Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

Design, synthesis, and DNA binding properties of photoisomerizable azobenzene-distamycin conjugates: an experimental and computational study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene-distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.     

Magnitude and quality of vaccine-elicited T-cell responses in the control of immunodeficiency virus replication in rhesus monkeys

While a diversity of immunogens that elicit qualitatively different cellular immune responses are being assessed in clinical human immunodeficiency virus vaccine trials, the consequences of those varied responses for viral control remain poorly understood. In the present study, we evaluated the induction of virus-specific T-cell responses in rhesus monkeys using a series of diverse vaccine vectors. We assessed both the magnitude and the functional profile of the virus-specific CD8⁺ T cells by measuring gamma interferon, interleukin-2, and tumor necrosis factor alpha production. We found that the different vectors generated virus-specific T-cell responses of different magnitudes and with different functional profiles. Heterologous prime-boost vaccine regimens induced particularly high-frequency virus-specific T-cell responses with polyfunctional repertoires. Yet, immediately after a pathogenic simian-human immunodeficiency virus (SHIV) challenge, no significant differences were observed between these cohorts of vaccinated monkeys in the magnitudes or the functional profiles of their virus-specific CD8⁺ T cells. This finding suggests that the high viral load shapes the functional repertoire of the cellular immune response during primary infection. Nevertheless, in all vaccination regimens, higher frequency and more polyfunctional vaccine-elicited virus-specific CD8⁺ T-cell responses were associated with better viral control after SHIV challenge. These observations highlight the contributions of both the quality and the magnitude of vaccine-elicited cellular immune responses in the control of immunodeficiency virus replication.     

Role of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaB-mediated innate antiviral response against human respiratory syncytial virus

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-alpha/beta-independent, innate antiviral response against RSV that was dependent on activation of NF-kappaB. We demonstrated that NF-kappaB inducing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) confers potent antiviral function against RSV in an NF-kappaB-dependent fashion, independent of interferon-alpha/beta. During our efforts to study this pathway, we identified HBD2 (human beta-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-kappaB-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-kappaB-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine beta-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-kappaB-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.     

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies

Abstract

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV–vis absorption studies. The binding constant (K_b) of the HEWL–6HF complex was observed to be 6.44?±?0.09?×?10⁴ M^?1 at 293?K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = ?11.91?±?1.02?kJ mol^?1) along with a positive entropy change (ΔS = +51.36?±?2.43 J K^?1 mol^?1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster’s theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL.

Communicated by Ramaswamy H. Sarma     

Synthesis, characterization, conformational analysis of a cyclic conjugated octreotate peptide and biological evaluation of 99mTc-HYNIC-His3-Octreotate as novel tracer for the imaging of somatostatin receptor-positive tumors

Peptides are attracting increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. We therefore synthesized new cyclic octapeptides conjugated with HYNIC by Fmoc solid-phase peptide synthesis. These were purified and analyzed by RP-HPLC, MALDI mass, ¹H NMR, ¹³C NMR, HSQC, HMBC, COSY and IR spectroscopy. Conformational analysis of the peptides was performed by circular dichroism spectroscopy, in pure water and trifluoroethanol–water (1:1), revealed the presence of strong secondary structural features like β-sheet and random coils. Labeling was performed with ^99mTc using Tricine and EDDA as coligands by SnCl₂ method to get products with excellent radiochemical purity >99.5 %. Metabolic stability analysis did not show any evidence of breaking of the labeled compounds and formation of free ^99mTc. Internalization studies were done and IC₅₀ values were determined in somatostatin receptor-expressing C6 glioma cell line and rat brain cortex membrane, and the results compared with HYNIC-TOC as standard. The IC₅₀ values of ^99mTc-HYNIC-His³-Octreotate (21 ± 0.93 nM) and ^99mTc-HYNIC-TOC (2.87 ± 0.41 nM) proved to be comparable. Biodistribution and image study on normal rat under gamma camera showed very high uptake in kidney and urine, indicating kidney as primary organ for metabolism and route of excretion. Biodistribution and image study on rats bearing C6 glioma tumor found high uptake in tumor (1.27 ± 0.15) and pancreas (1.71 ± 0.03). Using these findings, new derivatives can be prepared to develop ^99mTc radiopharmaceuticals for imaging somatostatin receptor-positive tumors.     

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background

Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH₂CH₂OH) and oligo-oxyethylene -(CH₂CH₂O)_n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.

Methodology/Principal Findings

To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH₂-CH₂-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.

Conclusions/Significance

-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.     

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC₅₀ value in micromolar concentrations.     

Expression patterns of MDA-9/syntenin during development of the mouse embryo

Melanoma differentiation associated gene-9 (MDA-9)/syntenin is a PDZ domain-containing adaptor protein involved in multiple diverse cellular processes including organization of protein complexes in the plasma membrane, intracellular trafficking and cell surface targeting, synaptic transmission, and cancer metastasis. In the present study, we analyzed the expression pattern of MDA-9/syntenin during mouse development. MDA-9/syntenin was robustly expressed with tight regulation of its temporal and spatial expression during fetal development in the developing skin, spinal cord, heart, lung and liver, which are regulated by multiple signaling pathways in the process of organogenesis. Recent studies also indicate that MDA-9/syntenin is involved in the signaling pathways crucial during development such as Wnt, Notch and FGF. Taken together, these results suggest that MDA-9/syntenin may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation through modulating multiple signaling pathways as a crucial adaptor protein. Additionally, temporal regulation of MDA-9/syntenin expression may be required during specific stages and in specific tissues during development.