Discovery and SAR of 2-aminothiazole inhibitors of cyclin-dependent kinase 5/p25 as a potential treatment for Alzheimer's disease

High-throughput screening with cyclin-dependent kinase 5 (cdk5)/p25 led to the discovery of N-(5-isopropyl-thiazol-2-yl)isobutyramide (1). This compound is an equipotent inhibitor of cdk5 and cyclin-dependent kinase 2 (cdk2)/cyclin E (IC(50)=ca. 320nM). Parallel and directed synthesis techniques were utilized to explore the SAR of this series. Up to 60-fold improvements in potency at cdk5 and 12-fold selectivity over cdk2 were achieved.     

Reconstructing Roma History from Genome-Wide Data

The Roma people, living throughout Europe and West Asia, are a diverse population linked by the Romani language and culture. Previous linguistic and genetic studies have suggested that the Roma migrated into Europe from South Asia about 1,000–1,500 years ago. Genetic inferences about Roma history have mostly focused on the Y chromosome and mitochondrial DNA. To explore what additional information can be learned from genome-wide data, we analyzed data from six Roma groups that we genotyped at hundreds of thousands of single nucleotide polymorphisms (SNPs). We estimate that the Roma harbor about 80% West Eurasian ancestry–derived from a combination of European and South Asian sources–and that the date of admixture of South Asian and European ancestry was about 850 years before present. We provide evidence for Eastern Europe being a major source of European ancestry, and North-west India being a major source of the South Asian ancestry in the Roma. By computing allele sharing as a measure of linkage disequilibrium, we estimate that the migration of Roma out of the Indian subcontinent was accompanied by a severe founder event, which appears to have been followed by a major demographic expansion after the arrival in Europe.     

An immunocytochemical approach to detection of mitochondrial disorders.

Mitochondrial disorders can lead to a confusing array of symptoms, which frequently makes a diagnosis difficult. Traditional approaches to such diagnoses are based on enzyme activity assays, with further characterization provided by genetic analysis. However, these methods require relatively large sample sizes, are time-consuming, labor-intensive, and show variability between laboratories. Here, we report an immunocytochemical test that makes use of monoclonal antibodies to subunits from each of the oxidative phosphorylation complexes and pyruvate dehydrogenase to aid in the detection of mitochondrial disorders. It can be completed and data analyzed in less than 4 hr. We have used this test to study fibroblast cultures from patients with mitochondrial disorders arising from both mitochondrial DNA and nuclear DNA defects. We have also examined cases of Leigh syndrome arising from different genetic causes. We show that patients can be categorized on the basis of which complexes are affected and whether or not the defect being studied shows a mosaic distribution, an indicator of whether the causal mutation(s) is/are in the mitochondrial or nuclear genome. Immunocytochemical analysis as described here should be considered as an initial screen for mitochondrial disorders by which to direct (and limit) the subsequent enzymatic and genetic tests required to make an unambiguous diagnosis.     

Global synthesis of the documented and projected effects of climate change on inland fishes

Although climate change is an important factor affecting inland fishes globally, a comprehensive review of how climate change has impacted and will continue to impact inland fishes worldwide does not currently exist. We conducted an extensive, systematic primary literature review to identify English-language, peer-reviewed journal publications with projected and documented examples of climate change impacts on inland fishes globally. Since the mid-1980s, scientists have projected the effects of climate change on inland fishes, and more recently, documentation of climate change impacts on inland fishes has increased. Of the thousands of title and abstracts reviewed, we selected 624 publications for a full text review: 63 of these publications documented an effect of climate change on inland fishes, while 116 publications projected inland fishes’ response to future climate change. Documented and projected impacts of climate change varied, but several trends emerged including differences between documented and projected impacts of climate change on salmonid abundance (P = 0.0002). Salmonid abundance decreased in 89.5% of documented effects compared to 35.7% of projected effects, where variable effects were more commonly reported (64.3%). Studies focused on responses of salmonids (61% of total) to climate change in North America and Europe, highlighting major gaps in the literature for taxonomic groups and geographic focus. Elucidating global patterns and identifying knowledge gaps of climate change effects on inland fishes will help managers better anticipate local changes in fish populations and assemblages, resulting in better development of management plans, particularly in systems with little information on climate change effects on fish.     

Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.The bacterial type IV prepilin peptidase (TFPP) is a well-characterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of N-terminal signal peptides from prepilins and pseudopilins, prior to their incorporation into the type IV pilus structure (22, 30, 31). The prepilin peptidase is also responsible for the processing of prepilin-like proteins needed for type II secretion (22). In Archaea, the existence of bacterial TFPP-like enzymes has also been reported, and they have been most extensively studied in relation to the assembly of the archaeal flagellum. In the euryarchaeotes Methanococcus maripaludis and Methanococcus voltae, the preflagellin peptidase FlaK was demonstrated to be responsible for cleaving the N-terminal signal peptide from the preflagellin prior to its incorporation into the growing flagellar filament, a step essential to flagellar assembly (6, 7, 26). In Sulfolobus solfataricus, an acidophilic crenarchaeote, the equivalent enzyme, PibD, was also shown to process preflagellins (4). Site-directed mutagenesis of FlaK and PibD demonstrated that both aspartic acid residues that aligned with aspartic acid residues essential for bacterial TFPP activity were also essential in the archaeal enzymes (6, 32), indicating that the two archaeal peptidases belong with the bacterial TFPPs in this novel family of aspartic acid proteases (20). More recently, an additional archaeal TFPP was found to be required for cleavage of the prepilin substrates (33) that are assembled into the unique pili of M. maripaludis (37).The substrate specificity of the archaeal preflagellin peptidase remains an open question. Like prepilin peptidases, FlaK in M. voltae has stringent requirements for the amino acids surrounding the cleavage site of the substrate, especially the −1 glycine, −2 and −3 lysines, and the +3 glycine (numbers given relative to the cleavage site) (35); the last position was conserved in all archaeal flagellins (25). Upon N-terminal sequence alignment of all available archaeal flagellin amino acid sequences at the predicted cleavage site, it was found that most archaeal preflagellin signal peptides are quite conserved in length, with the typical flagellin signal peptide being 11 to 12 amino acids in length (Table ​(Table1).1). It is speculated that while a certain amount of flexibility might exist, some optimum and minimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane (18). A recent study examining possible type IV pilin-like substrates in archaea using the FlaFind program indicated that such substrates may be more widespread than initially thought (33). Since in Methanococcus the pilins are processed by a second TFPP (EppA) (33), it is very possible that the preflagellins might be the only substrates of FlaK in these archaea.

TABLE 1.

N-terminal amino acid alignment of selected archaeal flagellin sequences^a

Modeling ensembles of transmembrane beta-barrel proteins

Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. Despite their importance, very few nonhomologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane proteins. We introduce the program partiFold to investigate the folding landscape of TMBs. By computing the Boltzmann partition function, partiFold estimates inter-beta-strand residue interaction probabilities, predicts contacts and per-residue X-ray crystal structure B-values, and samples conformations from the Boltzmann low energy ensemble. This broad range of predictive capabilities is achieved using a single, parameterizable grammatical model to describe potential beta-barrel supersecondary structures, combined with a novel energy function of stacked amino acid pair statistical potentials. PartiFold outperforms existing programs for inter-beta-strand residue contact prediction on TMB proteins, offering both higher average predictive accuracy as well as more consistent results. Moreover, the integration of these contact probabilities inside a stochastic contact map can be used to infer a more meaningful picture of the TMB folding landscape, which cannot be achieved with other methods. Partifold's predictions of B-values are competitive with recent methods specifically designed for this problem. Finally, we show that sampling TMBs from the Boltzmann ensemble matches the X-ray crystal structure better than single structure prediction methods. A webserver running partiFold is available at http://partiFold.csail.mit.edu/.     

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.     

Three Approaches to the Study of Language and Gender

Gender across Languages: The Linguistic Representation of Women and Men. Vol. 2. Marlis Hellinger and Hadumod Bußmann eds. Amsterdam: John Benjamins, 2002. 348 pp.
Gender Identity and Discourse Analysis. Lia Litosseliti and Jane Sunderland eds. Amsterdam: John Benjamins, 2002. 335 pp.
Talking Gender and Sexuality. Paul McIlvenny. ed. Amsterdam: John Benjamins, 2002. 327 pp.