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The human genome is rife with the proviral remains of many ancient retroviruses. The past year has seen significant progress in understanding the structure, distribution and potential function of many of these elements. Although hypotheses concerning the potential effects of these elements are common, however, incisive experiments to test any functions remain much less so.
Since its discovery 10 years ago, the potential functions of protein kinase B (PKB)/AKT have been catalogued with increasing efficiency. The physiological relevance of some of the proposed mechanisms by which PKB/AKT mediates many of its effects has been questioned, and recent work using new reagents and approaches has revealed some cracks in our understanding of this important molecule, and also hinted that these effects may involve other players.
To analyze potential functional consequences of myosin heavy chain (MHC) mutations identified in patients with familial hypertrophic cardiomyopathy (FHC), we have assessed the stability of the mutant MHCs and their ability to form thick filaments. Constructs encoding wild-type rat alpha MHC and seven corresponding FHC missense mutants were transfected into COS cells. Immunoblot analysis suggested that FHC mutations do not grossly alter protein stability. Wild-type alpha MHC transfected into COS cells forms structures previously shown to be arrays of thick filaments, which also resemble myosin structures observed early in differentiation of muscle cells. Surprisingly, up to 29% of COS cells transfected with the FHC mutants failed to form filamentous structures. To assess whether this phenotype was specific for the FHC mutants and not generalizable to any myosin mutation, COS cells were transfected with a construct encoding an MHC with a 168-amino acid deletion of the hinge/rod region. This deletion construct formed filamentous structures with the same frequency as wild-type MHC. Biochemical analysis of one FHC mutant (Arg-249-->Gln) demonstrates that the structures formed by the mutant are solubilized at a lower ionic strength than those formed by wild-type MHC. We conclude that although the FHC mutant MHC is not labile, its assembly properties may be impaired.
Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.
The cubic nonlinear Schrödinger equation with repulsive nonlinearity and an elliptic function potential models a quasi-one-dimensional repulsive dilute gas Bose-Einstein condensate trapped in a standing light wave. New families of stationary solutions are presented. Some of these solutions have neither an analog in the linear Schrödinger equation nor in the integrable nonlinear Schrödinger equation. Their stability is examined using analytical and numerical methods. All trivial-phase stable solutions are deformations of the ground state of the linear Schrödinger equation. Our results show that a large number of condensed atoms is sufficient to form a stable, periodic condensate. Physically, this implies stability of states near the Thomas-Fermi limit.
Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.
RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated "core" RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1(c/c)) mice. Significant B and T cell numbers are generated in RAG1(c/c) mice, showing that core RAG1, despite missing approximately 40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1(c/c) mice. Correspondingly, there are reduced numbers of peripheral RAG1(c/c) B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1(c/c) mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1(c/c) B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1(c/c) B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.
Density functional theory geometry optimizations and reduction potential calculations are reported for all five known oxidation states of [Fe(4)S(4)(SCH(3))(4)](n)()(-) (n = 0, 1, 2, 3, 4) clusters that form the active sites of iron-sulfur proteins. The geometry-optimized structures tend to be slightly expanded relative to experiment, with the best comparison found in the [Fe(4)S(4)(SCH(3))(4)](2)(-) model cluster, having bond lengths 0.03 A longer on average than experimentally observed. Environmental effects are modeled with a continuum dielectric, allowing the solvent contribution to the reduction potential to be calculated. The calculated protein plus solvent effects on the reduction potentials of seven proteins (including high potential iron proteins, ferredoxins, the iron protein of nitrogenase, and the "X", "A", and "B" centers of photosystem I) are also examined. A good correlation between predicted and measured absolute reduction potentials for each oxidation state of the cluster is found, both for relative potentials within a given oxidation state and for the absolute potentials for all known couples. These calculations suggest that the number of amide dipole and hydrogen bonding interactions with the Fe(4)S(4) clusters play a key role in modulating the accessible redox couple. For the [Fe(4)S(4)](0) (all-ferrous) system, the experimentally observed S = 4 state is calculated to lie lowest in energy, and the predicted geometry and electronic properties for this state correlate well with the EXAFS and Mössbauer data. Cluster geometries are also predicted for the [Fe(4)S(4)](4+) (all-ferric) system, and the calculated reduction potential for the [Fe(4)S(4)(SCH(3))(4)](1)(-)(/0) redox couple is in good agreement with that estimated for experimental model clusters containing alkylthiolate ligands.
