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Background: Metabolic syndrome in adolescence has been associated with adverse cardiometabolic outcomes in adulthood. Preliminary data suggest that boys may have worsened metabolic syndrome components compared to girls. Yet, little is known about the physical health of military dependents, a potentially at-risk population., Objective: Examine sex differences in metabolic syndrome components in a sample of adolescent military dependents., Methods: Participants were adolescents (N = 139; 14.4 ± 1.6 years; 45.3% male; 41.0% non-Hispanic White, 19.4% non-Hispanic Black; BMI-z: 1.9 ± 0.4) at-risk for adult obesity and binge-eating disorder due to an age- and sex-adjusted BMI ≥85th percentile and loss-of-control eating and/or elevated anxiety. A multivariate analysis of covariance was conducted to compare objectively measured metabolic syndrome components across boys and girls. Covariates were age, race, loss-of-control eating status, anxiety symptoms, and BMI-z., Results: Metabolic syndrome components differed by sex (P = .01). Boys had higher systolic blood pressure (P = .049), lower high-density lipoprotein cholesterol (P = .01), and higher glucose (P = .001) than girls. Waist circumference, diastolic blood pressure, and triglycerides did not differ between boys and girls (P > .05)., Conclusions: Future research should prospectively examine these relationships into adulthood. If the current findings are supported, prevention programs should consider targeting cardiometabolic health particularly among male adolescent military dependents., (© 2020 World Obesity Federation.)

Objective: Adolescent military dependents may be at higher risk for psychosocial stressors and disordered eating compared with civilian youths, but the mechanisms underlying these risks are unclear. Interpersonal theory proposes that difficult relationships lead to negative affect, thereby promoting emotional eating, which has been linked to and predictive of disordered eating. The interpersonal model may have particular relevance for understanding disordered eating among adolescent military dependents, given the unique stressors related to their parents' careers. This study aimed to examine the premise of the interpersonal model (that negative emotions mediate the association between multiple aspects of social functioning and emotional eating) among a cohort of adolescent military dependents., Methods: Military dependents (N=136; 56% female, mean±SD age=14±2 years, body mass index adjusted for age and sex [BMI z ]=2.0±0.4) at risk for adult obesity and binge eating disorder, as indicated by reported loss-of-control eating and/or anxiety symptoms, were assessed prior to participation in a study of excess weight-gain prevention. Bootstrapped mediation analyses were conducted to examine depressive symptoms as a potential mediator of the relationship between social functioning and emotional eating. Analyses were adjusted for age, sex, race-ethnicity, BMI z, and presence of reported loss-of-control eating and anxiety., Results: Depressive symptoms were a significant mediator of the relationship between multiple domains of social functioning, including loneliness, social adjustment related to family and friends, attachment to father and peers, and emotional eating (p<0.05)., Conclusions: The interpersonal model may contribute to our understanding of excess weight gain and binge eating disorder among adolescent military dependents. Prospective data are needed to determine the utility of interpersonal theory in predicting treatment response and outcomes among this population.

Objective: Parental military deployment can lead to stress in the family system due to concerns about the deployed service-member's safety and increased responsibilities for those not deployed. Parent-related stress can impact adolescent disordered eating. Given the important role that stress plays in disordered eating and obesity, it is crucial to understand the impacts of unique stressors to which vulnerable populations are exposed., Method: We studied 126 adolescent (14.3 ± 1.6 years; 59.5% girls; 44.4% non-Hispanic White; BMI-z, 1.91 ± .39) military dependents prior to entering an obesity and binge-eating disorder prevention trial. The Eating Disorder Examination was used to assess adolescent disordered eating. Parents self-reported their own distress and family deployment history that occurred during the adolescent's lifetime., Results: Parental distress interacted with frequency of parental deployments such that for those with high parental distress, more frequent deployment was associated with greater adolescent shape and weight concerns (β = .21, p = .012) and global eating pathology (β = .18, p = .024)., Discussion: In this hypothesis-generating study, the combination of number of deployments and parental distress may be associated with disordered eating among adolescent military dependents seeking prevention of binge-eating disorder and adult obesity. If these preliminary findings are supported longitudinally, interventions to reduce parental stress related to deployment may be warranted to reduce disordered eating in adolescent dependents., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Weight-based teasing (WBT) by family members is commonly reported among youth and is associated with eating and mood-related psychopathology. Military dependents may be particularly vulnerable to family WBT and its sequelae due to factors associated with their parents' careers, such as weight and fitness standards and an emphasis on maintaining one's military appearance; however, no studies to date have examined family WBT and its associations within this population. Therefore, adolescent military dependents at-risk for adult obesity and binge-eating disorder were studied prior to entry in a weight gain prevention trial. Youth completed items from the Weight-Based Victimization Scale (to assess WBT by parents and/or siblings) and measures of psychosocial functioning, including the Beck Depression Inventory-II, The Rosenberg Self-Esteem Scale, and the Social Adjustment Scale. Eating pathology was assessed via the Eating Disorder Examination interview, and height and fasting weight were measured to calculate BMI z . Analyses of covariance, adjusting for relevant covariates including BMI z , were conducted to assess relationships between family WBT, eating pathology, and psychosocial functioning. Participants were 128 adolescent military dependents (mean age: 14.35 years old, 54% female, 42% non-Hispanic White, mean BMI z : 1.95). Nearly half the sample (47.7%) reported family WBT. Adjusting for covariates, including BMI z , family WBT was associated with greater eating pathology, poorer social functioning and self-esteem, and more depressive symptoms ( ps ≤ 0.02). Among military dependents with overweight and obesity, family WBT is prevalent and may be linked with eating pathology and impaired psychosocial functioning; prospective research is needed to elucidate the temporal nature of these associations., Competing Interests: The authors declare no conflict of interest.

