Regulators of IAP function: coming to grips with the grim reaper

Inhibitor of apoptosis proteins (IAPs) are a conserved class of proteins that control apoptosis in both vertebrates and invertebrates. They exert their anti-apoptotic function through inhibition of caspases, the principal executioners of apoptotic cell death. Recent advances in vertebrates and Drosophila have demonstrated that IAPs use ubiquitin conjugation to control the stability, and thus the activity, of select target proteins. The Drosophila IAP1 gene is an instructive example: it employs at least two distinct ubiquitin-dependent mechanisms of protein destruction. The apoptosis-inducing genes grim, reaper and hid modulate these mechanisms, and determine the outcome.     

Microbial activities in soils of a healthy and a declining reed stand

Microbial processes were investigated in the soil of a declining, more eutrophic (Romberk West) and a healthy looking, less eutrophic (Romberk East) freshwater reed stand. Soil was sampled monthly from June to September 1997. Glucose induced carbon dioxide (CO₂) production in oxic and anoxic conditions, methane (CH₄) production, nitrification and denitrification activities were measured in laboratory conditions in suspensions prepared from homogenised soil samples. Within a stand the proportion of anaerobic (as opposed to aerobic) microbial activity was greatest in June. Potential methanogenesis was highest in June and decreased later in both stands. Methane production was approximately the same in June at both stands but it was higher at Romberk East than at Romberk West stand in later months. Denitrifying activity was higher in August than July at both stands. Nitrifying activity was undetectable at both stands over the entire study period. Generally Romberk West was more anaerobic than Romberk East, with lower redox potential, higher amounts of oxygen-consuming organic matter and a lower ratio of CO₂ production in oxic conditions to CO₂ production in anoxic conditions. Microbial activity was apparently restricted at Romberk West stand in comparison to Romberk East. The shift from aerobic to anaerobic microbial metabolism and a coinciding restriction of metabolic activities at Romberk West are thought to be indicative of a strengthened oxygen stress in the soil, associated with accumulation of metabolites toxic to both the microorganisms and the reed. Possible links between eutrophication, microbial characteristics and reed performance are discussed.     

Contributions of CD8+ T cells and viral spread to demyelinating disease

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.     

Primary structure of cytochrome c′ of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c′-type cytochromes

Cytochrome c′ of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome c ₅₅₅. This cytochrome is spectrally similar to other cytochromes c′ but is larger (16,000 Da) and has a lower midpoint potential (–205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c′ from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c′, only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c′ from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c′-type cytochrome which developed a covalent cross-link between a lysine residue and the c′-heme. Received: 26 May 1999 / Accepted: 9 September 1999     

Cytokine-modulated regulation of helper T cell populations

Helper T (Th) cells are a crucial component of the adaptive immune system and are of fundamental importance in orchestrating the appropriate response to pathogenic challenge. They fall into two broad categories defined by the cytokines each produces. Th1 cells produce interferon- gamma and are required for effective immunity to intracellular bacteria, viruses and protozoa whereas Th2 produce IL-4 and are required for optimal antibody production to T-dependent antigens. A great deal of experimental data on the regulation of Th1 and Th2 differentiation have been obtained but many essential features of this complex system are still not understood. Here we present a mathematical model of Th1/Th2 differentiation and cross regulation. We model Fas-mediated activation-induced cell death (AICD) as this process has been identified as an important mechanism for limiting clonal expansion and resolving T cell responses. We conclude that Th2 susceptibility to AICD is important for stabilizing the two polarized arms of the T helper response, and that cell-cell killing, not suicide, is the dominant mechanism for Fas-mediated death of Th1 effectors. We find that the combination of the anti-proliferative effect of the cytokine TGF- beta and the inhibiting influence of IL-10 on T cell activation are crucial controls for Th2 populations. We see that the strengths of the activation signals for each T helper cell subset, which are dependent on the antigen dose, co-stimulatory signals and the cytokine environment, critically determine the dominant helper subset. Switches from Th1- to Th2-dominance may be important in chronic infection and we show that this phenomenon can arise from differential AICD susceptibility of T helper subsets, and asymmetries in the nature of the cross-suppressive cytokine interactions. Our model suggests that in some senses a predominantly type 2 reaction may well be the "default" pathway for an antigen-specific immune response, due to these asymmetries.     

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea.

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.     

Heritability of scrotal circumference adjusted and unadjusted for body weight in Nellore bulls, using univariate and bivariate animal models

Mixed model methodology under univariate and bivariate animal models was used to estimate genetic parameters of scrotal circumference (SC) unadjusted (h2U) and adjusted (h2A) for body weight, from field data on 875 Nellore males sired by 80 bulls. The models included random effect of animal, the fixed effects of age of dam, year of birth, and month and year of measurement, and the covariate age of the animal. In addition, one of the models also included the covariate body weight. Using univariate models, SC estimates of h2U were 0.18, 0.65, 0.77 and 0.60, and of h2A they were 0.15, 0.60, 0.71 and 0.50, at 9, 12, 18 and 24 m.o. of age, respectively. Bivariate estimates of heritability ranged from 0.18 to 0.77 for h2U, and from 0.13 to 0.70 for h2A. The results suggest that it would be better to consider unadjusted SC rather than adjusted SC in selection programs. Bivariate body weight estimates of heritability ranged from 0.15 to 0.45. Genetic correlations between SC and body weight ranged from 0.58 to 0.71 when body weight was not included in the model, and from 0.33 to 0.64 when body weight was included. These results suggest that simultaneous selection for body weight and SC is possible in Nellore cattle.     

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium

 Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1–4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1–4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4. Accepted: 23 December 1997     

Susceptibility of Paulownia elongata to Agrobacterium and production of transgenic calli and hairy roots by in vitro inoculation

Susceptibility of Paulownia elongata S.Y. Hu (princess tree) to Agrobacterium tumefaciens and A. rhizogenes was demonstrated by inoculating in vitro shoots. Shoots had a gall formation frequency of ≥83% when inoculated with any of three A. tumefaciens strains (542, A281, or C58). Timing of gall appearance and type of callus proliferation differed among A. tumefaciens strains. Rapidly proliferating callus was produced from explants that were inoculated with A. tumefaciens. Hairy roots were produced directly from wound sites on 33% of shoots inoculated with A. rhizogenes strain R1601. Rapidly growing detached roots were produced from explants that were inoculated with A. rhizogenes. Opine analyses demonstrated the expression of foreign genes in proliferating galls/hairy roots shortly after emergence from wound sites and in callus and roots after 12 weeks of in vitro culture. Southern analyses demonstrated the presence of tDNA in long-term callus and root cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.