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Abstract Background Determining the functions of uncharacterized proteins is one of the most pressing problems in the post-genomic era. Large scale protein-protein interaction assays, global mRNA expression analyses and systematic protein localization studies provide experimental information that can be used for this purpose. The data from such experiments contain many false positives and false negatives, but can be processed using computational methods to provide reliable information about protein-protein relationships and protein function. An outstanding and important goal is to predict detailed functional annotation for all uncharacterized proteins that is reliable enough to effectively guide experiments. Results We present AVID, a computational method that uses a multi-stage learning framework to integrate experimental results with sequence information, generating networks reflecting functional similarities among proteins. We illustrate use of the networks by making predictions of detailed Gene Ontology (GO) annotations in three categories: molecular function, biological process, and cellular component. Applied to the yeast Saccharomyces cerevisiae, AVID provides 37,451 pair-wise functional linkages between 4,191 proteins. These relationships are ~65–78% accurate, as assessed by cross-validation testing. Assignments of highly detailed functional descriptors to proteins, based on the networks, are estimated to be ~67% accurate for GO categories describing molecular function and cellular component and ~52% accurate for terms describing biological process. The predictions cover 1,490 proteins with no previous annotation in GO and also assign more detailed functions to many proteins annotated only with less descriptive terms. Predictions made by AVID are largely distinct from those made by other methods. Out of 37,451 predicted pair-wise relationships, the greatest number shared in common with another method is 3,413. Conclusion AVID provides three networks reflecting functional associations among proteins. We use these networks to generate new, highly detailed functional predictions for roughly half of the yeast proteome that are reliable enough to drive targeted experimental investigations. The predictions suggest many specific, testable hypotheses. All of the data are available as downloadable files as well as through an interactive website at http://web.mit.edu/biology/keating/AVID. Thus, AVID will be a valuable resource for experimental biologists.

DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O 6 -dG-derived ICLs. Although less abundant, O 6 -alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O 6 -2'-deoxyguanosine-butylene-O 6 -2'-deoxyguanosine (O 6 -dG-C4-O 6 -dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O 6 -dG-C4-O 6 -dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol κ is completely inhibited by O 6 -dG-C4-O 6 -dG ICLs. Moderate bypass activities are observed for human pol η and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol η exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol η in O 6 -dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol η. Together, this study provides the first set of biochemical evidence for the mutagenicity of O 6 -dG-derived ICLs.

Vascular endothelial growth factor (VEGF) is an angiogenic factor shown to be a critical secreted cytokine in tumorigenesis and retinal neovascularization (NV). Currently, there are two anti-VEGF agents, pegaptanib and ranizumab, approved by the United States Food and Drug Administration (FDA) for intravitreal use in the treatment of wet age-related macular degeneration (AMD). Bevacizumab is FDA-approved for intravenous administration in the treatment of several cancers and is in widespread use, off-label, as an intravitreal injection to treat a variety of retinal pathologies. Animal studies demonstrate the role of VEGF in corneal NV. There are now a number of human case series reporting the use of anti-VEGF agents, primarily bevacizumab, to treat corneal NV. This review summarizes reports to date on the use of anti-VEGF agents in the treatment of corneal NV in humans, noting the limitations of current data and the need for further studies. The experience of one clinician with the use of an anti-VEGF drug in the treatment of active corneal NV is presented.

Bcor (BCL6 corepressor) is a widely expressed gene that is mutated in patients with X-linked Oculofaciocardiodental (OFCD) syndrome. BCOR regulates gene expression in association with a complex of proteins capable of epigenetic modification of chromatin. These include Polycomb group (PcG) proteins, Skp-Cullin-F-box (SCF) ubiquitin ligase components and a Jumonji C (Jmjc) domain containing histone demethylase. To model OFCD in mice and dissect the role of Bcor in development we have characterized two loss of function Bcor alleles. We find that Bcor loss of function results in a strong parent-of-origin effect, most likely indicating a requirement for Bcor in extraembryonic development. Using Bcor loss of function embryonic stem (ES) cells and in vitro differentiation assays, we demonstrate that Bcor plays a role in the regulation of gene expression very early in the differentiation of ES cells into ectoderm, mesoderm and downstream hematopoietic lineages. Normal expression of affected genes (Oct3/4, Nanog, Fgf5, Bmp4, Brachyury and Flk1) is restored upon re-expression of Bcor. Consistent with these ES cell results, chimeric animals generated with the same loss of function Bcor alleles show a low contribution to B and T cells and erythrocytes and have kinked and shortened tails, consistent with reduced Brachyury expression. Together these results suggest that Bcor plays a role in differentiation of multiple tissue lineages during early embryonic development.

