Bread Staling: Molecular Basis and Control.

The molecular basis of staling is examined by reviewing what is known about the components of wheat flour, factors that affect staling rate, and the various mechanisms that have been proposed. The conclusion reached is that bread staling is a complex phenomenon in which multiple mechanisms operate. Polymer crystallizations with the formation of supermolecular structures are certainly involved. The most plausible hypothesis is that retrogradation of amylopectin occurs, and because water molecules are incorporated into the crystallites, the distribution of water is shifted from gluten to starch/amylopectin, thereby changing the nature of the gluten network. The role of additives may be to change the nature of starch protein molecules, to function as plasticizers, and/or to retard the redistribution of water between components. Nothing more definite can be concluded at this time.

Nuclear (DNA, RNA, histone and non-histone protein) and nucleolar changes during growth and senescence of may apple leaves.

Quantitative interference microscopy was used to determine changes in nuclear and nucleolar indices (dry mass and cross-sectional area) in upper and lower epidermal cells and adjacent leaf-margin hair cells of the May apple (Podophyllum peltatum L.) leaves over a 42-day period (after leaves emerged above the ground litter). These indices decreased in a highly correlated manner. A ploidy variation may exist between epidermal cells and leaf-margin hair cells. Using the leaf-margin hair cells model, six nuclear macromolecule indices (total nucleic acid, DNA, RNA, total nuclear protein, histone and non-histone protein), nuclear volume, nucleolar volume and perinucleolar volume (measured using quantitative epifluorescence-phase contrast microscopy) all declined with age (42-day study) in a highly correlated manner. The degeneration of the nucleus and nucleolus in the three leaf locations studied followed the patterns observed for programmed cellular senescence and death (necrosis) in epidermal cells of onion leaf bases (stored tissue; leaf bases did not contain chlorophyll) and human epithelial cells (buccal; cervical). We conclude that the epidermal cells and leaf-margin hair cells from green leaves of the May Apple are ideal for the study of programmed cell senescence and death in plants, especially for the partitioning of this process into the study of: the point-of-no-return (solubilization of the karyoskeleton and loss of non-histone proteins and RNA associated with the karyoskeleton from the nucleus); nuclear pycnosis (loss of nuclear dry mass and volume and loss of nuclear internal support structure); chromatin condensation, margination along the inner nuclear envelope; and DNA-histone degeneration; degeneration of the nucleolus and loss of the perinucleolar zone of exclusion. The characterization of chlorenchyma cells during the 42-day period should now be undertaken (leaf senescence as indicated by the beginning of yellowing about 35 days after emergence) to determine whether these cells with functional chloroplasts undergo nuclear changes like those lacking functional chloroplasts.

A nitro sugar derivative route to 2-thioepisophorose and 2-thiosophorose and their remarkable facile epimerization.

The addition of 1-thio-D-glucose sodium salt to per-O-acetylated 1,2-dideoxy-1-nitro-D-arabino-hex-1-enitol, readily available from D-arabinose, afforded the corresponding 2-S-glycosylated 1-deoxy-1-nitro-D-mannitol and -D-glucitol peracetates. These, after deacetylation, were transformed by the Nef reaction to 2-thioepisophorose and 2-thiosophorose, respectively. The 2-thiodisaccharides easily epimerize in aqueous sodium bicarbonate at ambient temperature to a 1:4 equilibrium mixture. The predominant 2-thiosophorose was obtained crystalline. A 1H NMR study of the epimerization in deuterium oxide showed that the reaction involves an H-2 proton exchange mechanism.

Calcium ion-induced structural changes in bacteriophage phi X174.

Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods. Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion. Two sets of sites are associated with the F capsid protein. One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands. The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands. The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins. A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117. Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+. This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+. The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.

Suicide-substrate inactivation of beta-galactosidase by diazomethyl beta-D-galactopyranosyl ketone.

Diazomethyl beta-D-galactopyranosyl ketone (1) has been proven to be a mechanism-based, irreversible (suicide-substrate) inactivator of Aspergillus oryzae beta-D-galactosidase, but not an inactivator of E. coli lacZ beta-D-galactosidase. Compound 1 is stable in buffers of normal physiological pH. It is decomposed by H+, but not by nucleophiles. Inactivation of A. oryzae beta-D-galactopyranosyl ketone (2) nor diazomethyl alpha-D-galactopyranosyl ketone inactivated the enzyme and therefore inactivation is stereospecific, excess inhibitor could be separated from inactive enzyme without regain of activity and therefore it is bound irreversibly, and a second pulse of enzyme is inactivated at the same rate as enzyme inactivated to 95% activity by the first pulse. Diazomethyl beta-D-glucopyranosyl ketone (2) inhibited sweet almond beta-D-glucosidase.

