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Properties of smaller and larger cells from the 16-cell mouse embryo: increase in cell adhesiveness in the smaller cells cultured up to late 16-cell stage.

Blastomeres isolated from 16-cell mouse embryos consist of larger cells and smaller cells. In the intact embryo, the larger cells tend to differentiate to the trophectoderm, while the smaller cells give rise to the inner cell mass. The mode of phenotypic alteration of isolated blastomeres from early 16-cell embryos was examined by culturing them as single cells in vitro. The smaller blastomeres showed an increased tendency to be engulfed, as revealed by aggregation experiments during a 15 h culture period just prior to division into the 32-cell stage, while the larger cells remained showing high engulfing activity during this period. The present result demonstrates that the smaller blastomere continues to adopt a selected differentiation program for a certain period, even after its environment is changed.

Animals↗

Selection in guinea pigs: II. Direct response for litter size and body weight.

Four generations of selection for litter size, 13-wk weight and an index combining litter size and 13-wk body weight were conducted in a guinea pig population formed by intercrossing several local lines from different areas of Peru. A random selection control line was maintained also. Responses in the selected traits per generation were .069 +/- .069, 19.1 g +/- 16.2 and 2.5 +/- 20.3 for the litter size, 13-wk weight and index lines, respectively. Realized heritabilities were estimated by two methods. Estimates obtained from regression of response on cumulative selection differentials were .01 +/- .45 for litter size, .27 +2- .06 for 13-wk weight and -.09 +/- .13 for index values. Estimates from weighted multiple regression procedures were .07 +/- .01, .18 +/- .004 and .04 +/- .01 for litter size, 13-wk weight and index, respectively.

Animals↗

Controlling the phenotype and function of mesenchymal stem cells in vitro by adhesion to silane-modified clean glass surfaces.

The behaviour of human mesenchymal stem cells (hMSC) when cultured in contact with a range of silane-modified surfaces was examined to determine if changing the surface chemistry affected the early differentiation potential of mesenchymal stem cells in vitro over a 7-day period. Cells were cultured for 1 and 7 days in direct contact with glass which had been functionalized by surface treatment to provide a range of different surfaces: -CH(3), -NH(2), -SH, -OH, and -COOH modified surfaces and a clean glass reference (TAAB). Viable cell adhesion was quantified by Lactate Dehydrogenase assay, and morphology and viability was qualitatively evaluated using calcein AM, ethidium homodimer, cytoskeletal (F Actin), extra-cellular matrix (fibronectin and vitronectin) and Hoechst staining (nucleus). The expression of selected differentiation markers, Collagen II (chondrocytes), CBFA1 (bone transcription factor), Collagen I (MSC marker) and TGF-beta3 (extra-cellular matrix production) was determined using real time polymerase chain reaction. The expression of ornithine decarboxylase was evaluated as a marker of proliferation. Surfaces of the -NH(2) group demonstrated the greatest level of cell adhesion by the 7-day period, and mRNA expression profiles indicated osteogenic differentiation, increased CBFA1 and decreased Collagen II expression. Cells cultured in contact with the -COOH surfaces displayed different cell morphologies, fibronectin and vitronectin spatial distributions compared with the cells in contact with the -NH(2) surfaces, in addition to an increase in Collagen II expression, indicative of chondrogenic differentiation. The modifications to the surface chemistry of glass did affect cell behaviour, both in terms of viable cell adhesion, morphology and profiles of mRNA expression, providing the means to alter the differentiation potential of the MSCs.

Cell Adhesion↗

Selection for postweaning growth in inbred Hereford cattle: the Fort Keogh, Montana line 1 example.

