Moisture sorption, compressibility and caking of lactose polymorphs.

The aim of this study was to conduct storage studies on the moisture sorption and caking properties of lactose powder containing different polymorphs (i.e. alpha-monohydrate, alpha-anhydrous unstable, alpha-anhydrous stable, beta-anhydrous) and spray-dried lactose. The dry sample was compacted using a texture analyzer in paper cylinders and stored at relative humidity (RH) of 33%, 43%, 57% and 75% (25 degrees C, for 3 months). The samples were monitored for weight gain, moisture content, alpha/beta balance and hardness. A simple new method of powder compression for measuring the degree of hardness of caked lactose was developed using a texture analyzer. Clear distinctions were found in the storage behavior of the five different samples. Storage at various RHs caused severe caking to beta-lactose anhydrous and spray-dried lactose. The beta-lactose anhydrous was hygroscopic at 75% RH. The spray-dried lactose, which contained some amorphous lactose, was hygroscopic at all RHs studied. Its moisture sorption behavior differed from that of its major component, alpha-lactose monohydrate, by initially absorbing moisture then desorbing. alpha-Lactose monohydrate was less hygroscopic at 75% RH and it formed friable cakes. The alpha-lactose anhydrous stable was hygroscopic at 75% RH and initially formed hard cakes which became friable during storage. The unstable form of anhydrous alpha-lactose was hygroscopic at all levels of RH studied but did not cake.

Differential association of uracil DNA glycosylase with SIVSM Vpr and Vpx proteins.

The HIV-1 Vpr protein is a virion-associated protein which has been shown to facilitate infection of nondividing macrophages and additionally to alter cell cycle and proliferation status of the infected host cell. HIV-1 Vpr also was recently shown to associate with the DNA repair enzyme uracil DNA glycosylase (UDG). This association with a DNA repair enzyme is intriguing given that nonprimate lentiviruses encode a dUTPase, which, like UDG, minimizes the misincorporation of uracil into DNA and is important for virus replication in primary nondividing macrophages but not in dividing cells. This raises the possibility that the dependence upon Vpr for infection of nondividing macrophages may relate to its ability to interact with UDG. Members of the HIV-2/SIVSM group encode, in addition to Vpr, a related protein called Vpx. We previously demonstrated (Fletcher et al., 1996) that Vpx of HIV-2/SIVSM is necessary and sufficient for infection of primary macaque macrophages, while Vpr is not required for macrophage infection but governs cell cycle arrest. Here, we extend on these observations by demonstrating that Vpr, but not Vpx of HIV-2/SIVSM, associates with UDG, which suggests that Vpx facilitates infection of macrophages by a UDG-independent mechanism.

Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay for enzyme inhibition: detection of natural inhibitors of trypsin and chymotrypsin.

A technique for quickly detecting nanogram quantities of low- and high-molecular-weight inhibitors of some serine proteases is described. The inhibitor solutions are spotted onto agar films which contain either L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or tosyl lysine chloromethyl ketone (TLCK)-chymotrypsin. Enzyme inhibition is visualized as colorless zones on a pink background after the films were stained with the chromogenic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester. The method is used for rapidly testing both high-performance liquid chromatography fractions and thin-layer chromatograms to identify the inhibitors of trypsin and chymotrypsin in complex microbial extracts. The assay is quantitative so that it is possible to compare the specificity of the inhibitory fractions for trypsin and chymotrypsin. Results with standard inhibitors demonstrate the high sensitivity of the method, e.g., inhibition is detected with 1 ng of soybean trypsin inhibitor and 0.3 ng of antipain or chymostatin.

Formation of complexes between lecithin and apovitellenin I, an avian egg-yolk apoprotein.

In a study of lipid-protein interactions in egg yolk, it was found that L-alpha-dipalmitoyl lecithin gave two distinct noncovalent complexes (A and B) with apovitellenin I, an apoprotein in the major yolk lipoprotein. Interaction took place under widely varied conditions, and yolk lecithin gave similar complexes. Complex A, which was formed within minutes, consisted of round particles of about 9 nm diameter. Complex B, which was formed more slowly, consisted of larger particles, possibly resembling curved discs, with diameter of 30-40 nm. The preparation and some properties of these complexes are described. It is suggested that they may be suitable for an extensive study of phospholipid-protein interactions in yolk.

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A.

A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of Mr 18 000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 micrograms/kg with 0.10 micrograms corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60 degrees C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.