A galactomannan from Crotalaria medicaginea seeds.

A polysaccharide isolated from the seeds of Crotalaria medicaginea is composed of D-galactose and D-mannose in the molar ratio of 10:31. Structural studies were performed by methylation analysis, partial acid hydrolysis, chromic oxide oxidation, mild hydrolysis with dilute oxalic acid and 13CNMR analysis of the polymer.

Inhibition of glycoprotein oligosaccharide processing in vitro and in influenza-virus-infected cells by alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene.

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.

The crystal structure of diazomethyl beta-D-galactopyranosyl ketone.

The title compound (C8H12N2O6) crystallizes in the orthorhombic space group P2(1)2(1)2(1) (Z = 4), with a = 4.871(1), b = 11.136(2), c = 18.301(2) A. The structure was solved by the multi-solution technique and refined by full-matrix least-squares to a final R-index of 0.042. The compound adopts the 4C1(D) conformation. Bond lengths in the diazoacetyl group are consistent with the presence of a zwitterion.

Structural analysis of papaya polysaccharide II.

The structure of papaya polysaccharide II (PP II) isolated by Chandrasekaran et al. [Carbohydr. Res., 60 (1978) 105-115] has been investigated by methylation analysis of the carboxyl-reduced polymer and by partial hydrolysis of both the intact (arabinose, 31.0; rhamnose, 13.3; galactose, 42.6; glucuronic acid, 10.3; and 4-O-methylglucuronic acid, 2.8%), and carboxyl-reduced polymers. Methylation analysis of carboxyl-reduced PP II indicated a very highly branched structure in which approximately 39% of the galactopyranose units are disubstituted, 24% are monosubstituted, 20% are trisubstituted, and 17% are nonreducing end units. Methylation analysis of products of partial hydrolysis of both intact and carboxyl-reduced polymers indicated that the backbone of the polysaccharide is made up of galactosyl residues substituted at either O-3 or -6, that the principal aldobiouronic acid fragment is 6-O-(glucopyranosyluronic acid)galactose, that the rhamnosyl units are substituted at O-3 with either terminal arabinofuranosyl or galactopyranosyl groups, and that the rhamnosyl residues are themselves linked to glucuronic acid residues through O-4. From this information, a possible statistical fragment with six arabinofuranose and two galactopyranose nonreducing end-groups per 19 sugar units [five units in the main chain of (1----3)-linked galactopyranose units] is proposed.

Effect of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene on hepatic degradation and processing of the N-linked oligosaccharide chains of alpha 1-acid glycoprotein.

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (alpha-ManMNT) on the degradation and biosynthesis of oligosaccharide chains on alpha 1-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver prevented the complete degradation of endocytosed N-acetyl[14C]glucosamine-labeled asialo-AGP and caused the accumulation of Man2GlcNAc1 fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally derived [14C]GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the alpha-ManMNT-treated hepatocytes remained sensitive to digestion with endoglycosidase H, were resistant to neuraminidase, and consisted of Man9-7GlcNAc2 structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since alpha-ManMNT is likely to irreversibly inactivate alpha-D-mannosidases, the return of normal AGP secretory forms after 24 h probably resulted from synthesis of new processing enzymes.

Changes in DNA and in nuclear and nucleolar dry mass and area in senescing parenchyma cells of corn cob and stalk tissues.

Corn (Zea mays L.) cob and stalk parenchyma tissues were used for a study of senescence using quantitative interference microscopy and quantitative Feulgen cytophotometry. In both parenchyma tissues, nuclear dry mass (NDM), nucleolar dry mass (NoDM), nuclear area (NA), and nucleolar area (NoA) increased during cell elongation and then decreased prior to cell death. These changes were more distinct in corn cob parenchyma tissue than in stalk parenchyma tissue. DNA content (determined for cob parenchyma, center section, only) decreased over the entire study period, slowly at first, them more rapidly. Because NoDM and NoA trends in cob parenchyma tissue were similar to those of NDM and NA but with earlier decreases, we conclude that nucleolar degeneration is an early event in corn parenchyma cell senescence. Loss of DNA also occurs earlier than loss of NDM in cob parenchyma tissue. It is further suggested that the increases in NDM as DNA content decreased represents nuclear protein and RNA content increases prior to senescence. However, no indication of nucleoli degeneration was found in stalk parenchyma cells even though NDM and NA decreased prior to cell death.

Aromatic and heterocyclic 1-C-substituted derivatives of 1,5-anhydro-D-glucitol.

