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PubMed2026

Genome-resolved analysis reveals disruption of gut microbial vitamin B and K2 biosynthesis during Toxoplasma gondii infection in mice.

UNLABELLED: Toxoplasma gondii infection remodels the gut microbiome, yet its impact on microbial vitamin biosynthetic potential and host redox metabolism remains unclear. Here, we integrated mouse gut metagenomes with publicly available metagenome-assembled genomes (MAGs) to construct a genome-resolved atlas of B-vitamin and vitamin K2 biosynthesis. From 45,697 MAGs, we curated 4,771 representative genomes, of which 2,682 met high-quality criteria (completeness ≥90%, contamination <5%). Functional annotation identified 229,717 vitamin-related genes corresponding to 177 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs across de novo pathways for eight B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and vitamin K2. Among the high-quality genomes, 1,665 encoded complete de novo pathways for at least one vitamin, highlighting functional specialization and community-level complementarity. Transcripts per million-normalized metagenomic read counts revealed significant differences in KEGG ortholog abundances across six of the nine vitamin pathways. Reanalysis of metagenomic data from infected mice (acute, chronic, and control; n = 10 per group) revealed a stage-dependent reduction in α-diversity of vitamin biosynthesis pathways during acute infection, and a clear β-diversity separation from chronic and control groups. Core niacin biosynthesis genes (nadB, nadA, nadC) displayed phylum-specific redistribution, indicating selective remodeling of microbial NAD+ precursor production under infection-induced metabolic stress. These results suggest that T. gondii infection disrupts cooperative vitamin biosynthetic networks while specifically modulating niacin pathways linked to host NAD+ metabolism. IMPORTANCE: Gut microbes can synthesize essential vitamins, but how infection alters this function is poorly understood. By integrating mouse gut metagenomes with genome-resolved microbial data, we show that Toxoplasma gondii infection reshapes the vitamin biosynthetic potential of the gut microbiome in a stage-dependent manner. Acute infection reduces the diversity of vitamin biosynthesis pathways and shifts the taxonomic distribution of key niacin biosynthesis genes involved in microbial NAD+ precursor production. These findings identify vitamin metabolism, especially niacin-related pathways, as a sensitive functional axis of microbiome remodeling during infection. Our work links microbial taxonomic changes to functional metabolic consequences and suggests that microbiome-mediated regulation of NAD+-related metabolism may contribute to host redox adaptation during T. gondii infection.

B vitamins↗

PubMed2006

Aboveground and belowground responses to quality and heterogeneity of organic inputs to the boreal forest.

Leaf litter and other organic resources returned to the soil are important regulators of ecological processes in forest ecosystems, and their ecological impacts may be strongly influenced both by their quality and by interactions between coexisting resource types. To date, most studies on effects of resource identity and mixing have only involved leaf litter, despite the fact that other resource types constitute a major input to the soil. We investigated how quality and heterogeneity of organic substrates found in boreal forests affects the activity and community structure of soil microbes, and plant growth. Six organic substrates (wood, charcoal, berries, sporocarps, vertebrate faeces and leaf litter) were added singly or in mixtures of two, three and six resource types to pots containing forest soil (with or without tree seedlings of Betula pendula Roth). The largest positive effects of single substrates on microbial basal respiration (BR), substrate-induced respiration (SIR) and microbial metabolic quotient (qCO(2)) were found for nutrient-rich substrates (faeces and sporocarps) or substrates with high sugar-content (berries). Mixing of substrates had no effect on BR or SIR, but decreased qCO(2) or altered the microbial community structure for specific combinations of substrates. In contrast to the niche complementarity hypothesis, microbial catabolic diversity was not stimulated by greater diversity of resources. Seedling growth responses to single substrates were neutral or negative; the inhibition of growth probably resulted largely from microbial competition for nutrients. Substrate mixing enhanced seedling nutrient-uptake and growth for all mixtures containing sporocarps and leaf litter. Overall, plants responded more strongly to resource heterogeneity than microbes, and synergistic effects only occurred when nutrient-rich substrates were present within the substrate mixtures. In particular, our results demonstrate a role for complex and non-additive interactions among previously overlooked resource types returned to the soil in influencing ecosystem functions such as nutrient cycling and plant productivity.

Analysis of Variance↗

PubMed1987

Mutant U2 snRNAs of Xenopus which can form an altered higher order RNA structure are unable to enter the nucleus.

