The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579

The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.     

Long-term physical impairment and functional outcomes after complex facial fractures

To develop an understanding of the expected functional outcomes after facial trauma, a retrospective cohort study of patients with complex facial fractures was conducted. A cohort of adults aged 18 to 55 years who were admitted to the R. Adams Cowley Shock Trauma Center between July of 1986 and July of 1994 for treatment of a Le Fort midface fracture (resulting from blunt force) was retrospectively identified.Outcomes of interest included measures of general health status and psychosocial well being in addition to self-reported somatic symptoms. General health status was ascertained using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). The Body Satisfaction Scale was used to define patient concerns about altered body image and shape. To determine whether complex maxillofacial trauma and facial fractures contributed to altered social interactions, the Social Avoidance and Distress scale was used. In addition, information about a patient, his or her injury, and its treatment were ascertained from the medical records.Using the methods described above, 265 patients with Le Fort fractures were identified. These individuals were matched to a similar group of 242 general injury patients. A total of 190 of the Le Fort patients (72 percent of those eligible for the study) and 144 (60 percent) general injury patients were successfully located, and long-term interview data were acquired.Le Fort fracture patients as a group had similar health status outcomes when compared with the group of general injury patients. However, when outcomes were examined by the complexity of the Le Fort fracture, the authors found that study subjects with severe, comminuted Le Fort injuries (group D) had significantly lower SF-36 scores (worse outcomes) for the two dimensions related to role limitations: role limitations due to physical problems and role limitations due to emotional problems (p < 0.05). SF-36 scores for all other dimensions except physical function were also lower for comminuted versus less complex Le Fort fractures, although differences were not statistically significant.Specifically, there was a direct relationship between severity of facial injury and patients reporting work disability. Of group C and D Le Fort patients (severely comminuted fractures) only 55 and 58 percent, respectively, had returned to work at the time of follow-up interview. These figures are significantly lower than the back-to-work percentage of patients with less severe facial injury (70 percent).When study participants were asked if they were experiencing specific somatic symptoms at the time of the interview that they had not experienced before the injury, a significantly larger percent of the Le Fort fracture patients (compared with the general injury patients) responded in the affirmative. Differences between the Le Fort fracture and general injury groups were statistically significant (p < 0.05) for all 11 symptoms.The percentage of patients reporting complaints increased with increasing complexity of facial fracture in the areas of visual problems, alterations in smell, difficulty with mastication, difficulty with breathing, and epiphora, and these differences reached statistical significance.Patients sustaining comminuted Le Fort facial fractures report poorer health outcomes than patients with less severe facial injury and substantially worse outcomes than population norms. It is also this severely injured population that reports the greatest percentage of injury-related disability, preventing employment at long-term follow-up. The long-term goal of centralized tertiary trauma treatment centers must be to return the patient to a productive, active lifestyle.     

Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase: the roles of glutamine 100 and aspartate 125

The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H(4)folate and 10-formyl-H(4)folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H(4)folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions.     

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.     

ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.     

Effect of delivery method and population size of Trichoderma harzianum on growth response of unrooted chrysanthemum cuttings

In a previous study, addition of Trichoderma harzianum Rifai isolate T-12 to a propagative medium resulted in improved performance of chrysanthemum cuttings. However, root and shoot growth of one cultivar, 'Dark Bronze Charm', were more responsive to a lower (5 g T-12/kg medium) than higher (25 g T-12/kg medium) rate of fungal propagules, suggesting potential phytotoxicity at higher concentrations. The objectives of this study were to investigate higher rates of T-12 medium amendment for phytotoxicity, and to examine an alternative method of delivering the fungus to the propagative medium in order to obtain a more uniform response from cuttings. Isolate T-12 was added to the propagative medium as either a powdered peat-bran amendment (0, 5, or 50 g T-12/kg medium) or as alginate prills (80 or 800 g T-12/kg medium). There were no differences among treatments on day seven, but by day 21, shoot fresh weight and heights were significantly greater for plants treated with prills at 800 g T-12/kg medium. Both prill treatments resulted in greater shoot height on day 14 and 21 than all other treatments, which were similar to controls. Amendment with T-12 powder at 50 g/kg increased root length, but 80 g/kg medium added as prills decreased root dry weight compared to the control. The highest rate of T-12 (800 g prills/kg medium) had no effect on root growth. This suggests that moderate, rather than high rates of T-12 are more effective in promoting rooting of unrooted chrysanthemum, and that there is a potential for phytotoxic effects on root growth with higher rates.     

Evidence of Type I and Type II Transforming Growth Factor-β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia

Abstract: The peptides of the transforming growth factor-β (TGF-β) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-β receptors and their potential ligands (TGF-β1, TGF-β2, and TGF-β3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-β isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-βs and their receptors. We show that the three TGF-β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-β2. We have demonstrated that both type I and type II TGF-β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-β1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-β receptor was not altered by the ischemic insult, the pattern of the type II TGF-β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.