Stressors unique to military families may place dependents of military service members of both sexes at high-risk for disordered-eating. Yet, there are no data examining sex-related differences in eating pathology and distress among this population. Therefore, we examined disordered-eating attitudes and associated psychosocial characteristics in adolescent military dependents at high-risk for both eating disorders and adult obesity (i.e., BMI ≥ 85th percentile and elevated anxiety symptoms and/or loss-of-control eating). One-hundred-twenty-five (55.2% female) adolescent (12-17 y) military dependents were studied prior to entry in an eating disorder and obesity prevention trial. Youth were administered the Eating Disorder Examination interview to determine disordered-eating attitudes, and completed questionnaires to assess self-esteem, social functioning, and depression. Girls and boys did not differ in BMIz (p = .66) or race/ethnicity (p = .997/p = .55). Adjusting for relevant covariates, girls and boys did not differ significantly with regard to disordered-eating global scores (p = .38), self-esteem (p = .23), or social functioning (p = .19). By contrast, girls reported significantly more symptoms of depression (p = .001). Adolescent male and female dependents at high-risk for eating disorders and adult obesity reported comparable levels of eating-related and psychosocial stress. Data are needed to elucidate how adolescent military dependents respond to intervention and whether sex moderates outcome., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH resulted in the release of NAD+ cofactors, explaining the loss of the enzymatic activity of SAHH. Further investigation by an ESR probe and computational simulation suggested that Cu2+ could bind at the central channel and interrupt the subunit interactions of SAHH, resulting in a large decrease in affinity to the NAD+ cofactor. This effect of Cu2+ resembled that of enzyme mutations at the C-terminal domain or Asp244 [Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme, J. Biol. Chem. 275, 32147-32156]. The mechanism of action of Cu2+ on SAHH suggested a possible regulative role for Cu2+ on the intracellular activity of SAHH. This could be helpful in understanding the biological effects of copper compounds and suggest a potential coupling mechanism between biomethylation and the redox states of cells.

Cu-catalyzed addition of alkylzinc reagents to a range of allylic phosphates is promoted efficiently and with high enantioselectivity to afford tertiary as well as quaternary carbon centers (up to 98% ee). Reactions proceed to completion with 0.5-5 mol % catalyst loading and are best promoted by commercially available CuCl2.2H2O. The X-ray structure of the chiral NHC-Ag(I) complex used in the study as well as that of a catalytically active NHC-Cu(II) complex are also reported; both complexes are air-stable and are formed in >/=95% isolated yield. The isolated Cu complex, which can be handled in air, is catalytically active. The present report provides the first precedent for efficient Cu-catalyzed allylic alkylations with chiral NHC ligands.

The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestris pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the xpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into the xpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsN gene or by introducing extra copies of wild-type xpsL or xpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL or xpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with the xpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of the xpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of the xpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestris pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)(6)-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two.

The EMB30 gene is involved in apical-basal pattern formation in the Arabidopsis embryo. Mutations in this locus produce mutants with a wide range of seedling phenotypes, but all of the mutants lack a root and a true hypocotyl. We have cloned the EMB30 gene, and it encodes a protein that has similarity to the yeast Sec7 protein and to two other open reading frames identified in clones from humans and C. elegans. We refer to the region of similarity among these four sequences as the Sec7 domain. The emb30-1 allele has a mutation in the Sec7 domain that alters a residue conserved in all four of these sequences, suggesting that this domain may be important for EMB30 function. Molecular data and microscopy studies of emb30 seedlings presented here indicate that EMB30 affects cell division, elongation, and adhesion and functions in seedling and adult plants as well as during embryogenic pattern formation.

