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PubMed2003

Formation of vinylogous compounds in model Maillard reaction systems.

The thermal degradation over temperature and time of selected amino acids (Asp, Gln, and Glu) in the presence of reducing sugars was investigated in low moisture model systems. Copyrolysis of glucose-Asp mixtures led to the release of acrylic acid, attaining >5 mmol/mol Asp at 230 degrees C after 5 min. Spurious amounts of 3-butenamide were detected upon heating Gln together with a carbonyl source. Apparently, intramolecular cyclization is favored to procure 2-pyrrolidinone, reaching levels >3 mmol/mol above 230 degrees C. 2-Pyrrolidinone was also formed in comparable amounts in pyrolyzed sugar-Glu mixtures, indicating that the Maillard reaction may be an important contributor to the formation of 2-pyrrolidinone in certain cooked foods. The chemical route to acrylic acid and 3-butenamide is probably analogous to that described for acrylamide recently. Evidence is also presented that acrylic acid may be an intermediate in the formation of acrylamide, and yields could be augmented by coincubation of fructose-Asp with certain amino acids such as Gln, reaching approximately 5% of the yield obtained by the Asn route. A computational study to determine the reactivity of the vinylogous products indicated a reduced ability of 3-butenamide as compared to acrylamide to form stable intermediates by Michael nucleophilic addition. Acrylamide and acrylic acid exhibited a similar theoretical reactivity potential toward nucleophiles. No information is as yet available on the occurrence of acrylic acid in cooked foods. Extensive toxicological evaluation indicates that acrylic acid is of no concern at the amounts to be expected in foods.

Acrylamide↗

PubMed2006

Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity.

We have developed an acrylamide copolymerization strategy to immobilize acrylamide labeled peptides and proteins into a hydrogel surface and detect their modifications using MALDI-TOF mass spectrometry. Copolymerization into hydrogels is robust, compatible with "off-the-shelf" chemistry, and yields materials and surfaces that are stable to aqueous or organic solvents, drying, high or low temperature, high or low pH, oxidizing agents, sonication, mechanical contact, etc. The use of acrylamide hydrogels allows immobilization of substrates in a hydrated environment that can be used both as a biological reaction matrix and as a MALDI target. In our strategy, a substrate peptide was designed in a modular fashion to include both modification site and affinity domains. It was labeled with an acrylamide functionality using a generalized chemistry and covalently attached to the surface with a photocleavable linker, allowing for aggressive washing to remove any fouling, followed by selective release for MALDI-TOF analysis. Using this system we were able to analyze and compare v-Abl (truncated) and c-Abl (full-length) kinase activity on a peptide substrate with an affinity domain specific for the full-length kinase, observing excellent overall reproducibility in the extent of phosphorylation detected. This work serves as proof of principle for modular substrate design strategies for mass spectrometry-readable biosensors.

Acrylamides↗

PubMed2004

Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag). Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS [5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid]. Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2. Large changes in the microenvironment of the AEDANS [5-(2-aminoethylamino)-1-naphthalenesulphonic acid] probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values. This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins. As dictated by the bimolecular quenching constant (k(q)) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop. All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein. From these data, a low-resolution model of the toxin-eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin.

ADP Ribose Transferases↗

PubMedDate not supplied

Ultrastructural studies of the dying-back process. IV. Differential vulnerability of PNS and CNS fibers in experimental central-peripheral distal axonopathies.

