Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.     

Structure of the human 26S proteasome: subunit radial displacements open the gate into the proteolytic core

The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.     

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.     

Perkinsus olseni detected in Vietnamese aquacultured reef clams Tridacna crocea imported to the USA, following a mortality event

Morbidity and mortality were observed in a group of 30 reef clams Tridacna crocea that were imported to Florida, USA, from a Vietnamese culture facility and held in research facility aquaria. Clinical signs included an incompletely extended mantle, slow mantle responses to stimuli, and sloughing of byssal tissue beginning 2 to 5 d prior to death. Necropsy findings included emaciation, visceral mass edema, and rare multifocal 1 mm off-white to light-tan gill nodules. Histopathology revealed marked inflammation and necrosis within the visceral mass and gills, with interstitial edema and atrophy of glandular, gonadal, and muscular tissues. Inflamed tissues contained large numbers of 10 to 15 microm extracellular round organisms consistent with Perkinsus sp. trophozoites. The organisms often formed clusters of 1 to 4 cells and were surrounded by a 1 to 3 microm rim of eosinophilic material variably forming a radiating corona pattern and by 3 to 4 host hemocytes with dense round nuclei. Polymerase chain reaction assays indicated the presence of Perkinsus sp. DNA in these animals, and species-specific assays indicated the presence of P. olseni, and possibly other Perkinsus spp., but not P. marinus. Identification of Perkinsus spp. other than P. marinus in T. crocea imported from Vietnam confirms that importation of untested and unquarantined ornamental reef clams has possibly allowed incursion of P. olseni into the USA.     

Solution NMR structures of the antimicrobial peptides phylloseptin-1, -2, and -3 and biological activity: the role of charges and hydrogen bonding interactions in stabilizing helix conformations

Phylloseptins are antimicrobial peptides of 19-20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2> or =PS-1>PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions.     

Candidate genes and sensory functions in health and irritable bowel syndrome

Adrenergic and serotonergic (ADR-SER) mechanisms alter gut (GI) function; these effects are mediated through G protein transduction. Candidate genetic variations in ADR-SER were significantly associated with somatic scores in irritable bowel syndrome (IBS) and gastric emptying but not small bowel or colonic transit. Our aim was to assess whether candidate ADR-SER genes are associated with motor and sensory GI functions in IBS and subgroups on the basis of bowel dysfunction. In 122 patients with IBS and 39 healthy controls, we assessed gastrointestinal somatic symptoms and affect by validated questionnaires. We measured: gastric volume (GV), maximum tolerated volume, rectal compliance, sensation thresholds and ratings, and genetic variations including alpha2A (C-1291G), alpha2C (Del 332-325), GNbeta3 (C825T), and 5-HTTLPR. Demographics and genotype distributions were similar in the patients with IBS subgrouped on bowel function. There were significant associations between 5-HTTLPR SS genotype and absence of IBS symptoms and between 5-HTTLPR LS/SS genotype and increased rectal compliance and increased pain ratings, particularly at 12 and 24 mmHg distensions. GNbeta3 was associated only with fasting GV; we did not detect associations between alpha2A genotype and the gastrointestinal sensory or motor functions tested. We concluded that 5-HTTLPR LS/SS genotype is associated with both increased pain sensation and increased rectal compliance though the latter effect is unlikely to contribute to increased pain sensation ratings with LS/SS genotype. The data suggest the hypotheses that the endophenotype of visceral hypersensitivity in IBS may be partly related to genetic factors, and the association of GNbeta3 with fasting GV may explain, in part, the reported association of GNbeta3 with dyspepsia.     

Genetic variation in endocannabinoid metabolism, gastrointestinal motility, and sensation

Cannabinoid agonist inhibits gastrointestinal motility. The endocannabinoid, anandamide, is inactivated by fatty acid amide hydrolase (FAAH). A single nucleotide polymorphism in the human FAAH gene (C385A) reduces FAAH expression. Our aim was to evaluate associations between FAAH genotype variation and symptom phenotype, gastric emptying and volume, colonic transit, and rectal sensation in patients with functional gastrointestinal disorders (FGID). 482 FGID patients [Rome II positive, 159 constipation disorders, 184 diarrhea disorders (D-IBS), 86 mixed bowel function (M-IBS), 20 chronic abdominal pain (CAP), 33 functional dyspepsia], and 252 healthy volunteers (HV) underwent questionnaires and studies of phenotype and genotype from 2000 to 2007: 250 gastric emptying, 210 fasting and postprandial gastric volume, 152 colonic transit, and 123 rectal sensation. All had FAAH genotype [CC vs. polymorphic (CA/AA)] determined by TaqMan. FAAH genotype distribution of FGID patients and HV did not deviate from Hardy-Weinberg equilibrium. There was a significant association of FAAH genotype with FGID phenotype (overall chi(2), P = 0.011) and with specific individual phenotypes (P = 0.048). Thus FAAH CA/AA increases the odds (relative to HV) for D-IBS (P = 0.008), M-IBS (P = 0.012), and, possibly, CAP (P = 0.055). There was a significant association of FAAH CA/AA genotype with accelerated colonic transit in D-IBS (P = 0.037). There was no association of FAAH genotype with rectal sensation thresholds or ratings. The association of genetic variation in metabolism of endocannabinoids with symptom phenotype in D-IBS and M-IBS and with faster colonic transit in D-IBS supports the hypothesis that cannabinoid mechanisms may play a role in the control of colonic motility in humans and deserve further study.     

Structural characterization of cold extracted fraction of soluble sulfated polysaccharide from red seaweed Gracilaria birdiae

Water soluble polysaccharide from Gracilaria birdiae cultivated along the northeast coast of Brazil was characterized by infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. The composition of the polysaccharide in wt% was determined as: β-d-galp (50.3%), 3,6-anhydro--l-galp (40.5%) and --l-galp-6 sulfate (9.2%). The ratio of l/d units (β-d-galp units and 3,6-anhydro--l-galp + -l-galp-6 sulfate) is that of an ideal agarose. The sulfate content calculated by S% accounts for 6.4%. 1D and 2D NMR techniques were employed in order to assign the spin system of polysaccharide without partial degradation. The structure is composed of → 4-3,6-anhydro--l-galp (1 → 3)β-d-galp 1 → segments, with the possibility of a -l-galp unit substituted at the 6-position by sulfate ester.     

Auxiliary proteins involved in the assembly and sustenance of photosystem II

Chloroplast proteins that regulate the biogenesis, performance and acclimation of the photosynthetic protein complexes are currently under intense research. Dozens, possibly even hundreds, of such proteins in the stroma, thylakoid membrane and the lumen assist the biogenesis and constant repair of the water splitting photosystem (PS) II complex. During the repair cycle, assistance is required at several levels including the degradation of photodamaged D1 protein, de novo synthesis, membrane insertion, folding of the nascent protein chains and the reassembly of released protein subunits and different co-factors into PSII in order to guarantee the maintenance of the PSII function. Here we review the present knowledge of the auxiliary proteins, which have been reported to be involved in the biogenesis and maintenance of PSII.     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.