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A Frolov

Publications and source records attributed to A Frolov.

32 records · Page 2Linked to original sources
PubMed1998

Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion.

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Isoforms of rat liver fatty acid binding protein differ in structure and affinity for fatty acids and fatty acyl CoAs.

Although native rat liver fatty acid binding protein (L-FABP) is composed of isoforms differing in isoelectric point, their comparative structure and function are unknown. These properties of apo- and holo-L-FABP isoforms were resolved by circular dichroism, time-resolved fluorescence spectroscopy, and binding/displacement of fluorescent ligands. Both apo-isoforms had similar hydrodynamic radii of 18.5 A, but apo-isoform I had a greater alpha-helical content and exhibited a longer Tyr lifetime, indicative of secondary and tertiary structural differences from isoform II. Isoforms I and II both had two fatty acid or fatty acyl CoA binding sites. Ligand binding decreased the isoform hydrodynamic radii by 3-4 A and increased Tyr rotational motions in a more restricted range. Fatty acyl CoAs were more effective than fatty acids in altering the isoform structures. Scatchard analysis showed that both isoforms bound cis- parinaric acid with high affinity (Kd values 41 and 60 nM, respectively) and bound trans-parinaric acid with 2- and 7-fold, respectively, higher affinity than for cis-parinaric acid. In contrast, isoform I had higher affinity for cis- and trans-parinaroyl CoAs (Kd values of 33 and 14 nM) than did isoform II (Kd values of 110 and 97 nM), thereby resulting in biphasic plots of parinaroyl-CoA binding to native L-FABP. Finally, displacement studies indicated that each isoform displayed distinct specificities for fatty acid/fatty acyl CoA chain length and unsaturation. Thus, rat L-FABP isoforms differ markedly in both structure and ligand binding function.

Acyl Coenzyme A↗
PubMed1997

The sterol carrier protein-2 fatty acid binding site: an NMR, circular dichroic, and fluorescence spectroscopic determination.

The interaction and orientation of fatty acids with recombinant human sterol carrier protein-2 (SCP-2) were examined by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence techniques. 13C-NMR spectroscopy of stearic acid and oleic acid as well as fluorescence spectroscopy of cis-parinaric acid demonstrated that SCP-2 bound naturally occurring fatty acids with near 1:1 stoichiometry. Several findings indicated that the fatty acid was oriented in the binding site with its methyl end buried in the protein interior and its carboxylate exposed at the surface: the chemical shift of bound [18-13C]-stearate; dicarboxylic/monocarboxylic acid cis-parinaric acid displacement; complete ionization of the carboxylate group of SCP-2 bound [1-13C]stearate at neutral pH; lack of electrostatic interactions between 13C-fatty acids with SCP-2 cationic residues: pH titratability of the SCP-2 bound [1-13C]stearate carboxylate group. SCP-2 did not undergo global structural changes upon ligand binding or pH decrease as indicated by the absence of significant changes in NMR and only small alterations in time resolved fluorescence parameters. However, SCP-2 did undergo secondary structural changes detected by CD in the pH range 5-6. While these changes in secondary structure did not alter the fatty acid:SCP-2 binding stoichiometry, the affinity for fatty acid was increased severalfold at lower pH. In summary, 13C-NMR, CD, and fluorescence spectroscopy provided a detailed understanding of the interaction of fatty acids with SCP-2 and further showed for the first time the orientation of the fatty acid within the binding site. The pH-induced changes in SCP-2 secondary structure and ligand binding activity may be important to the mechanism whereby this protein interacts with membrane surfaces to enhance lipid binding/transfer.

Binding Sites↗
PubMed1997

Time-resolved fluorescence of intestinal and liver fatty acid binding proteins: role of fatty acyl CoA and fatty acid.

The effect of fatty acyl CoA and fatty acid on the solution structure and dynamics of two intestinal enterocyte fatty acid binding proteins, intestinal (I-FABP) and liver (L-FABP), was examined by time-resolved fluorescence of FABP aromatic amino acid residues. I-FABP Trp displayed two rotational correlation times, 6.6 and 0.4 ns. reflecting motion of the protein as a whole and segmental mobility of Trp. Neither oleoyl CoA, oleic acid, nor CoASH altered overall I-FABP rotational correlation time. However, oleic acid and CoASH increased I-FABP Trp segmental mobility, while oleoyl CoA and CoASH decreased I-FABP Trp limiting anisotropy (order). The angle of I-FABP Trp "wobbling in a cone" was increased by ligands in the order oleoyl CoA > CoASH > oleic acid. L-FABP Trp segmental mobility. L-FABP overall rotational motion, in contrast to that of I-FABP, was significantly increased by ligands in the order oleoyl CoA > oleic acid > CoASH. cis-Parinaric acid and cis-parinaroyl CoA bound to L-FABP also reflected overall L-FABP motion but yielded longer rotational correlation times, 8.2 and 10.7 ns, than the respective apo-FABPs. Such effects were not observed with I-FABP. Finally, both cis-parinaric acid and cis-parinaroyl CoA were much less ordered in the I-FABP ligand binding site than with L-FABP. These observations suggest that the rotational dynamics of L-FABP and its conformation are more sensitive to ligands than I-FABP. Further, ligands such as fatty acids, fatty acyl CoAs, and/or CoASH differentially modulate the I-FABP and L-FABP dynamics, and the ligand binding sites of these proteins differ in their ability to order the ligands.

