Artificial covalent linkage of bacterial acyl carrier proteins for fatty acid production.

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

An Open-Label Pilot Study of Duloxetine in Patients With Irritable Bowel Syndrome and Comorbid Major Depressive Disorder.

Major depressive disorder (MDD) and irritable bowel syndrome (IBS) frequently co-occur, yet treating their comorbid presentation is challenging. Low-dose tricyclic antidepressants are efficacious for IBS, but higher doses to treat depressive symptoms present tolerability problems, whereas selective serotonin reuptake inhibitors are more tolerable but show inconsistent efficacy for IBS. If efficacious, serotonin-norepinephrine reuptake inhibitors like duloxetine would provide a useful alternative. We explored efficacy, tolerability, and time to onset of action of duloxetine in comorbid IBS-MDD in an open-label, 12-week trial. Repeated-measures mixed-effects regression analysis with the intent-to-treat sample assessed rate of change of the clinician-administered Gastrointestinal Symptoms Rating Scale, Montgomery-Åsberg Depression Rating Scale, and other clinician-administered and self-report scales. Seventeen Hispanic adults with current MDD and comorbid IBS meeting Rome III criteria entered the study. Medical and laboratory assessment ruled out alarm symptoms and signs inconsistent with IBS. Duloxetine led to significant improvement in Gastrointestinal Symptoms Rating Scale and Montgomery-Åsberg Depression Rating Scale scores and 71.4% and 64.3% intent-to-treat response rates for IBS and MDD, respectively. Abdominal pain severity decreased by 56%. Contrary to expectation of rapid analgesic effects, based on duloxetine studies for neuropathic pain, both IBS and MDD symptoms improved gradually; differences in slopes of improvement were nonsignificant. Duloxetine was moderately well tolerated at a mean endpoint dose of 60 mg/d. Study limitations include the lack of placebo control, modest sample size, single ethnic group, and high attrition rate. Duloxetine efficacy for comorbid IBS-MDD should be studied under placebo-controlled conditions with larger and more diverse samples.

Toward a cultural adaptation of pharmacotherapy: Latino views of depression and antidepressant therapy.

Relative to non-Latino Whites, Latinos in the United States with major depressive disorder (MDD) show low engagement in antidepressant therapy, whether engagement is defined as pharmacotherapy access, medication initiation, pill-taking, or treatment retention. One potential reason for this disparity in depression care is the low cultural congruence of pharmacotherapy for this population. To examine Latinos' views of depression and antidepressant therapy, we conducted qualitative interviews with 30 Latino outpatients initiating antidepressants prior to their first treatment visit using the semistructured Treatment Adherence and Retention Questionnaire. These baseline interviews were randomly selected from data collected for a randomized controlled trial testing a novel intervention to enhance engagement by depressed Latino outpatients. Participant narratives were analyzed using open coding and the iterative analytical approach derived from grounded theory. Patient views about depression addressed stigmatizing views held by others in their social circle. Most participants directly refuted these views by providing alternate explanations to depression experiences. Antidepressant therapy narratives also revealed marked stigmatization, but participants tended not to refute these views. Instead, patients expressed concerns about antidepressants and showed marked ambivalence about seeking psychiatric care. Participants, however, did suggest ways in which clinicians and patients might collaborate to address their concerns about antidepressants. Some cultural views, such as concerns about addiction to or dependence on medication, may be negotiable barriers to treatment. Prescribing clinicians should address cultural views and concerns in order to improve Latino engagement in antidepressant therapy.

Potential epigenetic dysregulation of genes associated with MODY and type 2 diabetes in humans exposed to a diabetic intrauterine environment: an analysis of genome-wide DNA methylation.

OBJECTIVE: The aim of this study is to investigate the potential role of DNA methylation in mediating the increased risk of developing type 2 diabetes in offspring of mothers who had diabetes during pregnancy. MATERIALS AND METHODS: Peripheral blood leukocytes were collected from non-diabetic Pima Indians who were either offspring of diabetic mothers (ODM; n=14) or offspring of nondiabetic mothers (ONDM; n=14). The two groups were matched for age, sex, age of mother, and fraction of Pima ethnicity. Differentially methylated regions were determined using a MeDIP-chip assay on an Affymetrix Human Tiling 2.0R Array. Data were analyzed using the model based analysis of tiling arrays (MAT) algorithm, and 4883 regions overlapping with putative promoters, were identified as differentially methylated, having met an empirically derived threshold (nominal p<0.0077). The list of genes with differentially methylated promoters was subjected to KEGG pathway analysis to determine canonical metabolic pathways enriched by these genes. RESULTS: Pathway analysis of genes with differentially methylated promoters identified the top 3 enriched pathways as maturity onset diabetes of the young (MODY), type 2 diabetes, and Notch signaling. Several genes in these pathways are known to affect pancreatic development and insulin secretion. CONCLUSIONS: These findings support the hypothesis that epigenetic changes may increase the risk of type 2 diabetes via an effect on ß-cell function in the offspring of mothers with diabetes during pregnancy.

Whole exome sequencing identifies variation in CYB5A and RNF10 associated with adiposity and type 2 diabetes.

