area 12 km from Unit 4 were high, whereas levels in a decontaminated area 12 km from Unit 4 and another contaminated area 15 km from Unit 4 were comparatively low. On the other hand, the effective doses around SNTS were below the public dose limit. These findings suggest that the environmental contamination and effective doses on the ground definitely decrease with decontamination such as removing surface soil, although the effective doses of the sampling points around CNPP in the present study were all over the public dose limit. Thus, the remediation of soil as a countermeasure could be an extremely effective method not only for areas around CNPP and SNTS but also for areas around the Fukushima Dai-ichi Nuclear Power Plant (FNPP), and external exposure levels will be certainly reduced. Long-term follow-up of environmental monitoring around CNPP, SNTS, and FNPP, as well as evaluation of the health effects in the population residing around these areas, could contribute to radiation safety and reduce unnecessary exposure to the public.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na⁺- or Li⁺-dependent manner. Moreover, Na⁺ efflux via PdrM was observed when acriflavine was added to Na⁺-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na⁺/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.