The tryptophan hydroxylase-2 gene (TPH2) codes for the enzyme of serotonin (5-HT) synthesis in the brain and variation of TPH2 has been implicated in disorders of emotion regulation. Here, we used functional magnetic resonance imaging (fMRI) to demonstrate that a potentially functional variant of TPH2 modulates amygdala responsiveness to emotional stimuli of both negative and positive valence.
It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2+ release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.
The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in transformation. The expression of the IGF-IR gene is negatively regulated by a number of transcription factors, including the WT1 and p53 tumor suppressors. Previous studies have suggested both physical and functional interactions between the WT1 and p53 proteins. The potential functional interactions between WT1 and p53 in control of IGF-IR promoter activity were addressed by transient coexpression of vectors encoding different isoforms of WT1, together with IGF-IR promoter-luciferase reporter constructs, in p53-null osteosarcoma-derived Saos-2 cells, wild-type p53-expressing kidney tumor-derived G401 cells, and mutant p53-expressing, rhabdomyosarcoma-derived RD cells. Similar studies were also performed to compare p53-expressing Balb/c-3T3 and clonally derived p53-null, (10)1 fibroblasts and the colorectal cancer cell line HCT116 +/+, which expresses a wild-type p53 gene, and its HCT116 -/- derivative, in which the p53 gene has been disrupted by homologous recombination. WT1 splice variants lacking a KTS insert between zinc fingers 3 and 4 suppressed IGF-IR promoter activity in the absence of p53 or in the presence of wild-type p53. WT1 variants that contain the KTS insert are impaired in their ability to bind to the IGF-IR promoter and are unable to suppress IGF-IR promoter. In the presence of mutant p53, WT1 cannot repress the IGF-IR promoter. Coimmunoprecipitation experiments showed that p53 and WT1 physically interact, whereas electrophoretic mobility shift assay studies revealed that p53 modulates the ability of WT1 to bind to the IGF-IR promoter. In summary, the transcriptional activity of WT1 proteins and their ability to function as tumor suppressors or oncogenes depends on the cellular status of p53.
A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 A) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two alpha/beta domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of L-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 3(10)-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum.
Carriers of mutations in the BRCA2 gene have a high risk of developing breast and other cancers. The BRCA2 gene, which is located on human chromosome 13, encodes a very large protein of only poorly understood function. To define regions of sequence conservation and highlight potentially functionally important domains, we have cloned and characterized the chicken BRCA2 gene, the first non-mammalian BRCA2 gene to be described. The gene is organized similarly to the human BRCA2 gene, but is more compact and is localized to the subtelomeric region of chicken chromosome 1q, within a region that contains other genes from human chromosome 13. The chicken BRCA2 gene encodes a protein of 3399 amino acids, which is poorly conserved with mammalian BRCA2 proteins, having only 37% amino acid identity overall with human BRCA2. However, certain domains are much more highly conserved, indicating functional significance. We describe genes with some of these conserved domains in organisms as diverse as intracellular parasites, mosquitoes and plants. The evolutionarily divergent chicken BRCA2 sequence may also be useful in assigning the large number of sequence variants that have been described in the human BRCA2 gene which are of unknown significance in disease causation.
The subject of the present study is the Monte Carlo path-integral evaluation of the moments of spectral functions. Such moments can be computed by formal differentiation of certain estimating functionals that are infinitely differentiable against time whenever the potential function is arbitrarily smooth. Here, I demonstrate that the numerical differentiation of the estimating functionals can be more successfully implemented by means of pseudospectral methods (e.g., exact differentiation of a Chebyshev polynomial interpolant), which utilize information from the entire interval . The algorithmic detail that leads to robust numerical approximations is the fact that the path-integral action and not the actual estimating functional are interpolated. Although the resulting approximation to the estimating functional is nonlinear, the derivatives can be computed from it in a fast and stable way by contour integration in the complex plane, with the help of the Cauchy integral formula (e.g., by Lyness' method). An interesting aspect of the present development is that Hamburger's conditions for a finite sequence of numbers to be a moment sequence provide the necessary and sufficient criteria for the computed data to be compatible with the existence of an inversion algorithm. Finally, the issue of appearance of the sign problem in the computation of moments, albeit in a milder form than for other quantities, is addressed.
