Curcumin inhibits ultraviolet light induced human immunodeficiency virus gene expression

Recently, we reported that the herbal drug St. John's Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat/HeLa cells [35]. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-B are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism by which curcumin inhibits UV-activated HIV-LTR gene expression. We found that treatment of HIVcat/HeLa cells with micromolar concentrations of curcumin completely abolished UV activation of HIV gene expression. Curcumin treatment at similar doses as those used to inhibit HIV gene expression also effectively blocked UV activation of NF-B, as demonstrated by electrophoretic mobility shift assay. In contrast, curcumin did not inhibit UV-induced phosphorylation of p38 MAP kinase. This observation was also supported by findings that curcumin did not inhibit UV-induced phosphorylation of CREB/ATF-1 and ATF-2. Although curcumin was ineffective in preventing UV-induced p44/42 MAP kinase phosphorylation, the JNK (1 and 2) and AP-1 activation were efficiently blocked by curcumin in HeLa cells. We conclude that the mechanism by which curcumin modulates UV activation of HIV-LTR gene expression mainly involves the inhibition of NF-B activation.     

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1

We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.     

Noninvasive determination of ulnar stiffness from mechanical response--in vivo comparison of stiffness and bone mineral content in humans

An approach referred to as Mechanical Response Tissue Analysis (MRTA) has been developed for the noninvasive determination of mechanical properties of the constituents of the intact limb. Of specific interest in the present study is the bending stiffness of the ulna. The point mechanical impedance properties in the low frequency regime, between 60 and 1,600 Hz are used. The procedure requires a proper design of the probe for good contact of the skin at midshaft and proper support of the proximal and distal ends of the forearm to obtain an approximation to "simple support" of the ulna. A seven-parameter model for the mechanical response is then valid, which includes the first mode of anterior-posterior beam bending of the ulna, the damping and spring effect of the soft tissue between probe and bone, and the damping of musculature. A dynamic analyzer (HP3562A) provides in seconds the impedance curve and the pole-zero curve fit. The physical parameters are obtained from a closed-form solution in terms of the curve-fit parameters. The procedure is automated and is robust and analytically reliable at about the five percent level. Some 80 human subjects have been evaluated by this mechanical response system and by the Norland single photon absorptiometer, providing for the first time in vivo, a comparison of elastic bending stiffness (ulna) and bone mineral content (radius). Three functional parameters of potential clinical value are the cross-sectional bending stiffness EI, the axial load capability Pcr (Euler buckling load) and the bone "sufficiency" S, defined as the ratio of Pcr to body weight. The correlation between EI and bone mineral (r = 0.81) is only slightly less than previous in vitro results with both measurements on the same bone (r = 0.89). When sufficiency is taken into consideration, the correlation of Pcr and bone mineral content is improved (r = 0.89). An implication is that "quality" of bone is a factor which is not indicated by bone mineral content but which is indicated by stiffness. Bone mineral is necessary for proper stiffness but not sufficient. Therefore mechanical measurement should provide a new dimension to be used toward a better understanding of the factors related to bone health and disease.     

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.     

Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

Homonuclear and heteronuclear transition metal complexes by syn-proportionation reactions

Redox processes consisting of disproportionation and syn-proportionation are reviewed with special attention to metal complexes containing carbon-based ligands, i.e. carbon monoxide or unsaturated hydrocarbons. An introduction and a survey of reactions aimed to show the large applicability of syn-proportionation reactions in the field of coordination chemistry, is followed by examples of the use of these redox processes for the preparation of catalytic precursors. The latter studies derive from the idea that if a syn-proportionation reaction can be carried out between two complexes containing different metals in different oxidation states, inter-metallic systems could be formed which may act as active catalysts, e.g. for polymerization reactions.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions

Gram‐positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT‐1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in‐frame deletion mutants, Lactococcus expressing heterologous FCT‐1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter‐bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three‐dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection.     

“Bacillus thermoantarcticus” sp. nov., from Mount Melbourne,Antarctica: a novel thermophilic species

 A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63^°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellular α-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G+C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572). Received : 3 February 1995/Accepted : 12 May 1995