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PubMed2004

Nucleation near the spinodal: limitations of mean field density functional theory.

We investigate the diverging size of the critical nucleus near the spinodal using the gradient theory (GT) of van der Waals and Cahn and Hilliard and mean field density functional theory (MFDFT). As is well known, GT predicts that at the spinodal the free energy barrier to nucleation vanishes while the radius of the critical fluctuation diverges. We show numerically that the scaling behavior found by Cahn and Hilliard for these quantities holds quantitatively for both GT and MFDFT. We also show that the excess number of molecules Deltag satisfies Cahn-Hilliard scaling near the spinodal and is consistent with the nucleation theorem. From the latter result, it is clear that the divergence of Deltag is due to the divergence of the mean field isothermal compressibility of the fluid at the spinodal. Finally, we develop a Ginzburg criterion for the validity of the mean field scaling relations. For real fluids with short-range attractive interactions, the near-spinodal scaling behavior occurs in a fluctuation dominated regime for which the mean field theory is invalid. Based on the nucleation theorem and on Wang's treatment of fluctuations near the spinodal in polymer blends, we infer a finite size for the critical nucleus at the pseudospinodal identified by Wang.

Journal Article↗

PubMed1997

Diverse transcription factors are involved in the quantitative regulation of transcriptional activation of kappa promoters.

Immunoglobulin kappa promoters show sequence divergence but conserved function between different subgroups. Here we show that three separate 5' elements are required for synergistic stimulation of transcription with the decamer in a kappa promoter. These sites are a 5' E-box, a 3' AT-rich region in the pentadecamer (pd) element, and the kappa-Y element. Elf-1 is a novel kappa-Y element ligand induced upon mitogenic stimulation of resting B lymphocytes. Furthermore, the 5' E2A-like E-box in the pd element could be substituted by an upstream stimulatory factor motif with conservation of function. Thus, the synergistic activation requirements of kappa transcription is strictly dependent on the quantitative presence of transcription factor-binding motifs 5' of the decamer, but these differ qualitatively in that they may bind an array of proteins with conserved function.

Animals↗

PubMed2003

Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling.

Bone formation is presumably a complex and well-orchestrated process of osteoblast lineage-specific differentiation. As members of the TGFbeta superfamily, bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation. Several BMPs are able to induce de novo bone formation. Although significant progress has recently been made about the transcriptional control of osteoblast differentiation, detailed molecular events underlying the osteogenic process remain to be elucidated. In order to identify potentially important signaling mediators activated by osteogenic BMPs but not by non-osteogenic BMPs, we sought to determine the transcriptional differences between three osteogenic BMPs (i.e., BMP-2, BMP-6, and BMP-9) and two inhibitory/non-osteogenic BMPs (i.e., BMP-3 and BMP-12). Through the microarray analysis of approximately 12,000 genes in pre-osteoblast progenitor cells, we found that expression level of 203 genes (105 up-regulated and 98 down-regulated) was altered >2-fold upon osteogenic BMP stimulation. Gene ontology analysis revealed that osteogenic BMPs, but not inhibitory/non-osteogenic BMPs, activate genes involved in the proliferation of pre-osteoblast progenitor cells towards osteoblastic differentiation, and simultaneously inhibit myoblast-specific gene expression. BMP-regulated expression of the selected target genes was confirmed by RT-PCR, as well as by the CodeLink Bioarray analysis. Our findings are consistent with the notion that osteogenesis and myogenesis are two divergent processes. Further functional characterization of these downstream target genes should provide important insights into the molecular mechanisms behind BMP-mediated bone formation.

Adenoviridae↗

PubMed2005

Post-transcriptional gene regulation by gamma herpesviruses.

