Capping of Viral RNA in Cultured Spodoptera frugiperda Cells Infected with Autographa californica Nuclear Polyhedrosis Virus

Viral RNA from fall armyworm (Spodoptera frugiperda) cells infected with Autographa californica nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [³H]methionine or ³²P_i cochromatographed on DEAE-cellulose with the −5 charge marker; a minor component appeared at −4 net charge. The former is probably a cap 1 structure (m⁷GpppX^m_pYp), and the latter is probably a cap 0 (m⁷GpppXp). On the basis of relative labeling of the two caps with [³H]adenosine and [³H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m⁷GpppAp. Cleavage of [³H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m⁷Gp, confirming the m⁷GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of ³²P_i incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.     

Active H+ transport in the turtle urinary bladder. Coupling of transport to glucose oxidation

The turtle urinary bladder acidifies the contents of its lumen by actively transporting protons. H+ secretion by the isolated bladder was measured simultaneously with the rate of 14CO2 evolution from [14C]glucose. The application of an adverse pH gradient resulted in a decline in the rate of H+ secretion (JH) and in the rate of glucose oxidation (JCO2). The changes in JH and JCO2 were linear functions of the pH difference across the membrane. Hence, JH and JCO2 were linearly related to each other. The slope, deltaJH/deltaJCO2 was found to be similar in half-bladders from the same animal but was seen to vary widely in a population of turtles. To investigate the effect of pH gradients on deltaJH/deltaJCO2, two experiments were performed in each of 14 hemibladders. In one, JH and JCO2 were altered by changing the luminal pH. In the other, they were altered by changing the ambient pCO2 while the luminal pH was kept constant. The average slope, deltaJH/deltaJCO2, in the presence of pH gradients was 14.45 eq-mol-1. In the absence of gradients in the same hemibladders it was 14.72, delta = 0.27 +/- 1.46. The results show that H+ transport is organized in such a way that leaks to protons in parallel to the pump are negligible. Analysis of the transport system by use of the Essig-Caplan linear irreversible thermodynamic formalism shows that the system is tightly coupled. The degree of coupling, q, given by that analysis was measured and found to be at or very near the maximum theoretical value.     

Insulin stimulation of heart glycogen synthase D phosphatase (protein phosphatase).

Insulin rapidly produced an increase in per cent of total heart glycogen synthase in the I form in fed rats. In fasted rats the response was diminished and delayed. In diabetic animals there was no response over the 15-min time period studied. Since synthase phosphatase activity is necessary for synthase D to I conversion, the phosphatase activity was determined in extracts from these groups of animals. In the fasted and diabetic rats phosphatase activity was less than one-half of that in fed animals. Administration of insulin to fasting animals increased synthase phosphatase activity to a level approaching that of fed animals by 15 min. In diabetic animals insulin also stimulated an increase in synthase phosphatase activity but 30 min were required for full activation. Insulin had no effect in normal fed animals. Insulin activation of synthase phosphatase activity in heart extracts from fasted animals was still present after Sephadex G-25 chromatography and ammonium sulfate precipitation. Thus insulin had induced a stable modification of the phosphatase itself or of its substrate synthase D rendering the latter a more favorable substrate for the reaction. A difference in sensitivity of the reaction to glycogen inhibition was present between fed and fasted animals. Increasing concentrations of glycogen had only a slight inhibitory effect in extracts from fed animals but considerably reduced activity in extracts from fasted animals. Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition. This could explain, at least in part, the increased phosphatase activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.     

The origin of the Adams-Schuster difference spectrum of oxyhemoglobin.

The characteristic difference spectrum reported by Adams and Schuster (Biochem. Biophys. Res. Commun. 1974, $\text{58}$, 525) on the addition of inositol hexaphosphate to oxyhemoglobin is similar to the difference spectrum between (i) isolated α- and β-chains, (ii) α- and β-semihemoglobins, (iii) addition of inorganic phosphate to oxyhemoglobin, (v) change in temperature of a solution of oxyhemoglobin, (v) change in pH of carp carboxyhemoglobin and (vi) addition of inositol hexaphosphate to α-semihemoglobin. The spectrum may also be generated by differentiation of the spectra of oxyhemoglobin and carboxyhemoglobin, implying that the common feature of the results reported above is a shift in the position of the absorption bands. This shift may arise from several causes and so its interpretation is uncertain.     

Ultraviolet-mediated antibiotic activity of thiophene compounds of Tagetes

Two intensely mauve UV fluorescent compounds isolated from Tagetes root were found to be phototoxic to Candida albicans. By chromatography on alumina followed by gel filtration on Sephadex LH-20, the compounds were identified as 5-(3-buten-1-ynyl)-2,2′-bithienyl and α-terthienyl.     

JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum

Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma. Because the parasite’s eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied to S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg–induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis.     

Genetic Polymorphisms of Pneumocystis Jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. Open in a separate window     

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)‐related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site‐directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand‐binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR‐inducing effector in V. dahliae that contributes to the activation of plant immunity.     

Water Striders:The Biomechanics of Water Locomotion and Functional Morphology of the Hydrophobic Surface(Insecta:Hemiptera-Heteroptera)

Water striders are insects living on the water surface, over which they can move very quickly and rarely get wetted. We measured the force of free walking in water striders, using a hair attached to their backs and a 3D strain gauge. The error was calculated by comparing force and data derived from geometry and was estimated as 13%. Females on average were stronger (1.32 mN) than males (0.87 mN), however, the ratio of force to weight was not significantly different. Compared with other lighter species, Aquarius paludum seems stronger, but the ratio of force to weight is actually lower. A. paludum applies about 0.3 mN·cm-1 to 0.4 mN·cm-1 with its mid-legs, thus avoiding penetrating the surface tension layer while propelling itself rapidly over the water surface.We also investigated the external morphology with SEM. The body is covered by effectively two layers of macro-and micro-hairs, which renders them hydrophobic. The setae are long (40 um-60 um) and stiff, being responsible for waterproofing, and the microtrichia are much smaller (<10 um), slender, and flexible, holding a bubble over the body when submerged.