Demographic characteristics and genetic trends in birth weight and pre- and postweaning ADG were examined in a population of Hereford cattle (Line 1). Line 1 was founded largely from two paternal half-sib sires and has been selected for postweaning growth. There were pedigree records on 951 members of the base population that predated 1935, when data collection began. Numbers of records analyzed using mixed-model methodology were 4,716 birth weight, 4,427 preweaning ADG, and 3,579 postweaning ADG. Birth weight and preweaning ADG were considered to have direct and maternal genetic components. Inbreeding accumulated rapidly from 1935 to 1960 and more slowly (.22%/yr) thereafter. Any reduction in additive genetic variance due to inbreeding and selection may have been offset by a concurrent reduction in generation interval that was observed as time progressed. Expected selection differential for 365-d weight, averaged over sexes, was 31.2 kg per generation. For birth weight, annual genetic trends in direct and maternal effects were 42 +/- 3 g and 15 +/- 3 g, respectively. Annual direct and maternal genetic trends for preweaning ADG were .70 +/- .06 g/d and .63 +/- .06 g/d, respectively. Direct response in postweaning ADG was linear and equal to 5.3 +/- .6 g.d-1.yr-1. As a result, estimated breeding values of birth weight, 200-d weight, and 365-d weight increased by 3.2 kg, 14.5 kg, and 62.4 kg, respectively, from 1935 to 1989. Selection within Line 1 was effective in increasing genetic potential for growth over 13 generations. No selection plateau was observed in any of the traits examined.

Age Factors↗

Correlated responses of carcass and reproductive traits to selection for rate of lean growth in swine.

Mass selection for an index of increased postweaning average daily gain and decreased backfat thickness was practiced for five generations. Litter size and weight for 221 gilt litters, birth weight and nipple number for 2,242 piglets and weaning weight at 42 d of age for 2,111 pigs were recorded. Carcass measurements were taken on 331 pigs. Differences between means of the lines (select control) were regressed on cumulative selection differential of the index. These regression coefficients were negative (P greater than .10) for total number born, number born alive, number weaned per litter, nipple number and carcass backfat thickness. Coefficients were positive (P greater than .10) for individual pig and litter weights at birth and weaning and for the carcass traits of length, longissimus muscle area and percentage of ham and loin. Absolute values of realized genetic correlations of index with traits evaluated were all .35 or less except the correlation with carcass backfat, which was -.84. None of these was significant; therefore, index selection for lean growth should have little effect on litter size and weight but may have a beneficial effect on carcass backfat.

Adipose Tissue↗

A genetic analysis of sow productivity traits.

Genetic and phenotypic parameters for sow productivity traits were derived from analyses of records for 999 purebred Duroc and Yorkshire litters at the Beltsville Agricultural Research Center. Included were 682 spring-farrowed litters and 317 fall-farrowed litters. Primary traits examined were number of pigs born alive in the litter (NA), litter size at 21 d of age (N21), litter weight at 21 d (W21) and an index (I21) combining the three traits. Also analyzed were total number of pigs farrowed alive or stillborn, litter weight of pigs born alive and litter size and litter weight at 42 d of age (weaning). Selection was based mainly on the sow productivity index among litters in the spring season and mainly on performance traits among fall-farrowed pigs. Select and control lines were maintained in both breeds in both seasons. Yorkshires started at higher levels of productivity than Durocs in spring 1975, showed more fluctuation and ended in spring 1982 at about their starting levels. Durocs started at lower productivity levels and recorded generally significant increases by 1982, with comparable selection differentials for the two breeds. Heritabilities, estimated from daughter-biparental (mid-dam, paternal granddam) regressions using records expressed as deviations from contemporary breed-line means, varied widely among the breed-line groups. Suggested composite heritability estimates were 5, 10 and 20% for NA, N21 and W21, respectively. Genetic correlations, computed from daughter-biparental covariances, were high and positive (.7 or above) among all traits. Based on estimated genetic gains, it was proposed that W21, alone or in combination with N21 and(or) NA in an index, be used as the primary selection criterion to bring about genetic improvement in sow productivity traits.

Animals↗

Characterization and multipotentiality of human fetal femur-derived cells: implications for skeletal tissue regeneration.

To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1-enriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor-gamma detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur-derived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.

Adipogenesis↗

Assessment of selective inhibition of rat cerebral cortical calcium-independent and calcium-dependent phosphodiesterases in crude extracts using deoxycyclic AMP and potassium ions.