Kinetics of mouse T-cell gamma gene rearrangements in ontogeny were determined as an approach to understanding the possible role of these genes in the development of fetal thymocytes. Two of these genes (C gamma 1 and C gamma 2) rearranged rapidly during days 14 to 17 of the gestational period in BALB/c mice. Moreover, these rearrangements seemed to be tightly synchronized with rearrangements of T-cell receptor beta chain genes in the same cells. It is suggested that the early transcriptional activity of gamma genes, which precedes that of beta chain genes, may not reflect the functional activation of these genes. Nevertheless, productive and therefore potentially functional gamma gene rearrangements precede surface expression of T-cell receptors in the thymus by 2 to 3 days, which is compatible with a role for gamma gene products in thymocyte development prior to antigen-specific stages.
The widening gap between known protein sequences and their functions has led to the practice of assigning a potential function to a protein on the basis of sequence similarity to proteins whose function has been experimentally investigated. We present here a critical view of the theoretical and practical bases for this approach. The results obtained by analyzing a significant number of true sequence similarities, derived directly from structural alignments, point to the complexity of function prediction. Different aspects of protein function, including (i) enzymatic function classification, (ii) functional annotations in the form of key words, (iii) classes of cellular function, and (iv) conservation of binding sites can only be reliably transferred between similar sequences to a modest degree. The reason for this difficulty is a combination of the unavoidable database inaccuracies and the plasticity of protein function. In addition, analysis of the relationship between sequence and functional descriptions defines an empirical limit for pairwise-based functional annotations, namely, the three first digits of the six numbers used as descriptors of protein folds in the FSSP database can be predicted at an average level as low as 7.5% sequence identity, two of the four EC digits at 15% identity, half of the SWISS-PROT key words related to protein function would require 20% identity, and the prediction of half of the residues in the binding site can be made at the 30% sequence identity level.
Chronic lymphocytic leukemia (CLL), the most frequent leukemia in the Western world, is characterized by a profound dysregulation of the host immune system that has a marked impact on the clinical course of the disease. To date, the competence of the circulating dendritic cell (DC) compartment in CLL patients has not been investigated. To address this issue, we sorted DC precursors from the peripheral blood of CLL patients and found a profoundly altered compartment as compared with normal donors. CLL DCs proved a morphologically and phenotypically immature population, lacking the maturation antigen CD83 and the costimulatory molecule CD80, unable to induce a significant proliferative response in allo-mixed lymphocyte reaction, with a reduced ability to release interleukin 12 and to drive a type 1 T-cell response. To investigate whether these defects could be ascribed to inhibiting soluble factors released by the leukemic clone, DCs were generated in vitro from normal monocytes in the presence of allogeneic CLL cells. The addition of CLL cells induced similar markers of abnormal maturation and functional impairment with an inhibition in the expression of costimulatory molecules and a reduction of their allo-stimulatory ability. The blocking of interleukin 6 activity was able to revert the inhibition in a proportion of patients. Taken together, these findings indicate that mechanisms of tumor-induced DC inhibition are operational in CLL patients, resulting in both maturative and functional defects in the circulating DC compartment, with a potential functional impact in the regulation of in vivo T-cell immune responses.
Small leucine-rich proteoglycans (SLRPs) regulate extracellular matrix organization. In order to investigate the distribution and potential functions of decorin, biglycan (BGN), and fibromodulin (3 SLRPs, potentially related to dentinogenesis), we performed light and electron immunochemistry on teeth from rats, and on wild-type and biglycan knockout mice (BGN KO). Immunohistochemical data demonstrate that chondroitin sulfate/dermatan sulfate (CS/DS) and keratan sulfate (KS) distributions displayed reverse gradients in predentin. The decrease of CS/DS labeling from the proximal to the distal predentin contrasted with the sharp decorin increase observed in the distal predentin near the predentin/dentin transition, an effect possibly attributable to the deglycosylation action of stromelysin-1. In contrast, BGN concentration was apparently constant throughout the whole predentin. Additional immunolabelings showed, for the first time, the presence of fibromodulin in predentin. Compared with the wild-type mouse, the mean diameter of collagen fibrils in the BGN KO was smaller in the proximal predentin but larger in the central and distal predentin, the metadentin was broader, and the dentin mineralization appeared altered and heterogeneous. Altogether, our data suggest an important role for BGN in dentin formation and mineralization.