Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeY_Las was characterized as an endoribonuclease with activity on 3′ and 5′ termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeY_Las protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeY_Las. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeY_Las. Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.     

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.     

A steady-state model for the evaluation of disk rotational speed influence on RBC kinetic: model presentation

The physical and biological mechanisms of attached-biomass growth were analyzed and a steady-state model was proposed to determine the soluble carbonaceous removal in an RBC unit for different organic loading rates in the reactor. The objective of the model was the prediction of the organic loading rate corresponding to the maximum removal capacity in the system. A system of equations was solved where the influent soluble carbonaceous substrate concentration was the main variable. Monod's rate law was used for the growth of microorganism: the soluble carbonaceous substrate was the limiting substrate. Endogenous decay was neglected. The influence of disk rotational speed on the RBC removal capacity was investigated, the disk rotational speed being a parameter acting on oxygen transfer in the biofilm. The criteria for the evaluation of the kinetic parameter in the model were proposed.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Prebiotic chemistry: chemical evolution of organics on the primitive Earth under simulated prebiotic conditions.

A series of prebiotic mixtures of simple molecules, sources of C, H, N, and O, were examined under conditions that may have prevailed during the Hadean eon (4.6-3.8 billion years), namely an oxygen-free atmosphere and a significant UV radiation flux over a large wavelength range due to the absence of an ozone layer. Mixtures contained a C source (methanol, acetone or other ketones), a N source (ammonia or methylamine), and an O source (water) at various molar ratios of C : H : N : O. When subjected to UV light or heated for periods of 7 to 45 days under an argon atmosphere, they yielded a narrow product distribution of a few principal compounds. Different initial conditions produced different distributions. The nature of the products was ascertained by gas chromatographic-mass spectral analysis (GC-MS). UVC irradiation of an aqueous methanol-ammonia-water prebiotic mixture for 14 days under low UV dose (6 x 10(-2) Einstein) produced methylisourea, hexamethylenetetramine (HMT), methyl-HMT and hydroxy-HMT, whereas under high UV dose (45 days; 1.9 x 10(-1) Einstein) yielded only HMT. By contrast, the prebiotic mixture composed of acetone-ammonia-water produced five principal species with acetamide as the major component; thermally the same mixture produced a different product distribution of four principal species. UVC irradiation of the CH(3)CN-NH(3)-H(2)O prebiotic mixture for 7 days gave mostly trimethyl-s-triazine, whereas in the presence of two metal oxides (TiO(2) or Fe(2)O(3)) also produced some HMT; the thermal process yielded only acetamide.     

Atomic level description of the domain closure in a dimeric enzyme: thermus thermophilus 3-isopropylmalate dehydrogenase

The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 ? resolution, in complexes with both Mn(2+) and 3-isopropylmalate (IPM), as well as with both Mn(2+) and NADH, were determined at resolutions ranging from 2.0 to 2.5 ?. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn(2+) and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn(2+) and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 ? upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.