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The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin-and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin.

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.

Introduction Although breastfeeding has been shown to improve health outcomes for infants, African American women initiate and continue breastfeeding at lower rates than women from other racial groups. This scoping review was conducted to assess the effect racism, bias, and discrimination have on breastfeeding care, support, and outcomes for African American women. Methods A scoping review was performed of the literature published between January 2010 through December 2019 using databases MEDLINE via PubMed, CINAHL, Cochrane Library, PsycINFO, and Sociological Abstracts. Studies that examined racism, bias, or discrimination with breastfeeding as an outcome were included. After a review of titles and abstracts of the articles using exclusion and inclusion criteria, 5 full‐text articles were included in the scoping review. Results The qualitative and quantitative studies reviewed provide the perspectives of pregnant and postpartum African American women as well as those of health care providers. African American women's experiences of racism adversely affected both breastfeeding initiation and duration. Health care providers’ biased assumption that African American women would not breastfeed affected the quality of breastfeeding support provided to them. Specifically, African American women received fewer referrals for lactation support and more limited assistance when problems developed. This scoping review provides evidence that African American women experience racism, bias, and discrimination affecting breastfeeding care, support, and outcomes. Discussion Racism, bias, and discrimination are modifiable barriers that adversely affect breastfeeding among African American women. Researchers and health care providers are encouraged to consider the effect of racism, bias, and discrimination on breastfeeding care, support, and outcomes.

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8a-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.Succinate dehydrogenase (SDH) is a membrane-bound enzyme of the citric acid cycle that catalyses the oxidation of succinate to fumarate. The reducing equivalents are subsequently donated to the electron-transport chain. A closely related enzyme of bacterial membranes is fumarate reductase (FRD). FRD catalyses the reverse reaction, the reduction of fumarate to succinate, allowing cells to grow anaerobically with fumarate as the final electron acceptor. Both SDH and FRD can catalyse succinate oxidation and fumarate reduction but with different efficiencies (Cole et al., 1985 ;Hederstedt and Ohnishi, 1992).SDH and FRD are usually composed of four non-identical subunits : a flavoprotein, an iron-sulfur protein, and two smaller integral membrane proteins. The active site is located in the flavoprotein subunit, which also possesses the covalently bound FAD. The iron-sulfur-protein subunit contains three iron-sulfur clusters : [2Fe-2S], [3Fe-4S], and [4Fe-4S], centres 1, 3, and 2, respectively. The flavoprotein and ironsulfur-protein subunits form a catalytic dimer that is attached to the membrane by the two hydrophobic membrane anchoring proteins that may co-ordinate a b-type heme (except in Escherichia coli FRD) and interact with quinone substrates (Ackrell et al., 1992;Cole et al., 1985). However, voltammeCorrespondence to B. D. Lemire,

Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histonebinding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor. Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution. In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease. We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo.Eukaryotes package their genomic DNA into a complex nucleoprotein structure referred to as chromatin. The fundamental repetitive element of chromatin is the nucleosome, which is composed of 146 bp of DNA wrapped around an octamer of histone proteins: two dimers of histone H2A and H2B and a tetramer of histones H3 and H4 (56). This chromatinized template DNA is the substrate for in vivo reactions such as transcription, replication, recombination, and repair. Indeed, proper chromatin assembly or modification is necessary for the accurate execution and regulation of these processes.The cell uses a number of mechanisms to build nucleosomes. Nucleosomes are assembled in a DNA replication-coupled (RC) manner following the replication fork during S phase and are assembled onto DNA during gap-repair in response to DNA damage (28,35). In both cases, nascent DNA is packaged into chromatin. Alternatively, it has been demonstrated that nucleosomes can be assembled or reassembled independently of DNA replication. Replication-independent (RI) assembly is not limited to the S phase but occurs continually throughout the cell cycle (2, 4), perhaps functioning as a backup to RC assembly (44). RI assembly can introduce specific histone variants, such as the H3 variant Cid, at centrosomes (1) or histone H3.3 in transcriptionally active regions of the genome (2). By extension it has been proposed that RI assembly may replace histones that have been irreversibly modified by methylation (23); otherwise, the only way to change the histone methylation signal would be by gradual dilution of methylated with unmethylated histones in the course of cell proliferation. RI assembly also occurs in nondividing cells. For example, in nerve cells of higher eukaryotes infected with herpes simplex virus, RI assembly quickly packa...

Saccharomyces cerevisiae is a facultative anaerobe capable of meeting its energy requirements by fermentation and is thus an ideal system for studying the biogenesis of respiring mitochondria. We have isolated a respiration-deficient mutant exhibiting a pleiotropic loss of the mitochondrial electron transport chain. The corresponding wild-type gene, COQ5, was cloned, sequenced, and able to restore respiratory growth. Deletion of the chromosomal COQ5 gene results in a respiration deficiency and reduced levels of respiratory protein components. Exogenously added decylubiquinone can partially restore electron transport chain function to mitochondrial membranes from the deletion mutant. The COQ5 nucleotide sequence predicts a polypeptide of 307 amino acids containing a mitochondrial targeting signal. COQ5p is 43% identical to the polypeptide predicted by the Escherichia coli open reading frame, o251 (1). The COQ5 gene, when introduced into E. coli, complements the respiratory deficiency of an ubiE mutant that maps near o251, suggesting that it is the yeast homolog of the ubiE gene product. We conclude that the COQ5 gene encodes the mitochondria-localized 2-hexaprenyl-6-methoxy-1,4-benzoquinone methyltransferase of the yeast ubiquinone biosynthetic pathway.

HCP and parent collaboration could lead to strategies to increase supports for children with autism spectrum disorder in the hospital to decrease their frustration and challenging behaviors.