The Epstein-Barr virus (EBV) SM protein is a member of a highly conserved family of proteins present in most mammalian herpes viruses. There is a significant amount of functional and sequence divergence among the homologs encoded by the human herpes viruses, including differences in mechanism of action and varying effects on splicing and transcription. Nevertheless, in those cases where it has been studied, these proteins are essential for lytic replication of the virus. The mechanism by which SM regulates gene expression operates at the level of mRNA stability, processing, and export. SM enhances expression of EBV lytic genes and has both positive and negative effects on cellular gene expression. In addition to enhancing accumulation of EBV gene mRNAs, SM has important effects on cellular mRNAs, altering the host cell gene expression profile to facilitate viral replication. This article describes the current state of knowledge regarding the role of EBV SM in cellular and viral gene regulation and summarizes some of the similarities and differences with the ORF57 homolog from Kaposi's sarcoma-associated herpes virus (KSHV/HHV8).

Animals↗

PubMed1994

IL-4 and IL-13 have overlapping but distinct effects on HIV production in monocytes.

In HIV-1-infected monocytes and monocytoid cell lines, viral expression can be observed as high-level production, restricted (chronic low-level) expression, and latency (no viral expression). Interleukin-13 (IL-13) and IL-4, which have remarkedly similar deactivating effects on inflammatory monocyte functions, were studied for their regulation of HIV expression in monocytes. Pretreatment of peripheral monocytes for 48-72 h with IL-13 markedly decreased acute HIV infection, whereas IL-4 increased it. Similar effects were seen when the U1 and R-THP-1 monocytoid cell lines with restricted HIV expression were treated with these cytokines. However, when these continuously producing cell lines were chronically treated with cytokines, IL-13 increased HIV production. Neither IL-4 nor IL-13 stimulated HIV expression in latently infected cells. In chronically infected cells, several cytokines reduced viral mRNA. Both IL-4 and IL-13 increased monocyte aggregate formation, but only IL-4 ultimately stimulated cytolysis of HIV-infected monocytes as well as increased apoptosis of U1. In the presence of tumor necrosis factor alpha or IL-6, which upregulate HIV expression, IL-13 could no longer suppress HIV expression. These results indicate that IL-4 and IL-13, although closely related in modulating monocyte function, can have divergent effects on HIV expression in monocytes. Collectively, these data suggest that there exists a complex cytokine tissue environment with positive regulators of HIV expression able to override negative regulators.

Cells, Cultured↗

PubMed1995

A consensus prediction of the secondary structure for the 6-phospho-beta-D-galactosidase superfamily.

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-beta-D-galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (alpha-beta class), with the core beta strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel beta sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C+D) and (D+E), respectively, and an analogous beta-alpha unit (strand G and helix 7) toward the end of the sequence. The resemblance of the supersecondary model to the tertiary structure formed by 8-fold alpha-beta barrel proteins is almost certainly not coincidental.

Amino Acid Sequence↗

PubMed1996

Pseudogenes for the human uracil-DNA glycosylase on chromosomes 14 and 16.

Two clones containing nonfunctional pseudogenes for the human uracil-DNA glycosylase gene have been isolated. The sequences of the two clones that are homologous to the UNG cDNA span 670 and 580 bp, respectively. In the longest of these, a full length Sx type Alu sequence interrupts the homologous sequence. Chromosomal mapping locates the clones to chromosomes 16 and 14. Comparison of the pseudogene sequences to the cDNA sequence indicates that the pseudogenes diverged from the functional gene approximately 31 and 22 million years ago, which is before the point in evolution when great apes and hominides separated.

Base Sequence↗

PubMed2000

Xenopus and chicken sperm contain a cytosolic soluble protein factor which can trigger calcium oscillations in mouse eggs.

There is evidence showing that at fertilization the sperm introduces into egg cytoplasm a protein-based cytosolic factor, which serves as the physiological trigger for inducing Ca(2+) oscillations in mammalian eggs. Here we show that sperm of nonmammalian vertebrates also contain a cytosolic protein factor that can induce Ca(2+) oscillations when introduced into mammalian eggs. We have observed that cytosolic extracts derived from Xenopus or chicken sperm could induce mouse eggs to undergo Ca(2+) oscillations similar to those induced by bovine sperm extracts. The factor responsible for inducing Ca(2+) oscillations was of high molecular weight and heat- or proteinase K-labile. We show that 0.5 chicken sperm-equivalents or 1-2 Xenopus sperm-equivalents of the extracts had enough activity to trigger Ca(2+) oscillations in mouse eggs. Our findings illustrate that although Xenopus, chicken, and mammals are evolutionarily divergent species, the function of the sperm protein factor in triggering Ca(2+) oscillations in mammalian eggs appears not to be species specific in vertebrates.

