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At least 1,117 records · Page 62Linked to original sources
PubMed1998

The role of transferrin-receptor variation in the host range of Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. brucei to cope with the large sequence diversity in the transferrins of its hosts.

Animals↗
PubMed2006

The role of heparan sulphate in inflammation.

The polysaccharide heparan sulphate is ubiquitously expressed as a proteoglycan in extracellular matrices and on cell surfaces. Heparan sulphate has marked sequence diversity that allows it to specifically interact with many proteins. This Review focuses on the multiple roles of heparan sulphate in inflammatory responses and, in particular, on its participation in almost every stage of leukocyte transmigration through the blood-vessel wall. Heparan sulphate is involved in the initial adhesion of leukocytes to the inflamed endothelium, the subsequent chemokine-mediated transmigration through the vessel wall and the establishment of both acute and chronic inflammatory reactions.

Animals↗
PubMed2026

Integrated computational and experimental benchmarking of Bacillus phage endolysins reveals the relationship between peptidoglycan-fragment recognition descriptors and antibacterial performance.

Protein-based antibacterials such as bacteriophage endolysins offer a targeted therapeutic strategy against Gram-positive pathogens. However, prioritizing the most effective candidates from the large sequence diversity available remains a significant challenge. Here we present a standardized computational-experimental benchmarking framework that evaluates seven phage-derived endolysin variants (E1, E2, E3, E7, E10, E12, and E15) identified from Bacillus genomes. We combined molecular docking and residue-level interaction mapping against muramyl dipeptide (MDP), a minimal conserved peptidoglycan motif, with 1000-ns molecular dynamics simulations, MM/PBSA binding free-energy estimation, and matched functional inhibition assays against Staphylococcus aureus and Micrococcus luteus. Computational analyses revealed generally favorable MDP recognition across variants, albeit with notable differences in contact patterns and complex stability profiles. Experimental screening identified E2 as the most potent antibacterial agent against both species, while E7 and E1 performed strongly in selected computational metrics. Integrated analysis showed only modest correlations between computational descriptors of fragment recognition/stability and observed antibacterial performance. This study establishes a practical comparative benchmarking platform for endolysin candidate prioritization, nominates E2 and E7 as promising candidates for further development, and highlights E1 as a potential structural scaffold for rational engineering, while explicitly demonstrating both the utility and the current limitations of using minimal peptidoglycan fragments as proxies for full cell-wall recognition in lysin benchmarking.

Endopeptidases↗
PubMed2004

Quantitative measures of the effect of the melanocortin 1 receptor on human pigmentary status.

Variation in human hair and skin color is the most striking visible aspect of human genetic variation. The only gene known to exert an effect on pigmentary within the normal population is the melanocortin-1 receptor (MC1R). Previous studies have used a Mendelian framework to relate MC1R genotype to phenotype, by measuring pigmentary status using categorical scales. Such approaches are inadequate. We report results using direct measures of hair color using objective colorimetric dimensions and HPLC determined hair melanins. We have linked MC1R genotype with chemical measures of melanin quantity and type and objective phenotype measures of color. MC1R genotype was predictive of hair melanin expressed as the ratio of the loge of eumelanin to pheomelanin ratio, with a dosage effect evident: MC1R homozygote mean, 1.46; heterozygote, 4.44; and wild type, 5.81 (p<0.001). Approximately 67% of the variance in this model could be accounted for in terms of MC1R genotype. There was also a relation between MC1R status and hair color, most prominently for the b* axis (p<0.001), but also for the a* and L* scales (L*a*b*, CIE). We show for one of the most polymorphic human traits that it is possible to demonstrate meaningful relations between various physical characteristics: DNA sequence diversity, hair-wavelength-specific reflectance patterns, and chemical melanin assays.

Female↗
PubMed1999

The hepatic flaviviridae: summary.