Although mania was not reported as an adverse event in the pivotal trials of ziprasidone, there have been 7 reports of ziprasidone-induced mania in 12 patients. We now report 2 additional cases wherein the introduction of ziprasidone resulted in new-onset manic episodes. In 1 case, the patient required hospitalization and lost his job. In the other, time to mania was 5 months, considerably longer than previously reported. Of the 14 cases, 9 were in a depressive episode when ziprasidone was prescribed, and 8 had a history of current or past exposure to antidepressants. Ziprasidone, like many antidepressants, can block reuptake of norepinephrine and serotonin, although mania has developed in patients treated with atypical antipsychotics that are less potent in this regard. However, ziprasidone and other atypical antipsychotics have in common a high ratio of 5-HT2a to D2 receptor blockade, which may also play a role in this phenomenon. Clinicians and patients need to be aware of the potential for the induction of mania with ziprasidone, even after lengthy exposure.

Activators of eukaryotic transcription often function over a range of distances. It is commonly hypothesized that the intervening DNA between the transcription start site and the activator binding sites forms a loop in order to allow the activators to interact with the basal transcription apparatus, either directly or through mediators. If this hypothesis is correct, activation should be sensitive to the presence of intrinsic bends in the intervening DNA. Similarly, the precise helical phasing of such DNA bends and of the activator binding sites relative to the basal promoter should affect the degree of transcription activation. To explore these considerations, we designed transcription templates based on the adenovirus E4 promoter supplemented with upstream Gal4 activator binding sites. Surprisingly, we found that neither insertion of intrinsically curved DNA sequences between the activator binding sites and the basal promoter, nor alteration of the relative helical alignment of the activator binding sites and the basal promoter significantly affected in vitro transcription activation in HeLa cell nuclear extract. In all cases, the degree of transcription activation was a simple inverse function of the length of intervening DNA. Possible implications of these unexpected results are discussed., (Copyright 2000 Academic Press.)

Pathogenic Neisseria species elaborate type IV pili, which are considered important for virulence. In this study, we examined pilin-encoding expression loci (pilE) in nonpathogenic Neisseria species. PCR based screening detected homology to a conserved N-terminal region of pilE in 12 of 15 Neisseria species, including all human commensal isolates. The three species failing to display homology were isolated from nonhuman sources. We have also characterized complete pilE loci from the human commensal species N. lactamica and N. cinerea. As anticipated, the predicted protein sequences from these species display features typical of all type IV pilins. In addition, these commensal pilins possess two highly conserved regions, SV2 and CYS2, which are shared among all neisserial pilins. However, a comparative analysis of pilE loci from pathogenic and nonpathogenic Neisseria species reveals two distinct structural groups, one composed of the pilin genes from N. lactamica, N. cinerea, and the class II pilin-producing subset of N. meningitidis isolates, the other of gonococcal and meningococcal class I pilin-encoding genes. Since both class I and class II pilin-producing meningococci can act as pathogens, structural relationships among neisserial pilin genes do not obviously reflect either species membership or ability to cause human disease., (Copyright 2000 Academic Press.)

This paper re-examines the theoretical concept of severe brain injury focusing on the duration of coma as a precise indicator of the clinical profile. A retrospective hospital chart study of 361 traumatic brain-injured patients was undertaken to determine the homogeneity of the subsample of the severely brain-injured (defined as 2 or more days of coma) with respect to the probability of four types of impairment: ataxia, contractures, paralysis and speech impairment. The current concept of severity assumes homogeneity among the 'severely brain-injured'. However, our results indicate significant differences in impairment within this population. The authors feel strongly that future studies must describe coma duration in finer gradations, and test for homogeneity within samples before inferences are made. Improvements in life-sustaining technologies have resulted in longer coma durations. The need to use coma days as an indicator of impairment rather than a broad category of severity is emphasized.