Human atrial beta1L-adrenoceptor but not beta3-adrenoceptor activation increases force and Ca2+ current at physiological temperature

BACKGROUND AND PURPOSE It has been proposed that BRL37344, SR58611 and CGP12177 activate b3-adrenoceptors in human atrium to increase contractility and L-type Ca2+ current (ICa-L). b3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the b1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective b-adrenoceptor subtype antagonists to clarify cardiostimulant b-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on ICa-L. Contractile effects were determined on right atrial trabeculae. KEY RESULTS BRL37344 increased force which was antagonized by blockade of b1- and b2-adrenoceptors but not by blockade of b3-adrenoceptors with b3-adrenoceptor-selective L-748,337 (1 mM). The b3-adrenoceptor agonist SR58611 (1 nM–10 mM) did not affect atrial force. BRL37344 and SR58611 did not increase ICa-L at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and ICa-L at both 24°C and 37°C which was prevented by (-)-bupranolol (1–10 mM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS We conclude that the inotropic responses to BRL37344 are mediated through b1- and b2-adrenoceptors. The inotropic and ICa-L responses to (-)-CGP12177 are mediated through the low affinity site b1L-adrenoceptor of the b1-adrenoceptor. b3-adrenoceptor-mediated increases in ICa-L are restricted to low temperatures. Human atrial b3-adrenoceptors do not change contractility and ICa-L at physiological temperature.

Degradation of poly-L-lactide. Part 1: in vitro and in vivo physiological temperature degradation

Poly-L-Lactide is a bioresorbable polymer which degrades through hydrolysis of its ester linkage influenced by initial molecular weight and degree of crystallinity. Polymers belonging to the aliphatic polyester family currently represent the most attractive group of polymers that meet the medical and physical demands for safe clinical applications. Compression moulded PLLA pellets were produced as rods, sterilized and degraded both in vitro and in vivo (sub-dermal implantation model). The material molecular weight, crystallinity, mechanical strength and thermal properties were evaluated. In both in vitro and in vivo environments, degradation proceeded at the same rate and followed the general sequence of aliphatic polyester degradation, ruling out enzymes accelerating the degradation rate in vivo. By 44 weeks duration of implantation the PLLA rods were still biocompatible, before any mass loss was observed.

A Quantitative Analysis of Growth and Size Regulation in Manduca sexta: The Physiological Basis of Variation in Size and Age at Metamorphosis.

Body size and development time are important life history traits because they are often highly correlated with fitness. Although the developmental mechanisms that control growth have been well studied, the mechanisms that control how a species-characteristic body size is achieved remain poorly understood. In insects adult body size is determined by the number of larval molts, the size increment at each molt, and the mechanism that determines during which instar larval growth will stop. Adult insects do not grow, so the size at which a larva stops growing determines adult body size. Here we develop a quantitative understanding of the kinetics of growth throughout larval life of Manduca sexta, under different conditions of nutrition and temperature, and for genetic strains with different adult body sizes. We show that the generally accepted view that the size increment at each molt is constant (Dyar's Rule) is systematically violated: there is actually a progressive increase in the size increment from instar to instar that is independent of temperature. In addition, the mass-specific growth rate declines throughout the growth phase in a temperature-dependent manner. We show that growth within an instar follows a truncated Gompertz trajectory. The critical weight, which determines when in an instar a molt will occur, and the threshold size, which determines which instar is the last, are different in genetic strains with different adult body sizes. Under nutrient and temperature stress Manduca has a variable number of larval instars and we show that this is due to the fact that more molts at smaller increments are taken before threshold size is reached. We test whether the new insight into the kinetics of growth and size determination are sufficient to explain body size and development time through a mathematical model that incorporates our quantitative findings.

Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological temperature of 37°. However, recent research has shown that the use of low-temperature conditions (30-33°) as a platform for cell-culture results in changes in cell characteristics, such as increased specific productivity and extended periods of cell viability, that can potentially improve the production of recombinant proteins. Furthermore, many recent reports have focused on investigating low-temperature mammalian cell culture of Chinese hamster ovary (CHO) cells, one of the principal cell-lines used in industrial production of recombinant proteins. Exposure to low ambient temperatures exerts an external stress on all living cells, and elicits a cellular response. This cold-stress response has been observed in bacteria, plants and mammals, and is regulated at the gene level. The exact genes and molecular mechanisms involved in the cold-stress response in prokaryotes and plants have been well studied. There are also various reports that detail the modification of cold-stress genes to improve the characteristics of bacteria or plant cells at low temperatures. However, there is very limited information on mammalian cold-stress genes or the related pathways governing the mammalian cold-stress response. This project seeks to investigate and characterise cold-stress genes that are differentially expressed during low-temperature culture of CHO cells, and to relate them to the various changes in cell characteristics observed in low-temperature culture of CHO cells. The gene information can then be used to modify CHO cell-lines for improved performance in the production of recombinant proteins.

The effects of temperature and busulfan (Myleran) on the yellowtail tetra Astyanax altiparanae (Pisces, Characiformes) spermatogenesis

We aimed to standardize a protocol to suppress spermatogenesis in the characiform fish, Astyanax altiparanae, for future use as a host in germ cell transplant research, opening opportunities for a range of studies, such as spermatogenesis analyses and transgenesis because this species presents livestock characteristics to be used as a biological model. The effects of the chemotherapeutic busulfan (formulated as Myleran), which is used as medicine, therefore not as toxic to humans manipulation as analytical grade busulfan (Fluka) used in previous studies, were evaluated at physiological temperature of 28 °C, ideal for growth and reproduction of A altiparanae, and also at increased temperature 35 °C. The temperature groups were divided into three treatment groups: busulfan, DMSO only, and an untreated control. Macroscopic, histologic, stereological, and ultrastructure analysis showed that, at 28 °C, busulfan did not cause depletion of germ cells in A altiparanae. However, at 35 °C, sterilization was observed 3 weeks after the initial application. Similar results were obtained with maintenance of fish at 35 °C for a longer period with no accompanying Myleran treatment. This procedure allows reduction in stress and lower mortality resulting from manipulation during busulfan injection and is also suitable for mass treatment because large numbers of fish can be incubated in warm water.

Recovery of Yeast Saccharomyces cerevisiae after Thermal Insult

All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.

Structure, tensile properties and cytotoxicity assessment of sebacic acid based biodegradable polyesters with ricinoleic acid

A new family of ricinoleic acid based polyesters was synthesized using catalyst free melt-condensation polymerization with sebacic acid, citric acid, mannitol and ricinoleic acid as precursors. The use of FT-IR and NMR characterisation techniques confirms the presence of ester linkages in the as-synthesized polymers. Depending on the precursor combination, their relative amount and the degree of curing, a broad range of elastic modulus (22-327 MPa) and tensile strength (0.7-12.7 MPa) can be obtained in the newly synthesized biopolymers. The polymers show rubbery behaviour at a physiological temperature (37 degrees C) and the contact angles of the synthesized polymers fall in the range of 42 degrees to 71 degrees, making them ideal substrates to study delivery of drugs through polymer scaffolds. The cytocompatibility assessment of the cured polymers confirmed good cell attachment and growth of smooth muscle cells (C2C12 myoblast cells). Importantly, oriented cell growth was observed after culturing myoblast cells for 3 days. The in vitro degradation in PBS indicates that the mild cured polymers follow a first order reaction kinetics and have degradation rate constants in the range of 0.009-0.038 h(-1), depending on the relative proportions of monomers. Overall, the results of our study indicate that the physical properties can be tailored by varying the composition of the monomers and curing conditions in the newly developed polyesters. Hence, they may be used as potential substrates for tissue engineering scaffolds and for localized drug delivery.

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. D-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stern cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release.

