Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.     

A comprehensive examination of protein sequences for evidence of internal gene duplication

Summary We have implemented a routine procedure for screening protein sequences for evidence of intragenic duplications. We tested 163 protein sequences representing 116 superfamilies of unrelated proteins. Twenty superfamilies contain proteins with internal gene duplications. The intragenic duplications detected can be divided into two major types. (1) One or more duplications of all or part of a gene produce a protein with two or several detectable regions of sequence homology. Sequences from 18 superfamilies contained this type of duplication. (2) Repeated reduplication of a small DNA segment can produce a protein that is repetitive over most of its length. Three superfamilies contain such repetitive sequences. We also investigated the limits of detection of ancient duplications using sequences derived by random mutation of a model sequence consisting of ten 10-residue repeats. The original repetitive nature of the sequence was usually detected after 250 point mutations even though the ancestral segment could not be accurately reconstructed.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

The Protein Information Resource

The Protein Information Resource (PIR) is an integrated public resource of protein informatics that supports genomic and proteomic research and scientific discovery. PIR maintains the Protein Sequence Database (PSD), an annotated protein database containing over 283 000 sequences covering the entire taxonomic range. Family classification is used for sensitive identification, consistent annotation, and detection of annotation errors. The superfamily curation defines signature domain architecture and categorizes memberships to improve automated classification. To increase the amount of experimental annotation, the PIR has developed a bibliography system for literature searching, mapping, and user submission, and has conducted retrospective attribution of citations for experimental features. PIR also maintains NREF, a non-redundant reference database, and iProClass, an integrated database of protein family, function, and structure information. PIR-NREF provides a timely and comprehensive collection of protein sequences, currently consisting of more than 1 000 000 entries from PIR-PSD, SWISS-PROT, TrEMBL, RefSeq, GenPept, and PDB. The PIR web site (http://pir.georgetown.edu) connects data analysis tools to underlying databases for information retrieval and knowledge discovery, with functionalities for interactive queries, combinations of sequence and text searches, and sorting and visual exploration of search results. The FTP site provides free download for PSD and NREF biweekly releases and auxiliary databases and files.     

Ethics guidelines for research with the recently dead

The objective of the multidisciplinary expert Consensus Panel on Research with the Recently Dead (CPRRD) was to craft ethics guidelines for research with the recently dead. The CPRRD recommends that research with the recently dead: (i) receive scientific and ethical review and oversight; (ii) involve the community of potential research subjects; (iii) be coordinated with organ procurement organizations; (iv) not conflict with organ donation or required autopsy; (v) use procedures respectful of the dead; (vi) be restricted to one procedure per day; (vii) preferably be authorized by first-person consent, though both general advance research directives and surrogate consent are acceptable; (viii) protect confidentiality; (ix) not impose costs on subjects' estates or next of kin and not involve payment; (x) clearly explain ultimate disposition of the body.     

The 50s Cliff: Perceptuo-Motor Learning Rates across the Lifespan

We recently found that older adults show reduced learning rates when learning a new pattern of coordinated rhythmic movement. The purpose of this study was to extend that finding by examining the performance of all ages across the lifespan from the 20 s through to the 80 s to determine how learning rates change with age. We tested whether adults could learn to produce a novel coordinated rhythmic movement (90° relative phase) in a visually guided unimanual task. We determined learning rates to quantify changes in learning with age and to determine at what ages the changes occur. We found, as before, that learning rates of participants in their 70 s and 80 s were half those of participants in their 20 s. We also found a gradual slow decline in learning rate with age until approximately age 50, when there was a sudden drop to a reduced learning rate for the 60 though 80 year olds. We discuss possible causes for the “50 s cliff” in perceptuo-motor learning rates and suggest that age related deficits in perception of complex motions may be the key to understanding this result.     

Analysis and Organization of Protein Sequence Data: A Retrospective Spanning Four Decades

Protein sequence data are as useful and valuable today as was envisioned by pioneering sequencers and by the organizers of the first sequence database. Sequence analysis was first the province of specialists who developed search, comparison, and tree-building methods. Microcomputers, communication satellites, and the Internet have made these methods accessible to any scientist. The rapid increase in the data has driven a succession of changes in how databases are compiled, distributed, and accessed. Large public databases have become international collaborations. Although they need to develop still more efficient ways to accumulate, organize, annotate, and standardize huge amounts of data, inadequate support is available for such efforts. Thus there will be greater reliance on direct input from the scientific community. The World Wide Web is essential but not sufficient for integrated access to related databases.     

Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen–glucose deprivation

Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen–glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5–10 min of HS and decreased to baseline levels or lower by 30–60 min. Decrease in nuclear CHIP at 30–60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen–glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute changes in subcellular localization of CHIP in response to cellular stress suggest that cellular changes that occur shortly after exposure to stress ultimately impact on the capacity and capability of a cell to recover and survive.     

Behavioral and neuronal interactions between exercise and alcohol: Sex and genetic differences

Alcohol use disorders (AUDs) lead to early death and many devastating consequences for individuals, families and society. Currently, few effective treatments are available, but emerging research suggests exercise might be beneficial in some individuals. To develop the most effective exercise treatment program, more research on intensity, type, timing, stage of addiction, drug involved, sex of subject and subject population is needed. This review highlights the complexity of the interaction between alcohol behaviors and exercise, with a focus on the role of sex and genetics. Moreover, we describe a variety of rodent models used to investigate the neuronal physiology changes that underlie alcohol consumption and exercise. Specifically, current data indicate that moderate exercise may ameliorate neuronal damage caused by alcohol consumption. Additionally, we describe studies of rodent models in the context of hedonic substitution to draw broad conclusions about shared underlying neurobiological mechanisms. Until recently, most studies in rodents were performed only in males, and few studies have utilized different genetic strains of mice or rats. Comparing similar behavioral paradigms across sex and strain, it has become clear that major sex and genetic differences exist for each behavioral context alone (alcohol consumption and exercise) and combined. Therefore, future research in this area should be developed with careful study design and attention to address both of these factors.     

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-γ-carotene and γ-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a β-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the β-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and ∼18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.