Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.     

Evolutionary control of leaf element composition in plants

Leaf nitrogen (N) and phosphorus (P) concentrations are correlated in plants. Higher-level phylogenetic effects can influence leaf N and P. By contrast, little is known about the phylogenetic variation in the leaf accumulation of most other elements in plant tissues, including elements with quantitatively lesser roles in metabolism than N, and elements that are nonessential for plant growth. Here the leaf composition of 42 elements is reported from a statistically unstructured data set comprising over 2000 leaf samples, representing 670 species and 138 families of terrestrial plants. Over 25% of the total variation in leaf element composition could be assigned to the family level and above for 21 of these elements. The remaining variation corresponded to differences between species within families, to differences between sites which were likely to be caused by soil and climatic factors, and to variation caused by sampling techniques. While the majority of variation in leaf mineral composition is undoubtedly associated with nonevolutionary factors, identifying higher-level phylogenetic variation in leaf elemental composition increases our understanding of terrestrial nutrient cycles and the transfer of toxic elements from soils to living organisms. Identifying mechanisms by which different plant families control their leaf elemental concentration remains a challenge.     

Solid cancer incidence in atomic bomb survivors: 1958-1998

This is the second general report on radiation effects on the incidence of solid cancers (cancers other than malignancies of the blood or blood-forming organs) among members of the Life Span Study (LSS) cohort of Hiroshima and Nagasaki atomic bomb survivors. The analyses were based on 17,448 first primary cancers (including non-melanoma skin cancer) diagnosed from 1958 through 1998 among 105,427 cohort members with individual dose estimates who were alive and not known to have had cancer prior to 1958. Radiation-associated relative risks and excess rates were considered for all solid cancers as a group, for 19 specific cancer sites or groups of sites, and for five histology groups. Poisson regression methods were used to investigate the magnitude of the radiation-associated risks, the shape of the dose response, how these risks vary with gender, age at exposure, and attained age, and the evidence for inter-site variation in the levels and patterns of the excess risk. For all solid cancers as a group, it was estimated that about 850 (about 11%) of the cases among cohort members with colon doses in excess of 0.005 Gy were associated with atomic bomb radiation exposure. The data were consistent with a linear dose response over the 0- to 2-Gy range, while there was some flattening of the dose response at higher doses. Furthermore, there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15 Gy or less. The excess risks for all solid cancers as a group and many individual sites exhibit significant variation with gender, attained age, and age at exposure. It was estimated that, at age 70 after exposure at age 30, solid cancer rates increase by about 35% per Gy (90% CI 28%; 43%) for men and 58% per Gy (43%; 69%) for women. For all solid cancers as a group, the excess relative risk (ERR per Gy) decreases by about 17% per decade increase in age at exposure (90% CI 7%; 25%) after allowing for attained-age effects, while the ERR decreased in proportion to attained age to the power 1.65 (90% CI 2.1; 1.2) after allowing for age at exposure. Despite the decline in the ERR with attained age, excess absolute rates appeared to increase throughout the study period, providing further evidence that radiation-associated increases in cancer rates persist throughout life regardless of age at exposure. For all solid cancers as a group, women had somewhat higher excess absolute rates than men (F:M ratio 1.4; 90% CI 1.1; 1.8), but this difference disappears when the analysis was restricted to non-gender-specific cancers. Significant radiation-associated increases in risk were seen for most sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin, breast, ovary, bladder, nervous system and thyroid. Although there was no indication of a statistically significant dose response for cancers of the pancreas, prostate and kidney, the excess relative risks for these sites were also consistent with that for all solid cancers as a group. Dose-response estimates for cancers of the rectum, gallbladder and uterus were not statistically significant, and there were suggestions that the risks for these sites may be lower than those for all solid cancers combined. However, there was emerging evidence from the present data that exposure as a child may increase risks of cancer of the body of the uterus. Elevated risks were seen for all of the five broadly classified histological groups considered, including squamous cell carcinoma, adenocarcinoma, other epithelial cancers, sarcomas and other non-epithelial cancers. Although the data were limited, there was a significant radiation-associated increase in the risk of cancer occurring in adolescence and young adulthood. In view of the persisting increase in solid cancer risks, the LSS should continue to provide important new information on radiation exposure and solid cancer risks for at least another 15 to 20 years.     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Characteristics of type IV collagen unfolding under various pH conditions as a model of pathological disorder in tissue

The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.     

Physiological changes of premotor nonspiking interneurons in the central compensation of eyestalk posture following unilateral sensory ablation in crayfish

We investigated how the physiological characteristics and synaptic activities of nonspiking giant interneurons (NGIs), which integrate sensory inputs in the brain and send synaptic outputs to oculomotor neurons innervating eyestalk muscles, changed after unilateral ablation of the statocyst in order to clarify neuronal mechanisms underlying the central compensation process in crayfish. The input resistance and membrane time constant in recovered animals that restored the original symmetrical eyestalk posture 2 weeks after operation were significantly greater than those immediately after operation on the operated side whereas in non-recovered animals only the membrane time constant showed a significant increase. On the intact side, both recovered and non-recovered animals showed no difference. The frequency of synaptic activity showed a complex pattern of change on both sides depending on the polarity of the synaptic potential. The synaptic activity returned to the bilaterally symmetrical level in recovered animals while bilateral asymmetry remained in non-recovered ones. These results suggest that the central compensation of eyestalk posture following unilateral impairment of the statocyst is subserved by not only changes in the physiological characteristics of the NGI membrane but also the activity of neuronal circuits presynaptic to NGIs.