BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.     

Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3' residue

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3' residue elective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.     

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

Stability of reconstructed paralyzed shoulders using a reflected long head biceps technique

A new tendon transfer technique is proposed for the reconstruction of the paralyzed shoulders secondary to Brachial Plexus Injury (BPI). In this tendon transfer, the long head of the biceps tendons is utilized as a bridging tendon graft. It is reflected at the exit of the bicipital groove, passed through the deltoid and directed to the trapezius. The technique is referred to here as the Reflected Long Head Bicepts (RLHB) technique. This study evaluated the effect of this tendon transfer on the anterior, posterior, and inferior stability of the reconstructed should using cadaveric specimens. It was shown that loading of the RLHB contributed significantly to anterior stability of the reconstructed shoulder for 90 deg elevation in the scapula plane. The mean displacement was reduced by 56 percent with RLHB loaded (p<0.01), by 56 percent with the rotator cuff loaded (p <0.005), and by 67 percent with both the RLHB and the rotator cuff loaded (p<0.004). For the post-operation conditions, variation of the directions of RLHB had no significant effect on joint displacement in response to anterior loading. The RLHB tendon also contributed to the posterior and inferior stability for the low and middle elevations in the plane of scapula. Two variations of the RLHB tendon transfer procedures, namely the "Sub-Deltoid" and the "Through-Deltoid" techniques, were introduced and studied. These two techniques did not seem to have significantly different effects on the displacement of the humeral head in response to both posterior and inferior loading. The results of this study seemed to support the clinical feasibility of this tendon transfer approach as far as the biomedical stability of the reconstruction is concerned.     

Vitamin C prevents hyperoxia-mediated vasoconstriction and impairment of endothelium-dependent vasodilation

High arterial blood oxygen tension increases vascular resistance, possibly related to an interaction between reactive oxygen species and endothelium-derived vasoactive factors. Vitamin C is a potent antioxidant capable of reversing endothelial dysfunction due to increased oxidant stress. We tested the hypotheses that hyperoxic vasoconstriction would be prevented by vitamin C, and that acetylcholine-mediated vasodilation would be blunted by hyperoxia and restored by vitamin C. Venous occlusion strain gauge plethysmography was used to measure forearm blood flow (FBF) in 11 healthy subjects and 15 congestive heart failure (CHF) patients, a population characterized by endothelial dysfunction and oxidative stress. The effect of hyperoxia on FBF and derived forearm vascular resistance (FVR) at rest and in response to intra-arterial acetylcholine was recorded. In both healthy subjects and CHF patients, hyperoxia-mediated increases in basal FVR were prevented by the coinfusion of vitamin C. In healthy subjects, hyperoxia impaired the acetylcholine-mediated increase in FBF, an effect also prevented by vitamin C. In contrast, hyperoxia had no effect on verapamil-mediated increases in FBF. In CHF patients, hyperoxia did not affect FBF responses to acetylcholine or verapamil. The addition of vitamin C during hyperoxia augmented FBF responses to acetylcholine. These results suggest that hyperoxic vasoconstriction is mediated by oxidative stress. Moreover, hyperoxia impairs acetylcholine-mediated vasodilation in the setting of intact endothelial function. These effects of hyperoxia are prevented by vitamin C, providing evidence that hyperoxia-derived free radicals impair the activity of endothelium-derived vasoactive factors.     

Multicompartmental distribution of the tuberous sclerosis gene products,hamartin and tuberin

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Treatment of experimental adjuvant arthritis with the combination of methotrexate and lyophilized Enterococcus faecium enriched with organic selenium

The efficacy of combination therapy with methotrexate (MTX) and probiotic bacteriaEnterococcus faecium enriched with organic selenium (EFSe) in rats with adjuvant arthritis was determined. Rats with adjuvant arthritis were given MTX (0.3 mg/kg 2-times weekly, orally); lyophilizedE. faecium enriched with Se (15 mg/kg, 5 d per week, orally); and a combination of MTX plus EFSe for a period of 50 d from the immunization. Levels of serum albumin, serum nitrite/nitrate concentrations, changes in hind paw swellling, arthrogram score, bone erosions, whole body bone mineral density (BMD) and bone mineral content (BMC) were assayed in the rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX and with the combination MTX+EFSe significantly inhibited markers of both inflammation and arthritis. Significant differences in favor of combination therapy with MTX+EFSe as compared to MTX alone were seen in serum albumin concentration, hind paw swelling and arthrogram score. Reductions in radiographic scores were also more pronounced in the combination therapy group. Combination therapy, but not MTX alone, inhibited the reduction of BMD and BMC; treatment with lyophilized EFSe alone had no significant effect on adjuvant arthritis in rats. The potent therapeutic effect of low dosage MTX therapy in combination with lyophilized EFSe on adjuvant arthritis in rats was shown.