Animals↗

PubMed1995

Solution structure of the GTPase activating domain of alpha s.

We have used heteronuclear three-dimensional NMR spectroscopy to determine the solution structure of a 141 residue protein containing the GTPase activating domain from the alpha chain of the heterotrimeric G protein Gs. The domain contains six alpha-helices and is stable and structured in solution despite having been excised from the intact Gs protein. The N-terminal ten and C-terminal 11 residues of the protein are unstructured in solution while the core is well determined by the 2483 distance and torsion restraints derived from the NMR spectra. The final ensemble of 14 structures, generated with a hybrid distance geometry/simulated annealing protocol, have an average to-the-mean backbone root-mean-square deviation of 0.39 A for the core residues 89 to 201. The majority of the structure is remarkably similar to that observed for the cognate domains in crystal structures of the homologous proteins alpha t and alpha i1. However, the orientations of the second helix and the subsequent interhelical loops differ markedly among the three proteins. This structural divergence, along with functional studies of chimeric proteins, suggests that this region of the domain interacts with either the downstream effector adenylyl cyclase or with some other intermediary protein.

Amino Acid Sequence↗

PubMed1996

A molecular phylogenetic survey of the nightjars and allies (Caprimulgiformes) with special emphasis on the potoos (Nyctibiidae).

A 656-pb fragment of the mitochondrial cytochrome b gene was sequenced for six species of Central and South American potoos (genus Nyctibius, Nyctibiidae) as well as for selected representatives of all other caprimulgiform families. Sequence divergence among potoos was much higher (11.1-16.2%) than has typically been observed among congeneric species of birds, suggesting that the species of Nyctibius are quite old. Divergence among families was also quite high (13.7-21.8%), confirming recent DNA-DNA hybridization studies. Such high genetic divergences in a functionally constrained gene like cyt b indicate that many of those sites which are free to vary will have experienced multiple substitutions. We therefore performed phylogenetic analysis using parsimony under a variety of weighting schemes intended to reduce the effect of multiple substitutions. The monophyly of all the traditional caprimulgiform families was confirmed and a number of new hypotheses of relationship emerged. From our analysis, it appears that the oilbird (Steatornis) is an ancient member of the order, and it is not closely related to the potoos. We also note the close link between Aegothelidae and Caprimulgidae, and the basal position of the Podargidae/Batrachostomidae clade in the phylogeny. These results are in accordance with several classical works of the first half of the century. The relationships of the various Nyctibius species to each other have not been fully resolved; however, a close link between N. leucopterus and N. maculosus appears to be highly probable from our data.

Amino Acid Sequence↗

PubMed1982

[Dissociation of serum kinetics of amylase and trypsin following stimulation with secretin and pancreozymin].

The diagnostic value of basal serum trypsin and amylase values in the assessment of pancreatic exocrine function is limited. Secretin injection evokes a significantly different response in the serum kinetics of trypsin and amylase, due probably to their difference in molecular weight (21,000 vs 52,000 Daltons). Serum trypsin increases in 70% of people with normal secretin-pancreozymin test after stimulation with secretin, whereas amylase remains unchanged. The post-stimulatory rise in trypsin is lower in mild exocrine insufficiency and almost completely abolished in severe exocrine insufficiency. The diagnosis of severe exocrine insufficiency is confirmed in 93% and that of mild insufficiency in 54% by low basal and post-stimulatory levels of serum trypsin. In diabetics with low basal values the post-stimulatory rise in serum trypsin confirmed normal pancreatic function. The poststimulatory kinetics of serum amylase shows no clear correlation to pancreatic function. From the divergent serum kinetics of trypsin and amylase it may be concluded that trypsin is primarily of ductular and amylase primarily of acinar origin.