A companion paper in this issue (46) described the evolution of peripheral nervous system dying-back disease of the giant axonal type in animals chronically intoxicated with the neurotoxic hexacarbons n-hexane (CH3CH2CH2CH2CH2CH3), methyl n-butyl ketone or MBK (CH3COCH2CH2CH2CH3), and 2,5-hexanedione (CH3COCH2CH2COCH3). The present study compares the distribution and pattern of peripheral (PNS) and central nervous system (CNS) dying-back disease produced by these three neurotoxic hexacarbons with that produced by acrylamide (CH2CHCONH2), and, in addition, employs these compounds to address unresolved issues in the dying-back process. In the PNS, large myelinated fibers in tibial nerve branches supplying calf muscles were especially sensitive in rats intoxicated with hexacarbons. These nerve branches and sensory plantar nerves in the hindfeet were equally vulnerable in acrylamide-treated rats. In both conditions, fibers located at these sites commenced degeneration before the distal regions of much longer and smaller diameter nerve fibers in nerve branches supplying the flexor digitorum brevis muscle and, in rats intoxicated with hexacarbons, before equivalent regions of plantar sensory branches to the digits. Pacinian corpuscles sited in the hindfeet of intoxicated cats were much less vulnerable to MBK than to acrylamide. Rats and cats intoxicated with hexacarbons displayed giant axonal swellings in vulnerable regions of the PNS degeneration in these animals was accompanied by pronounced endoneurial edema. In the CNS, rostral regions of long, ascending tracts (dorso-spino-cerebellar, gracile and, later, the cuneate) and the caudal end of long, descending tracts (lateral colums, ventrolateral and ventromedial tracts) of hexacarbon-treated animals were especially vulnerable. After prolonged intoxication of cats with MBK, giant axonal swelling was also found in preterminal and terminal axons in Rexed laminae V-VII at spinal levels C4 through S3-Neurofilament proliferation without giant axonal swelling was seen in CNS fibers of rats intoxicated with acrylamide. Taken in concert, the findings underline the importance of axon diameter and length in determining the hierarchy of fiber vulnerability and indicate the common sensitivity of selected regions of the PNS and CNS. The term central-peripheral distal axonopathy is introduced to emphasize the widespread, distal distribution of disease in these and in similar experimental conditions. It is suggested that certain human neuropathies (toxic, nutritional, uremic, diabetic and some hereditary polyneuropathies, and the neuropathy associated with multiple myeloma) are additional examples of central-peripheral distal axonopathies.

Acrylamides↗

PubMed2002

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies.

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.

Acrylamide↗

PubMed2005

Hydrolytic stability of self-etching adhesive systems.

PURPOSE: The aim of this investigation was to determine the hydrolytic stability of conventional methacrylates in comparison with a new acrylic ether phosphonic acid and a bis(acrylamide) cross-linking monomer under acidic aqueous conditions and to confirm the potential of these new monomers as components in a self-etching enamel-dentin primer, based on shear bond strength measurements. MATERIALS AND METHODS: Two new monomers were synthesized, an acrylic ether phosphonic acid and a bis(acrylamide). Characterization of the two monomers was carried out by 1H-NMR, and 13C-NMR spectroscopy and of the phosphonic acid by 31P-NMR spectroscopy. The hydrolytic stability of these monomers was evaluated by 1H-NMR spectroscopy or high performance liquid chromatography (HPLC), and was compared with the hydrolytic stability of conventional adhesive methacrylates, ie, MDP, 4-META, TEGDMA and GDMA. Shear bond strength to enamel and dentin was determined using the acrylic ether phosphonic acid and bis(acrylamide) formulated into what is now being marketed as a self-etching primer (AdheSE, Ivoclar Vivadent), and compared with the total-etch adhesives Prime & Bond NT (Dentsply) and Excite (Ivoclar Vivadent) and the self-etching systems One-Up Bond F (Tokuyama), Touch & Bond (Parkell), i-Bond (Heraeus Kulzer), Prompt-L-Pop (3M ESPE), and Clearfil SE Bond (Kuraray). Initial and 24-h bond strength values of AdheSE were compared with those after 16 weeks of storage at 42 degrees C. RESULTS: Stability studies have shown that conventional methacrylate monomers undergo rapid hydrolysis under acidic aqueous conditions (up to 90% degradation after 16 weeks at 42 degrees C). However, the newly formulated strongly acidic acrylic phosphonic acid and cross-linking bis(acrylamide) are stable under these conditions. Moreover, a self-etching enamel-dentin primer based on these new monomers showed high bond strength values. CONCLUSION: The shelf life of dental adhesives can be increased by using ether- or amide-linked polymerizable analogues instead of conventional, less stable ester derivatives. This should improve the performance of adhesive bonds made with these more hydrolytically stable comonomers.

Acid Etching, Dental↗

PubMedDate not supplied

A fibre-optic calcium ion sensor using a calcein derivative.