Reaction of anomeric 1-O-acyl and 1-halide derivatives of 2,3,4,6-tetra-O-benzyl-D-glucose with anisole, ferrocene, thiophene, furan, and 1,3,5-trimethoxybenzene in the presence of a Lewis acid gives the corresponding C-beta-D-glucopyranosyl derivatives.

Determination of the nuclear RNA content of cells from donors of three different ages during in vitro senescence.

Diploid fibroblasts from male donors aged 8, 40 and 84 years were grown in vitro and their nuclear RNA content was determined at early and late doublings by absorption cytophotometry after staining with Azure B for 2 hours at 40 degree C and pH 4. It was found that the average nuclear RNA content of the early doublings was lower than that of the last doubling in all three strains. This difference became smaller as donor age increased. Also, as the age of the donor increased the percentage of early passage cells with a relative nuclear RNA content of less than 6 arbitrary units decreased, those with a relative nuclear RNA content of more than 6 units increased and the diversity in nuclear RNA content increased. The distribution pattern of relative RNA content for late passage cells was similar for the three donors.

Nuclear area changes in senescing human diploid fibroblasts.

The pattern of mean nuclear area changes was determined in human fibroblast cell strains from male donors of three different ages (8,40 and 84 years) during their in vitro lifespan. There was a statistically significant increase in mean nuclear area of cells of all three strains during their aging in vitro. A gradual increase of the subpopulation of cells with larger nuclei was also observed. Evidence for a positive correlation between aging in vitro, as reflected by mean nuclear areas at the tenth doubling from the end of in vitro doubling activity, and donor age is presented.

Isolation, partial characterization, and biological properties of polysaccharides from crude papain.

Two polysaccharides have been isolated from crude papain by precipitation of papain with ammonium sulfate, further precipitation of other proteins with trichloroacetic acid, and chromatography of the supernatant on DEAE-cellulose. The first polysaccharide to be eluted, designated PP-I, contained D-glucuronic acid, D-glucose, D-galactose, L-arabinose, and L-rhamnose, in the approximate molar ratios of 4:1:12:10:4. The other (PP-II), eluted at a higher salt-concentration, contained the same sugars (with about one-third less glucose and more uronic acid) in the approximate molar ratios of 13:1:40:26:12. Reduction of the uronic acid groups of PP-II produced a polysaccharide (PP-II-R) containing the same sugars in the approximate molar ratios of 2:11:37:28:12. Hydrolysis of a mixture of the two polysaccharides yielded an aldobiouronic acid, D-glucosyluronic acid-D-galactose. Neither polysaccharide preparation contained protein. These polysaccharides dramatically affected aggregation and alignment of normal human fibroblasts but had no effect on a mouse embryo fibroblast aneuploid cell-line that does not exhibit contact inhibition of growth or movement. In aggregating cells, these polysaccharides caused the cells to behave as contact-inhibited cells, that is, cell division and nuclear area were decreased.

Relationship of nuclease activity and synthesis to senescence of corn (Zea mays L.) stalk pith, cob parenchyma and first developed leaf tissues.

An increase in total RNase activity was associated with three patterns of cell senescence in corn (Zea mays L.) (cv. WF9 X 38-11) cob parenchyma during the first two weeks following silking, stalk pith tissue after internode elongation and the first developed leaf of seedlings. Stalk pith tissue had two RNase activities, one inhibited by EDTA and one not. Both remained in approximately equal amounts in young to old pith tissue. In the first developed leaf of seedlings, the activity not inhibited by EDTA remained at a constant low level during the period studied, while the other activity varied. No inhibition by EDTA was found in cob parenchyma tissue. Incubation of sections of cob parenchyma and stalk pith tissues suggested that the total RNase activity of cob parenchyma is very stable and that of stalk pith tissue is relatively stable. An age-related increase in DNase activity was found in stalk pith tissue and in the first developed leaf of seedlings, but not in cob parenchyma tissue.

Relationship of senescence of corn (Zea mays L.) stalk pith, cob parenchyma and first developed leaf tissues to RNA content and synthesis.

In stalk parenchyma tissue of WF9 X 38-11 single corn (Zea mays L.), there was a per cell increase in RNA synthesis and a slight increase in total RNA as cells became older. In cob parenchyma tissue of WF9 X 38-11 single cross corn, both total RNA content and RNA synthesis per cell decreased with the age of cells. In first developed leaf tissue of FR43 X FR14A single cross corn, RNA synthesis increased steadily but only slightly over its life span. The pattern of RNA synthesis and destruction in senescing leaf tissue of seedlings and two sources of parenchyma tissue of maturing plants appeared to differ.