Adult↗

PubMed1996

Directed evolution studies with combinatorial libraries of T4 lysozyme mutants.

Gene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of > 10(7) mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection in lacZ or pheA deficient strains of E. coli to investigate whether they sample sufficient diversity to contain mutants with acquired beta-galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novel E. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4-6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min-1 for the lacZ or pheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from future E. coli complementation schemes.

Bacteriophage T4↗

PubMed2001

Identification, mapping, and phylogenetic analysis of three novel chicken CC chemokines.

We have identified three novel chicken CC chemokine genes among cDNA clones derived from lipopolysaccharide-stimulated cells of the chicken macrophage cell line HD11. Two of these chemokines show DNA sequence homology to the mammalian genes SCYA20 (MIP-3alpha) and SCYA5 (RANTES), while the third shows similar levels of homology to several mammalian CC chemokines. Sequencing of genomic DNA showed that all three chicken chemokines possess the three-exon structure and conserved intron positions typical of mammalian CC chemokines. Genetic mapping of the three chicken chemokines locates them in three chromosomal regions which correspond to regions containing homologous chemokines in humans. Phylogenetic analysis of the currently known chicken and human chemokines suggests that individual chicken and human chemokines derive from common ancestral genes in patterns that reflect their genomic positions, indicating that the diversity of chemokine genes pre-dated avian-mammalian divergence. Since the function of the chemokines is principally to act as intermediates between stimulated cells and specific subsets of responding immune cells, this suggests that the complex organization of the immune system and diversity of responding cells were largely in place at that time.

Animals↗

PubMed2001

Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook.

This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

Amino Acid Sequence↗

PubMed2001

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7.

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.

Base Sequence↗

PubMed1986

Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12.

phoB encodes a positive regulator for a number of the genes belonging to the phosphate regulon of Escherichia coli, including phoA, phoS, phoE and ugpAB. We have determined the nucleotide sequence of the chromosomal segment containing the promoter and the coding region of the phoB gene. The sequence data combined with the known amino-terminal amino acid sequence of a PhoB-LacZ hybrid protein suggest that the PhoB protein consists of 228 amino acid residues with a Mr of 26,302. In the regulatory region of the gene, a consensus nucleotide sequence shared by the regulatory regions of the phoA, phoS and phoE genes, which we name the "phosphate box", was found. Since these genes are positively regulated by the product of phoB, this suggests that transcription of the phoB gene is also regulated positively by its own product. Extensive homology was found in the amino acid sequences of the products of phoB, ompR and dye, all of which are positive regulatory genes for a number of genes coding for envelope proteins. This implies that these genes were originally duplications of a protogene that evolved to have divergent but related functions.

Amino Acid Sequence↗

PubMed1987

High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits.

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.

Adenosine Triphosphatases↗

PubMed1993

Comparison of pore-forming peptides from pathogenic and nonpathogenic Entamoeba histolytica.

Similar to the findings obtained with pathogenic Entamoeba histolytica, nonpathogenic isolates were found to kill mammalian cells in vitro, and cell extract caused pore formation in liposome membranes. A pore-forming peptide termed APnp was isolated from a nonpathogenic isolate using the schedule developed for the purification of APp or amoebapore, the homologous peptide of the pathogenic isolate HM-1:IMSS. Compared to APp, the specific activity of APnp in pore formation was 60% lower. cDNA sequencing indicated 95% identity of the primary structures of APnp and APp, and secondary structure predictions revealed a high degree of similarity. Notably, a glutamic acid residue at position 2 of APp is in APnp replaced by proline, which shortens one of the two amphipathic alpha-helices considered crucial for the pore-forming function. This structural divergence of the two peptides might explain the difference in their pore-forming activities.

Amino Acid Sequence↗