OBJECTIVE: Few coding variants in genes associated with type 2 diabetes (T2D) have been identified, and the underlying physiologic mechanisms whereby susceptibility genes influence T2D risk are often unknown. The objective of this study was to identify coding variation that increases risk for T2D via an effect on a pre-diabetic trait. METHODS: Whole exome sequencing was done in 177 Pima Indians. Selected variants (N = 345) were genotyped in 555 subjects characterized for body fatness, glucose disposal rates during a clamp, acute insulin response to glucose, and 2-h plasma glucose concentrations during an OGTT, and were also genotyped in up to 5,880 subjects with longitudinal measures of BMI. Variants associated with quantitative traits were assessed for association with T2D in 7,667 subjects. RESULTS: rs7238987 in CYB5A associated with body fatness (P = 7.0 × 10(-6) ). This SNP and a novel SNP in RNF10 also associated with maximum recorded BMI (P = 6.2 × 10(-7) and P = 7.2 × 10(-4) ) and maximum childhood BMI z-score (P = 5.9 × 10(-4) and P = 8.5 × 10(-7) ). The BMI increasing alleles increased the risk for T2D (P = 0.01; OR = 1.13 [1.03-1.24] and 9.5 × 10(-3) ; OR = 1.49 [1.10-2.02]). CONCLUSIONS: CYB5A, which has a role in stearyl-CoA-desaturase activity, and RNF10, with an unknown role in weight regulating pathways, associated with adiposity and nominally increased the risk for T2D in American Indians.

ß-Puromycin selection of modified ribosomes for in vitro incorporation of ß-amino acids.

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against ß-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated ß-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing ß-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of ß-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence ß-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. ß-Puromycin, a putative mimetic for ß-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of ß-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to ß-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of ß-alanyl-tRNA(CUA).

The crystal structure of auracyanin A at 1.85 A resolution: the structures and functions of auracyanins A and B, two almost identical "blue" copper proteins, in the photosynthetic bacterium Chloroflexus aurantiacus.

Auracyanins A and B are two closely similar "blue" copper proteins produced by the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus. Both proteins have a water-soluble 140-residue globular domain, which is preceded in the sequence by an N-terminal tail. The globular domains of auracyanins A and B have sequences that are 38% identical. The sequences of the N-terminal tails, on the other hand, are distinctly different, suggesting that auracyanins A and B occupy different membrane sites and have different functions. The crystal structure of auracyanin A has been solved and refined at 1.85 A resolution. The polypeptide fold is similar to that of auracyanin B (Bond et al. in J Mol Biol 306:47-67, 2001), but the distribution of charged and polar residues on the molecular surface is different. The Cu-site dimensions of the two auracyanins are identical. This is unexpected, since auracyanin A has a shorter polypeptide loop between two of the Cu-binding residues, and the two proteins have significantly different EPR, UV-visible and resonance Raman spectra. The genes for the globular domains of auracyanins A and B have been cloned in a bacterial expression system, enabling purification of large quantities of protein. It is shown that auracyanin A is expressed only when C. aurantiacus cells are grown in light, whereas auracyanin B is expressed under dark as well as light conditions. The inference is that auracyanin A has a function in photosynthesis, and that auracyanin B has a function in aerobic respiration.

Cellular apoptosis and cytotoxicity of phenolic compounds: a quantitative structure-activity relationship study.

In this comprehensive study on the caspase-mediated apoptosis-inducing effect of 51 substituted phenols in a murine leukemia cell line (L1210), we determined the concentrations needed to induce caspase activity by 50% (I50) and utilized these data to develop the following quantitative structure-activity relationship (QSAR) model: log 1/I50 = 1.06 B5(2) + 0.33 B5(3) - 0.18pi(2,4) - 0.92. B5(3) and B5(2) represent steric terms, while pi(2,4) represents the hydrophobic character of the substituents on the ring. The strong dependence of caspase-mediated apoptosis on mostly steric parameters suggests that the process is a receptor-mediated interaction with caspases or mitochondrial proteins being the likely targets. Conversely, cytotoxicity studies of 65 electron-releasing phenols in the L1210 cell line led to the development of the following equation: log 1/ID50 = -1.39sigma+ - 0.28 B5(2,6) + 0.16 log P - 0.58I(2) - 1.04I(1) + 3.90. The low coefficient with log P may pertain to cellular transport that may be enhanced by a modest increase in overall hydrophobicity, while the presence of sigma+ is consistent with the suggestion that radical stabilization is of prime importance in the case of electron-releasing substituents. On the other hand, the QSAR for the interactions of 27 electron-attracting phenols in L1210 cells, log 1/ID50 = 0.56 log P - 0.30 B5(2) + 2.79, suggests that hydrophobicity, as represented by log P is of critical importance. Similar cytotoxicity patterns are observed in other mammalian cell lines such as HL-60, MCF-7, CCRF-CEM, and CEM/VLB. The significant differences between the cytotoxicity and apoptosis QSAR for electron-releasing phenols suggest that cytotoxicity involves minimal apoptosis in most of these substituted monophenols.

New class of bacterial membrane oxidoreductases.

A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purified from the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus, and putative operons for similar complexes were identified in a wide range of bacteria. In most cases, the presence of the new complex is anticorrelated with the cytochrome bc or bf electron-transfer complex, suggesting that it replaces it functionally. This appears to be a widespread yet previously unrecognized protein complex involved in energy metabolism in bacteria.