Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

Oxidative stress in cardiovascular disease: myth or fact?

Oxidative stress is a mechanism with a central role in the pathogenesis of atherosclerosis, cancer, and other chronic diseases. It also plays a major role in the aging process. Ischemic heart disease is perhaps the human condition in which the role of oxidative stress has been investigated in more detail: reactive oxygen species and consequent expression of oxidative damage have been demonstrated during post-ischemic reperfusion in humans and the protective role of antioxidants has been validated in several experimental studies addressing the pathophysiology of acute ischemia. Although an impressive bulk of experimental studies substantiate the role of oxidative stress in the progression of the damage induced by acute ischemia, not a single pathophysiologic achievement has had a significant impact on the treatment of patients and randomized, controlled clinical trials, both in primary and secondary prevention, have failed to prove the efficacy of antioxidants in the treatment of ischemic cardiovascular disease. This dichotomy, between the experimental data and the lack of impact in the clinical setting, needs to be deeply investigated: certainly, the pathophysiologic grounds of oxidative stress do maintain their validity but the concepts of the determinants of oxidative damage should be critically revised. In this regard, the role of intermediate metabolism during myocardial ischemia together with the cellular redox state might represent a promising interpretative key.     

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.     

Influence of stage of maturation of bovine oocytes at time of vitrification on the incidence of diploid metaphase II at completion of maturation

The present study was to verify the incidence of bovine diploid oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughterhouse and then divided into five groups: control group (unvitrified oocytes), 0 h group (composed of oocytes vitrified before the onset of maturation) and 8, 12, and 22 h groups (vitrified respectively at 8, 12 and 22 h after the onset of maturation). The oocytes remained vitrified for 24 h, and then were thawed. In all groups, the oocytes completed 24 h of maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences (P>0.05) in the incidence of diploid metaphase II oocytes were observed between the control, non-vitrified group (2.4%, 1/41) and oocytes vitrified at 12 h (6.9%, 3/43) or 22 h (2%, 1/50). However, significantly (P<0.05) more diploid oocytes were detected after vitrification at 0 h (28.5%, 10/35) or 8 h (35.4%, 11/31) of maturation. These results suggest that the nuclear stage at which bovine oocytes are vitrified may affect the incidence of diploid oocytes, especially in oocytes vitrified before maturation or 8 h after the onset of maturation.     

Serum levels of tartrate-resistant acid phosphatase-5b in breast cancer patients treated with pamidronate

A novel immunoassay specific for the osteoclast-produced TRAP isoform 5b has been developed recently. By means of this assay we studied the usefulness of serum TRAP-5b in monitoring the response to palliative treatment with pamidronate in breast cancer patients with bone metastases. We correlated serum TRAP-5b levels with pain intensity and intake of analgesics to assess the possible utility of the marker in identifying patients who could benefit from pamidronate treatment. Twenty-eight advanced breast cancer patients with bone metastases entered the study. Patients were treated according to the following schedule: two two-week cycles of 60 mg/week pamidronate IV, with a three-week interval in between (six infusions over seven weeks), followed by one infusion every three weeks for a total of 24 infusions over a treatment period of 61 weeks. Blood samples were taken before the start of treatment and before each infusion during two treatment cycles. To measure serum TRAP levels we employed the new immunoassay kit BoneTRAP produced by Suomen Bioanalytiikka Oy (SBA), Oulu, Finland. In order to assess the usefulness of this marker in evaluating the response to pamidronate treatment we divided patients into two groups (group A, worsened; group B, improved) with respect to pain trend and analgesic intake. Our results did not show any statistically significant difference in baseline serum TRAP levels in the two groups. However, one week after the first pamidronate infusion TRAP-5b serum levels decreased by 39% and 18% in groups A and B, respectively (p=0.01); these levels persisted throughout the treatment period. In conclusion, a decrease in TRAP-5b serum levels may reflect the pharmacological activity of pamidronate and seems to predict pain relief and a reduction in analgesic consumption.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

Selective ligand-induced stabilization of active and desensitized parathyroid hormone type 1 receptor conformations

For many G protein-coupled receptors, agonist-induced activation is followed by desensitization, internalization, and resensitization. In most cases, these processes are dependent upon interaction of agonist-occupied receptor with cytoplasmic beta-arrestins. The ligand-induced intramolecular rearrangements of the receptor responsible for the desensitized versus active conformational states, which dictate both the pharmacological properties of ligands and the biological activity of G protein-coupled receptors, have not been fully elucidated. Here, we identify specific interactions between parathyroid hormone (PTH)-related protein and the human PTH type 1 receptor (PTH1Rc) and the related receptor conformational changes that lead to beta-arrestin-2-mediated desensitization. PTH-related protein analogs modified at position 1 induced selective stabilization of the active G protein-coupled state of the receptor, resulting in lack of beta-arrestin-2 recruitment to the cell membrane, sustained cAMP signaling, and absence of ligand-receptor complex internalization. Mechanistically, the ligands modified at position 1, interacting with the extracellular end of helix VI of PTH1Rc, produced a translocation of transmembrane helices V and VI that differed from that induced by the cognate agonist, resulting in significantly different conformations of the third intracellular loop. These results show how specific interactions between PTH1Rc and its ligands may stabilize distinct conformational states, representing either the active G protein-coupled or a desensitized beta-arrestin-coupled receptor state. In addition, they establish that sustained biological activity of PTH1Rc may be induced by appropriately designed agonist ligands.