Conformational homogeneity and excited-state isomerization dynamics of the bilin chromophore in phytochrome Cph1 from resonance Raman intensities

The ground-state structure and excited-state isomerization dynamics of the P_r and P_fr forms of phytochrome Cph1 are investigated using resonance Raman intensity analysis. Electronic absorption and stimulated resonance Raman spectra of P_r and P_fr are presented; vibronic analysis of the Raman intensities and absorption spectra reveals that both conformers exist as a single, homogeneous population of molecules in the ground state. The homogeneous and inhomogeneous contributions to the overall electronic broadening are determined, and it is found that the broadening is largely homogeneous in nature, pointing to fast excited-state decay. Franck-Condon displacements derived from the Raman intensity analysis reveal the initial atomic motions in the excited state, including the highly displaced, nontotally symmetric torsional and C₁₅–H HOOP modes that appear because of symmetry-reducing distortions about the C₁₄–C₁₅ and C₁₅=C₁₆ bonds. P_fr is especially well primed for ultrafast isomerization and torsional Franck-Condon analysis predicts a <200 fs P_fr → P_r isomerization. This time is significantly faster than the observed 700 fs reaction time, indicating that the P_fr S₁ surface has a D-ring rotational barrier caused by steric interactions with the protein.     

Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain

APP intracellular C-terminal domain (AICD-47), generated upon γ-secretase cleavage of amyloid precursor's protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine-binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the "conformational switching" of AICD-47 becomes evident. In order to understand different binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spectroscopy has revealed an overall random coil like structure in different pHs while TFE (2'-2'-2'-trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced α-helicity to a certain extent. Binding of Tyr phosphorylated AICD-47 ((P)AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (ΔH=-197.5±6.2kcalmole(-1) at 25°C) and an unfavorable entropic contribution (ΔS=-631calmol(-1)deg(-1) at 25°C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer's disease phenotype.     

Immunodominant "asymptomatic" herpes simplex virus 1 and 2 protein antigens identified by probing whole-ORFome microarrays with serum antibodies from seropositive asymptomatic versus symptomatic individuals

Herpes simplex virus 1 (HSV-1) and HSV-2 are medically significant pathogens. The development of an effective HSV vaccine remains a global public health priority. HSV-1 and HSV-2 immunodominant "asymptomatic" antigens (ID-A-Ags), which are strongly recognized by B and T cells from seropositive healthy asymptomatic individuals, may be critical to be included in an effective immunotherapeutic HSV vaccine. In contrast, immunodominant "symptomatic" antigens (ID-S-Ags) may exacerbate herpetic disease and therefore must be excluded from any HSV vaccine. In the present study, proteome microarrays of 88 HSV-1 and 84 HSV-2 open reading frames(ORFs) (ORFomes) were constructed and probed with sera from 32 HSV-1-, 6 HSV-2-, and 5 HSV-1/HSV-2-seropositive individuals and 47 seronegative healthy individuals (negative controls). The proteins detected in both HSV-1 and HSV-2 proteome microarrays were further classified according to their recognition by sera from HSV-seropositive clinically defined symptomatic (n = 10) and asymptomatic (n = 10) individuals. We found that (i) serum antibodies recognized an average of 6 ORFs per seropositive individual; (ii) the antibody responses to HSV antigens were diverse among HSV-1- and HSV-2-seropositive individuals; (iii) panels of 21 and 30 immunodominant antigens (ID-Ags) were identified from the HSV-1 and HSV-2 ORFomes, respectively, as being highly and frequently recognized by serum antibodies from seropositive individuals; and (iv) interestingly, four HSV-1 and HSV-2 cross-reactive asymptomatic ID-A-Ags, US4, US11, UL30, and UL42, were strongly and frequently recognized by sera from 10 of 10 asymptomatic patients but not by sera from 10 of 10 symptomatic patients (P < 0.001). In contrast, sera from symptomatic patients preferentially recognized the US10 ID-S-Ag (P < 0.001). We have identified previously unreported immunodominant HSV antigens, among which were 4 ID-A-Ags and 1 ID-S-Ag. These newly identified ID-A-Ags could lead to the development of an efficient "asymptomatic" vaccine against ocular, orofacial, and genital herpes.     

Discovery of potential diagnostic and vaccine antigens in herpes simplex virus 1 and 2 by proteome-wide antibody profiling

Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1- or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1- and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli.     

Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication

The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.     

Photoinduced DNA cleavage by anthracene based hydroxamic acids

Two different series of naphthalene and anthracene based hydroxamic acids having amino acid derivatives were synthesized. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroxamic acids by UV light (≥350nm). Both reactive oxygen species (ROS) and generated radicals from hydroxamic acids were shown to be responsible for the DNA cleavage. Further, DNA cleaving ability of hydroxamic acids was found to be dependent on its concentration and on its structure.     

Non inflammatory boronate based glucose-responsive insulin delivery systems

Boronic acids, known to bind diols, were screened to identify non-inflammatory cross-linkers for the preparation of glucose sensitive and insulin releasing agglomerates of liposomes (Agglomerated Vesicle Technology-AVT). This was done in order to select a suitable replacement for the previously used cross-linker, ConcanavalinA (ConA), a lectin known to have both toxic and inflammatory effects in vivo. Lead-compounds were selected from screens that involved testing for inflammatory potential, cytotoxicity and glucose-binding. These were then conjugated to insulin-encapsulating nanoparticles and agglomerated via sugar-boronate ester linkages to form AVTs. In vitro, the particles demonstrated triggered release of insulin upon exposure to physiologically relevant concentrations of glucose (10 mmoles/L-40 mmoles/L). The agglomerates were also shown to be responsive to multiple spikes in glucose levels over several hours, releasing insulin at a rate defined by the concentration of the glucose trigger.     

Dopamine regulates mobilization of mesenchymal stem cells during wound angiogenesis

Angiogenesis is an important step in the complex biological and molecular events leading to successful healing of dermal wounds. Among the different cellular effectors of wound angiogenesis, the role of mesenchymal stem cells (MSCs) is of current interest due to their transdifferentiation and proangiogenic potentials. Skin is richly innervated by sympathetic nerves which secrete dopamine (DA) and we have recently shown that concentration of DA present in synaptic cleft can significantly inhibit wound tissue neovascularization. As recent reports indicate that MSCs by mobilizing into wound bed play an important role in promoting wound angiogenesis, we therefore investigated the effect of DA on the migration of MSCs in wound tissues. DA acted through its D(2) receptors present in the MSCs to inhibit their mobilization to the wound beds by suppressing Akt phosphorylation and actin polymerization. In contrast, this inhibitory effect of DA was reversed after treatment with specific DA D(2) receptor antagonist. Increased mobilization of MSCs was demonstrated in the wound site following blockade of DA D(2) receptor mediated actions, and this in turn was associated with significantly more angiogenesis in wound tissues. This study is of translational value and indicates use of DA D(2) receptor antagonists to stimulate mobilization of these stem cells for faster regeneration of damaged tissues.