Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes

Lys(63)-linked polyubiquitin (poly-Ub) chains appear to play a nondegradative signaling and/or recruitment role in a variety of key eukaryotic cellular processes, including NF-kappaB signal transduction and DNA repair. A protein heterodimer composed of a catalytically active ubiquitin-conjugating enzyme (Ubc13) and its homologue (Mms2 or Uev1a) forms a catalytic scaffold upon which a noncovalently associated acceptor Ub and thiolester-linked donor Ub are oriented such that Lys(63)-linked poly-Ub chain synthesis is facilitated. In this study, we have used (1)H-(15)N nuclear magnetic resonance spectroscopy, in combination with isothermal titration calorimetry, to determine the thermodynamics and kinetics of the interactions between various components of the Lys(63)-linked poly-Ub conjugation machinery. Mms2 and Uev1a interact in vitro with acceptor Ub to form 1/1 complexes with macroscopic dissociation constants of 98 +/- 15 and 213 +/- 14 microM, respectively, and appear to bind Ub in a similar fashion. Interestingly, the Mms2.Ubc13 heterodimer associates with acceptor Ub in a 1/1 complex and binds with a dissociation constant of 28 +/- 6 microM, significantly stronger than the binding of Mms2 alone. Furthermore, a dissociation constant of 49 +/- 7 nM was determined for the interaction between Mms2 and Ubc13 using isothermal titration calorimetry. In connection with previous structural studies for this system, the thermodynamics and kinetics of acceptor Ub binding to the Mms2.Ubc13 heterodimer described in detail in this study will allow for a more thorough rationalization of the mechanism of formation of Lys(63)-linked poly-Ub chains.     

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.     

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.     

Education and training for radiation scientists: radiation research program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland, May 12-14, 2003

Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204-223, 2002; Radiat. Res. 159, 812-834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences.     

Altered Sonic hedgehog signaling is associated with morphological abnormalities in the penis of the BB/WOR diabetic rat

Erectile dysfunction (ED) is a common and debilitating pathological development that affects up to 75% of diabetic males. Neural stimulation is a crucial aspect of the normal erection process. Nerve injury causes ED and disrupts signaling of the Sonic hedgehog (Shh) cascade in the smooth muscle of the corpora cavernosa. Shh and targets of its signaling establish normal corpora cavernosal morphology during postnatal differentiation of the penis and regulate homeostasis in the adult. Interruption of the Shh cascade in the smooth muscle of the corpora cavernosa results in extensive changes in corpora cavernosal morphology that lead to ED. Our hypothesis is that the neuropathy observed in diabetics causes morphological changes in the corpora cavernosa of the penis that result in ED. Disruption of the Shh cascade may be involved in this process. We tested this hypothesis by examining morphological changes in the penis, altered gene and protein expression, apoptosis, and bromodeoxyuridine incorporation in the BB/WOR rat model of diabetes. Extensive smooth muscle and endothelial degradation was observed in the corpora cavernosa of diabetic penes. This degradation accompanied profound ED, significantly decreased Shh protein in the smooth muscle of the corpora cavernosa, and increased penile Shh RNA expression in the intact penis (nerves, corpora, and urethra). Localization and expression of Shh targets were also disrupted in the corpora cavernosa. Increasing our understanding of the molecular mechanisms that regulate Shh signaling may provide valuable insight into improving treatment options for diabetic impotence.     

Effects of dipole position,orientation and noise on the accuracy of EEG source localization

Background  

The electroencephalogram (EEG) reflects the electrical activity in the brain on the surface of scalp. A major challenge in this field is the localization of sources in the brain responsible for eliciting the EEG signal measured at the scalp. In order to estimate the location of these sources, one must correctly model the sources, i.e., dipoles, as well as the volume conductor in which the resulting currents flow. In this study, we investigate the effects of dipole depth and orientation on source localization with varying sets of simulated random noise in 4 realistic head models.     

Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.     

Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.     

Immunogenicity study of glycoprotein-deficient rabies virus expressing simian/human immunodeficiency virus SHIV89.6P envelope in a rhesus macaque

Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (DeltaG) expressing a simian/human immunodeficiency virus SHIV(89.6P) Env ectodomain and transmembrane domain fused to the RV G CD (DeltaG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with DeltaG-89.6P-RVG developed SHIV(89.6P) virus-neutralizing antibodies and SHIV(89.6P)-specific cellular immune responses after challenge with SHIV(89.6P). There was no evidence of CD4(+) T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.