Long-lived oscillatory incoherent electron dynamics in molecules: trans-polyacetylene oligomers

We identify an intriguing feature of the electron-vibrational dynamics of molecular systems via a computational examination of trans-polyacetylene oligomers. Here, via the vibronic interactions, the decay of an electron in the conduction band resonantly excites an electron in the valence band, and vice versa, leading to oscillatory exchange of electronic population between two distinct electronic states that lives for up to tens of picoseconds. The oscillatory structure is reminiscent of beating patterns between quantum states and is strongly suggestive of the presence of long-lived molecular electronic coherence. Significantly, however, a detailed analysis of the electronic coherence properties shows that the oscillatory structure arises from a purely incoherent process. These results were obtained by propagating the coupled dynamics of electronic and vibrational degrees of freedom in a mixed quantum-classical study of the Su-Schrieffer-Heeger Hamiltonian for polyacetylene. The incoherent process is shown to occur between degenerate electronic states with distinct electronic configurations that are indirectly coupled via a third auxiliary state by vibronic interactions. A discussion of how to construct electronic superposition states in molecules that are truly robust to decoherence is also presented

The antioxidative effect of icariin in human erythrocytes against free-radical-induced haemolysis

Icariin (2-(4'-methoxyl phenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3'-methyl-2-butyleny"chromanone) is the major component in Herba Epimedii used in traditional Chinese medicine for the treatment of atherosclerosis. This work focuses on the antioxidative effect of icariin on freeradical-induced haemolysis of human erythrocytes, in which the initial free radical derives from the decomposition of 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) at physiological temperature. To reveal the structure-activity relationship of icariin, the antioxidant effects of two structural analogues of icariin, acacetin (2-(4'-methoxylphenyl)-5,7-dihydroxylchromone) and norwogonin (2-phenyl-5,7,8-trihydroxylchromone), on the same experimental system were examined as well. It was found that all these chromone derivatives (Chm-OHs) dose-dependently protected human erythrocytes against free-radical-induced haemolysis. The order of antioxidative activity was nonvogoni-n > acacetin > icariin by the analysis of the relationship between the concentration of Chm-OHs and the prolongation percentage of the lag time of haemolysis (PP%). It was also proved that the phenyl hydroxyl group attached to the chromone ring at 7-position cannot trap the free radical- On the contrary, phenyl hydroxyl groups at the 5- and 8-position in nonvogonin made it a significant antioxidant in AAPH-induced haemolysis.

Reversible B/Z-DNA transition under the low salt condition and non-B-form polydApolydT selectivity by a cubane-like europium-L-aspartic acid complex

We report here that a cubane-like europium-L-aspartic acid complex at physiological pH can discriminate between DNA structures as judged by the comparison of thermal denaturation, binding stoichiometry, temperature-dependent fluorescence enhancement, and circular dichroism and gel electrophoresis studies. This complex can selectively stabilize non-B-form DNA polydApolydT but destabilize polydGdCpolydGdC and polydAdTpolydAdT. Further studies show that this complex can convert B-form polydGdCpolydGdC to Z-form under the low salt condition at physiological temperature 37 degrees C, and the transition is reversible, similar to RNA polymerase, which turns unwound DNA into Z-DNA and converts it back to B-DNA after transcription. The potential uses of a left-handed helix-selective probe in biology are obvious. Z-DNA is a transient structure and does not exist as a stable feature of the double helix. Therefore, probing this transient structure with a metal-amino acid complex under the low salt condition at physiological temperature would provide insights into their transitions in vivo and are of great interest.

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N-epsilon-(carboxymethyl)lysine; CML) wereanalyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N-epsilon-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.

