The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using ³¹P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2′,3′-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by ¹H/¹⁵N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

Amyloid-Beta Immunization in Alzheimer's Disease Transgenic Mouse Models and Wildtype Mice

Alzheimer's disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid- deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid- (A) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive A immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral A levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of A immunization studies in mice, a clinical trial was initiated for A vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.     

Protein Phosphorylation and APP Metabolism

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimer's amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimer's disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed A. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and A production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.     

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.     

Recognition of nucleic acid bases and base-pairs by hydrogen bonding to amino acid side-chains

Sequence-specific protein-nucleic acid recognition is determined, in part, by hydrogen bonding interactions between amino acid side-chains and nucleotide bases. To examine the repertoire of possible interactions, we have calculated geometrically plausible arrangements in which amino acids hydrogen bond to unpaired bases, such as those found in RNA bulges and loops, or to the 53 possible RNA base-pairs. We find 32 possible interactions that involve two or more hydrogen bonds to the six unpaired bases (including protonated A and C), 17 of which have been observed. We find 186 "spanning" interactions to base-pairs in which the amino acid hydrogen bonds to both bases, in principle allowing particular base-pairs to be selectively targeted, and nine of these have been observed. Four calculated interactions span the Watson-Crick pairs and 15 span the G:U wobble pair, including two interesting arrangements with three hydrogen bonds to the Arg guanidinum group that have not yet been observed. The inherent donor-acceptor arrangements of the bases support many possible interactions to Asn (or Gln) and Ser (or Thr or Tyr), few interactions to Asp (or Glu) even though several already have been observed, and interactions to U (or T) only if the base is in an unpaired context, as also observed in several cases. This study highlights how complementary arrangements of donors and acceptors can contribute to base-specific recognition of RNA, predicts interactions not yet observed, and provides tools to analyze proposed contacts or design novel interactions.     

Molecular and cellular cognitive studies of the role of synaptic plasticity in memory

Synaptic plasticity has a central role in nearly all models of learning and memory. Besides experiments documenting changes in synaptic function during learning, most of the evidence supporting a role for synaptic plasticity in memory comes from manipulations that either enhance or lesion synaptic processes. In the last decade, mouse transgenetics (knock outs and transgenics) have provided compelling evidence that the molecular mechanisms responsible for the induction and stability of synaptic changes have a critical role in the acquisition and storage of information. Here, I will review this literature, with a special focus on studies of hippocampal-dependent learning and memory.     

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.