Hepatitis C envelope proteins (E1, E2) induce protective neutralizing antibodies. The extent of sequence diversity reflects the host's ability to control viral populations and the response to antiviral therapy. Attempts to prepare effective vaccines against HCV are foiled by lack of prolonged protective immunity. Plasmid vaccines and the use of uninfectious virus-like particles are being developed. HCV induces a cellular humoral immune response, but this is inadequate to clear the virus and the disease becomes chronic. In any patient, the natural history of HCV infection depends on the age when infected, and the presence of other diseases. The transfusion-related disease has a worse prognosis than that transmitted by syringes and needles. The outlook in 'healthy blood donors' is uncertain. Interferon therapy for 3 or preferably 6 months results in a sustained response in about 30% of patients. Negative serum HCV RNA and normal AST values after 3 months of therapy indicates that there may be a sustained response. Whether or not to stop treatment at that time if HCV is still positive remains a matter of debate. The role of interferon treatment in preventing progression to cirrhosis and hepatocellular cancer is still uncertain. Ribavirin therapy alone reduces transaminases and hepatic histology improves. Improved results follow the combination of ribavirin with interferon. Ribavirin may have immuno-modularity and anti-inflammatory actions. Hepatitis G virus (HGV) is unlikely to play a significant role in liver disease in man.

Animals↗
PubMed2001

Host specificity and incidence of Trypanosoma in some African rainforest birds: a molecular approach.

Studies of host-parasite interactions in birds have contributed greatly to our understanding of the evolution and ecology of disease. Here we employ molecular techniques to determine the incidence and study the host-specificity of parasitic trypanosomes in the African avifauna. We developed a polymerase chain reaction (PCR)-based diagnostic test that amplified the small subunit ribosomal RNA gene (SSU rRNA) of Trypanosoma from avian blood samples. This nested PCR assay complements and corroborates information obtained by the traditional method of blood smear analysis. The test was used to describe the incidence of trypanosomes in 479 host individuals representing 71 rainforest bird species from Cameroon, the Ivory Coast and Equatorial Guinea. Forty-two (59%) of these potential host species harboured trypanosomes and 189 individuals (35%) were infected. To examine host and geographical specificity, we examined the morphology and sequenced a portion of the SSU rRNA gene from representative trypanosomes drawn from different hosts and collecting locations. In traditional blood smear analyses we identified two trypanosome morphospecies, T. avium and T. everetti. Our molecular and morphological results were congruent in that these two morphospecies had highly divergent SSU rRNA sequences, but the molecular assay also identified cryptic variation in T. avium, in which we found seven closely allied haplotypes. The pattern of sequence diversity within T. avium provides evidence for widespread trypanosome mixing across avian host taxa and across geographical locations. For example, T. avium lineages with identical haplotypes infected birds from different families, whereas single host species were infected by T. avium lineages with different haplotypes. Furthermore, some conspecific hosts from geographically distant sampling locations were infected with the same trypanosome lineage, but other individuals from those locations harboured different trypanosome lineages. This apparent lack of host or geographical specificity may have important consequences for the evolutionary and ecological interactions between parasitic trypanosomes and their avian hosts.

Africa↗
PubMed2003

Effects of habitat fragmentation and changes of dispersal behaviour after a recent population decline on the genetic variability of noncoding and coding DNA of a monogamous Malagasy rodent.

While interactions among demography, behaviour and genetic structure are well-documented for neutral genetic markers, the role of these parameters and the effects of genetic drift and selection are considerably less well understood in functional genes, such as the major histocompatibility complex (MHC). In this study, the consequences of habitat fragmentation and the effects of a current population decline on noncoding (mitochondrial DNA) and two coding MHC loci (DQA, DRB) with different functional importance were investigated in the small remnant subdivided population of the endangered Malagasy giant jumping rat (Hypogeomys antimena). Both neutral and selective markers revealed a significant genetic differentiation between the two remnant subpopulations. The FST values were much lower in the MHC DQA and DRB genes than in the mitochondrial data. The MHC DRB loci display the effects of both balancing selection (high sequence diversity, four times higher nonsynonymous than synonymous substitutions in the functionally important antigen-binding site positions, twice the average heterozygosity of individual amino acids at the positions identified as part of the antigen-binding site (ABS) than those outside the ABS and nonselective forces including genetic drift. Simultaneously with a current population decline offspring reduced their dispersal distances. No substantial effects were detected within the first 6 years of reduced gene flow in either mitochondrial or MHC markers.