Self-referencing thermometry in the nanoscale

Na ´ultima d´ecada emergiu uma linha de investiga¸c˜ao muito activa em term´ometros n˜ao invasivos e precisos que possam determinar temperatura `a escala nanom´etrica. Esta investiga¸c˜ao foi fortemente estimulada pelas numerosas solicita¸c˜oes da nanotecnologia e da biomedicina, por exemplo. Uma das abordagens mais promissoras prop˜oe o uso de i˜oes trivalentes de lantan´ıdeos que apresenta propriedades fotoluminescentes que dependem da temperatura. Neste trabalho demonstra-se que esta t´ecnica combina as vantagens de te um limite de detec¸c˜ao de 0.5 graus com sensibilidade at´e 4.5 % · K−1. Este term´ometro molecular pode ser processado em filmes finos ou nanopart´ıculas, abrindo os campos de aplica¸c˜ao a diferentes utiliza¸c˜oes. As nanopart´ıculas de s´ılica produzidas s˜ao caracterizadas na presen¸ca e na ausˆencia de i˜oes lantan´ıdeos. Sem o metal, as nanopart´ıculas de APTES/TEOS demonstram ser luminescentes sob excita¸c˜ao UV sem necessidade de utilizar qualquer tratamento t´ermico. O rendimento quˆantico de emiss˜ao depende apenas da propor¸c˜ao dos silanos e pode atingir o valor de 0.15 ± 0.02. A co-dopagem destas nanopart´ıculas com Eu3+ e Tb3+ permite obter sondas com resposta raciom´etrica, com a possibilidade de ajustar a gama de temperaturas de opera¸c˜ao e a sensibilidade, via desenho inteligente da matriz de suporte e dos ligandos de β-dicetona que est˜ao coordenados ao i˜ao met´alico. Quando processados como filmes, este term´ometro permite o mapeamento de temperaturas com resolu¸c˜ao espacial 1.8 μm. A racionaliza¸c˜ao da dependˆencia de temperatura ´e uma ferramenta ´util para desenvolver term´ometros que operam em gamas de temperatura espec´ıficos (e.g. gama de temperatura fisiol´ogica, 290-340 K) com sensibilidade acima de 0.5 % · K−1. A combina¸c˜ao de esfor¸cos de um grande n´umero de diversas disciplinas ir´a previsivelmente permitir o surgimento de term´ometros moleculares novos e sofisticados, preenchendo os principais requisitos das nanociencias.

