Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Effect of Phosphate on the Corrosion of Carbon Steel and on the Composition of Corrosion Products in Two-Stage Continuous Cultures of Desulfovibrio desulfuricans

A field isolate of Desulfovibrio desulfuricans was grown in defined medium in a two-stage continuous culture apparatus with different concentrations of phosphate in the feed medium. The first state (V1) was operated as a conventional chemostat (D = 0.045 h⁻¹) that was limited in energy source (lactate) or phosphate. The second stage (V2) received effluent from V1 but no additional nutrients, and contained a healthy population of transiently starved or resting cells. An increase in the concentration of phosphate in the medium fed to V1 resulted in increased corrosion rates of carbon steel in both V1 and V2. Despite the more rapid corrosion observed in growing cultures relative to that in resting cultures, corrosion products that were isolated under strictly anaerobic conditions from the two culture modes had similar bulk compositions which varied with the phosphate content of the medium. Crystalline mackinawite (Fe₉S₈), vivianite [Fe₃(PO₄)₂ · 8H₂O], and goethite [FeO(OH)] were detected in amounts which varied with the culture conditions. Chemical analyses indicated that the S in the corrosion product was almost exclusively in the form of sulfides, while the P was present both as phosphate and as unidentified components, possibly reduced P species. Some differential localization of S and P was observed in intact corrosion products. Cells from lactate-limited, but not from phosphate-limited, cultures contained intracellular granules that were enriched in P and Fe. The results are discussed in terms of several proposed mechanisms of microbiologically influenced corrosion.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

D-propranolol and DL-propranolol both decrease conversion of L-thyroxine to L-triiodothyronine.

The effects of propranolol (DL-propranolol) and D-propranolol on thyroid hormone metabolism were studied in six euthyroid volunteers receiving L-thyroxine (T4) and six hypothyroid patients receiving T4 replacement. D-propranolol as well as propranolol decreased L-triiodothyronine (T3) concentrations and the ratio of T3 to T4 in the euthyroid subjects, and D-propranolol decreased these variables in the subjects with hypothyroidism (propranolol was not given to this group). It is concluded from this study and from parallel invitro investigations that the effect of propranolol on the conversion of T4 to T3 is unrelated to its beta-adrenergic blocking activity, and that at low therapeutic doses propranolol may exert appreciable "membrane-stabilising" effects in vivo.     

Miniature two-dimensional thin-layer chromatographic separation of lecithin and sphingomyelin

A miniature two-dimensional thin-layer chromatographic procedure employing silica gel impregnated glass-microfiber chromatography sheets (commercial product, ITLC-type SG sheets) has been developed for the separation of lecithin (L) and sphingomyelin (S) from a standard lipid mixture containing L, S, lysolecithin, phosphatidyl inositol (PI), phosphatidyl serine (PS), phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The newly developed procedure eliminates possible interference from PI and PS. Complete separation of L and S was easily achieved with chromatographic solvent migration times of approximately 3 and 2 min for the first and second dimensions, respectively. The lipids were visualized by charring and fluorescent staining techniques. The procedure has been adapted for the separation of L and S from amniotic fluid samples.