The transcriptional response to six commonly-abused drugs was assessed in the mouse brain revealing common modules of drug-induced genes.

We applied a new multi-step protocol to predict the structures of all targets during CASP5, regardless of their potential category. 1) We used diverse fold-recognition (FR) methods to generate initial target-template alignments, which were converted into preliminary full-atom models by comparative modeling. All preliminary models were evaluated (scored) by VERIFY3D to identify well- and poorly-folded fragments. 2) Preliminary models with similar 3D folds were superimposed, poorly-scoring regions were deleted and the "average model" structure was created by merging the remaining segments. All template structures reported by FR were superimposed and a composite multiple-structure template was created from the most conserved fragments. 3). The average model was superimposed onto the composite template and the structure-based target-template alignment was inferred. This alignment was used to build a new (intermediate) comparative model of the target, again scored with VERIFY3D. 4) For all poorly scoring regions series of alternative alignments were generated by progressively shifting the "unfit" sequence fragment in either direction. Here, we considered additional information, such as secondary structure, placement of insertions and deletions in loops, conservation of putative catalytic residues, and the necessity to obtain a compact, well-folded structure. For all alternative alignments, new models were built and evaluated. 5) All models were superimposed and the "FRankenstein's monster" (FR, fold recognition) model was built from best-scoring segments. The final model was obtained after limited energy minimization to remove steric clashes between sidechains from different fragments. The novelty of this approach is in the focus on "vertical" recombination of structure fragments, typical for the ab initio field, rather than "horizontal" sequence alignment typical for comparative modeling. We tested the usefulness of the "FRankenstein" approach for non-expert predictors: only the leader of our team had considerable experience in protein modeling - he registered as a separate group (020) and submitted models built only by himself. At the onset of CASP5, the other five members of the team (students) had very little or no experience with modeling. They followed the same protocol in a deliberately naïve way. In the fourth step they used solely the VERIFY3D criterion to compare their models and the leader's model (the latter regarded only as one of the many alternatives) and generated the hybrid or selected only one model for submission (group 517). In order to compare our protocol with the traditional "one target-one template-one alignment" approach, we submitted (as a separate group 242) models selected from those automatically generated by all CAFASP servers (i.e. obtained without any human intervention). Here, we compare the results obtained by the three "groups", describe successes and failures of the "FRankenstein" approach and discuss future developments of comparative modeling. The automatic version of our multi-st...

The memory of fear extinction is context dependent: fear that is suppressed in one context readily renews in another. Understanding of the underlying neuronal circuits is, therefore, of considerable clinical relevance for anxiety disorders. Prefrontal cortical and hippocampal inputs to the amygdala have recently been shown to regulate the retrieval of fear memories, but the cellular organization of these projections remains unclear. By using anterograde tracing in a transgenic rat in which neurons express a dendriticallytargeted PSD-95:Venus fusion protein under the control of a c-fos promoter, we found that, during the retrieval of extinction memory, the dominant input to active neurons in the lateral amygdala was from the infralimbic cortex, whereas the retrieval of fear memory was associated with greater hippocampal and prelimbic inputs. This pattern of retrieval-related afferent input was absent in the central nucleus of the amygdala. Our data show functional anatomy of neural circuits regulating fear and extinction, providing a framework for therapeutic manipulations of these circuits.gene expression | hippocampus | prefrontal cortex | learning and memory T here is an increasing interest in the neural mechanisms underlying extinction of learned fear, in part because fear extinction is a useful model for exposure-based therapies for the treatment of human anxiety disorders, such as phobias and posttraumatic stress disorder (1). During fear extinction, a previously conditioned stimulus (CS) is repeatedly presented in the absence of the unconditioned stimulus (US), a procedure that induces a progressive decrease in the magnitude and probability of learned fear responses, including freezing behavior. However, extinction does not erase the original fear memory; rather, it promotes the formation of a new inhibitory memory that reduces fear to the CS (2). Extinguished fear is highly context dependent, insofar as CS presentation outside the extinction context results in the recovery of the previously conditioned fear response, a phenomenon known as fear renewal (3). The return of fear after extinction is a considerable challenge for the efficacy of exposure-based therapies (4). Therefore, identification of brain structures and neuronal circuits selectively implicated in extinction vs. renewal of fear is of great importance.Owing to substantial progress toward understanding the neural mechanisms underlying the context specificity of fear extinction, there is now a general consensus that, for auditory fear conditioning, extinction involves three main structures: the amygdala, hippocampus (HIPP), and prefrontal cortex (PFC) (2, 5-8). However, the neuronal interactions between these structures that underlie contextual retrieval of fear memory after extinction remain to be elucidated. This problem is further complicated by the fact that neither the amygdala nor the PFC is a homogeneous structure. Among the substructures of the amygdala, the central, basal, and lateral nuclei (Ce, Ba, and La, respectively) have been impl...

The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

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The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC--C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed ‘yeast Endonuclease G (EndoG)’, is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5′–3′ exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog ‘EXOG’, a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5′–3′ exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.

In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts.