Radiographic depiction of intra-abdominal fat in newborns: a marker of infants born to diabetic mothers.

OBJECTIVE: To examine whether the presence of intra-abdominal fat (IAF) in newborns is diagnostic of infants of diabetic mothers (IDMs), and determine whether IAF is merely the consequence of increased body size. STUDY DESIGN: Abdominal radiographs of 277 neonates >34 weeks gestational age (147 male and 130 female) were reviewed to determine the presence of IAF. Unpaired t-test and regression analyses were used to examine the influence of gestational age, birth weight, birth length and maternal diabetes on the prevalence and thickness of IAF. RESULT: The prevalence of IAF was higher in IDMs (41.2% vs 13.2%; P<0.0001)-an association that persisted even after accounting for sex, gestational age and weight. Both birth weight and maternal diabetic status influenced the amount of IAF. Values of IAF thickness in IDMs were, however, more than threefold greater than those in non-IDMs (2.53±2.08 vs 0.81±0.29 mm; P<0.0001). An IAF thickness >1.5 mm was indicative of an IDM. CONCLUSION: The depiction of IAF in radiographs is significantly more prevalent in IDMs when compared with non-IDMs, regardless of body size. A thickness of IAF >1.5 mm is a marker that should encourage work-up for the physiological, metabolic and congenital complications associated with IDM.

Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice.

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1(tm1Bc) to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1(tm1Bc) mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.

Length variation in HV2 of the human mitochondrial DNA control region.

Hair samples were typed from three individuals who exhibited length heteroplasmy in the homopolymeric cytosine stretches (C-stretch) in hypervariable region 2 (HV2). The study demonstrated that for different hairs within an individual, the HV2 C-stretch region can vary with respect to the number of cytosines and/or proportion of C-stretch length variants. Length heteroplasmy may occur regardless of the prominent length variant present in this region. Differences in the number of cytosines at the C-stretch region, or a variation in the relative amounts of heteroplasmic length variants, cannot be used to support an interpretation of exclusion.

Comparison of the efficacy of extended-release clarithromycin tablets and amoxicillin/clavulanate tablets in the treatment of acute exacerbation of chronic bronchitis.

BACKGROUND: Clarithromycin has established efficacy and safety in the treatment of respiratory infections. OBJECTIVE: This study examined the efficacy and safety of a new extended-release formulation of clarithromycin compared with amoxicillin/clavulanate in the treatment of acute exacerbation of chronic bronchitis (AECB). METHODS: This phase IIIB, multicenter, randomized, parallel-group, investigator-blinded study in patients with AECB and productive cough with purulent sputum compared treatment with extended-release clarithromycin (two 500-mg tablets once daily for 7 days) and amoxicillin/clavulanate (one 875-mg tablet twice daily for 10 days). Assessments were performed before treatment, between study days 10 and 12 (or within 48 hours after premature discontinuation), and between study days 17 and 21 (test of cure). RESULTS: Of 287 patients randomized and treated, 270 were clinically evaluable (137 clarithromycin, 133 amoxicillin/clavulanate). Treatment groups were well matched in terms of demographic characteristics, medical condition, and history. Among clinically evaluable patients at test of cure, 85% and 87% of clarithromycin- and amoxicillin/clavulanate-treated patients, respectively, demonstrated clinical cure (as defined in 1998 draft US Food and Drug Administration guidelines); among clinically and bacteriologically evaluable patients, 92% versus 89%, respectively, demonstrated bacteriologic cure. Overall pathogen eradication rates were similar in the 2 groups (88% clarithromycin, 89% amoxicillin/clavulanate). Rates of premature discontinuation of study drug for any reason differed between treatments: 3% (4/142) of clarithromycin-treated patients versus 12% (17/145) of amoxicillin/clavulanate-treated patients (P = 0.005). One percent (2/142) and 6% (8/145) of the respective treatment groups discontinued study drug because of adverse events. Adverse events generally occurred with a similar frequency in the 2 groups; however, taste alteration was more common with clarithromycin (9/142 [6%]) than with amoxicillin/clavulanate (1/145 [1%]; P = 0.01). Mean severity scores for gastrointestinal adverse events showed a significant difference between groups (1.16 for clarithromycin-treated patients and 1.58 for amoxicillin/clavulanate-treated patients: P = 0.016). CONCLUSIONS: The results of this study demonstrate the clinical and bacteriologic equivalence and improved gastrointestinal tolerability of a 7-day course of once-daily extended-release clarithromycin relative to a 10-day course of twice-daily amoxicillin/clavulanate in the treatment of AECB.

M-ABC2, a new human mitochondrial ATP-binding cassette membrane protein.

We have isolated a human cDNA encoding a novel ATP-binding cassette (ABC) protein whose gene was previously localized to chromosome 1q42 [Allikmets et al. (1995) Mamm. Genome 6, 111-117]. The gene transcript is expressed in all human tissues examined, with the highest levels in bone marrow. A non-expressed pseudogene also exists at chromosome 15q13-14. The new protein, which is most similar to the mitochondrial (M)-ABC1 protein, was also localized to mitochondria and therefore designated 'M-ABC2'. The N-terminus of M-ABC2 was shown to contain a mitochondrial-targeting signal sequence.

A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity.

As part of a large scale effort to discover novel secreted proteins, a cDNA encoding a novel cytokine was identified. Alignments of the sequence of the new protein, designated IL-17B, suggest it to be a homolog of the recently described T cell-derived cytokine, IL-17. By Northern analysis, EST distribution and real-time quantitative polymerase chain reaction analysis, mRNA was detected in many cell types. A novel type I transmembrane protein, identified in an EST data base by homology to IL-17R, was found to bind specifically IL-17B, as determined by surface plasmon resonance analysis, flow cytometry, and co-immunoprecipitation experiments. Readily detectable transcription of IL-17BR was restricted to human kidney, pancreas, liver, brain, and intestines and only a few of the many cell lines tested. By using a rodent ortholog of IL-17BR as a probe, IL-17BR message was found to be drastically up-regulated during intestinal inflammation elicited by indomethacin treatment in rats. In addition, intraperitoneal injection of IL-17B purified from Chinese hamster ovary cells caused marked neutrophil migration in normal mice, in a specific and dose-dependent manner. Together these results suggest that IL-17B may be a novel proinflammatory cytokine acting on a restricted set of target cell types. They also demonstrate the strength of genomic approaches in the unraveling of novel biological pathways.

Characterization of ABCB9, an ATP binding cassette protein associated with lysosomes.

We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a "half" ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two "half" ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.

Cigarette smoking and thyroid hormone levels in males.

BACKGROUND: Cigarette smoking has been linked to thyroid disease, although studies of this problem have not shown consistent affects, with some studies linking smoking to increased thyroid hormone levels, and others to decreased thyroid hormone levels. METHODS: We performed a secondary analysis of information collected from 4462 Vietnam-era male US Army veterans aged 31-49 years who participated in the Vietnam Experience Study in 1985-1986. The study group consisted of 1962 current smokers and 2406 current non-smokers who had no thyroid abnormalities on physical examination, no current use of thyroid medicine, and no history of thyroid disease. RESULTS: We found that current smokers have higher thyroxine levels and lower thyroid stimulating hormone levels than never smokers and former smokers. The higher thyroxine levels that we detected in smokers, compared to non-smokers, diminished when we controlled for thyroxine-binding globulin and testosterone. We also found that heavy smokers had a smaller increase in thyroxine levels than did light smokers, when compared to non-smokers. CONCLUSIONS: Our findings suggest at least two distinct mechanisms for the effect of tobacco smoke on thyroid function; one related to higher levels of thyroxine-binding globulin and testosterone among smokers compared to non-smokers and another related to higher levels of thyrotoxins in tobacco smoke in heavy smokers compared to light and moderate smokers.

Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase.

Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions.

Correction of the NMR structure of the ETS1/DNA complex.

The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761-771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456-460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix-turn-helix element and minor groove recognition via protein side-chain intercalation. The presence of intercalation was deduced on the basis of numerous NOEs between several amino acids in the protein and a resonance at 12.33 ppm originally assigned to a DNA imino proton. New NMR experiments now conclusively demonstrate that this resonance, which is located within the DNA imino proton region of the spectrum, arises from the hydroxyl proton of Tyr86. Realization of this error necessitated reanalysis of the intermolecular NOEs. This revealed that the orientation of the ETS1-DBD in the complex is opposite to that originally reported and that a tryptophan residue does not intercalate into the DNA. The calculation of a new ensemble of structures based on the corrected data indicates that the structure of the ETS1-DBD/DNA complex is indeed similar to the X-ray structure of the Pu.1-DBD/DNA complex.

The solution structure of the human ETS1-DNA complex reveals a novel mode of binding and true side chain intercalation.

The solution structure of a 24.4 kDa specific complex of the DNA-binding domain (DBD) of the human ETS1 (hETS1) oncoprotein with a 17-mer DNA has been solved by NMR. The interaction of the hETS1 DBD with DNA reveals a surprising twist on the general features of helix-turn-helix (HTH)-DNA interactions. Major groove recognition involves the C-terminal two thirds of the HTH recognition helix, while minor groove recognition occurs via true intercalation of the side chain of Trp-28, which extends from the minor to the major groove. This results in a sharp kink of approximately 60 degrees and a widening of the minor groove over one-half turn of the DNA. The orientation of the HTH element of the hETS1 DBD with respect to the major groove is significantly rotated relative to other HTH proteins. These observations establish the ETS family of DNA-binding proteins as a distinct family of HTH proteins.

Anti-immunoglobulin M activates nuclear calcium/calmodulin-dependent protein kinase II in human B lymphocytes.

We and others have previously shown that the nuclear protein, Ets-1, is phosphorylated in a calcium-dependent manner after ligation of immunoglobulin (Ig) M on B lymphocytes. As this phosphorylation was independent of protein kinase C activity, we tested whether a calcium/calmodulin-dependent protein kinase (CaM kinase) might phosphorylate the Ets-1 protein after elevation of intracellular free calcium concentrations. The dephosphorylated form of Ets-1 has been shown to bind to chromatin, suggesting that the operative kinase should be detectable in the nucleus. We prepared nuclear extracts from two human B cell lines in which increased intracellular free calcium levels correlated with increased phosphorylation of the Ets-1 protein. Activity of the CaM kinases was determined using a synthetic peptide substrate both in the absence and presence of an inhibitor specific for the CaM kinase family, KN-62. Stimulation of cells with anti-IgM led to increased activity of a nuclear kinase that could phosphorylate the peptide, and this activity was reduced by 10 microM KN-62. Kinase activity was reduced in lysates preadsorbed using an antibody specific for CaM kinase II. Two-dimensional phosphopeptide maps of the Ets-1 protein from cells incubated with ionomycin or anti-IgM contained two unique phosphopeptides that were absent in untreated cells. Incubation of isolated Ets-1 protein with purified CaM kinase II produced phosphorylation of peptides that migrated identically to those found in cells incubated with either anti-IgM or ionomycin. These data suggest a model of signal transduction by the antigen receptor on B lymphocytes in which increased intracellular free calcium can rapidly activate nuclear CaM kinase II, potentially resulting in phosphorylation and regulation of DNA-binding proteins.

Alternative to polyacrylamide gels improves the electrophoretic mobility shift assay.

In this paper we outline a simplified protocol for the electrophoretic mobility shift assay utilizing TreviGel 500, a nontoxic alternative to polyacrylamide. The TreviGel 500 matrix combines the strength and resolution of polyacrylamide with the simplicity and flexibility of agarose in the casting of gels. Therefore, this method provides a simple, rapid and nontoxic alternative to current protocols for the investigation of protein: DNA interactions.

Possible structural implications of 20 mutations in the protein C protease domain.

Analysis of naturally occurring protein mutations yields valuable insights into functionally important sequences. Characterizing mutations responsible for protein C deficiency at the molecular level has been the subject of intensive investigation. In a previous study, a three-dimensional model of the serine protease domain of protein C was used to analyze the set of protease domain mutations previously available. The mutations were largely found to fall into a limited number of categories. A recently updated protein C mutation data base has provided a number of new mutations which have been analyzed for structural predictions.

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: computational and experimental evaluation by mutational analysis.

Cu,Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu,Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited protonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (> 10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu,Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143.

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: patterns revealed by three-dimensional molecular modelling of mutations of the protease domain.

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, know protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites.

Rational design and expression of a heparin-targeted human superoxide dismutase.

In order to improve the therapeutic effectiveness of human Cu,Zn superoxide dismutase (HSOD) by targeting it to cell surfaces and increasing its circulatory half-life, we have designed and expressed a heparin-binding derivative of HSOD. This design was based on the idea that structurally independent protein units, HSOD and the heparin-binding A+ helix from protein C inhibitor, could be combined with a carefully chosen linker, GlyProGly, to form a stable, bifunctional protein. The chimeric HSOD-GlyProGly-A+ protein was expressed and secreted to the periplasm of E. coli and had normal SOD activity. HSOD-GlyProGly-A+ had a significantly increased retention time relative to wild-type HSOD on a heparin affinity column, indicating that it was successfully targeted to heparin, and this binding was maintained at physiological ionic strength. When administered to mice, HSOD-GlyProGly-A+ had a half-life of approximately 15 minutes, twice that of wild-type HSOD. Our rational design approach should be generally applicable to the creation of bifunctional chimeric molecules.

Influence of the Ig H chain locus on autoantibody production in autoimmune mice.

Autoimmune disease is influenced by multiple genes. In this study, we investigated the role of one genetic locus, Ig H chain. IgG2a antichromatin, anti-ssDNA, and antihistone autoantibodies (autoAb) from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr), (Ighj/b); (C57BL/6-lpr/lpr x C57BL/6-lpr/lpr-Igha), (Ighb/a); and (MRL/Mp-lpr/lpr x MRL/Mp-lpr/lpr-Ighb), (Ighj/b) mice were determined using allotype-specific ELISA. Strikingly, antichromatin and antihistone antibodies (Ab) were comprised of significantly more b allotype than either a or j allotype in all cohorts of F1 mice examined. In mice that produced anti-Sm Ab, the b allotype was used preferentially for these autoAb as well. However, no allotype skewing was observed in IgG2a Ab directed against TNP or DNA, or for total IgG2a. An Igh recombinant locus was utilized to examine the genetic control of b allotype skewing in lpr mice and in chronic graft vs host disease. In both models, the VH region did not appear to be responsible for the preferential use of b allotype. These results indicate a contribution to autoimmunity by the Igh locus and raise the possibility that Ig allotype may influence autoimmune disease by its effect on the production of certain autoAb.