Neutrophil restraint by green tea: inhibition of inflammation,associated angiogenesis,and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (-)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.     

CTLA-4 blockade enhances the therapeutic effect of an attenuated poxvirus vaccine targeting p53 in an established murine tumor model

p53 is overexpressed by half of all cancers, and is an attractive target for a vaccine approach to immunotherapy. p53 overexpression is frequently the result of point mutations, which leaves the majority of the protein in its wild-type form. Therefore, the majority of p53 sequence is wild type, making it a self-protein for which tolerance plays a role in limiting immune responses. To overcome tolerance to p53, we have expressed wild-type murine p53 in the nonpathogenic attenuated poxvirus, modified vaccinia virus Ankara (recombinant modified vaccinia virus Ankara expressing wild-type murine p53 (rMVAp53)). Mice immunized with rMVAp53 vaccine developed vigorous p53-specific CTL responses. rMVAp53 vaccine was evaluated for its ability to inhibit the outgrowth of the syngeneic murine sarcoma Meth A, which overexpresses mutant p53. Mice were inoculated with a lethal dose (5 x 10(5) cells injected s.c.) of Meth A tumor cells and vaccinated by i.p. injection 3 days later with 5 x 10(7) PFU of rMVAp53. The majority of mice remained tumor free and resistant to rechallenge with Meth A tumor cells. We wished to determine whether rMVAp53 immunization could effect the rejection of an established, palpable Meth A tumor. In subsequent experiments, mice were injected with 10(6) Meth A tumor cells, and treated 6 days later with anti-CTLA-4 Ab (9H10) and rMVAp53. The majority of treated mice had complete tumor regression along with lasting tumor immunity. In vivo Ab depletion confirmed that the antitumor effect was primarily CD8 and to a lesser extent CD4 dependent. These experiments demonstrate the potential of a novel cell-free vaccine targeting p53 in malignancy.     

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus permits replication of terminal repeat-containing plasmids

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus can associate with mitotic chromosomes and promote latent episome maintenance and segregation. Here we report that LANA also mediates the replication of plasmid DNAs bearing viral terminal repeats. The predicted secondary structure of LANA's C terminus reveals striking similarity to the known structure of the DNA-binding domain of Epstein-Barr virus EBNA1, despite the absence of primary sequence homology between these proteins, suggesting conservation of the key mechanistic features of latent gammaherpesvirus DNA replication.     

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.     

A single site in human beta-hexosaminidase A binds both 6-sulfate-groups on hexosamines and the sialic acid moiety of GM2 ganglioside

Human beta-hexosaminidase A (Hex A) (alphabeta) is composed of two subunits whose primary structures are approximately 60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay-Sachs disease, alpha deficiency, and Sandhoff disease, beta deficiency. Whereas both subunits contain active sites only the alpha-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the alphaArg(424) as playing a critical role in the binding of 6-sulfate-containing substrates, and betaAsp(452) as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an alphaArg(424)Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[3H]-GM2 ganglioside complex as a substrate. The mutant showed a approximately 3-fold increase in its K(m) for the complex. Next a form of Hex B (betabeta) containing a double mutation, betaAspLeu(453)AsnArg (duplicating the alpha-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this alpha-site is critical for binding both types of negatively charge substrates.     

Probing Ca2+-induced conformational changes in porcine calmodulin by H/D exchange and ESI-MS: effect of cations and ionic strength

We applied a new method, "protein-ligand interaction using mass spectrometry, titration, and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determine the conformational changes, binding stoichiometry, and binding constants for Ca(2+) interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca(2+)-binding, as revealed by the PLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca(2+) binding. The Ca(2+)-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational change in apo-CaM. Furthermore, Ca(2+)-saturated CaM exhibits larger protection and higher Ca(2+) affinity in media of low rather than high ionic strength. Both Ca(2+) and Mg(2+) bind to CaM with different affinities, causing different conformational changes. K(+), if it does bind, causes no detectable conformational change, and interactions of Ca(2+) with CaM in the presence of Li(+), Na(+), and K(+) occur with similar affinities and associated changes in solvent accessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metal ions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast, intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein-ligand interactions.     

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.     

Heart rate-arterial blood pressure relationship in conscious rat before vs. after spinal cord transection

This experiment quantified the initial disruption and subsequent adaptation of the blood pressure (BP)-heart rate (HR) relationship after spinal cord transection (SCT). BP and HR were recorded for 4 h via an implanted catheter in neurally intact, unanesthetized rats. The animals were then anesthetized, and their spinal cords were severed at T(1)-T(2) (n = 5) or T(4)-T(5) (n = 6) or sham lesioned (n = 4). BP was recorded for 4 h daily over the ensuing 6 days. The neurally intact rat showed a positive cross correlation, with HR leading BP at the peak by 1.8 +/- 0.8 (SD) s. The cross correlation in unanesthetized rats (n = 2) under neuromuscular blockade was also positive, with HR leading. After SCT at T(1)-T(2), the cross correlation became negative, with BP leading HR, and did not change during the next 6 days. The cross correlation also became negative 1-3 days after SCT at T(4)-T(5), but in four rats by day 6 and thereafter the cross correlation progressively reverted to a positive value. We propose that the positive cross correlation with HR leading BP in the intact rat results from an open-loop control that depends on intact supraspinal input to sympathetic preganglionic neurons in the spinal cord. After descending sympathetic pathways were severed at T(1)-T(2), the intact vagal pathway to the sinoatrial node dominated BP regulation via the baroreflex. We suggest that reestablishment of the positive correlation after SCT at T(4)-T(5) was attributable to the surviving sympathetic outflow to the heart and upper vasculature reasserting some effective function, perhaps in association with decreased spinal sympathetic hyperreflexia. The HR-BP cross correlation may index progression of sympathetic dysfunction in pathological processes.     

Slow rate during AF improves ventricular performance by reducing sensitivity to cycle length irregularity

Atrial fibrillation (AF) is characterized by short and irregular ventricular cycle lengths (VCL). While the beneficial effects of heart rate slowing (i.e., the prolongation of VCL) in AF are well recognized, little is known about the impact of irregularity. In 10 anesthetized dogs, R-R intervals, left ventricular (LV) pressure, and aortic flow were collected for >500 beats during fast AF and when the average VCL was prolonged to 75%, 100%, and 125% of the intrinsic sinus cycle length by selective atrioventricular (AV) nodal vagal stimulation. We used the ratio of the preceding and prepreceding R-R intervals (RR(p)/RR(pp)) as an index of cycle length irregularity and assessed its effects on the maximum LV power, the minimum of the first derivative of LV pressure, and the time constant of relaxation by using nonlinear fitting with monoexponential functions. During prolongation of VCL, there was a pronounced decrease in curvature with the formation of a plateau, indicating a lesser dependence on RR(p)/RR(pp). We conclude that prolongation of the VCL during AF reduces the sensitivity of the LV performance parameters to irregularity.