The kinase activity of PKR represses inflammasome activity

The protein kinase R (PKR) functions in the antiviral response by controlling protein translation and inflammatory cell signaling pathways. We generated a transgenic, knock-in mouse in which the endogenous PKR is expressed with a point mutation that ablates its kinase activity. This novel animal allows us to probe the kinase-dependent and -independent functions of PKR. We used this animal together with a previously generated transgenic mouse that is ablated for PKR expression to determine the role of PKR in regulating the activity of the cryopyrin inflammasome. Our data demonstrate that, in contradiction to earlier reports, PKR represses cryopyrin inflammasome activity. We demonstrate that this control is mediated through the established function of PKR to inhibit protein translation of constituents of the inflammasome to prevent initial priming during innate immune signaling. These findings identify an important role for PKR to dampen inflammation during the innate immune response and caution against the previously proposed therapeutic strategy to inhibit PKR to treat inflammation.     

Bacteriocin activity and probiotic activity of Aeromonas media

Three strains of Aeromonas media (161, A164 and A199) were shown to be active in-vitro producers of bacteriocin-like inhibitory substances (BLIS). For example, the producer strain, Aer. media A199, displayed antagonistic activity against all strains tested of Aer. caviae, Aer. hydrophila, Aer. salmonicida, Aer. veronii var. sobria, Listonella anguillarum, Photobacterium damsella, eight species of Vibrio and Yersinia ruckeri. Because of this wide-ranging activity against fish/shellfish pathogens, A199 was chosen for the probiotic work. By contrast, however, the BLIS produced by A199 did not inhibit the growth of Enterococcus seriolicida. The aim of the project was to ascertain whether or not the activity observed in vitro could be repeated in vivo. The ability of BLIS-producing strain A199 to act as a probiotic was assessed on the host animal, Crassostrea gigas, by testing whether or not strain A199 could prevent death of the oyster larvae when challenged with V. tubiashit. Whereas larvae, challenged with the Vibrio, died within 5 days, the presence of both the pathogen and the probiotic strain, together, did not affect the viability of the larvae over the same time period; the viability of larvae challenged with A199 alone was also unaffected when compared with the viability of unchallenged larvae (controls). These findings have important, economic implications for those engaged in the oyster producing industry where heavy losses can be experienced as a result of an infectious outbreak. At this stage, the association between BLIS activity and probiotic activity is circumstantial and, hence, future work will involve the use of non-BLIS-producing strains of Aer. media and BLIS-negative variants of the producer. Moreover, extension of the project will involve the use of other BLIS-producing strains (A161, and A164), hosts (salmon, crayfish, scallops and abalone) and pathogens.     

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

Antagonizing activity of chick Grg4 against tectum-organizing activity

Alar plate of chick mesencephalon differentiates into the optic tectum. It has been shown that factors expressed in the mes-metencephalic boundary induce the tectum and give positional specificity. Chick Grg4 is expressed at first in the anterior neural fold. The expression localizes from the posterior diencephalon to the mesencephalon by stage 10. To investigate the function of Grg4 in mesencephalic development, Grg4 overexpression was carried out by in ovo electroporation. After Grg4 overexpression, expression of En-2, Pax5, Fgf8, and EphrinA2 was repressed, and Pax6 was upregulated in the mesencephalic region. Grg4 overexpression caused the morphological change; mesencephalic swelling became smaller and the di-mesencephalic boundary shifted posteriorly, that is, the anterior limit of tectum shifted posteriorly. Importantly, cotransfection of Grg4 with Pax5 canceled the tectum-inducing activity of Pax5. These results suggest that Grg4 works as an antagonist against tectum-organizing activity. It was also shown that transfected N-terminal domains of Grg4 induced En-2 expression. Since N-terminal domains were transported to the nucleus in the neuroepithelium, they could act as dominant negative for endogenous Grg4. These results indicate that Grg4 has repressing activity against the organizing molecules and suggest that Grg4 plays important roles in formation of anterior tectal boundary and polarity.     

Antiplasmodial activity of hydroxyethylamine analogs: Synthesis,biological activity and structure activity relationship of plasmepsin inhibitors