Animals↗
PubMed1998

The primary antibody repertoire of kappa-deficient mice is characterized by non-stochastic Vlamda1 + V(H) gene family pairings and a higher degree of self-reactivity.

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv kappa-deficient (JCkappaD) mice, in order to understand the contributions of the lambda-light chain, in the absence of an otherwise predominant kappa-light chain, to the development of humoral immunity. The expression of Vlambda1 gene (lambda1 and lambda3 subtypes) and the Vlambda1 + V(H) (J558, 36-60, V(H)11 and S107) gene family associations were studied in 7.43 x 10(3) mitogen-activated splenic B-lymphocyte clones of JCkappaD origin. Furthermore, the functional significance of the exclusive expression of the lambda-light chain, in the peripheral B-cell repertoire of JCkappaD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available Vlambda genes at the lambda locus, the Vlambda1 gene is the one that is expressed most frequently (59.9%); second, non-random Vlambda1 + V(H) (J558, 36-60) gene family pairings occur in kappa-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the lambda-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in kappa-deficient (129/Sv JCkappaD) mice. These observations suggest that the high murine kappa/lambda ratio in mice may, apart from high sequence diversity at the kappa-locus, be a result of endogenous selection against the lambda-light chain to restrict self-reactivity within the homeostatic threshold.

Animals↗
PubMed2006

Introduction of a nuclear marker for phylogenetic analysis of Nepenthaceae.

Nepenthaceae, the pitcher plants of the Old World tropics show a remarkable diversity in SE Asia, especially on the islands of Borneo and Sumatra. This region is considered as a secondary center of diversity. Sequence analysis of the cpDNA TRNK intron supports this hypothesis showing the species of the Malay Archipelago as neighbour group to the isolated species from Sri Lanka, the Seychelles, and Madagascar. Based on phylogenetic reconstructions an origin of recent Nepenthaceae in the Indian subcontinent is assumed. A recent investigation focused on a non-plastid, translocated copy of the TRNK intron has revealed an incongruence to tree topology based on the cpDNA TRNK intron. Although the translocated copy emerged as insufficient for phylogenetic reconstruction of Nepenthaceae some taxa showed, contrary to the cpDNA dataset, relatively high distances to the rest of the taxa. These results indicated that the phylogeny of the TRNK intron could not reflect true phylogenetic relationships. We investigated the peptide transferase 1 (PTR1), to develop a phylogenetic marker that is based on a nuclear low copy gene in Nepenthes. All sequences obtained were probably functional, indicated by the ratio of point mutations of the single codon positions in exon and intron regions. Comparative analysis showed that this locus is of similar variability as the cpDNA TRNK intron and, contrary to the translocated copy of TRNK, potential useful for phylogenetic reconstruction. While in parts congruent to the plastid TRNK intron phylogeny, a higher divergence of some sequences in PRT1 and in the previously reported, non cpDNA dataset indicates that remnants of an older species stock persisted east of Wallace's line and on the Sunda Shelf. This suggests that plastid haplotypes existing today in the main distribution center of the Nepenthaceae could be descendants of more recently dispersed lineages that had been transmitted to an old species stock.

Asia↗
PubMed2004

Computational design of receptors for an organophosphate surrogate of the nerve agent soman.

