Impact of dengue virus (serotype DENV-2) infection on liver of BALB/c mice: A histopathological analysis

In this research, we characterized the histopathological impact of dengue virus (serotype DENV-2) infection in livers of BALB/c mice. The mice were infected with different doses of DENV-2 via intraperitoneal injection and liver tissues were processed for histological analyses and variation was documented. In the BALB/c mouse model, typical liver tissues showed regular hepatocyte architecture, with normal endothelial cells surrounding sinusoid capillary. Based on histopathological observations, the liver sections of BALB/c mice infected by DENV-2 exhibited a loss of cell integrity, with a widening of the sinusoidal spaces. There were marked increases in the infiltration of mononuclear cells. The areas of hemorrhage and micro- and macrovesicular steatosis were noted. Necrosis and apoptosis were abundantly present. The hallmark of viral infection, i.e., cytopathic effects, included intracellular edema and vacuole formation, cumulatively led to sinusoidal and lobular collapse in the liver. The histopathological studies on autopsy specimens of fatal human DENV cases are important to shed light on tissue damage for preventive and treatment modalities, in order to manage future DENV infections. In this framework, the method present here on BALB/c mouse model may be used to study not only the effects of infections by other DENV serotypes, but also to investigate the effects of novel drugs, such as recently developed nano-formulations, and the relative recovery ability with intact immune functions of host.     

Monoterpene ‘thermometer’ of tropical forest‐atmosphere response to climate warming

Tropical forests absorb large amounts of atmospheric CO₂ through photosynthesis but elevated temperatures suppress this absorption and promote monoterpene emissions. Using ¹³CO₂ labeling, here we show that monoterpene emissions from tropical leaves derive from recent photosynthesis and demonstrate distinct temperature optima for five groups (Groups 1–5), potentially corresponding to different enzymatic temperature‐dependent reaction mechanisms within β‐ocimene synthases. As diurnal and seasonal leaf temperatures increased during the Amazonian 2015 El Niño event, leaf and landscape monoterpene emissions showed strong linear enrichments of β‐ocimenes (+4.4% °C^?1) at the expense of other monoterpene isomers. The observed inverse temperature response of α‐pinene (?0.8% °C^?1), typically assumed to be the dominant monoterpene with moderate reactivity, was not accurately simulated by current global emission models. Given that β‐ocimenes are highly reactive with respect to both atmospheric and biological oxidants, the results suggest that highly reactive β‐ocimenes may play important roles in the thermotolerance of photosynthesis by functioning as effective antioxidants within plants and as efficient atmospheric precursors of secondary organic aerosols. Thus, monoterpene composition may represent a new sensitive ‘thermometer’ of leaf oxidative stress and atmospheric reactivity, and therefore a new tool in future studies of warming impacts on tropical biosphere‐atmosphere carbon‐cycle feedbacks.     

Initial reactions in the fungal degradation of guaiacylglycerol-β-coniferyl ether,a lignin substructure model

Fusarium solani M-13-1 was shake-cultured in a medium containing guaiacylglycerol-β-coniferyl ether (I), a model compound representing the arylglycerol-β-aryl ether linkage in lignin, as sole carbon source. From the culture filtrate guaiacylglycerol-β-coniferyl aldehyde ether (II) and guaiacylglycerol-β-ferulic acid ether (III) were isolated as metabolic products. Incubation with (III) resulted in formation of guaiacylglycerol-β-vanillin ether (IV), which was further metabolized to guaiacyglycerol-β-vanillic acid ether (V). The results indicate that the cinnamyl alcohol group of (I) is initially oxidized to an aldehyde group, which is further oxidized to a carboxyl group, yielding (II) and (III). Compound (III) is converted to (IV) by the release of a C₂ fragment, and the aldehyde group of (IV) is further oxidized to a carboxyl group, giving (V). In the pathway from (I) to (V), neither oxidation of the benzylic secondary alcohol to ketone nor cleavage of the arylglycerol-β-aryl ether linkage was observed. The fungus was found to attack both erythro and threo form without distinction.     

A lymphocyte chemotactic peptide released from immunoglobulin G by neutrophil neutral thiol protease.

A thermostable and dialyzable peptide, released from rabbit IgG by rabbit neutrophil neutral thiol protease, exhibited a distinct chemotactic activity for rat lymphocytes; it was assumed to be derived from the Fc fragment (but not from the Fab fragment) by the enzyme. This substance seemed to be effective for adherent cells (B cells) from rat spleen, but not for nonadherent cells (T cells). The chemotactic peptide was purified by gel filtration on Sephadex G-50 and G-15 and then by high-voltage paper electrophoresis. As previously described, the IgG residue after release of dialyzable peptide(s) exhibited chemotactic activity for neutrophils but not for macrophages.     

Identification of major proteins in maize egg cells

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.     

Characterization of the unfolding process of lipocalin-type prostaglandin D synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.     

Validity of NIR spectroscopy for quantitatively measuring muscle oxidative metabolic rate in exercise

The purpose of this studywas to examine the validity of the quantitative measurement of muscleoxidative metabolism in exercise by near-infrared continuous-wavespectroscopy (NIRcws). Twelve male subjects performed two bouts ofdynamic handgrip exercise, once for the NIRcws measurement and once forthe ³¹P-magnetic resonance spectroscopy (MRS) measurementas a standard measure. The resting muscle metabolic rate (RMRmus) wasindependently measured by ³¹P-MRS during 15 min of arterialocclusion at rest. During the first exercise bout, the quantitativevalue of muscle oxidative metabolic rate at 30 s postexercisewas evaluated from the ratio of the rate of oxyhemoglobin/myoglobindecline measured by NIRcws during arterial occlusion 30 s afterexercise and the rate at rest. Therefore, the absolute values of muscleoxidative metabolic rate at 30 s after exercise[O_2NIR(30)] wascalculated from this ratio multiplied by RMRmus. During the secondexercise bout, creatine phosphate (PCr) resynthesis rate was measuredby ³¹P-MRS at 30 s postexercise[Q₍₃₀₎] under the same conditions but without arterial occlusion postexercise. To determine the validity ofNIRcws, O_2NIR(30) wascompared with Q₍₃₀₎. There was a significant correlation betweenO_2NIR(30), which rangedbetween 0.018 and 0.187 mM ATP/s, and Q₍₃₀₎,which ranged between 0.041 and 0.209 mM ATP/s (r = 0.965, P < 0.001). This result supports theapplication of NIRcws to quantitatively evaluate muscle oxidativemetabolic rate in exercise.

     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.     

Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion

We investigated whether and how rat liver thioredoxin reductase spares alpha-tocopherol in biomembranes. Purified hydroperoxides of beta-linoleoyl-gamma-palmitoylphosphatidylcholine were decreased 35% by treatment with thioredoxin reductase and 54% by thioredoxin reductase plus E. coli thioredoxin. Thioredoxin reductase also halved the amount of hydroperoxides that had been formed during photoperoxidation of liposomes composed of beta-linoleoyl-gamma-palmitoylphosphatidylcholine, and of emulsions of both cholesterol and cholesteryl linolenate. In erythrocyte ghosts, thioredoxin reductase spared alpha-tocopherol from oxidation by both soybean lipoxygenase and ferricyanide. Thioredoxin reductase also decreased F(2)-isoprostanes in ghosts oxidized by ferricyanide, suggesting that its ability to spare alpha-tocopherol relates to reduction of lipid hydroperoxides.