We studied the nuclear targeting of U snRNAs by microinjection of wild-type and mutant U2 small nuclear RNA transcripts into the cytoplasm of Xenopus oocytes. It has previously been shown that a mutant U2 RNA (delta C) which does not bind certain common U snRNP proteins, some of which carry epitopes recognized by anti-Sm antisera, does not enter the nucleus. We show here that several mutant U2 RNAs which bind to Sm antigens do not enter the nucleus, demonstrating that this RNA-protein interaction is insufficient to produce a nuclear targeting signal. Computer predictions of the secondary structures of the RNAs, derived from minimal energy calculations, show that those which are unable to enter the nucleus have the potential to form an additional secondary structure interaction due to base complementarity between sequences near to their 5' and 3' ends. The data suggest that this structural feature inhibits nuclear targeting.

Animals↗

PubMed1989

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model.

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.

Animals↗

PubMed1999

Novel approach to predicting P450-mediated drug metabolism: development of a combined protein and pharmacophore model for CYP2D6.

A combined protein and pharmacophore model for cytochrome P450 2D6 (CYP2D6) has been derived using various computational chemistry techniques. A combination of pharmacophore modeling (using 40 substrates), protein modeling, and molecular orbital calculations was necessary to derive a model which incorporated steric, electronic, and chemical stability properties. The initial pharmacophore and protein models used to construct the combined model were derived independently and showed a high level of complementarity. The combined model is in agreement with experimental results concerning the substrates used to derive the model, with site-directed mutagenesis data available for the CYP2D6 protein, and takes into account the site-directed mutagenesis results for a variety of other 2-family P450s.

Amino Acid Sequence↗

PubMed1987

Complementarity of colestipol, niacin, and lovastatin in treatment of severe familial hypercholesterolemia.

OBJECTIVE: To compare the effectiveness of the ternary-drug combination of colestipol, niacin, and lovastatin with binary combinations of those drugs in treating patients with familial hypercholesterolemia. DESIGN: An open sequential study of serum lipoprotein responses in patients receiving diet alone (mean duration, 4 months); colestipol and niacin with diet (mean duration, 9 months); and colestipol, niacin, and lovastatin with diet (mean duration, 15 months). SETTING: Metabolic ward and lipid clinic of a university medical center. PATIENTS: Twenty-two patients with clinical characteristics of familial hypercholesterolemia (low-density-lipoprotein cholesterol, greater than 8.48 mmol/L; 21 of 22 with tendon xanthomas). INTERVENTIONS: Diet: less than 200 mg/d of cholesterol and less than 8% of total calories from saturated fat; colestipol, 30 g/d; lovastatin, 40 to 60 mg/d; and niacin, 1.5 to 7.5 g/d. MEASUREMENTS AND MAIN RESULTS: Mean total serum cholesterol and low-density-lipoprotein cholesterol levels of 4.86 +/- 0.62 mmol/L (188 +/- 24 mg/dL SD) and 2.89 +/- 0.54 mmol/L (112 +/- 21 mg/dL SD), respectively, were significantly lower during ternary-drug treatment than during colestipol-niacin treatment (p less than 0.003) or during treatment in which other possible binary combinations were given. The cholesterol content of very low-density-lipoproteins was lower and high-density-lipoprotein cholesterol levels higher during this phase than during the colestipol-niacin phase. CONCLUSIONS: Colestipol, lovastatin, and niacin are mutually complementary in treating hypercholesterolemia. This regimen produces reductions in serum cholesterol levels similar to those associated with regression of atheromatous plaques in animal studies.

Adult↗

PubMed2006

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry.

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).

Amino Acid Sequence↗

PubMed2026

Dual recognition drives site-directed G-quadruplex stabilization: Oligonucleotide design in G4 ligand-oligonucleotide conjugates.

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among G4 motifs. G4 Ligand-Oligonucleotides conjugates (GL-Os) address this challenge by combining small-molecule G4 ligands with the sequence specificity of oligonucleotides, targeting sequences flanking the intended G4 target. Here, we systematically investigate how oligonucleotide length, backbone composition, and sequence complementarity govern GL-O binding, selectivity, and G4 stabilization. We show that effective G4 recognition depends on the interdependence between oligonucleotide hybridization and G4 ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Longer oligonucleotides promote more stable complexes and stronger G4 stabilization, whereas central mismatches disrupt this dual-recognition mechanism. Replacement of DNA with peptide nucleic acids (PNAs) enhances binding strength, thermal stability, and metabolic stability. Importantly, ligand conjugation redirects PNA oligonucleotides from nonspecific polymerase stalling toward selective G4 stabilization. Finally, we demonstrate receptor-mediated cellular uptake of modified GL-Os, supporting the feasibility of cellular delivery while highlighting remaining delivery barriers. Together, these findings show the molecular design principles governing GL-O behavior and provide a foundation for the future development and evaluation of selective G4-targeting therapeutics.

