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Papers by Paul Black

Biochemical Journal, Sep 1, 1995

Journal of Bacteriology, 1991

The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central r... more The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central role in the uptake of exogenous long-chain fatty acids. The nucleotide sequence of the fadL gene revealed a single open reading frame of 1,344 bp encoding a protein with 448 amino acid residues and a molecular weight of 48,831. The transcriptional start, analyzed by primer extension, was shown to be 95 bp upstream from the translational start. Apparent -10 and -35 regions were found at -12 and -37 bp upstream from the transcriptional start. Three regions with hyphenated dyad symmetry (two between the transcriptional start and the translational start and one upstream from the -10 and -35 regions) were identified that may play a role in the expression offadL. The protein product of the fadL gene contained a signal sequence and signal peptidase I cleavage site similar to that defined for other E. coli outer membrane proteins. The N-terminal sequence of the mature FadL protein was determined by automated amino acid sequencing of protein purified from the outer membrane of a strain harboring fadL under the control of a T7 RNA polymerase-responsive promoter. This amino acid sequence, Ala-Gly-Phe-Gln-Leu-Asn-Glu-Phe-Ser-Ser, verified the signal peptidase I cleavage site on pre-FadL and confirmed the N-terminal amino acid sequence of FadL predicted from the DNA sequence. Mature FadL contained 421 amino acid residues, giving a molecular weight of 45,969. The amino acid composition of FadL deduced from the DNA sequence suggested that this protein contained an abundance of hydrophobic amino acid residues and lacked cysteinyl residues. The hydrophobic amino acids within FadL were predicted to contribute to at least five regions of the protein with an overall hydrophobic character. The amino acid sequence of FadL was used to search GenBank for other proteins with amino acid sequence homology. These data demonstrated that FadL and the heat-modifiable outer membrane protein P1 of Haemophilus influenzae type b were 60.5% conserved and 42.0% identical over 438 amino acid residues.

Journal of Biological Chemistry, Sep 1, 2011

Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Sep 1, 2007

Journal of Biological Chemistry, Dec 1, 1997

In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the leve... more In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the level of transcription by FadR. FadR represses transcription of at least eight genes required for fatty acid transport and ␤-oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encoding the transcriptional regulator of the ace-BAK operon encoding the glyoxylate shunt enzymes, IclR. FadR-dependent DNA binding and transcriptional activation is prevented by long chain fatty acyl-CoA. In the present work, we provide physical and genetic evidence that FadR exists as a homodimer in solution and in vivo. Native polyacrylamide gel electrophoresis and glycerol gradient ultracentrifugation of the purified protein show that native FadR was a homodimer in solution with an apparent molecular mass of 53.5 and 57.8 kDa, respectively. Dominant negative mutations in fadR were generated by random and site-directed mutagenesis. Each mutation mapped to the amino terminus of the protein (residues 1-66) and resulted in a decrease in DNA binding in vitro. In an effort to separate domains of FadR required for DNA binding, dimerization, and ligand binding, chimeric protein fusions between the DNA binding domain of LexA and different regions of FadR were constructed. One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter sulA-lacZ, and ␤-galactosidase activities were derepressed by fatty acids, suggesting that the fusion protein had determinants both for dimerization and ligand binding. These studies support the conclusion that native FadR exists as a stable homo-dimer in solution and that determinants for DNA binding and acyl-CoA binding are found within the amino terminus and carboxyl terminus, respectively.

Biochemical and Biophysical Research Communications, Nov 1, 2013

Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Mar 1, 2008

Clinical & Experimental Metastasis

staining. (D) Quantitative analysis of proliferating tumor cell frequency per 200x field in the H... more staining. (D) Quantitative analysis of proliferating tumor cell frequency per 200x field in the HO-MET of the CMGs. (E) Ki67 staining for proliferating cells in the ductal wall of CMGs and (F) their frequency per 400x microscopic field. (G) F4/80 + macrophages in the ductal wall of CMGs and (H) their frequency per 200x microscopic field. (I) F4/80 + macrophages in adipose tissue of CMGs and (J) their frequency per 200x microscopic field. Images of (A) were taken at 100x. Images of (C), (G) and (I) were taken at 200x and Images of (E) were taken at 400x. *p < 0.05 mice fed ω-3 versus ω-6 diet and were analyzed using t-test. The authors would like to apologize for this error and state that these corrections do not change the scientific conclusions of the article in any way. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Biochemical Pharmacology, Apr 1, 2010

Journal of Bacteriology, Dec 1, 1992

Nature, Apr 17, 2019

Users may view, print, copy, and download text and data-mine the content in such documents, for t... more Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The FASEB Journal, 2014

A large scale in vivo high throughput screen (HTS) was performed to identify small compounds that... more A large scale in vivo high throughput screen (HTS) was performed to identify small compounds that stimulate lipid production and accumulation using the model organism Chlamydomonas reinhardtii. The HTS employed a 384‐well microplate format in which cells were allowed to grow in the presence of the compounds at 10 μM final concentration for 72 hours. Accumulation of intracellular lipids was assessed using the lipophilic dye Nile Red and growth was monitored at OD600. The screening library included 43,736 compounds (ChemBridge, Corp). A total of 367 active compounds were identified that stimulated lipid accumulation to > 2.5‐fold and did not affect cell viability to give a hit rate of 0.8%. Primary hits were “cherry picked” and the sub‐set of compounds were retested using an 8‐point dose response assay (0.25‐30 µM). Hits were further assessed visually using a Nikon Ti‐inverted microscope (60X) to reconfirm lipid droplet accumulation was induced.. The final set of hits included 306 ...