Malaria, particularly in endemic countries remains a threat to the human health and is the leading the cause of mortality in the tropical and sub-tropical areas. Herein, we explored new C₂ symmetric hydroxyethylamine analogs as the potential inhibitors of Plasmodium falciparum (P. falciparum; 3D7) in in-vitro cultures. All the listed compounds were also evaluated against crucial drug targets, plasmepsin II (Plm II) and IV (Plm IV), enzymes found in the digestive vacuole of the P. falciparum. Analog 10f showed inhibitory activities against both the enzymes Plm II and Plm IV (K_i, 1.93?±?0.29?µM for Plm II; K_i, 1.99?±?0.05?µM for Plm IV). Among all these analogs, compounds 10g selectively inhibited the activity of Plm IV (K_i, 0.84?±?0.08?µM). In the in vitro screening assay, the growth inhibition of P. falciparum by both the analogs (IC₅₀, 2.27?±?0.95?µM for 10f; IC₅₀, 3.11?±?0.65?µM for 10g) displayed marked killing effect. A significant growth inhibition of the P. falciparum was displayed by analog 12c with IC₅₀ value of 1.35?±?0.85?µM, however, it did not show inhibitory activity against either Plms. The hemolytic assay suggested that the active compounds selectively inhibit the growth of the parasite. Further, potent analogs (10f and 12c) were evaluated for their cytotoxicity towards mammalian HepG2 and vero cells. The selectivity index (SI) values were noticed greater than 10 for both the analogs that suggested their poor toxicity. The present study indicates these analogs as putative lead structures and could serve as crucial for the development of new drug molecules.     

A special deoxyribonuclease activity accompanying competence-inducing activity

A deoxyribonuclease activity accompanies the competence substance isolated from transformableDiplococcus pneumoniae even in well purified fractions. The deoxyribonuclease seems to exhibit a rather different kind of activity from the one found as a major nuclease in a partially purified competence substance. The products of interaction between the enzyme and double-stranded DNA would indicate that the enzyme might act as an “unwindase” on the double-stranded DNA.     

Separation of toxic activity and ADP-ribosylation activity of botulinum neurotoxin D

Neurotoxin from Clostridium botulinum type D strain South African (neurotoxin D) has shown ADP-ribosylation activity as well as toxic activity (Matsuoka, I., Sakuma, H., Syuto, B., Moriishi, K., Kubo, S., and Kurihara, K. (1989) J. Biol. Chem. 264, 706-712). Separation of these activities from each other was attempted by means of gel filtration, hydroxylapatite column chromatography, or immunoaffinity chromatography. Approximately 90% of toxic activity was recovered in each chromatography. Although ADP-ribosylation activity was incompletely separated from neurotoxin D by gel filtration, it was separated by hydroxylapatite column chromatography. In immunoaffinity chromatography with a column of Sepharose 4B coupling antibodies against botulinum ADP-ribosyltransferase, no ADP-ribosylation activity was detected by autoradiography in the unabsorbed toxic fraction. These results indicate that neurotoxin D does not have ADP-ribosylation activity.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

Synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity

Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found to show moderate antioxidant activity (IC(50) 13 microM in DPPH radical scavenging; 32 microM in superoxide anion scavenging). Modification of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity. Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norbergenin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at 10 microM on the primary culture of rat cortical neurons in DMEM supplemented with N2.     

Influence of water activity on the ice-nucleating activity of Pseudomonas syringae

Pseudomonas syringae is known as a biological ice-nucleating agent. The bacterium has the unusual property of increasing the temperature at which water freezes by a few degrees. However, the ice-nucleating activity (INA) always remains lower for in vitro cultivated cells, than for cells grown in planta. We examined the effects of the hydrophobic environment and of water availability, on the in vitro growth and INA of P. syringae. The hydrophobic environment was modified by addition of fatty acids, vegetable oils or silicone oil to the culture medium. Addition of olive oil (1%), or traces of silicone oil in the culture medium had a positive effect upon the expression of INA. Variations in water activity from 0.990 to 0.988 by addition of sugar beet fibres or sodium chloride in the culture medium were followed by an increase in INA. This study suggested that control of the medium’s water activity must be considered as an important parameter for optimization of INA in P. syringae. Received 16 June 1998/ Accepted in revised form 02 September 1999     

Synthesis and thromboxane A2 antagonistic activity activity of indane derivatives

A new series of indane derivatives were prepared and evaluated for their thromboxane A2 (TXA2, 1) antagonistic activity. Among these compounds, 24a (Z-335) was found to be a potent TXA2 antagonist in oral administration.     

Bifurcation of periodic activity from periodic activity in a molluscan neurone

The response of a molluscan neurone to intra-somatic micro-iontophoresis of TEA⁺ is a periodic discharge of action potentials that bifurcates into doublet, triplet and bursting discharges, and then through regular, small amplitude oscillations to a depolarized steady state. This response pattern is explained by the presence of two TEA⁺-sensitive, K⁺-selective conductances: we conjecture that the maximal fast, transient K⁺-conductance acts as the bifurcation parameter.     

Neuronal activity

The knowledge of the mechanisms regulating electric neuronal activity is fragmented by the wide variety of techniques and experimental models currently used in neurophysiological research. The interest and importance of the results obtained in any research is improved when interpreted in the perspective of the organism functioning as a whole in physiological conditions. Such interpretation, freed of the constraints imposed by the different techniques and experimental conditions used, is especially important when discussing together results obtained at the behavioral, cellular, and molecular level. This article outlines some of the key factors to consider when experiments from different models are interpreted together.