The effects of various inhibitors on the activity of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex were examined. While the agents varied greatly in their relative potency, each was found to be approximately equipotent in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP. In contrast, the inhibitors displayed a marked substrate specificity for the calcium-independent enzyme with ratios of IC50 values for inhibition of cyclic GMP hydrolysis when compared to cyclic AMP hydrolysis in decreasing order being: ZK 62711 (much greater than 100) greater than Ro 20-1724 (much greater than 25) papaverine (13) greater than 7-benzyl IBMX (4) greater than quercetin and kaempferol (2). The differential selectivity of the inhibitors for the two enzymes was most pronounced for ZK 62711 and Ro 20-1724 which were at least 25-100-times more potent in inhibiting the calcium-independent hydrolysis of cyclic AMP when compared to the calcium-dependent hydrolysis of cyclic AMP. In contrast, 7-benzyl IBMX, kaempferol and quercetin were 8-100-times more effective as inhibitors of cyclic GMP hydrolysis by the calcium-dependent phosphodiesterase while 7-benzyl IBMX and trimazosin displayed a similar enzyme selectivity using cyclic AMP as substrate. With the exception of papaverine, all agents were competitive inhibitors of the calcium-dependent phosphodiesterase. The type of inhibition observed with the calcium-independent enzyme was dependent on the substrate employed. The specificity of potassium ions in inhibiting the activity of the calcium-dependent phosphodiesterase and deoxycyclic AMP in inhibiting the calcium-independent enzyme was found to provide a convenient means to assess the effects of agents on these activities in crude extracts of cerebral cortex.

Animals↗

Cumulative selection and genetic change for weaning or yearling weight or for yearling weight plus muscle score in Hereford cattle.

Selection in three lines of Hereford cattle for 1) weaning weight (WWL), 2) yearling weight (YWL), and 3) an index of yearling weight and muscle score (IXL) was studied. Remnant foundation cows and semen from seven foundation sires were used to establish an unselected control line for the last 11 yr of the experiment. Performance data collected over a 23-yr period on birth weight (BWT), weaning weight (WWT), postweaning gain (PWG), yearling weight (YWT), muscle score (MSC), and an index (IDX) giving equal weight to standard deviations of yearling weight and muscle score were analyzed. Generation interval of midparents was about 4.16 yr in each selected line. Sire and dam selection differentials, in standard deviation units per generation, for primary criteria were, respectively, 1.59 and .33 for WWT in WWL, 1.75 and .25 for YWT in YWL, and 1.42 and .25 for IDX in IXL. Components of direct and maternal genetic variances, direct-maternal covariance, and dam permanent environmental variance were estimated by REML. The average annual response of males and females in actual units for each trait in WWL, YWL, and IXL was, respectively, BWT, .22, .24, and .27 kg; WWT, .98, .63, and 1.26 kg; YWT, 2.43, 2.64, and 3.44 kg; and MSC, .053, .009, and .104 scores. Average selection responses in BWT, WWT, YWT, MSC, and IDX per unit of primary criteria in each selection line (all in standard deviation units) were .22, .20, .31, .10, and .24 for WWT in WWL; .23, .12, .32, .04, and .21 for YWT in YWL; and .27, .22, .40, .20, and .36 for IDX in IXL. Responses in bold type are realized heritability and others are correlated responses. Realized genetic correlations were .78 for WWT and YWT, .87 for WWT and IDX, and .86 for YWT and IDX. Responses for all traits in IXL were greater than in other selected lines.

Animals↗

T cell receptor diversity in alloreactive responses against HLA-B27 (B*2705) is limited by multiple-level restrictions in both alpha and beta chains.

The T cell receptors (TCR) in HLA-B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non-random usage was found in V beta, N+D beta, V alpha, and J alpha, but not in J beta segments or N alpha-regions. V beta segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA-B27 molecule. In contrast, preferential V alpha usage was associated with particular individuals and fine specificities, indicating distinct V beta and V alpha recruitment and contribution to allorecognition. Recurrent N+D beta motifs and J alpha segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical beta and unrelated alpha chains. Similar alpha were associated with unrelated beta chains, and vice versa. CTL using V beta subgroup 4 did not globally show concomitant predominance of other TCR elements. V alpha 7, one of the preferred V alpha segments, was always associated with V beta subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.

Antigen Presentation↗

Comparison of media for the isolation of Enterobacter sakazakii.

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.

Agar↗

A strategy to reduce healthy worker effect in a cross-sectional study of asthma and metalworking fluids.