G-quadruplex DNA↗

PubMed1991

Aberrant expression of intestinal mucin antigens associated with colorectal carcinoma defined by a panel of monoclonal antibodies.

Small intestine mucin antigen (SIMA) is an oncofoetal antigen for the colon and is distinct from the normal large intestinal mucin antigen (LIMA). In the present study, a panel of anti-SIMA and anti-LIMA monoclonal antibodies (MAb) was used to charaterise altered mucin expression in colorectal adenocarcinomas, by immunohistochemistry and quantitative immunoassays of tissue extracts. These results are compared with CEA expression and correlated with various clinicopathological indices. All mucin MAb reacted with a high proportion of the 100 colon cancers of every stage, histological type (including non-mucinous cancers), differentiation, site, or size. Inappropriate SIMA production was detected by either anti-SIMA MAb 4D3 or 4A1, even in 85% of early stage cancers. MAb 4D3 reacted with a higher proportion of cancers of smaller size and better differentiation. At the subcellular level, both anti-SIMA MAb showed reactivity typical of normal mucin, i.e., goblet cell and extracellular mucin. The normal colonic antigen, LIMA, was also detectable in the majority of cases, but quantitatively overproduced in some cases and reduced in others. However, in contrast to SIMA, LIMA was detected in predominantly undifferentiated cancer cells but not in goblet cells. Heterogeneity of MAb reactivity between cases and complementarity within each cancer was frequently observed. Mucin reactive with at least one of the MAb was detected in all of the CEA-negative cancers. A high rate of inappropriate SIMA expression was also detected in the perineoplastic transitional mucosa (88%, c.f. CEA, 35%) and adjacent, morphologically normal mucosa (80% c.f. CEA, 24%), indicating biochemical changes similar to the cancer. This panel of anti-mucin MAb demonstrated altered mucin glycoprotein metabolism associated with the development and progression of most colorectal cancers, which emphasises their utility as indicators of neoplastic change in the colon, and their superiority to CEA.

Aged↗

PubMed2001

Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation.

This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.

Bacterial Proteins↗

PubMed2001

Bensingtonia thailandica sp. nov., a novel basidiomycetous yeast species isolated from plant leaves in Thailand.

Ten strains which were characterized by the formation of ballistoconidia, the absence of xylose in whole-cell hydrolysates, the presence of Q-9 as the major ubiquinone isoprenologue, the inability to ferment sugars and positive diazonium blue B and urease reactions were isolated from plant samples collected in Thailand. These isolates were closely related to Bensingtonia phyllada based on the analysis of 18S rDNA sequences. On the basis of the morphological, physiological and chemotaxonomic properties, the 10 isolates were assigned to the genus Bensingtonia. DNA complementarity showed that these isolates were genetically distinct from known species of the genus Bensingtonia. The isolates are described as Bensingtonia thailandica sp. nov. The type strain is strain TY-138T (= JCM 10651T).

Basidiomycota↗

PubMed1997

From replicators to reproducers: the first major transitions leading to life.

A classification of replicators is proposed: life depends on replicators that can exist in an indefinitely large number of forms (unlimited heredity), and whose replication is modular rather than processive. The first template replicators would have increased at a rate less than exponential, because of self-inhibition arising from molecular complementarity. The result would be the survival of a varied population of replicators, rather than the victory of one type. This variability was important, because inaccurate copying meant that individual replicators were small (Eigen's paradox). The origin of cooperation between replicators, and the problem of molecular parasites, are discussed. Today, cooperation depends on cellular compartments, and on the linkage of genes on chromosomes, but we argue that at an earlier stage surface metabolism, in which replicators react only with neighbours, was important. The origin of translation and the genetic code is discussed. The essential step is the binding of amino acids to specific oligonucleotides. We suggest that this binding originated, not as a step in protein synthesis, but in the formation of coenzymes in a metabolically complex RNA world. Existing organisms are not replicators (that is, new individuals do not arise by copying), but reproducers that contain replicators. We outline Griesemer's concept of a reproducer, which brings out the essential role of development in evolution.

Animals↗

PubMed2003

Sequential bio- and phototransformation of the herbicide methabenzthiazuron in water.

We investigated the transformation of methabenzthiazuron in water by microorganisms and solar light. This compound was very slowly phototransformed when irradiated at lambda > 290 nm, but it could be successfully oxidized into 6-hydroxymethabenzthiazuron by Aspergillus niger, as shown by nuclear magnetic resonance experiments. The toxicity of this metabolite, as determined by the standardized Microtox test, was sixfold lower than that of the parent molecule. The 6-hydroxymethabenzthiazuron was not further metabolized by A. niger but was photooxidized with ring cleavage of the aromatic ring and photodimerized on irradiation at lambda > 290 nm. In the presence of humic substances, the photodegradation was slower. We demonstrate that the transformations of methabenzthiazuron, observed either with the fungus A. niger or by the action of solar light, do not proceed via the urea chain N-dealkylation, as usually reported, but only via hydroxylation or cleavage of the benzene ring. This work shows the complementarity of both approaches, photo- and biodegradation, to study the fate of herbicides in the environment.

Aspergillus niger↗

PubMed1976

Amidated carboxyl groups in elastin.

Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.

Amides↗

PubMed1994

Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping.

In an effort to understand the mechanisms by which nonsense codons affect RNA metabolism in mammalian cells, nonsense mutations were generated within the gene for the secretory major urinary protein (MUP) of mice. The translation of MUP mRNA normally begins within exon 1 and terminates within exon 6, the penultimate exon. Through the use of Northern (RNA) blot hybridization and assays that couple reverse transcription and PCR, a nonsense mutation within codon 50 of exon 2 or codon 143 of exon 5 was found to reduce the abundance of fully spliced, nuclear MUP mRNA to 10 to 20% of normal without an additional reduction in the abundance of cytoplasmic mRNA. In contrast, a nonsense mutation within codon 172 of exon 5 was found to have no effects on the abundance of MUP mRNA. These findings suggest that a boundary between nonsense mutations that do and do not reduce the abundance of nuclear mRNA exists within the exon preceding the exon that harbors the normal site of translation termination. In this way, the boundary is analogous to the boundary that exists within the penultimate exon of the human gene for the cytosolic enzyme triosephosphate isomerase. Assays for exon skipping, i.e., the removal of an exon as a part of the flanking introns during the process of splicing, reveal that 0.1, 2.0, and 0.1% of MUP mRNA normally lack exon 5, exon 6, and exons 5 plus 6, respectively. Relative to normal, the two nonsense mutations within exon 5 increase the abundance of RNA lacking exon 5 on average 20-fold and increase the abundance of RNA lacking exons 5 plus 6 on average 5-fold. Since only one of these nonsense mutations also reduces the abundance of fully spliced nuclear mRNA to 10 to 20% of normal, the two mechanisms by which a nonsense mutation can alter nuclear RNA metabolism must be distinct. The analysis of missense mutations within codons 143 and 172, some of which retain the nonsense mutation, indicates that the reduction in the abundance of fully spliced nuclear mRNA is dependent upon the premature termination of MUP mRNA translation, whereas skipping is attributable to nonsense mutation-mediated changes in exon 5 structure rather than to the premature termination of translation. The increase in exon 5 skipping by either the nonsense or missense mutations within codon 172 correlates with a decrease in the complementarity of exon 5 to U1 snRNA. This suggests that a 5' splice site may extend as far as 12 nucleotides into the upstream exon, which is, to our knowledge, the largest extension.

Animals↗

PubMed1992

In vitro methods: their relevance and complementarity in ocular safety assessment.

Ocular irritation includes a wide variety of mechanisms some of which can be explored by in vitro methods. For example, the effects on epithelial cells that constitute the outer layers of both the conjunctiva and the cornea may result in direct cytotoxicity or impairment of cellular functions -such as impermeability-, phenomena that can be explored in vitro. Irritancy may also involve inflammation of the conjunctival connective tissue and of the corneal stroma with its vascular and cellular features; effects on the stroma can lead to the opacification of the cornea; this last phenomenon may be the consequence of mechanisms such as modification of the structure of proteins or changes in stroma hydration which in particular is closely related to corneal endothelium metabolic activity. Recovery after eye injury depends partly on the extent of ocular damage and on the residual mitotic activity of the remaining cells. We have studied 41 surfactants, lotions and shampoos in 6 to 8 in vitro methods each one exploring one or two endpoints that could be linked to the ocular irritancy phenomena described above. In vivo ocular irritancy data for these materials from previous studies were compared to in vitro results. The results obtained show that -among the techniques that were investigated and for the categories of substances that were studied- the Het-CAM test and more particularly the endpoint that is related to vascular effects gives the best assessment of acute ocular irritancy (Spearman's rho coefficients between in vivo and in vitro data greater than 0.90); however, cell culture methods, especially one based on short contact time between cells and products and on evaluation of early toxic effects, also proved interesting (Spearman's rho coefficients between in vivo and in vitro data greater than 0.85). Moreover, the isolated cornea opacity and permeability test gave complementary information more related to recovery from surfactant-induced damage. These encouraging results lead us to consider in vitro ocular safety assessment with optimism for the categories of products investigated.

