Digestibility of proteins of the histological components of cooked and raw rice.

The aleurone layer, grain coat and embryo which constitute rice bran are rich in vitamins, lipids, protein and lysine compared with the endosperm (milled rice). A method was developed to measure the in vitro protein digestibility of raw and cooked brown rice and their histological components. The protein digestibility of cooked endosperm by the in vitro method agreed with that of other workers using in vivo techniques. The protein digestibility of the aleurone layer and grain coat from raw rice was only 25% but increased to 65% from cooked rice, due to disruption of the cellulosic cell walls at 100 degrees, which was shown by electron microscopy. The decreased protein digestibility due to cooking was not the result of formation of epsilon-lysyl-gamma-glutamyl isopeptide cross-links, but may be due to formation of a cystine-rich core that is resistant to proteases. The protein digestibility of cooked brown rice was approximately the same as that of cooked milled rice, hence it is advantageous for those for whom rice is a staple food to consume brown rather than milled rice.

A chromatographic method for the purification of K99 pili from enterotoxigenic Escherichia coli.

Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0.02 M-Tris/HCl, pH 8.6 containing 0.05 M-NaCl and DEAE-Sephadex A-50 equilibrated with 0.05 M-phosphate buffer, pH 7.2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl2 precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.

A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization.

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN.

Ultrastructure of pigment granules in the eyes of adults of the ommochrome-deficient mutant yellow of the Australian sheep blowfly Lucilia cuprina.

Compound eyes from adult flies of the ommochrome deficient mutant yellow of the Australian sheep blowfly, Lucilia cuprina, are processed for transmission electron microscopy using conventional methods. The ultrastructure of the pigment granules in the primary and secondary pigment cells is compared with that in corresponding cells of the wild-type strain and of yellow adults raised from larvae fed a diet supplemented with 3-hydroxykynurenine (3-HK). The pigment granules in both cell types of yellow are shown to be of the "empty" type in contrast to those in the wild-type strain. The pigment granules in eyes of yellow adults raised from larvae fed 3-HK are effectively normalized. The results are discussed in terms of previous electron microscope studies on other dipteran eye-colour mutants.

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope.

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA3). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA3-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA3 and probably destined for secretion.

Metal accumulation by bacteria with particular reference to dissimilatory sulphate-reducing bacteria.

Dissimilatory sulphate-reducing bacteria, genera Desulfovibrio and Desulfotomaculum, exhibit a superior ability, over assimilatory organisms, to extract three amounts of metals from culture media. This property does not appear to be solely a function of the presence of H2S. In media containing elevated amounts of Fe, electron dense particles, provisionally identified as FeS, are deposited within the cells of dissimilatory bacteria.

Gibberellin-induced separation of cells in isolated endosperm of celery seed.

The mature seed of celery (Apium graveolens, L.) contains a small axile linear embryo surrounded by endosperm which occpies the bulk of the seed. The endosperm is living and consists of mostly large angular thick-walled cells containing aleurone grains (often with globoids) and lipid droplets. - Using de-embryonated seeds, it has been shown that the endosperm was induced to break down by gibberellin. The aleurone grains became swollen and lost their contents and the bulk of each cell wall was hydrolyzed. However, a thin resistant layer of wall remained around each protoplast. The wall hydrolysis caused the endosperm to break down into individual cells which could be plasmolyzed and therefore appeared to be still living. All cells of the endosperm responded to gibberellin in a similar way although the cells near the radicle appeared to degrade more rapidly than those elsewhere. There was no change in the absence of the hormone. The response was apparently specific to gibberellin and did not occur in the presence of ethylene, kinetin, abscisic acid and indole acetic acid. The results were the same in light and in darkness. - It has been thought that endosperm breakdown during germination of seed such as celery involved release of hydrolases from the expanding embryo. The results of this study indicate that endosperm breakdown might be caused by hydrolases arising in the endosperm itself in response to gibberellin released from the embryo.

The regreening of nitrogen-deficient Chlorella fusca II. Structural changes during synchronous regreening.

Chlorella fusca, strain 211-15, cells degreened in a nitrogen-deficient mineral growth medium in the light for 4-6 weeks were regreened for up to 24 hrs in a nitrogen rich medium that leads to synchronous cell division at 24-26 hrs. Structural changes in the plastid membranes during the regreening period were observed by thin section and freeze-fracture electron microscopy. Nitrogen-deficient plastids were found to have non-appressed lamellae, prolamellar body-like membrane aggregations, and only 2 types of freeze-fracture face. At this time no photosynthetic oxygen evolution could be demonstrated. After 6 hrs regreening the plastid lamellae had fused to form bands of appressed lamellae and the four types of freeze-fracture face, described previously, were visible. At this time photosynthetic oxygen evolution could be demonstrated. After 24 hrs regreening the plastids had an appearance typical of normally grown Chlorella and had commenced to divide. Supporting evidence for these developmental stages is presented from isolated chloroplast particle fractions. An unusual type of cell wall proliferation was observed in the nitrogen-deficient Chlorella cells that resulted in the laying down of several walls, each with a trilaminar component.

The structure and composition of aleurone grains in the barley aleurone layer.

Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.