This paper describes the development of a new fibre-optic calcium ion sensor based on the immobilization of the calcein derivative, calcein acrylamide (CA) onto an optical fibre. The method involves the covalent attachment of CA in a polymer which itself is covalently bonded to an optical fibre. The fluorescence of CA is pH-sensitive in the region 4--12 and also increases as a function of Ca(2+) concentration at pH > 12. Typically, the indicator is sensitive to Ca(2+) in the concentration range 0--20 micromol/L, with a linear region occurring when the concentration of calcium is equal to or below that of CA. The binding ratio of Ca(2+) to CA in the linear calibration region is 1:1 and beyond the linear region is 2:1. The calcium-sensitive polymer is prepared using photo-initiated polymerization of a stock solution containing CA, monomer, a crosslinker and a photo-initiator. The monomer units used were acrylamide and acrylamide doped with MAPTAC. The fluorescence characteristics of bound CA in acrylamide are similar to that of CA in solution. However, the cationic nature of the polymer doped with MAPTAC allows analysis of Ca(2+) to be carried out in pH 8.5 solution. The Ca(2+) sensing range of the sensor is typically 0--12 micromol/L.

Calcium↗

PubMed1993

Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations.

The efficiency of detection of single base substitutions by single-stranded conformation polymorphism (SSCP) analysis was tested on 86 randomly distributed point mutations in a 193-bp-long DNA fragment of the mouse beta-globin gene. Multiple parameters were varied, including electrophoresis temperature, buffer concentration, gel concentration, acrylamide-to-bis-acrylamide ratio, and/or addition of different compounds to the gel matrix. Gels with a higher concentration of acrylamide and lower crosslinking gave optimal separation, and all 86 mutations can be clearly distinguished from the wild type on a 5% or 7.5% (2.6% C) acrylamide gel at 4 degrees C. Most of the mutations are also resolvable from wild type on gels with 5% urea or formamide, or 10% dimethylsulfoxide or sucrose. The relative position of the purine and pyrimidine-rich single strands were followed by an asymmetric PCR-SSCP technique. We found that most of the informativity comes from the purine rich strand, which appears to be much more sensitive to changes in the gel. The position or type of mutation showed no correlation with its ability to be detected. However, the neighboring base sequence around the mutation appears to have an effect on mobility. For example, A-->G substitutions in GC-rich regions significantly increase the mobility shift of the purine-rich strand, while most G-->A changes decrease it. We conclude that SSCP is a very efficient method for the detection of point mutations, if the parameters that effect the separation are optimized for a particular DNA fragment.

Animals↗

PubMed2007

Biodegradation of high-toughness double network hydrogels as potential materials for artificial cartilage.

This study evaluated biodegradation properties of four novel high-toughness double network (DN) hydrogels as potential materials for artificial cartilage. Concerning each DN gel material, a total of 12 specimens were prepared, and 6 of the 12 specimens were examined to determine the mechanical properties without any treatments. The remaining 6 specimens were implanted into the subcutaneous tissue, using 6 mature female rabbits. At 6 weeks after implantation, the mechanical properties and the water content of the implanted specimens were measured. In the poly(2-acrylamide-2-methyl-propane sulfonic acid)/poly(N,N'-dimethyl acrylamide) DN gel, the ultimate stress and the tangent modulus were significantly increased from 3.10 and 0.20 MPa, respectively, to 5.40 and 0.37 MPa, respectively, with a significant reduction of the water content after implantation (94 to 91%). In the poly(2-acrylamide-2-methyl-propane sulfonic acid)/polyacrylamide DN gel and the cellulose/poly(dimethyl acrylamide) DN gel, the stress (11.4 and 1.90 MPa, respectively) and the modulus (0.30 and 1.70 MPa, respectively) or the water content rarely changed after implantation (90 and 85%, respectively). In the bacterial cellulose/gelatin DN gel, the ultimate stress was dramatically reduced from 4.30 to 1.98 MPa with a significant increase of the water content after implantation (78 to 86%). This study implied that these DN gels except for the cellulose/gelatin DN gel are potential materials that may meet the requirements of artificial cartilage.

Absorbable Implants↗

PubMed2002

A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis.