We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligand-binding pockets of two periplasmic binding proteins, glucose-binding protein and ribose-binding protein, were converted to bind PMPA instead of their cognate sugars. The designs introduce 9-12 mutations in the parent proteins. Twelve of 20 designs tested exhibited PMPA-dependent changes in emission intensity of a fluorescent reporter with affinities between 45 nM and 10 microM. The contributions to ligand binding by individual residues were determined in two designs by alanine-scanning mutagenesis, and are consistent with the molecular models. These results demonstrate that designed receptors with radically altered binding specificities and affinities that rival or exceed those of the parent proteins can be successfully predicted. The designs vary in parent scaffold, sequence diversity, and orientation of docked ligand, suggesting that the number of possible solutions to the design problem is large and degenerate. This observation has implications for the genesis of biological function by random processes. The designed receptors reported here may have utility in the development of fluorescent biosensors for monitoring nerve agents.

Binding Sites↗
PubMed1984

Membrane proteins unique to vertebrate olfactory cilia: candidates for sensory receptor molecules.

In search for olfactory receptor molecules, we carried out comprehensive electrophoretic mapping of membrane proteins in the cilia of frog olfactory epithelium. Seven polypeptides, extracted from isolated cilia by nonionic detergent, were unique to the sensory organelles, compared to nonsensory (respiratory) counterparts. Olfactory cilia contained 3-10 times more membrane-associated protein as compared to respiratory cilia, in agreement with reported densities of freeze-fracture intramembranous particles. Four of the olfactory polypeptides were major constituents of the ciliary membrane, each amounting to greater than 10% of its total protein. Three major and one minor specific polypeptide were glycosylated, whereas membranes of nonsensory cilia were practically devoid of glycoproteins. A clear difference in surface composition was also shown by microscopic visualization of fluoresceinated lectin bound to intact isolated cilia. Two of the olfactory glycoproteins displayed pronounced heterogeneity of apparent molecular weight, which could partly be due to protein sequence diversity, as expected for odorant receptor molecules. The recently described inhibition of odorant-evoked sensory potentials by the lectin concanavalin A is consistent with the hypothesis that one or more of the specific glycoproteins described here plays a role in olfactory reception.

Animals↗
PubMed1991

Role of diversifying selection and gene conversion in evolution of major histocompatibility complex loci.

Genes at the major histocompatibility complex (MHC) in mammals are known to have exceptionally high polymorphism and linkage disequilibrium. In addition, these genes form highly complicated gene families that have evolved through gene conversion and unequal crossing-over. It has been shown recently that amino acid substitution at the antigen recognition site (ARS) is more rapid than synonymous substitution, suggesting some kind of positive natural selection working at the ARS. It is highly desirable to know the interactive effect of gene conversion and natural selection on the evolution and variation of MHC gene families. A population genetic model is constructed that incorporates both selection and gene conversion. Diversifying selection is assumed in which sequence diversity is enhanced not only between alleles at the same locus but also between duplicated genes. Expressed and nonexpressed loci are assumed as in the class I gene family of MHC, with gene conversion occurring among all loci. Extensive simulation studies reveal that very weak selection at individual amino acid sites in combination with gene conversion can explain the unusual pattern of evolution and polymorphisms. Here both gene conversion and natural selection contribute to enhancing polymorphism.

Alleles↗
PubMed1992

Extensive genetic diversity in the HLA class II region of Africans, with a focally predominant allele, DRB1*1304.

Molecular HLA class II typing of greater than 1700 individuals from The Gambia in West Africa and Malawi in South-Central Africa revealed a striking diversity of HLA DRB-DQB haplotypes as defined by restriction fragment length polymorphism (RFLP); this diversity is twice as extensive as that found in northern Europeans. Despite this diversity, sequence and PCR/oligonucleotide analysis showed that the recently described variant DRB1*1304 is the commonest DRB1 allele in The Gambia. The sequence, geographical distribution, and RFLP association of this allele, together with homozygosity test results, suggest that DRB1*1304 may have arisen from DRB1*1102 and have reached its remarkably high frequency as a result of recent directional selection. The prevalence of this unusual allele has implications for trials of subunit vaccines in this area. The extensive and distinctive HLA class II region polymorphism in sub-Saharan Africans is consistent with evidence from other genetic loci implying an African origin of modern Homo sapiens.