Blood, 1989

Pretreatment of mice with recombinant murine (rM) colony-stimulating factor-granulocyte-macrophag... more Pretreatment of mice with recombinant murine (rM) colony-stimulating factor-granulocyte-macrophage (CSF-gm) or recombinant human (rH) CSF-g provides partial protection from the lethal effects of ionizing radiation or the alkylating agent cyclophosphamide (CTX). In addition, these agents can significantly prolong survival if administered following lethal doses of irradiation or CTX. To induce protective activity, cytokines were injected 20 hours before lethal irradiation or CTX administration. To accelerate recovery from lethal irradiation, the cytokines must be administered shortly following irradiation, and the induction of maximal levels of activity is dependent on chronic administration. In contrast, because of their longer half-lives, accelerated recovery from alkylating agents requires a delay of at least 24 to 48 hours to allow complete clearance of CTX before administration of a CSF. Studies quantitating peripheral blood leukocytes and bone marrow cellularity as well as colon...

International Immunopharmacology, 2018

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for p... more Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Journal of Bacteriology, 1994

TnphoA was used to mutagenize the chromosome in an effort to identify membrane-bound and exported... more TnphoA was used to mutagenize the chromosome in an effort to identify membrane-bound and exported components of the long-chain fatty acid transport system of Escherichia coli. This strategy identified three classes of fusions that were unable to grow or grew at reduced rates on minimal agar plates containing the long-chain fatty acid oleate (C18:1), (i) fadL-phoA, (ii) tolC-phoA, and (iii) tsp-phoA, fadL-phoA and tolC-phoA fusions were unable to grow on oleate as the sole carbon and energy source, while the tsp-phoA fusion had a markedly reduced growth rate. As expected, fadL-phoA fusions were unable to grow on oleate plates because the outer membrane-bound fatty acid transport protein FadL was defective. The identification of multiple fadL-phoa fusions demonstrated that this strategy of mutagenesis specifically targeted membrane-bound and exported components required for growth on long-chain fatty acids. tolC-phoA fusions were sensitive to fatty acids (particularly medium chain) an...

Plant Physiology, Jun 26, 2017

Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. Howe... more Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii, and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using gas chromatographymass spectrometry quantification of total fatty acids, and targeted TAG and galactolipid measurements were performed using liquid chromatography-multiple reaction monitoring/mass spectrometry. These results demonstrated that TAG accumulation does not necessarily proceed at the expense of galactolipid. Untargeted metabolite profiling provided important insights into pathway shifts due to five different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds also was demonstrated in three other algal species. These lipid-inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.

Multi-omics analyses reveal metabolic pathway activities induced by two synthetic compounds that ... more Multi-omics analyses reveal metabolic pathway activities induced by two synthetic compounds that increase lipid synthesis during microalgal growth. Author Contributions: CD and NW conceived the original research plan and were responsible for execution of the work, data analysis, and interpretation. NW, BT, GKR, and RG conducted biochemical, RNA-Seq, and metabolomics experiments. RC and JA provided oversight for proteomic and metabolomics analyses, respectively. PB assisted in data presentation and preparation of figures. All authors contributed to final manuscript preparation. CD agrees to serve as the author responsible for contact and ensures communication

A method of removing nitrogen-bound nitrate from at least one of groundwater, surface water, or w... more A method of removing nitrogen-bound nitrate from at least one of groundwater, surface water, or waste water is disclosed. The method includes providing contaminant-containing water from groundwater, surface water, and/or waste water sources. The method further includes adding the contaminant-containing water to an algal photobioreactor system. The method further includes adding an alga culture to the alga photobioreactor system. The method further includes adjusting temperature, CO2 concentration, pH, light wavelength, and/or light intensity in the algal photobioreactor system to optimize the growth of the algea. The method further includes separating the aglae from the water and harvesting algal biomass

The FASEB Journal, 2016

Third-Year Beckman Scholar (2015-2016); University of Nebraska-Lincoln Evaluation of Compounds th... more Third-Year Beckman Scholar (2015-2016); University of Nebraska-Lincoln Evaluation of Compounds that Inhibit Fatty Acid Uptake in Mice Lipotoxicity is characterized by apoptosis due to the toxic spillover of fatty acids from adipocytes into non-adipose tissue. Adipocytes function under high lipid concentrations and serve to regulate triglyceride levels in the body, but their storage capacity is limited, and adipocytes are frequently unable to regulate body triglyceride levels in cases of obesity. In such cases, lipid metabolic products (including ceramides and free fatty acids) become toxic at high concentrations. Lipotoxicity is seen in an array of cell types, including liver, kidney, heart, and pancreatic cells. My research will seek to extend previous work done in identifying and characterizing small molecule fatty acid uptake inhibitors as potential drug therapies. A high-throughput screen was first employed to identify inhibitors of the liver Fatty Acid Transporter, FATP2. Positive results were then evaluated in a cell-based system, and two compounds were selected for continued study. I will be examining the effects of these compounds when delivered orally in mice, with particular attention to assessing the compounds' toxicity, measuring inhibition of 13 C-oleate absorption, and quantifying lipids in the liver, bloodstream, and feces.