This report describes the reanalysis of a cross-sectional study of asthma in a large cohort of autoworkers with exposure to metalworking fluids (MWF). There is strong evidence from case reports, clinical studies, and medical surveillance data that exposure to MWF can cause asthma, yet no association was found in the original analysis. The central hypothesis of the reanalysis was that the absence of an association between asthma and MWF exposure was the result of bias caused by the self-selection of asthmatics out of exposed jobs. We addressed the potential job transfer bias by redefining exposure and disease status at the time of asthma onset, rather than at the time of the health survey. This permitted us to treat the cross-sectional study as if it were a historical cohort study, despite the fact that the population was a biased sample of the full cohort. This approach resulted in a significantly elevated incidence rate ratio of 3.2 (95% CI: 1.2-8.3) for synthetic MWF estimated in a Cox proportional hazards model. Although the cross-sectional design makes it impossible to document or control for differential selection out of the workforce, the approach described here provides a strategy for reducing the healthy-worker effect due to job transfer bias in cross-sectional studies.

Adult↗

F statistics in Drosophila buzzatii: selection, population size and inbreeding.

Drosophila buzzatii is confined to reproducing in a well defined patchy environment consisting of rotting cactus cladodes which are ephemeral, permitting at most three generations. Flies emerging from such rots were used to estimate the additive genetic variance within rots and the genetic variance between rots for body size and also were electrophoresed to determine their genotypes at six polymorphic loci. F statistics were estimated from body size and allozyme data. The FST derived from body size was significantly larger than the allozyme FST. It is proposed this is due to selective differentiation of body size. The allozyme FST is used to estimate effective population size: 10 < N < 50. It is suggested that the regularly observed positive FIS's could be due to partial sib mating, S. If so, the estimated lower bound is S = 0.258. Experiments are identified which could support or contradict these interpretations.

Animals↗

The ligand-selective domains of corticotropin-releasing factor type 1 and type 2 receptor reside in different extracellular domains: generation of chimeric receptors with a novel ligand-selective profile.

The nonselective human corticotropin-releasing factor (hCRF) receptor 1 (hCRFR1) and the ligand-selective Xenopus CRFR1 (xCRFR1), xCRFR2, and hCRFR2alpha were compared. To understand the interactions of hCRF, ovine CRF (oCRF), rat urocortin (rUcn), and sauvagine, ligands with different affinities for type 1 and type 2 CRFRs, chimeric and mutant receptors of hCRFR1, xCRFR1, hCRFR2alpha, and xCRFR2 were constructed. In cyclic AMP stimulation and CRF-binding assays, it was established that different extracellular regions of CRFR1 and CRFR2 conferred their ligand selectivities. The ligand selectivity of xCRFR1 resided in five N-terminal amino acids, whereas the N-terminus of both CRFR2 proteins did not contribute to their ligand selectivities. Chimeric receptors in which the first extracellular domain of hCRFR1 replaced that of hCRFR2alpha or xCRFR2 showed a similar pharmacological profile to the two parental CRFR2 molecules. Chimeric receptors carrying the N-terminal domain of xCRFR1 linked to hCRFR2alpha or xCRFR2 displayed a novel pharmacological profile. hCRF, rUcn, and sauvagine were bound with high affinity, whereas oCRF was bound with low affinity. Furthermore, when three or five residues of xCRFR1 (Gln76, Gly81, Val83, His88, Leu89; or Gln76, Gly81, Val83) were introduced into receptor chimeras carrying the N-terminus of hCRFR1 linked to xCRFR2, the same novel pharmacology was observed. These data indicate a compensation mechanism of two differentially selecting regions located in different domains of both xCRFR1 and CRFR2.

Amphibian Proteins↗

Indirect partner choice through manipulation of male behaviour by female fowl, Gallus gallus domesticus.