Acyl Coenzyme A↗
PubMed1997

Lipid specificity and location of the sterol carrier protein-2 fatty acid-binding site: a fluorescence displacement and energy transfer study.

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14-22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and L-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp50 and bound cis-parinaric acid was determined to be 40 A. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp50. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.

Animals↗
PubMed1996

Sterol carrier protein-2, a new fatty acyl coenzyme A-binding protein.

The ability of sterol carrier protein-2 (SCP-2) to interact with long chain fatty acyl-CoAs was examined. SCP-2 bound fluorescent fatty acyl-CoAs at a single site with high affinity. Kd values for cis- and trans-parinaroyl-CoA were 4.5 and 2.8 nM, respectively. Saturated 10-18-carbon and unsaturated 14-20-carbon fatty acyl-CoAs displaced SCP-2-bound fluorescent ligand. Oleoyl-CoA and oleic acid (but not coenzyme A) significantly altered SCP-2 Trp50 emission and anisotropy decay, thereby increasing SCP-2 rotational correlation time, SCP-2 hydrodynamic radius, and SCP-2 Trp50 remaining anisotropy up to 1.7-, 1.2-, and 1.3-fold, respectively. These changes were not accompanied by significant alterations in protein secondary structure as determined by circular dichroism. Finally, SCP-2 differentially altered the fluorescence emission and anisotropy decays of bound cis- and trans-parinaroyl-CoA. Both fluorescent fatty acyl-CoAs were located within a very ordered (limited cone angle of rotation) environment within SCP-2, as shown by a remaining anisotropy of 0.365 and 0.361 and a wobbling cone angle of 12 and 13 degrees , respectively. These anisotropy values were very close to those of such ligands in a propylene glass. However, the rotational relaxation times exhibited by SCP-2-bound cis- and trans-parinaroyl-CoA, 8.4-8.8 ns, were longer than those for the corresponding free fatty acid, 7.5-6.6 ns. These data show for the first time that SCP-2 is a fatty acyl-CoA-binding protein.

Acyl Coenzyme A↗
PubMed1996

Fluorescence studies of lipid association-induced conformational adaptations of an exchangeable amphipathic apolipoprotein.

The conformational adaptability of Manduca sexta apolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145 is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145 and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKa of Tyr145 in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the (lambda)max of absorbance of Tyr145 was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145 fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145 in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145 exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145 remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145 which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145 is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145 distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.

Animals↗
PubMed1996

Spontaneous and protein-mediated sterol transfer between intracellular membranes.

Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4-5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell.

Animals↗
PubMed1996

Fibroblast membrane sterol kinetic domains: modulation by sterol carrier protein-2 and liver fatty acid binding protein.

The mechanism(s) of intracellular sterol trafficking among subcellular organelle membranes is not well understood. Relative contributions of vesicular, sterol carrier protein, and membrane sterol domain pathways are not resolved. A sterol kinetic assay was used to resolve multiple sterol domains in microsome (MICRO), mitochondria (MITO), and plasma (PM) membrane: exchangeable, 20-40% of total; non-exchangeable, 60-80% of total. Spontaneous sterol transfer between dissimilar donor and acceptor membranes was vectorial and depended both on acceptor and donor membrane properties. For example, sterol transfer from PM to MICRO or to MITO, or from MICRO to MITO was 3- to 5-fold slower as compared to sterol movement in the opposite direction. Sterol carrier protein-2 (SCP-2) stimulated sterol transfer in most donor/acceptor membrane combinations by decreasing exchange half-time but not domain size. SCP-2 enhanced sterol transfer selectively: PM-MICRO (12-fold); MITO-MITO, MICRO-MICRO, MICRO-PM (3-fold); PM-PM (1.4-fold); PM-MITO, MICRO-MITO (no effect). Thus, SCP-2-mediated sterol movement was vectorial and not necessarily down a membrane sterol concentration gradient. In contrast, liver fatty acid binding protein (L-FABP) revealed a modest (2-fold) stimulatory effect on sterol transfer only between PM-MITO and MICRO-MICRO. In conclusion, in vitro studies of sterol transfer among isolated subcellular membranes provided kinetic evidence for sterol domains in microsomes and mitochondria as well as plasma membranes. Furthermore, both spontaneous and protein-mediated sterol transfer appeared vectorial and selective in nature.

