Sensing of cytoplasmic pH by bacterial chemoreceptors involves the linker region that connects the membrane-spanning and the signal-modulating helices.

The two major chemoreceptors of Escherichia coli, Tsr and Tar, mediate opposite responses to the same changes in cytoplasmic pH (pH(i)). We set out to identify residues involved in pH(i) sensing to gain insight into the general mechanisms of signaling employed by the chemoreceptors. Characterization of various chimeras of Tsr and Tar localized the pH(i)-sensing region to Arg(259)-His(267) of Tar and Gly(261)-Asp(269) of Tsr. This region of Tar contains three charged residues (Arg(259)-Ser(261), Asp(263), and His(267)) that have counterparts of opposite charge in Tsr (Gly(261)-Glu(262), Arg(265), and Asp(269)). The replacement of all of the three charged residues in Tar or Arg(259)-Ser(260) alone by the corresponding residues of Tsr reversed the polarity of pH(i) response, whereas the replacement of Asp(263) or His(267) did not change the polarity but altered the time course of pH(i) response. These results suggest that the electrostatic properties of a short cytoplasmic region within the linker region that connects the second transmembrane helix to the first methylation helix is critical for switching the signaling state of the chemoreceptors during pH sensing. Similar conformational changes of this region in response to external ligands may be critical components of transmembrane signaling.     

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes

In this study, we created porcine‐induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c‐Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA‐Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537–553, 2017. © 2016 Wiley Periodicals, Inc.     

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.     

Effects of negative air ions on activity of neural substrates involved in autonomic regulation in rats

The neural mechanism by which negative air ions (NAI) mediate the regulation of autonomic nervous system activity is still unknown. We examined the effects of NAI on physiological responses, such as blood pressure (BP), heart rate (HR), and heart rate variability (HRV) as well as neuronal activity, in the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus (LC), nucleus ambiguus (NA), and nucleus of the solitary tract (NTS) with c-Fos immunohistochemistry in anesthetized, spontaneously breathing rats. In addition, we performed cervical vagotomy to reveal the afferent pathway involved in mediating the effects of NAI on autonomic regulation. NAI significantly decreased BP and HR, and increased HF power of the HRV spectrum. Significant decreases in c-Fos positive nuclei in the PVN and LC, and enhancement of c-Fos expression in the NA and NTS were induced by NAI. After vagotomy, these physiological and neuronal responses to NAI were not observed. These findings suggest that NAI can modulate autonomic regulation through inhibition of neuronal activity in PVN and LC as well as activation of NA neurons, and that these effects of NAI might be mediated via the vagus nerves.     

Bisphenol A Does Not Affect Memory Performance in Adult Male Rats

Bisphenol A (BPA) is an estrogenic endocrine disruptor used for producing polycarbonate plastics and epoxy resins. This study investigated the effects of oral BPA administration on memory performance, general activity, and emotionality in adult male Sprague Dawley rats using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial memory performance. In addition, in order to confirm the effects of BPA on spatial memory performance, we examined whether intrahippocampal injection of BPA affects spatial memory consolidation. In the MAZE test, although oral BPA administration at 10 mg/kg significantly altered the number of entries into the incorrect area compared to those of vehicle-treated rats, male rats given BPA through either oral administration or intrahippocampal injection failed to show significant differences in latencies to reach the reward. Also, oral BPA administration did not affect fear-motivated memory performance in the step-through passive avoidance test. Oral BPA administration at 0.05 mg/kg, the lowest dose used in this study, was correlated with a decrease in locomotor activity in the open-field test, whereas oral administration at 10 mg/kg, the highest dose used in this study, was correlated with a light anxiolytic effect in the elevated plus-maze test. The present study suggests that BPA in adulthood has little effect on spatial memory performance in male rats.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Serosurvey of Smooth Brucella,Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.     

Augmentation of fatality of influenza in mice by inhibition of phagocytosis

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages, and this leads to the inhibition of virus propagation in vitro. To assess if this were also true in vivo, mice infected with influenza A/WSN (H1N1) virus were administered with phagocytosis inhibitors and examined for the progress of influenza. Administration of the inhibitors caused a decrease in the level of phagocytosis observed with bronchoalveolar lavage cells. We found that both the lethality in mice and the extent of inflammation in the lung were augmented in those mice. These results suggest that phagocytosis of virus-infected cells helps suppress the progress of influenza in mice.     

A possible balance of magnesium accumulations among bone, cartilage, artery, and vein in single human individuals

It is known that a large quantity of magnesium contains bones, and the magnesium contents in spongy bones decrease gradually with advancing age. To elucidate the relationships between a decrease of mineral contents in human bones and an accumulation of minerals in the other human tissues, the content of magnesium was analyzed by inductively coupled plasma-atomic emission spectrometry among human bones, arteries, veins, and cartilages in 27 subjects (17 men and 10 women). These were resected from the subjects who died in the age range 40–98 yr. Calcanei were chosen for analysis of magnesium contents in contrast with femoral, popliteal, and common carotid arteries, internal jugular and femoral veins, superior and inferior venae cavae, and pubic symphyses. The magnesium contents in the calcanei decreased gradually with aging, whereas they increased progressively in the arteries, veins, and pubic symphyses with aging. It was found that as the magnesium contents decreased in the calcanei, they increased in the arteries, such as the femoral, popliteal, and common carotid arteries, whereas they decreased inversely in the veins, such as the internal jugular and femoral veins and superior and inferior venae cavae. Furthermore, as the magnesium contents decreased in the calcanei, they hardly changed in the pubic symphyses. These suggest that magnesium released from bones is accompanied by accumulation of magnesium in the arteries.     

Increases of Calcium,Phosphorus, and Magnesium in Both the Right and Left Fibrous Trigones of Human Heart with Aging

We previously reported that reduced platelet endogenous antioxidant enzymes activities are related to the low plasma zinc level in patients with end-stage renal failure (ESRF). In this study, we attempt to evaluate whether dietary zinc deprivation reduces the activities of endogenous antioxidant and then enhances oxidative stress in the unstimulated platelet of normal and 5/6 nephrectomized (Nx) rats because increased platelet oxidative stress is suggested to involve in the incidence of thrombotic and atherosclerotic diseases. Male Sprague–Dawley rats (n = 48) were fed a zinc-deficient diet and deionized distilled water for 1 week to induce reduction of plasma zinc level. Half of the rats continued on this diet for 4 weeks as zinc-deplete group, and the other half were maintained on the same diet but with zinc-supplemented water (120 mg/L zinc sulfate solution) to correct the reduction of plasma zinc level as zinc-replete group. Half of each group underwent 5/6 Nx, while the other half underwent sham operation. Another 12 normal rats were fed standard rat chow (containing 23.4% protein and 50 ppm zinc) and drank deionized distilled water as normal control rats. In zinc-deplete rats including sham-operated and 5/6 Nx rats exhibited lower endogenous antioxidant enzymes activities such as reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione peroxidase (GPX) and higher malondialdehyde (MDA) levels than normal control rats in the unstimulated platelets. However, in zinc-replete rats including sham-operated and 5/6 Nx rats have a normal endogenous antioxidant enzymes activity and normal MDA levels in the unstimulated platelets. We suggest that in uremia, the low plasma zinc level may be a risk factor for thrombotic and atherosclerotic diseases because it reduces the activities of endogenous antioxidant enzymes and increases oxidative stress in the unstimulated platelet. Supported by grant 92-117 from Taipei Veterans General Hospital