Allantois↗

PubMed2003

Treatment of malignancy-associated hypercalcemia and cachexia with humanized anti-parathyroid hormone-related protein antibody.

Parathyroid hormone-related protein (PTHrP) plays a central role in humoral hypercalcemia of malignancy (HHM), which is one of the most frequent paraneoplastic syndromes. PTHrP produced by the tumor acts through a common PTH/PTHrP receptor to promote bone resorption, inhibit calcium excretion from the kidney, and induce hypercalcemia. Patients with HHM often develop cachexia associated with typical symptoms such as anorexia, malaise, nausea, constipation, polyuria, polydipsia, and confusion. The etiology of the cachexia is not fully understood but is thought to be caused by hypercalcemia and various cytokines such as interleukin-6, tumor necrosis factor-alpha, leukemia inhibitory factor, and others. In this study, we investigated the role of PTHrP in hypercalcemia and cachexia in HHM by using humanized anti-PTHrP antibody. A mouse monoclonal antibody that binds to PTHrP amino acid sequence 1-34 and inhibits PTHrP function has been humanized to create a specific and potent agent for the treatment of patients with HHM. The mouse monoclonal antibody has been shown to have antihypercalcemic activity against nude mice bearing human tumors. Because a mouse antibody is highly immunogenic in human patients, the complementarity-determining regions from the mouse antibody were grafted into a human antibody. The resulting humanized antibody specifically recognizes PTHrP(1-34) and neutralizes PTHrP functions in vitro and in vivo. The humanized anti-PTHrP antibody was administered intravenously to HHM model animals bearing tumors such as LC-6 human lung carcinoma. These animals showed symptoms similar to those of patients with HHM (eg, hypercalcemia and cachexia). The humanized anti-PTHrP antibody-treated animals responded with normalization of blood ionized calcium level through an improvement of bone metabolism and calcium excretion. Moreover, the treated animals also showed an improvement in body weight, ultromotivity, metabolic alkalosis, food consumption, water intake, serum phosphorus, and renal function. Consequently, the humanized antibody-treated animals experienced complete resolution of hypercalcemia and cachexia. These results suggest that the humanized antibody would be an effective and beneficial agent for patients with HHM, and that PTHrP is a major pathogenetic factor of hypercalcemia and cachexia in patients with HHM.

Animals↗

PubMed2005

High-quality homology models derived from NMR and X-ray structures of E. coli proteins YgdK and Suf E suggest that all members of the YgdK/Suf E protein family are enhancers of cysteine desulfurases.

The structural biology of proteins mediating iron-sulfur (Fe-S) cluster assembly is central for understanding several important biological processes. Here we present the NMR structure of the 16-kDa protein YgdK from Escherichia coli, which shares 35% sequence identity with the E. coli protein SufE. The SufE X-ray crystal structure was solved in parallel with the YdgK NMR structure in the Northeast Structural Genomics (NESG) consortium. Both proteins are (1) key components for Fe-S metabolism, (2) exhibit the same distinct fold, and (3) belong to a family of at least 70 prokaryotic and eukaryotic sequence homologs. Accurate homology models were calculated for the YgdK/SufE family based on YgdK NMR and SufE crystal structure. Both structural templates contributed equally, exemplifying synergy of NMR and X-ray crystallography. SufE acts as an enhancer of the cysteine desulfurase activity of SufS by SufE-SufS complex formation. A homology model of CsdA, a desulfurase encoded in the same operon as YgdK, was modeled using the X-ray structure of SufS as a template. Protein surface and electrostatic complementarities strongly suggest that YgdK and CsdA likewise form a functional two-component desulfurase complex. Moreover, structural features of YgdK and SufS, which can be linked to their interaction with desulfurases, are conserved in all homology models. It thus appears very likely that all members of the YgdK/SufE family act as enhancers of Suf-S-like desulfurases. The present study exemplifies that "refined" selection of two (or more) targets enables high-quality homology modeling of large protein families.

Animals↗