Alleles↗
PubMed1997

Design of polydactyl zinc-finger proteins for unique addressing within complex genomes.

Zinc-finger proteins of the Cys2-His2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.

Amino Acid Sequence↗
PubMed2001

Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.

Several species of streptococci produce extracellular polysaccharides in the form of secreted exopolysaccharides or cell-associated capsules. Although the biological properties and repeating unit structures of these polysaccharides are diverse, sequence analysis of the genes required for their production has revealed a surprising degree of conservation among five genes found in the capsule gene cluster of each of several polysaccharide-producing streptococci. To determine the function of these conserved genes, we characterized a series of isogenic mutants derived from a wild-type strain of type Ia group B Streptococcus by selectively inactivating each gene. Inactivation of cpsIaE resulted in an acapsular phenotype, consistent with previous work that identified the cpsIaE product as the glycosyltransferase that initiates synthesis of the polysaccharide repeating unit. Mutants in cpsIaA, cpsIaB, cpsIaC, or cpsIaD produced type Ia capsular polysaccharide, but in reduced amounts compared with the wild type. Analysis of the mutant polysaccharides and of capsule gene transcription in the mutant strains provided evidence that cpsIaA encodes a transcriptional activator that regulates expression of the capsule gene operon. Mutants in cpsIaC or cpsIaD produced polysaccharide of reduced molecular size but with an identical repeating unit structure as the wild-type strain. We conclude that CpsA to -D are not required for polysaccharide repeating unit biosynthesis but rather that they direct the coordinated polymerization and export of high molecular weight polysaccharide.

Bacterial Capsules↗
PubMed2003

Single-stranded DNA induces ataxia telangiectasia mutant (ATM)/p53-dependent DNA damage and apoptotic signals.

Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand breaks, cytochrome c release, and increase in reactive oxygen species production. Moreover, single-stranded DNA-induced apoptosis was reduced significantly in p53 null cells and in cells treated with ATM small interfering RNA. These results suggest that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.

3T3 Cells↗
PubMed2005

Why is the Arg5-Glu13 salt bridge conserved in mammalian alpha-defensins?

Mammalian alpha-defensins, expressed primarily in leukocytes and epithelia, kill a broad range of microbes, constituting one of the first lines of innate immune defense against infection. Nine amino acid residues, including six cysteines, one glycine, and a pair of oppositely charged residues Arg/Glu, are conserved in the otherwise diverse sequences of all known mammalian alpha-defensins. Structural analysis indicates that the two charged residues form a salt bridge, likely stabilizing a protruding loop in the molecule. To investigate the structural and functional roles of the conserved Arg5-Glu13 salt bridge in alpha-defensins, we chemically prepared human neutrophil alpha-defensin 2 (HNP2) and five HNP2 analogs, R5E/E13R, E13Q, E13R, R5T/E13Y, and R14A. In contrast to HNP2 and R14A-HNP2, none of the four salt bridge analogs was capable of folding into a native conformation in the context of isolated defensin domains. However, when covalently attached to the 45-residue pro-HNP2 propeptide, the salt bridge analogs of HNP2 in their pro-forms all folded productively, suggesting that the Arg5-Glu13 salt bridge is not required for correct pro-alpha-defensin folding. When assayed against both Escherichia coli and Staphylococcus aureus, the six alpha-defensins showed bactericidal activity that correlated with the number of net positive charges carried by individual molecules in the panel, irrespective of whether or not the Arg5-Glu13 salt bridge was decimated, suggesting that Arg5 and Glu13 are not functionally conserved. Proteolytic resistance analysis with human neutrophil elastase, one major protease contained in azurophils with HNPs, revealed that destabilization of the salt bridge dramatically accelerated defensin degradation by the enzyme. Thus, we propose that the Arg5-Glu13 salt bridge found in most mammalian alpha-defensins is conserved for defensin in vivo stability.

Alleles↗
PubMed2006

Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding.

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.

Chemokine CCL8↗