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Biomedical subjects

John Flanagan

Publications and source records attributed to John Flanagan.

12 recordsLinked to original sources
PubMed2006

Conjugation of sodium caseinate and gum arabic catalyzed by transglutaminase.

Conjugation of the milk protein sodium caseinate and a protein-containing polysaccharide, gum arabic, was achieved through the use of the cross-linking enzyme transglutaminase. The extent of conjugation was monitored by size exclusion separation coupled with a multiangle laser light scattering detector. The elution times of gum arabic solutions incubated with transglutaminase were unchanged over time, whereas incubation of sodium caseinate with transglutaminase resulted in shorter elution times as reaction time increased, indicating the formation of cross-linked caseinate polymers. However, when mixtures of caseinate and gum arabic were incubated with transglutaminase, the elution times were decreased markedly, indicating conjugation between the protein and polysaccharide. The molecular masses of the conjugates increased from approximately 950 to 1600 kDa. This method of protein-polysaccharide conjugation offers noticeable advantages over previously used methods, and the conjugates produced may exhibit unique functional properties.

Caseins↗
PubMed2006

Identification of depot-specific human fat cell progenitors through distinct expression profiles and developmental gene patterns.

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs.

Adipose Tissue↗
PubMed2006

Formation of stable nanoparticles via electrostatic complexation between sodium caseinate and gum arabic.

The formation of electrostatic complexes between sodium caseinate and gum arabic (GA) was studied as a function of pH (2.0-7.0), using slow acidification in situ with glucono-delta-lactone (GDL) or titration with HCl. The colloidal behavior of the complexes under specific conditions was investigated using absorbance measurements (at 515 or 810 nm) and dynamic light scattering (DLS). In contrast to the sudden increase in absorbance and subsequent precipitation of sodium caseinate solutions at pH < 5.4, the absorbance values of mixtures of sodium caseinate and GA increased to a level that was dependent on GA concentration at pH 5.4 (pH(c)). The absorbance values remained constant with further decreases in pH until a sudden increase in absorbance was observed (at pH(phi)). The pH(phi) was also dependent upon the GA concentration. Dynamic light scattering (DLS) data showed that the sizes of the particles formed by the complexation of sodium caseinate and GA between pH(c) and pH(phi) were between 100 and 150 nm and these nanoparticles were visualized using negative staining transmission electron microscopy (TEM). Below pH(phi), the nanoparticles associated to form larger particles, causing phase separation. zeta-Potential measurements of the nanoparticles and chemical analysis after phase separation showed that phase separation was a consequence of charge neutralization. The formation of complexes between sodium caseinate and GA was inhibited at high ionic strength (>50 mM NaCl). It is postulated that the structure of the nanoparticles comprises an aggregated caseinate core, protected from further aggregation by steric repulsion of one, or more, electrostatically attached GA molecules.

Caseins↗
PubMed2006

Microemulsions: a potential delivery system for bioactives in food.

Microemulsions are thermodynamically stable, transparent, low viscosity, and isotropic dispersions consisting of oil and water stabilized by an interfacial film of surfactant molecules, typically in conjunction with a cosurfactant. Microemulsions (so-called due to their small particle size; 5-100 nm) have found application in a wide variety of systems, such as pharmaceutical and oil recovery, but their application in food systems has been hindered by the types of surfactant permissible for use in food. The objective of this review is to provide an overview of the structures and phase behavior of microemulsions, methods of microemulsion formation, and techniques which may be used for characterization. A comprehensive review of previous work on both food-grade microemulsion systems, and non-food-grade systems of specific food interest is included. The application of microemulsions as reaction media, their ability to solubilize proteins and hence their use as a separation technique is also documented. In addition, attention is focused on the application of microemulsions as delivery systems for delivery of bioactive compounds, and the links between microemulsions and increased bioavailability. Future research, both applied and fundamental, should focus on surfactants which are not restricted for use in foods.

Chemical Phenomena↗
PubMed2006

Two new phosphoglycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain.

We report two previously undescribed mutations of the phosphoglycerate kinase gene (PGK1) leading to enzyme deficiency. In both cases, the patients were of Spanish origin and they exhibited a severe life-long chronic haemolytic anaemia associated with progressive neurological impairment. Sequence analysis of the first patient's entire PGK1 gene found a novel missense mutation (140T > A). This mutation caused an amino acid change of Ile to Asn at 46th position from the NH(2)-terminal serine residue (Ile46Asn), which has been called PGK-Barcelona based on the place of origin of the patient. In the second patient, a G to A transversion was discovered at nucleotide 958 (958G > A). This caused a Ser319Asn amino acid substitution. Since this mutation had not been previously described, the provisional name of PGK-Murcia was given to this deficient enzyme. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases, the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their consequence is related to a loss of enzyme stability rather than a decrease of enzyme catalytic function.

Anemia, Hemolytic↗
PubMed2005

PFG-NMR analysis of intercompartment exchange and inner droplet size distribution of W/O/W emulsions.