Animals↗
PubMed1994

Membrane ionic current photomodification by rose bengal and menadione: role of singlet oxygen.

Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.

Animals↗
PubMed1982

[Vertical displacements of the center of gravity in the cat during movement of limb flexion induced by motor cortex stimulation].

A limb movement and the corresponding postural adjustment results in a displacement of the body's center of gravity. The vertical component of this displacement has been calculated by measuring the variations of the sum of vertical forces at each limb. The displacements are upwards and are larger in the case of hindlimb flexion than forelimb flexion. Their amplitude is directly linked to the time course of the vertical force variations recorded at each limb. The feed-forward character of the postural adjustment appears to minimize this amplitude and this may allow us to discuss the functional significance of this postural adjustment.

Animals↗
PubMedDate not supplied

Combination of adenovirus-mediated thymidine kinase gene therapy with cytotoxic chemotherapy in bladder cancer in vitro.

We evaluated efficacy, toxicity and potential synergism of adenoviral-mediated thymidine kinase (tk)- ganciclovir (GCV) gene therapy in combination with 4 cytotoxic chemotherapeutic agents (doxorubicin, cisplatin, mitomycin C, and methotrexate) in 3 human bladder cancer cell lines. Cell lines were exposed to (1) 10 different concentrations of adenovirus expressing tk plus GCV; (2) 8 different concentrations of either doxorubicin, methotrexate, mitomycin C or cisplatin; or (3) combination treatment consisting of either low-, medium- or high-dose tk-GCV gene therapy plus 8 different concentrations of a single chemotherapeutic agent. Cell survival was determined using a MTT-based cell proliferation-assay. For most combinations, adding chemotherapy to tk-GCV gene therapy did not result in any therapeutic benefit. In some scenarios, we observed modest improvement with combinations of high-dose tk-GCV gene therapy and high-dose standard chemotherapy over tk-GCV monotherapy. Low concentrations of methotrexate enhanced the antitumor effects of low- and medium-dose tk-GCV gene therapy. Low level negative interference between tk-GCV gene therapy and chemotherapy occurred in some combinations but was overall negligible. In general, adding chemotherapy to tk-GCV gene therapy did not demonstrate significant therapeutic benefit in vitro. High doses of chemotherapeutic agents should be used in combination with tk-GCV gene therapy in order to take advantage of the occasional instance where modest improvement occurred with combination therapy. Additional studies exploring the role of methotrexate in enhancing the tk-GCV system are required. Investigation of other, potentially more synergistic chemotherapeutic agents in combination with tk-GCV is warranted.

Adenoviruses, Human↗
PubMedDate not supplied

Blood flow velocity and pulsatility index differences in patients with unilateral migraine.

OBJECTIVE: To evaluate blood flow velocity and pulsatility in unilateral migraine without aura during the headache-free period using transcranial Doppler (TCD) sonography. METHODS: Patients with unilateral headache were recruited during the headache-free period. Maximum mean flow velocity (MFV) and pulsatility index (PI) were measured in the middle cerebral (MCA) and basilar arteries. Controls were headache-free individuals without cerebrovascular disease. RESULTS: Twenty-five patients with right-sided migraine, 25 patients with left-sided migraine, and 19 controls were studied. The MCA PI was higher on the right headache side versus the left headache side (0.97 +/- 0.2 versus 0.86 +/- 0.1 cm/s, P =.02) and versus controls (0.9 +/- 0.2 cm/s, NS). The basilar artery MFV was higher in patients with right-sided headache versus left-sided headache (39.5 +/- 5.6 versus 34.7 +/- 8.2 cm/s, P =.02) and versus controls (38.2 +/- 8 cm/s, NS). No decrease in MFV with age was observed in patients with migraine. CONCLUSIONS: Middle cerebral artery flow pulsatility and basilar artery velocity are higher in patients with right-sided migraine compared with left-sided migraineurs, during the headache-free period. Although these parameters were similar to controls, the differences found during the headache-free period in migraineurs may indicate vascular involvement predisposing to the unilateral headache recurrence.

Adult↗