Gel electrophoresis is often used for the primary analysis and purification of proteins, and peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Another important aspect in many proteomic experiments is the determination of the relative protein quantities (e.g. comparison between control and altered states). Usually, this is obtained by comparing the spot intensities of two independent gels. This procedure is time-consuming and not very accurate. Recently, several methodologies using isotope labeling of proteins for quantitative proteomic studies have been introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). However, none of these methodologies is foolproof and there is still the need for simple and inexpensive alternatives for determining the relative quantities of proteins. Previously, we showed that a mixture of acrylamide and deuterated acrylamide could be used as cysteine alkylating reagent prior to electrophoresis, improving the coverage and the confidence of the protein identification procedure (Sechi S, Chait BT. Anal. Chem. 1998; 70: 5150). Here we show that a similar approach can be used to obtain relative quantitation at the femtomole level of proteins isolated by gel electrophoresis. Deuterated acrylamide is used to alkylate the cysteines in one sample and regular acrylamide is used to alkylate the cysteines in the second sample. The two samples are then mixed together in a 1:1 ratio and the relative protein quantities are determined from the ion intensity ratios of the two cysteine-containing peptides isotopic envelopes (regular/deuterated). The analysis of several proteins mixed in different ratios is reported showing that this approach can reliably be used for protein identification and quantification. Briefly, a simple and inexpensive method for quantifying and simultaneously identifying proteins isolated by gel electrophoresis using MALDI-MS is presented.

Alkylating Agents↗

PubMed1994

Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.

Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis.

DNA↗

PubMed1986

Vitamin E concentrations in different regions of the spinal cord and sciatic nerve of the rat.

Since the spinal cord and peripheral nerves are vulnerable to the effects of vitamin E deficiency, vitamin E concentrations in various discrete regions of these parts of the nervous system of the rat were determined. Furthermore, as acrylamide toxicity and vitamin E deficiency share some neuropathological features, tissue vitamin E concentrations in acrylamide-treated rats were also studied. Male Sprague Dawley rats (200 to 250 g body weight) were fed normal rat chow with or without 0.03% acrylamide in their drinking water. After 24 days, the animals were sacrificed and the tissues assayed for vitamin E by a liquid chromatographic method. Vitamin E concentrations decreased from cerebral cortex to spinal cord with no concentration gradients between different levels of the spinal cord. Sciatic nerve concentration of alpha tocopherol was as high as that of cerebral cortex, and the former also contained measurable amounts of gamma tocopherol. Vitamin E concentrations in the majority of nervous tissues samples remained unchanged with acrylamide treatment.

Animals↗

PubMed1988

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution.

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.

Electrophoresis, Polyacrylamide Gel↗

PubMed1988

A nondenaturing vertical isoelectric focusing polyacrylamide slab gel system suitable for silver staining and electrophoretic blotting.

We have devised a nondenaturing vertical isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) system which is amenable to silver staining and electroblotting. Apart from being accessible, inexpensive, and simple to use, this new methodology overcomes problems inherent in current IEF methods, for example, pH gradient drift, nonuniform cooling, restricted sample volume, and inability to perform electroblotting. Two photopolymerization gel formulas were derived: a 5% acrylamide formula using bisacrylamide (Bis) as the crosslinker and a 6% acrylamide formula using diallyltartdiamide (DATD) as the crosslinker. The 5% acrylamide Bis gel gave excellent resolution and separation of proteins whereas the 6% acrylamide DATD gel expanded slightly during silver staining, resulting in mild band distortions. At least 80 ng of protein per band could be detected by the silver staining protocol devised. Both the DATD and the Bis gels were suitable for electroblot transfer. Parameters to ensure the optimum conditions for reproducible, high resolution vertical IEF-PAGE are described. IEF-PAGE silver staining and electroblotting procedures and silver staining of the nitrocellulose electroblot procedures are also described. The advantages of this methodology over previously published methods are discussed.

Animals↗

PubMed1976

Isolation and characterization of four peptide hydrolases from the brush border of rat intestinal mucosa.