Synthesis and functionalization of hydroxyapatite nanoparticles

Graças aos desenvolvimentos na área da síntese de nanomaterais e às potentes técnicas de caracterização à nanoescala conseguimos hoje visualizar uma nanopartícula (NP) como um dispositivo de elevado potencial terapêutico. A melhoria da sua efectividade terapêutica requer no entanto o aprofundamento e sistematização de conhecimentos, ainda muito incipientes, sobre toxicidade, selectividade, efeitos colaterais e sua dependência das próprias características físico-químicas da NP em análise. O presente trabalho, elegendo como alvo de estudo uma substância considerada biocompatível e não tóxica, a hidroxiapatite (Hap), pretende dar um contributo para esta área do conhecimento. Definiram-se como metas orientadoras deste trabalho (i) estudar a síntese de nanoparticulas de Hap (Hap NP), e a modificação das características físico-químicas e morfológicas das mesmas através da manipulação das condições de síntese; (ii) estudar a funcionalização das Hap NP com nanoestruturas de ouro e com ácido fólico, para lhes conferir capacidades acrescidas de imagiologia e terapêuticas, particularmente interessantes em aplicações como o tratamento do cancro (iii) estudar a resposta celular a materiais nanométricos, com propriedades físico-químicas diversificadas. No que se refere à síntese de Hap NP, comparam-se dois métodos de síntese química distintos, a precipitação química a temperatura fisiológica (WCS) e a síntese hidrotérmica (HS), em meios aditivados com ião citrato. A síntese WCS originou partículas de tamanho nanométrico, com uma morfologia de agulha, pouco cristalinas e elevada área superficial especifica. A síntese HS à temperatura de 180ºC permitiu obter partículas de dimensões também nanométricas mas com área específica inferior, com morfologia de bastonete prismático com secção recta hexagonal e elevada cristalinidade. Com o objectivo de aprofundar o papel de algumas variáveis experimentais na definição das características finais das partículas de hidroxiapatite, designadamente o papel do ião citrato (Cit), variou-se a razão molar [Cit/Ca] da solução reagente e o tempo de síntese. Demonstrou-se que o ião citrato e outras espécies químicas resultantes da sua decomposição nas condições térmicas (180ºC) de síntese tem um papel preponderante na velocidade de nucleação e de crescimento dessas mesmas partículas e por conseguinte nas características físico-químicas das mesmas. Elevadas razões [Cit/Ca] originam partículas de dimensão micrométrica cuja morfologia é discutida no contexto do crescimento com agregação. Com o objectivo de avaliar a citotoxicidade in vitro das nanopartículas sintetizadas procedeu-se à esterilização das mesmas. O método de esterilização escolhido foi a autoclavagem a 121º C. Avaliou-se o impacto do processo de esterilização nas características das partículas, verificando-se contrariamente às partículas WCS, que as partículas HS não sofrem alterações significativas de morfologia, o que se coaduna com as condições de síntese das mesmas, que são mais severas do que as de esterilização. As partículas WCS sofrem processos de dissolução e recristalização que se reflectem em alterações significativas de morfologia. Este estudo demonstrou que a etapa de esterilização de nanopartículas para aplicações biomédicas, por autoclavagem, pode alterar substancialmente as propriedades das mesmas, sendo pois criticamente importante caracterizar os materiais após esterilização. Os estudos citotoxicológicos para dois tipos de partículas esterilizadas (HSster e WCSster) revelaram que ambas apresentam baixa toxicidade e possuem potencial para a modelação do comportamento de células osteoblásticas. Tendo em vista a funcionalização da superfície das Hap NP para multifunções de diagnóstico e terapia exploraram-se condições experimentais que viabilizassem o acoplamento de nanopartículas de ouro à superfície das nanopartículas de Hidroxiapatite (Hap-AuNP). Tirando partido da presença de grupos carboxílicos adsorvidos na superfície das nanopartículas de Hap foi possível precipitar partículas nanométricas de ouro (1,5 a 2,5 nm) na superfície das mesmas adaptando o método descrito por Turkevich. No presente trabalho as nanopartículas de Hap funcionaram assim como um template redutor do ouro iónico de solução, propiciando localmente, na superfície das próprias nanopartículas de Hap, a sua redução a ouro metálico. A nucleação do ouro é assim contextualizada pelo papel redutor das espécies químicas adsorvidas, designadamente os grupos carboxílicos derivados de grupos citratos que presidiram à síntese das próprias nanopartículas de Hap. Estudou-se também a funcionalização das Hap NP com ácido fólico (FA), uma molécula biologicamente interessante por ser de fácil reconhecimento pelos receptores existentes em células cancerígenas. Os resultados confirmaram a ligação do ácido fólico à superfície das diferentes partículas produzidas HS e Hap-AuNPs. Graças às propriedades ópticas do ouro nanométrico (efeito plasmão) avaliadas por espectroscopia vis-UV e às potencialidades de hipertermia local por conversão fototérmica, as nanoestruturas Hap-AuNPs produzidas apresentam-se com elevado interesse enquanto nanodispositivos capazes de integrar funções de quimio e terapia térmica do cancro e imagiologia. O estudo da resposta celular aos diversos materiais sintetizados no presente trabalho foi alvo de análise na tentativa de se caracterizar a toxicidade dos mesmos bem como avaliar o seu desempenho em aplicações terapêuticas. Demonstrou-se que as Hap NP não afectam a proliferação das células para concentrações até 500 g/ml, observando-se um aumento na expressão genética da BMP-2 e da fosfatase alcalina. Verificou-se também que as Hap NP são susceptíveis de internalização por células osteoblásticas MG63, apresentando uma velocidade de dissolução intracelular relativamente reduzida. A resposta celular às Hap-AuNP confirmou a não citotoxicidade destas partículas e revelou que a presença do ouro na superfície das Hap NP aumenta a taxa proliferação celular, bem como a expressão de parâmetros osteogénicos. No seu conjunto os resultados sugerem que os vários tipos de partículas sintetizadas no presente estudo apresentam também comportamentos interessantes para aplicações em engenharia de tecido ósseo.

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N-epsilon-(carboxymethyl)lysine; CML) wereanalyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N-epsilon-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.