Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid

Degradation and the antioxidative effect of Na-, Zn-, Co-, Cu-, and Mn-hyaluronic acid (HA) associates were studied. Our findings revealed the protective effect of certain counterions against ROS-induced HA degradation. We could also separate the antioxidative effect of certain counterions from that of the HA by examining the effect of the counterions in their free ionic forms. The result showed that metal ions with altering oxidative status (Co(2+), Cu(2+), Mn(2+)) proved to be effective in themselves or their effect added to that of HA when HA was also effective. Moreover, the effects of Co-HA against z.rad;O(2)(-) and of Mn-HA against ONOO(-) as well as the synergic effect of Zn-HA associates where Zn(2+) is of fixed oxidative status were attributed to the structure-stabilizing complex formed between certain counterions and HA. Our examination also concerned the influence of HA associates on the indirect antioxidation related to Fe(2+) chelating. The individual effects of Zn(2+), Co(2+), and Cu(2+) were only detectable, which could be explained by the competitive displacement of ferrous from its binding site.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Proteasome-cytochrome c interactions: a model system for investigation of proteasome host-guest interactions

Owing to its high thermal stability and structural simplicity, the archaebacterium Thermoplasma Acidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzyme complex consists of a barrel-shaped 20S core (approximately 700kDa) comprised of four stacked seven-membered rings with a alpha(7)beta(7)beta(7)alpha(7) subunit structure situated around a 7-fold symmetry axis. The hollow interior of the proteasome has three large interconnected chambers with narrow (13 A diameter) entrances from solution located at either end of the barrel. The 14 beta-subunit proteolytic sites are located on the inner surface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome c (Cyt c) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditions of temperature and denaturant which unfold Cyt c but do not alter the thermophilic proteasome, Cyt c is extensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasome digest. Analysis of the cleavage products established that unfolded Cyt c and its covalently attached heme prosthetic group are translocated to the central chamber where proteolysis occurs. In the presence of site-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome c can be sequestered inside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis of the complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cyt c is essentially intact within the inhibited proteasome. High-performance liquid chromatography data show that (1) complexes with an apparent stoichiometry of approximately one Cyt c per proteasome can be formed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt c occurs.     

RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli

The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Effects of age and anesthetic on plasma glucose and insulin levels and insulin sensitivity in spontaneously hypertensive and Wistar rats

We examined the effects of anesthetic, age, and strain on oral glucose tolerance tests (OGTT, 1 g/kg body weight) and intraperitoneal glucose tolerance tests (IPGTT, 2 g/kg body weight) in spontaneously hypertensive (SH) and Wistar rats. Pentobarbital anesthesia caused an elevation in basal glucose and insulin levels in Wistar rats at 9 and 16 weeks of age and in SH rats at 9 weeks. Anesthesia increased the insulin output during an OGTT in both strains of rats while glucose was unchanged. Anesthesia reduced the insulin sensitivity index calculated from the OGTT but not from the IPGTT data. The age of the rats (9-11 vs. 16-18 weeks) had no effect on the basal glucose or insulin levels, but older Wistar rats had a greater insulin output following oral glucose and older SH rats had a greater insulin output following intraperitoneal glucose. On the basis of the insulin sensitivity index, SH rats were clearly more insulin resistant than age-matched Wistar rats. The SH rats also had higher basal insulin levels, as well as higher insulin output, following both glucose challenges. In summary, SH rats are more insulin resistant than Wistar rats, and anesthesia, which elevated basal glucose and insulin levels and increased the insulin output in response to a glucose challenge, may increase insulin resistance.     

Standardization of the method proposed by the World Health Organization for determining the susceptibility levels of leishmaniasis vectors to insecticides

The WHO method for determining insecticide resistance was standardized for several species of Lutzomyia sand flies under laboratory and field conditions. The biological assays were applied solely to optimize the conditions for the control, i.e., without insecticide, and to estimate mortality due to handling or other unfavorable conditions. Adult female flies from 3 laboratory colonies and one field strain were tested: two laboratory strains of Lutzomyia longipalpis, one laboratory strain of Lutzomyia serrana and one field-collected strain of Lutzomyia quasitownsendi. The WHO method was compared with one modified in which, during the post-exposure period, the recommended plain tube apparatus was replaced with a plastic container layered with damp plaster of Paris. Three paper substrate types were compared under each condition: olive oil additive, silicon oil additive and plain paper. The measured variable was percent mortality in 24 h. For the WHO protocol, the L. longipalpis strains indicated a 0-10% mortality, L. serrana 20-80% and L. quasitownsendi 10-50%. With the modified WHO apparatus, the average mortality was < 4% for all species. No significant differences were observed among the paper treatments. These results indicate a strong species-specific effect of post-exposure conditions on sand flies. To establish baseline levels of insecticide resistance in Lutzomyia sand flies, the WHO method is recommended only for L. longipalpis, and the modified method for L. serrana, L. quasitownsendi and closely related species.     

Neurotoxic mechanisms triggered by Alzheimer's disease-linked mutant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin target

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.