Laser photolysis of carboxymethylated chitin derivatives in aqueous solution. Part 1. Formation of hydrated electron and a long-lived radical

Laser photolysis experiments on carboxymethylated chitin derivatives, such as carboxymethyl chitin (CM-chitin) and carboxymethyl chitosan (CM-chitosan), in aqueous solution by a 248 nm excimer laser were carried out for the first time. The transient absorption spectra of photolyzed CM-chitin or CM-chitosan solutions revealed a strong band with the maximum at 720 nm, which was assigned to the hydrated electron (eaq-). In the presence of argon, the eaq- decays by reacting with CM-chitin or CM-chitosan, and the rate constants are (6.1 +/- 0.1) x 10(7) M(-1) s(-1) and (3.7 +/- 0.1) x 10(7) M(-1) s(-1), respectively. Long-lived radicals with relatively weak absorption intensity were detected in the near-UV region. The absorption band was not notably characteristic and showed only an increasing absorption toward shorter wavelengths. It is similar to the signal of *CM-chitin or *CM-chitosan macroradicals formed by the reaction of CM-chitin or CM-chitosan with an OH* radical. It was assigned to *CM-chitin- or *CM-chitosan- macroradicals formed by eaq- + CM-chitin or CM-chitosan reaction. CM-chitin aqueous solutions were further examined by pulse radiolysis in order to confirm the site of the long-lived radical.     

Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor

The application of omega-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20% (w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70 degrees C. A plot of the natural logarithm of the frequency factor, In ka0, vs. the activation energy, Ea, demonstrated that In ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.     

Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.     

Role of side-chains in the cooperative beta-hairpin folding of the short C-terminal fragment derived from streptococcal protein G

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

Entrainment of Drosophila circadian rhythms by temperature cycles

Sleep and Biological Rhythms - The fruit fly, Drosophila melanogaster, has been a good organism for elucidating the molecular and cellular bases of circadian behavioral rhythms. The fly shows a...     

Optimal conditions for plaque assay of echoviruses in human embryonic lung fibroblast cells

The present paper gives optimal conditions for plaque assays tn human embryonic lung fibroblast cells with 45 echoviruses including 11 fresh isolates, covering 31 serotypes. All the viruses tested except echovirus type 23 produced plaques at a high titer in the same cells by the methods described in this communication. Plaque formations by a number of strains were markedly enhanced in size or number or both by the addition of polycation.     

Infection of XC cells by MLVs and Ebola virus is endosome-dependent but acidification-independent

Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells.     

Deletion in open reading frame 49 of varicella-zoster virus reduces virus growth in human malignant melanoma cells but not in human embryonic fibroblasts

The ORF49 gene product (ORF49p) of the varicella-zoster virus (VZV) is likely a myristylated tegument protein, and its homologs are conserved across the herpesvirus subfamilies. The UL11 gene of herpes simplex virus type 1 and of pseudorabies virus and the UL99 gene of human cytomegalovirus are the homologs of ORF49 and have been well characterized by using mutant viruses; however, little research on the VZV ORF49 gene has been reported. Here we report on VZV ORF49p expression, subcellular localization, and effect on viral spread in vitro. ORF49p was expressed during the late phase of infection and located in the juxtanuclear region of the cytoplasm, where it colocalized mainly with the trans-Golgi network-associated protein. ORF49p was incorporated into virions and showed a molecular mass of 13 kDa in VZV-infected cells and virions. To elucidate the role of the ORF49 gene, we constructed a mutant virus that lacked a functional ORF49. No differences in plaque size or cell-cell spread were observed in human embryonic fibroblast cells, MRC-5 cells, infected with the wild-type or the mutant virus. However, the mutant virus showed diminished cell-cell infection in a human malignant melanoma cell line, MeWo cells. Therefore, VZV ORF49p is important for virus growth in MeWo cells, but not in MRC-5 cells. VZV may use different mechanisms for virus growth in MeWo and MRC-5 cells. If so, understanding the role of ORF49p should help elucidate how VZV accomplishes cell-cell infections in different cell types.