The direct and indirect consequences of female copulatory behaviour for copulation success have seldom been quantified. In feral fowl, most copulations were forced by males and copulation success was determined by two factors. First, female differential resistance and solicitation directly affected copulation success and were displayed non-randomly with respect to male social status. Second, another female copulatory behaviour, the distress call, had an indirect effect on both copulation success and the quality of copulation partners. Distress calls triggered male attention to a copulation, which increased the probability of higher-ranking males than the copulating male disrupting the copulation and inseminating the calling female. Females preferentially uttered distress calls when mounted by low-ranking males. Both copulation resistance and distress calling influenced copulation success, but only distress calling increased the probability of copulation disruption by other males. Consistent with the effect of direct selection, differential distress calling indirectly biased copulation success in favour of dominant males. Female fowl may thus ameliorate the effect of male sexual coercion by manipulating male behaviour.

Animals↗

Correlated responses in reproductive and carcass traits to selection for 70-day weight in Landrace swine.

Correlated responses in reproductive and carcass traits were studied in 181 litters and 218 pigs from a line of Landrace pigs selected six generations for increased weight at 70 d of age and a contemporaneous, randomly selected control line. The reproductive and maternal traits studied included litter sizes born, born alive, and alive at 21 d and litter weight at birth and at 21 d. Carcass traits studied were carcass length, longissimus muscle area, average backfat thickness, 10th-rib backfat thickness, specific gravity, weights of closely trimmed ham, loin, and shoulder, belly weight, subjective scoring of the longissimus muscle for color and marbling, estimated percentage of muscle, and lean gain per day. Total weighted cumulative selection differential for 70-d weight was 30.2 kg. The realized heritability for 70-d weight was .13 +/- .06, and the change in 70-d weight was .65 +/- .29 kg per generation. The regression coefficient of litter size at 21 d on generation was .24 +/- .10 (P less than .10) pigs per generation. None of the other regression coefficients for the reproductive traits differed from zero. Carcass length, specific gravity, and ham weight decreased (P less than .10) -.075 +/- .036 cm, -.00054 +/- .00027, and -.102 +/- .048 kg, respectively, per generation. Color score and lean gain per day increased .046 +/- .021 points and .0032 +/- .0013 kg/d, respectively, each generation in response to the selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats.

There are numerous parallels between the heamatolymphopoietic and nervous systems in terms of the mechanisms regulating their development. We proposed that neural stem cells (NSCs) may respond to the microenvironmental signals provided by bone marrow stromal cells (BMSCs) which regulate the differentiation and maturation of hematolymphopoietic stem cells. First, we isolated and proliferated BMSCs from the femur and tibia, and NSCs from the midbrain of Sprague-Dawley (SD) rats, and then investigated the effects of BMSCs on the differentiation of NSCs into neurons, astrocytes and oligodendrocytes by directly plating neurospheres on BMSC monolayers in serum-free conditions. The results confirmed that BMSCs induced NSCs to differentiate selectively into neurons. The percentage of neurons significantly increased in 7 days in vitro co-cultures of NSCs and BMSCs as compared to NSCs cultures alone. When the duration of the cultures was extended to 12 days in vitro, BMSCs enhanced the survival of neurons derived from these NSCs; our investigation then focused on the underlying mechanism for this effect of BMSCs. NSCs were cultured with BMSC conditioned-medium and co-cultured with membrane fragments of live BMSCs or paraformaldehyde fixed BMSCs, the inducing activity of BMSCs was solely detectable in BMSC conditioned-medium, indicating that soluble factors secreted by BMSCs were responsible for its effect on the neuronal differentiation of NSCs. Therefore, BMSCs may provide a powerful tool for therapeutic neurological applications.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin A: preferential inhibition of glycation by nucleophiles at sites of low isomerization potential.

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A for in vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(beta), Val-1(alpha), Lys-66(beta), Lys-61(alpha), and Lys-16(alpha). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(alpha), Val-1(beta), Lys-66(beta), Lys-82(beta), Lys-61(alpha), and Val-1(alpha). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16(alpha) is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(alpha), Lys-82(beta), Lys-66(beta), Lys-61(alpha), Val-1(beta), and Val-1(alpha). The propensity of the microenvironment of Lys-16(alpha) to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1(alpha) and is about 50 times higher than that of Val-1(beta). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as the pH, concentration of aldotriose, and the concentration of the protein. The nucleophiles--such as tris, glycine ethyl ester, and amino guanidine--inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role of pKa of the site in dictating the glycation at that site.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