Presented is a novel application of pulsed field gradient (PFG)-NMR to the analysis of intercompartment exchange and the inner compartment droplet size distribution of a W/O/W multiple emulsion. The method involves monitoring the diffusional behavior of different components of the emulsion. Pfeuffer et al. [Pfeuffer, J.; Flogel, U.; Dreher, W.; Leibfritz, D. NMR Biomed. 1998, 11(1), 19-31.](1) and Price et al. [Price, W. S.; Barzykin, A. V.; Hayamizu, K.; Tachiya, M. Biophys. J. 1998, 74(5), 2259-2271.](2) proposed methods to extend Kärger's PFG-NMR model of exchange between two compartments to accommodate spherical inner compartments. Each model enables the prediction of the oil membrane permeability, the inner compartment volume fraction, and a representation of the inner compartment droplet size distribution. The models were fitted to PFG-NMR experimental data of W/O/W emulsions. The Pfeuffer et al. model provided the best description of the observed experimental data. Predicted values of permeability and swelling were consistent with those reported in the literature for W/O/W emulsions. The addition of sorbitol to either the inner or outer water compartment resulted in an increase in the oil membrane permeability. Inner compartment droplet size distribution measurements indicate that swelling, rupture, and coalescence are likely to have occurred during the secondary emulsification and emulsion ripening. In its present form, the method still constitutes a fast, noninvasive (no addition of a tracer), and in situ method for comparative analysis of the permeability, stability, and yield of different formulations of multiple emulsions with a single PFG-NMR experiment.

Journal Article↗
PubMed2004

Assessment of retinal hemodynamics with the Canon laser blood flowmeter after a single dose of 2% dorzolamide hydrochloride eyedrops.

BACKGROUND: Dorzolamide hydrochloride is a carbonic anhydrase inhibitor that reduces intraocular pressure (IOP) by decreasing the production of aqueous humour in the ciliary body. Theoretically, topical use of this agent has the potential to directly affect retinal vasculature through local induced acidosis. We performed a study to determine whether there are changes in retinal arteriole hemodynamics, as assessed with the Canon laser blood flowmeter, in healthy subjects following topical administration of dorzolamide. METHODS: We recruited 17 healthy volunteers, nine men and eight women aged 25 to 55 years (mean 31.4 [standard deviation (SD) 9.88] years). The inclusion criteria were Snellen visual acuity of 20/30 or better, normal anterior eye examination, IOP of 21 mm Hg or less, and a normal fundus appearance. One eye of each subject was randomly assigned to receive a drop of 2% dorzolamide. The contralateral eye of 10 of the subjects received a placebo drop (artificial tears). Before and 1 hour after drop administration, we obtained blood flow measurements from an inferotemporal arteriole approximately 1 disc diameter from the optic nerve head rim using the Canon laser blood flowmeter, model 100. The IOP was measured by means of Goldmann applanation tonometry before and 1 hour after drop administration. RESULTS: The mean IOP was significantly reduced in the dorzolamide-treated eyes, from 14.4 mm Hg (SD 2.94 mm Hg) to 11.7 mm Hg (SD 2.50 mm Hg) (p < 0.001). The IOP was also reduced in the placebo group (15.6 mm Hg [SD 3.41 mm Hg] vs. 14.6 mm Hg [SD 3.28 mm Hg]), but the difference was not significant. There was no significant difference in mean arteriole diameter, mean blood velocity or mean blood flow after drug administration in the dorzolamide-treated eyes. INTERPRETATION: Our results indicate that a single topical application of dorzolamide in healthy subjects has no effect on retinal arteriole diameter, blood velocity or blood flow, as measured with the Canon laser blood flowmeter. Longer-term studies of retinal hemodynamics in patients with glaucoma are warranted as evolving treatments aim to improve ocular blood flow as well as reduce IOP.

Administration, Topical↗
PubMed2004

Role of eIF5A in TNF-alpha-mediated apoptosis of lamina cribrosa cells.

PURPOSE: To determine the role of eukaryotic translation initiation factor 5A (eIF5A) in TNF-alpha-induced apoptosis of lamina cribrosa (LC) cells. METHODS: LC cells were isolated from optic nerve heads of eyes of two human donors. The cells were treated with TNF-alpha and camptothecin, a TNF synergist, and the incidence of apoptosis was scored by Hoechst staining. Expression of eIF5A protein in response to camptothecin or a combination of camptothecin and TNF-alpha was determined by Western blot analysis. The ability of small inhibitory (si)RNAs directed against eIF5A to protect LC cells from TNF-alpha-induced apoptosis was determined by Hoechst and TUNEL staining of transfected LC cells. RESULTS: TNF-alpha and camptothecin synergized to induce greater than two times more apoptosis in LC cells than when the cells were treated with TNF-alpha or camptothecin separately. Expression of eIF5A protein increased significantly after 8 hours of exposure to TNF-alpha and camptothecin, but not in response to camptothecin alone. siRNAs directed against eIF5A reduced apoptosis of LC cells in response to TNF-alpha and camptothecin by between 35% and 69%, as determined by Hoechst staining. An siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also reduced apoptosis of LC cells by 42%. TUNEL of transfected LC cells treated with TNF-alpha and camptothecin revealed an 80% reduction in apoptosis with siRNA against eIF5A. CONCLUSIONS: TNF-alpha, in synergy with camptothecin, induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF-alpha, and siRNAs against eIF5A protect LC cells from apoptosis. Thus, eIF5A appears to be a novel proapoptotic protein in the TNF pathway and a possible target for treatment of glaucoma.

Adolescent↗
PubMed2003

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o-phthaldialdehyde reagent.

The potential application of the o-phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH3, a by-product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH3 (at NH3 concentrations > 10 mM) was found to decrease during extended incubation at 23, 37 and 50 degrees C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH3 from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mm NH3 at 23 degrees C and pH 7.0 was found to be 88%. The release of NH3 and the decrease in epsilon-amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23 degrees C resulted in progressive increases and decreases, respectively, in NH3 and -amino group concentration with increased incubation time. These changes were dependent on TGase:NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase-catalysed cross-links. However, the observed decrease in epsilon-amino group concentration was higher than expected. This may be due to concealment of noncross-linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.

Ammonia↗
PubMed2002

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis.

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.

Bacillus↗