The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase). Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase. In this study, we investigated the role of histidine residues in the function of MelC1 by examining a series of substitution or deletion mutants. Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishment of the intracellular tyrosinase activity in both Streptomyces and Escherichia coli. Replacement of His-102 by Leu in the mutant M-102L also caused a 64-70% reduction of tyrosinase activity in Streptomyces and E. coli. These two mutations also affected the secretion of both MelC1 and MelC2 proteins. In vitro copper activation of the purified MelC1.MelC2 binary complex from these two mutants regained only 20-30% tyrosinase activity of the wild type. Biochemical characterization of the tyrosinases from these two mutants revealed that they were different in several aspects. The intracellular tyrosinase activity in M-117D, but not in M-102L, could be partially reactivated by copper ion or by the cell extract containing MelC1. The copper content and the specific activity of the tyrosinase purified from the culture supernatant from M-117D were only 40% of those in wild type and M-102L. Additionally, fast protein liquid chromatography analysis indicated that in these two mutants the copper activation process was defective, very likely due to the incompetent MelC1.MelC2 binary complex formed: reduced association in M-117D and elevated association in M-102L. Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme form in wild type could be differentiated by the proteinase K digestion pattern, and so did the conformation of MelC2 found in those of M-102L, but not in M-117D mutant. Taken together, our results demonstrate that MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred. This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were abolished in a MelC1-negative mutant, M-950. Taken together, these results demonstrate that a functional MelC1 is essential for tyrosinase secretion and activity. Furthermore, the results suggest that like other secretory proteins, basic and hydrophobic residues in the MelC1 signal sequence are an important feature of the signal-peptide and play a pivotal role in the secretion of both the MelC1 and MelC2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

The melanin operon (melC) of Streptomyces antibioticus is composed of two genes that encode MelC1 and MelC2 proteins. MelC1 has been suggested as a trans-activator which can facilitate the incorporation of copper into the apotyrosinase (MelC2) (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81). However, the molecular mechanism of the trans-activation or copper-transfer process mediated through MelC1 to MelC2 is not clear yet. In this study, we found apotyrosinase in both the extracellular fraction and cell extract from cells grown in copper-deficient medium. Using gel-filtration and immunoaffinity chromatographies, we demonstrated that apotyrosinase (MelC2) formed a stable complex with MelC1 in the intra- and extracellular fractions. Furthermore, addition of copper ion to the complex generated tyrosinase activity. The MelC1-MelC2 complex was purified to near homogeneity by DE52 and phenyl-agarose chromatographies. In conjunction with fast protein liquid gel filtration chromatography and NH2-terminal sequencing analysis, the results indicated that the stoichiometric ratio of MelC1 and MelC2 in the purified complex was 1:1. Essentially no copper was found in the complex. Addition of copper ion to the purified complex resulted in incorporation of approximately 2 molecules of copper ion and the mature active tyrosinase was gradually released from the complex. Taken together, these results demonstrate that the molecular mechanism of activation of Streptomyces apotyrosinase by its trans-activator MelC1 is initially mediated via a binary complex formation between these two proteins, followed by incorporation of copper ion. This activation mechanism accounts for the essential role of MelC1 in the expression of melanin operon.

The melanin locus (melC) from Streptomyces antibioticus was previously shown to be composed of two open reading frames (ORFs), melC1 and melC2. The melC2 ORF codes for the polypeptide chain of tyrosinase (apotyrosinase). The function of melC1 is not known except that insertional mutation within it abolishes the tyrosinase activity. Here, we show that in Streptomyces lividans TK64 harboring melC1 mutated and melC2 intact (melC1- melC2+) plasmids, while there was no tyrosinase activity, melC transcript was synthesized and apotyrosinase could be detected. The apotyrosinase could be activated to a limited degree by incubation with copper ions, or by mixing the mycelial extract from a culture harboring a melC1- melC2+ (pPF950) plasmid with that from a culture containing a melC1+ melC2- (pSA1) plasmid. Complementation analysis showed that melC1 acted in trans on the tyrosinase gene expression. Together, these results suggest that melC1 encodes or regulates a copper-transfer protein serving an in vivo copper-donor function in the biosynthesis of active tyrosinase.

Several approaches were used to study the transcriptional control region of the melanin-production locus (melC) of Streptomyces antibioticus. Filter-binding in combination with exonuclease III protection localized the 3' boundary of a Streptomyces RNA polymerase-binding site predominantly about 39 nucleotides (nt) upstream from the start codon of melC1, the first open reading frame in the melC locus. Deletion of nt 112-197 upstream from the melC1 start codon reduced melC expression to less than 10%, and deletion of nt 28-107 or 28-120 upstream from melC1 totally inactivated melC. High-resolution nuclease S1 mapping identified the in vitro transcriptional start point (tsp) at 33-34 nt upstream from the start codon of melC1. No sequence resembling the E. coli consensus promoter sequence was found in this region, and site-directed mutagenesis of such a sequence located 101-132 nt upstream from melC1 did not influence melC expression. These studies suggest that transcription of melC is principally from a single tsp and is positively regulated by a mechanism that involves sequences 87-163 nt upstream from the tsp.