Peptide hydrolases (EC 3.4.-.-) were solubilized from purified brush borders of rat intestinal mucosa by papain digestion. Three peptide hydrolases, I, II, and III, with different substrate specificities were isolated by means of DEAE-cellulose chromatography and preparative acrylamide gel electrophoresis. On repeat preparative acrylamide gel electrophoresis under slightly different conditions, enzyme II was resolved into two proteins, IIa and IIb, with vary similar, possibly identical, substrate specificities. Efforts to discover additional brush border peptide hydrolases revealed none. Studies using more than 50 substrates showed that enzyme I was most active against Met-Met, Met-Ala, and Met-Phe while enzyme II was most active against Phe-Gly, Phe-Ser, and Leu-Gly-Gly, and enzyme III most rapidly hydrolyzed Gly-Leu, Leu-Gly, and Met-Gly. Efforts to discover substrates which are highly discriminating for each enzyme were partly successful. Thus, a number of substrates including leucine amide, leucyl-beta-naphthylamide and Phe-Asp were hydrolyzed almost exclusively (95% or more) by enzyme II while Gly-Leu was similarly specific for enzyme III. No substrate highly discriminating for enzyme I was discovered. Ion-exchange chromatography resulted in increases in specific activity of 10- and 120-fold for enzymes II and III, respectively. By sequential use of ion-exchange chromatography and preparative acrylamide gel electrophoresis, each of the three enzymes was partially purified to the point that they were free of contaminating disaccharidases and enzymes I and II gave single dense bands on analytical acrylamide gel electrophoresis while enzyme III gave a single dense band plus one additional faint protein band. Under appropriate conditions, analytical gel electrophoresis also resolved enzyme II into two bands with enzyme activity. The three enzymes were isolated from intestinal brush borders of germ-free rats indicating that none of the enzymes is of bacterial origin. With Phe-Gly as substrate, pH optima for enzymes I, II, and III were 8.0, 8.0, and 8.5, respectively. Molecular weights determined by gel filtration were 283 000, 284 000, and 134 000, respectively. Studies of activation by metal ions and inhibition by metal ion chelators suggested that the activity of each of the enzymes is dependent on a relatively tightly bound metal cofactor. Peptide hydrolases of the intestinal mucosa play an essential role in protein digestion. The studies presented here help to clarify the total number and substrate specificities of these enzymes in the rat brush border.

Animals↗

PubMed1989

Selective degeneration of the parvocellular-projecting retinal ganglion cells in a New World monkey, Saimiri sciureus.

Selective degeneration of retinal ganglion cells projecting to parvocellular layers of the dorsal lateral geniculate nucleus (LGN) was observed in squirrel monkeys (Saimiri sciureus) exposed to a range of doses of acrylamide monomer. Similar acrylamide-induced neuronal loss has previously been reported in parvocellular-projecting ganglion cells of macaques, but no such selective degeneration has been found in acrylamide-dosed rats, squirrels, rabbits or cats. The extent of ganglion cell loss observed in the present study suggests that in the squirrel monkey, as in the macaque, a majority of ganglion cells project to parvocellular layers of the LGN. The locus of optic tract degeneration suggests that the squirrel monkey parvocellular pathway passes in dorsolateral optic tract, as does that of the macaque. Patterns of decreases in cytochrome oxidase activity confirm that, in both of these primates, geniculocortical pathways driven by these vulnerable neurons project to cortical layers 4A and 4C beta. These results suggest close parallels in the neuroanatomical projections and toxic vulnerability of the parvocellular-projecting pathway in New and Old World monkeys. They indicate that acrylamide intoxication can be used to selectively damage this pathway in order to study the functional roles of parallel visual pathways in both New and Old World monkeys.

Animals↗

PubMed1992

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.

Acrylic Resins↗

PubMed1991

Labeling of monoclonal antibody conjugates with 90Y.

An anti-carcinoembryonic antigen (CEA) antibody, NP-4, was labeled with 90Y using p-isothiocyanatobenzyl DTPA (SCN-Bz-DTPA) and its derivatives 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), 1-(2)-methyl-4- isothiocyanatobenzyl-DTPA (MX-DTPA) as the chelating agents. The 90Y conjugates were purified from unbound 90Y by two different methods, HPLC or acrylamide size exclusion gel chromatography, in order to evaluate the best purification method. Labeling efficiency, reaction kinetics and immunoreactivity were compared to the same antibodies labeled with [111In]citrate. Labeling efficiency, as determined by either HPLC or ITLC (instant thin layer chromatography), was consistently higher by ITLC than HPLC for 90Y-labeled MAb, but equal for 111In-labeled MAbs. Discrepancies between the 2 methods were linked to impurities in the 90Y that remained at the origin of ITLC plates. After purification by acrylamide gel filtration, recovery was 50-60% of loaded 90Y activity, but was more than 87% for the 111In compounds. Using HPLC, the recovery measured 85% for 90Y-labeled MAb and more than 93% for 111In-labeled conjugates. Immunoreactivity of the [90Y]MAb was comparable to the 111In-labeled conjugates. These studies indicate that HPLC purification of the [90Y]MAbs improves recovery of activity, and suggests that impurities found in the 90Y and metal-binding properties of acrylamide may have contributed to the poor recoveries from acrylamide gels.

Antibodies, Monoclonal↗