Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain

By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni‐CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni‐CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni‐CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni‐CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni‐CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.     

Global dynamics of two-compartment models for cell production systems with regulatory mechanisms

We present a global stability analysis of two-compartment models of a hierarchical cell production system with a nonlinear regulatory feedback loop. The models describe cell differentiation processes with the stem cell division rate or the self-renewal fraction regulated by the number of mature cells. The two-compartment systems constitute a basic version of the multicompartment models proposed recently by Marciniak-Czochra and collaborators [25] to investigate the dynamics of the hematopoietic system. Using global stability analysis, we compare different regulatory mechanisms. For both models, we show that there exists a unique positive equilibrium that is globally asymptotically stable if and only if the respective reproduction numbers exceed one. The proof is based on constructing Lyapunov functions, which are appropriate to handle the specific nonlinearities of the model. Additionally, we propose a new model to test biological hypothesis on the regulation of the fraction of differentiating cells. We show that such regulatory mechanism is incapable of maintaining homeostasis and leads to unbounded cell growth. Potential biological implications are discussed.     

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca²⁺ ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca²⁺. These findings suggest that even in the myosin-bound (open) state, Ca²⁺ may regulate actomyosin contractile properties via Tm.     

Central and peripheral des-acyl ghrelin regulates body temperature in rats

In the present study using rats, we demonstrated that central and peripheral administration of des-acyl ghrelin induced a decrease in the surface temperature of the back, and an increase in the surface temperature of the tail, although the effect of peripheral administration was less marked than that of central administration. Furthermore, these effects of centrally administered des-acyl ghrelin could not be prevented by pretreatment with [D-Lys3]-GHRP-6 GH secretagogue receptor 1a (GHS-R1a) antagonists. Moreover, these actions of des-acyl ghrelin on body temperature were inhibited by the parasympathetic nerve blocker methylscopolamine but not by the sympathetic nerve blocker timolol. Using immunohistochemistry, we confirmed that des-acyl ghrelin induced an increase of cFos expression in the median preoptic nucleus (MnPO). Additionally, we found that des-acyl ghrelin dilated the aorta and tail artery in vitro. These results indicate that centrally administered des-acyl ghrelin regulates body temperature via the parasympathetic nervous system by activating neurons in the MnPO through interactions with a specific receptor distinct from the GHS-R1a, and that peripherally administered des-acyl ghrelin acts on the central nervous system by passing through the blood–brain barrier, whereas it exerts a direct action on the peripheral vascular system.     

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

Contrasting calcium localization and speciation in leaves of the Medicago truncatula mutant cod5 analyzed via synchrotron X–ray techniques

Oxalate‐producing plants accumulate calcium oxalate crystals (CaOx_(c)) in the range of 3–80% w/w of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca concentrations to wild‐type (WT) plants, but lower oxalate and CaOx_(c) concentrations. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X–ray fluorescence mapping (SXRF). We observed a difference in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, i.e. the opposite of what is observed in WT. X–ray microdiffraction (μXRD) of M. truncatula leaves confirmed that crystalline CaOx_(c) (whewellite; CaC₂O₄·H₂O) was present in the WT only, in cells sheathing the secondary veins. Together with μXRD, microbeam Ca K–edge X–ray absorption near‐edge structure spectroscopy (μXANES) indicated that, among the forms of CaOx, i.e. crystalline or amorphous, only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets.     

Structure–activity relationships of bisphenol A analogs at estrogen receptors (ERs): Discovery of an ERα-selective antagonist

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERβ subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERβ and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.     

Biochemical Characterization of Lipoxygenase Lacking Mutants,L-l-less,L-2-less,and L-3-less Soybeans

In this paper, lipoxygenase lacking mutants were characterized in comparison with normal soybeans. The three lipoxygenase isozymes (L-l, L-2, and L-3) in crude seed extracts of normal soybeans were resolved clearly by an improved SDS-polyacrylamide gel electrophoresis. As expected, the three mutant types, L-l-less (P. I. 408251 and 133226), L-2-less (P. I. 86023), and L-3 less (Wasenatsu and Ichigowase) soybeans did not give L-l, L-2, and L-3 protein bands, respectively on a single dimension SDS gel.

An anti L-2 serum obtained from a rabbit reacted not only with the purified L-2 protein, but also partially with the purified L-l and L-3 proteins. By double immunodiffusion and immuno-disc gel electrofocusing analyses using the anti L-2 serum, L-l, L-2, and L-3 isozymes could not be detected in crude seed extracts from P.I. 408251, P. I. 86023, and Wasenatsu soybeans, respectively.

Three lipoxygenase activity peaks (L-l, L-2, and L-3 enzyme peaks) and a small unknown activity peak eluted right after the L-l peak were fractionated by DEAE-Sephacel column chromatography of crude seed extracts of Raiden (normal) soybeans. The chromatographic analyses have demonstrated that both the L-l and the unknown enzyme activities disappear completely in the L-l-less type soybean seeds, and that the L-2 and L-3 enzyme activities disappear completely in P. I. 86023 and the L-3-less type soybean seeds, respectively.