Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules

Targeting recycling endocytic receptors with specific Abs provides a means for introducing a variety of tumor-associated Ags into human dendritic cells (DCs), culminating in their efficient presentation to T cells. We have generated a human mAb (B11) against the mannose receptor that is rapidly internalized by DCs through receptor-mediated endocytosis. By genetically linking the melanoma Ag, pmel17, to Ab B11, we obtained the fully human fusion protein, B11-pmel17. Treatment of DCs with B11-pmel17 resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Thus, potent pmel17-specific T cells were cytotoxic toward gp100(+) HLA-matched melanoma targets, but not HLA-mismatched melanoma or gp100(-) nonmelanoma tumor lines. Importantly, competitive inhibition of lysis of an otherwise susceptible melanoma cell line by cold targets pulsed with known gp100 CD8 T cell epitopes as well as a dose-dependent proliferative response to Th epitopes demonstrates that DCs can process targeted Ag for activation of cytotoxic as well as helper arms of the immune response. Thus, the specific targeting of soluble exogenous tumor Ag to the DC mannose receptor directly contributes to the generation of multiple HLA-restricted Ag-specific T cell responses.     

Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry

The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.     

New clerodane diterpenoids from the aerial parts of Pulicaria wightiana

Two new ent-clerodane-type diterpenoids, compounds 1 and 2, were isolated from the aerial parts of Pulicaria wightiana, together with three known constituents. Their structures were established based on spectroscopic data, and their antibacterial activities were evaluated (Table 2).     

Estimation of arterial and cardiopulmonary total peripheral resistance baroreflex gain values: validation by chronic arterial baroreceptor denervation

Feedback control of total peripheral resistance (TPR) by the arterial and cardiopulmonary baroreflex systems is an important mechanism for short-term blood pressure regulation. Existing methods for measuring this TPR baroreflex mechanism typically aim to quantify only the gain value of one baroreflex system as it operates in open-loop conditions. As a result, the normal, integrated functioning of the arterial and cardiopulmonary baroreflex control of TPR remains to be fully elucidated. To this end, the laboratory of Mukkamala et al. (Mukkamala R, Toska K, and Cohen RJ. Am J Physiol Heart Circ Physiol 284: H947-H959, 2003) previously proposed a potentially noninvasive technique for estimating the closed-loop (dimensionless) gain values of the arterial TPR baroreflex (GA) and the cardiopulmonary TPR baroreflex (GC) by mathematical analysis of the subtle, beat-to-beat fluctuations in arterial blood pressure, cardiac output, and stroke volume. Here, we review the technique with additional details and describe its experimental evaluation with respect to spontaneous hemodynamic variability measured from seven conscious dogs, before and after chronic arterial baroreceptor denervation. The technique was able to correctly predict the group-average changes in GA and GC that have previously been shown to occur following chronic arterial baroreceptor denervation. That is, reflex control by the arterial TPR baroreflex was virtually abolished (GA = -2.1 +/- 0.6 to 0.3 +/- 0.2; P < 0.05), while reflex control by the cardiopulmonary TPR baroreflex more than doubled (GC = -0.7 +/- 0.4 to -1.8 +/- 0.2; P < 0.05). With further successful experimental testing, the technique may ultimately be employed to advance the basic understanding of TPR baroreflex functioning in both humans and animals in health and disease.     

Degeneracy and repertoire of the human HIV-1 Gag p17(77-85) CTL response

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db beta2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.     

Effect of sesame oil on diuretics or Beta-blockers in the modulation of blood pressure, anthropometry, lipid profile, and redox status

The study was undertaken to investigate the effect of sesame oil in hypertensive patients who were on antihypertensive therapy either with diuretics (hydrochlorothiazide) or ß-blockers (atenolol). Thirty-two male and 18 female patients aged 35 to 60 years old were supplied sesame oil (Idhayam gingelly oil) and instructed to use it as the only edible oil for 45 days. Blood pressure, anthropometry, lipid profile, lipid peroxidation, and enzymic and non-enzymic antioxidants were measured at baseline and after 45 days of sesame oil substitution. Substitution of sesame oil brought down systolic and diastolic blood pressure to normal. The same patients were asked to withdraw sesame oil consumption for another 45 days, and the measurements were repeated at the end of withdrawal period. Withdrawal of sesame oil substitution brought back the initial blood pressure values. A significant reduction was noted in body weight and body mass index (BMI) upon sesame oil substitution. No significant alterations were observed in lipid profile except triglycerides. Plasma levels of sodium reduced while potassium elevated upon the substitution of sesame oil. Lipid peroxidation (thiobarbituric acid reactive substances [TBARS]) decreased while the activities of superoxide dismutase (SOD), catalase (CAT), and the levels of vitamin C, vitamin E, ß-carotene, and reduced glutathione (GSH) were increased. The results suggested that sesame oil as edible oil lowered blood pressure, decreased lipid peroxidation, and increased antioxidant status in hypertensive patients.     

Indoleamines and calcium enhance somatic embryogenesis in <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr

Melatonin (MEL) and serotonin (SER) are important indoleamines that are involved in neural transmission in mammalian cells. They are also known to be present in various genera of plants. The role (s) of these indoleamines in plants are not well known. In this study, the effects of SER, MEL, calcium, and calcium ionophore (A23187), a calcium channel activator, on somatic embryogenesis in Coffea canephora have been investigated. Adding 100 μM of either SER or MEL to ½ strength Murashige and Skoog (MS) medium and 0.93 μM kinetin (KN) has resulted in enhanced induction of somatic embryogenesis, 85 ± 3 and 62 ± 6 embryos/callus, respectively. In the presence of either 5 mM calcium or 100 μM calcium ionophore A23187, number of somatic embryos/callus is also increased, with 56 ± 4 and 118 ± 10 somatic embryos/callus, respectively, compared to 25 ± 3 embryos/callus for control. The presence of 5 mM calcium chloride along with either 100 μM SER or 100 μM MEL, respectively, have also promoted somatic embryogenesis with induction of 105 ± 6 and 78 ± 2 somatic embryos/callus. While, addition of calcium ionophore A23187 along with either 100 μM SER or 100 μM MEL have produced 155 ± 12 or 135 ± 8 embryos/callus, respectively. In contrast, addition of such indoleamine inhibitors as 40 μM p-chlorophenylalanine (p-CPA), 20 μM fluoexitine hydrochloride (prozac), 1 mM verapamil hydrochloride (calcium channel blocker), and 1 mM ethylene glycol-bis (β-amino ethylether)-N, N, N′, N′-tetra acetic acid (EGTA) (a calcium chelator) individually, has inhibited induction of somatic embryos while reducing levels of endogenous pools of SER, MEL and indole-3-acetic acid (IAA) levels. Calcium imaging by laser scanning confocal microscopy (LSCM) has revealed high fluorescence intensity in callus treated with calcium and calcium ionophore A23187. Immunolocalization of SER in different tissues of C. canephora has revealed that it is localized in vascular tissues of stems, roots, and somatic embryos, as well as in endocarps (husks) of immature fruits.