Lipoproteins from the blood and egg yolk of the hen. The transfer of very-low-density lipoprotein to egg yolk and possible changes to apoprotein B.

As part of a study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low-density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very-low-density lipoprotein (VLDL) of blood are the most likely precursors of YLP, their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B (ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk, ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented.

Effect of particle size and lipid composition of bovine blood high density lipoprotein on its function as a carrier of beta-carotene.

As part of a study on the influence of dietary lipids on vitamin transport and metabolism in lactating cows, we have examined the beta-carotene content and other properties of fractions of the high-density lipoprotein (HDL, density 1.05-1.16 g/ml) of bovine blood. Our purpose was primarily to explain previous results indicating that feeding cows polyunsaturated lipids alters the properties of the HDL and increases the concentration of beta-carotene in the blood but not in the milk. Fractions of HDL of different particle size were prepared by gel-filtration chromatography and the particle diameters measured by electron microscopy. We found that large HDL particles contain more beta-carotene per unit weight than small particles. Furthermore the HDL from cows fed lipid-rich diets with a high proportion of linoleic-acid residues, which had been protected against microbial degradation in the rumen, had a high percentage of HDL particles with large diameters. The blood from these cows had a higher concentration of beta-carotene than before feeding polyunsaturated lipids, but their milk had a lower concentration. We suggest that HDL is the main store of beta-carotene in bovine blood. Moreover the concentration of beta-carotene in blood is increased by feeding polyunsaturated lipids largely because of the increase in the percentage of large HDL particles, which contain more beta-carotene. The effect on the concentration of beta-carotene in milk implies that the transfer mechanism is less efficient as a result of feeding polyunsaturated lipids. This lower efficiency may be due in part to the higher percentage of large HDL particles.

NMR studies of the phosphoserine regions of bovine alpha s1- and beta-casein. Assignment of 31P resonances to specific phosphoserines and cation binding studied by measurement of enhancement of 1H relaxation rate.

(1) High-resolution 31P-NMR was used to study the environment of the phosphoserine residues of the phosphoproteins, alpha s1-casein B, beta-casein A2 and beta-casein C. For reference purposes 31P-NMR spectra of phosvitin and ovalbumin were also collected. (2) 31P resonances were assigned to specific phosphoserine residues as a result of comparisons of the high-resolution 31P-NMR spectra for alpha s1- and beta-caseins and for peptide fragments of these proteins obtained by cyanogen bromide and trypsin cleavage. (3) Measurements of the enhancement of the relaxation rate for water protons (1H) on addition of Mn2+ to alpha s1-casein B and to a fragment alpha s1-CN3, obtained by cyanogen bromide cleavage, gave approximate pK values for the binding groups and suggest the possibility of a conformational change induced by varying the concentration of divalent cation.

Hydrophobic chromatography of proteins in urea solutions. The separation of apoproteins from a lipoprotein of avian egg yolk.

A method is described for the chromatographic separation of mixtures of egg-yolk proteins of low solubility by using a hydrophobic column (phenyl-Sepharose) and eluting with increasing concentrations of aqueous urea at low pH. The resolving power of the method was established by tests on proteins and protein fragments of known sequence. The theoretical basis for the method remains, however, unclear. Factors such as the aggregation of the protein often appeared to be more important than its hydrophobicity in determining the urea concentration needed for elution. The method was applied to the mixture of apoproteins from the low-density lipoprotein (density about 0.95 g/ml) of avian egg yolk. For the previously studied apoproteins from egg yolk of the hen (Gallus domesticus), hydrophobic chromatography provided a new and convenient method for isolating the main apoproteins (hen apovitellenins I-VI). For the hitherto unexplored apoproteins from egg yolk of the duck (Anas platyrhynchos) the method has now been used to isolate three new proteins, two of which were not readily separated by methods based on molecular size. The elution pattern obtained with duck egg-yolk apoproteins is not the same as that of the hen egg-yolk apoproteins, although we suggest a relationship for the three new apoproteins based on their amino acid compositions and other properties. Possible roles for the apoproteins in avian egg yolk are described.

The binding of beta-casein to hydroxyapatite: the effect of phosphate content and location.

The elution behaviour of beta-casein from columns of hydroxyapatite has been studied and the effect examined of the enzymic addition of an extra phosphate residue. When the additional phosphate is located near pre-existing phosphate residues stronger binding to hydroxyapatite is observed. When the additional phosphate is remote from the pre-existing phosphates no increase in strength of binding is observed. It is suggested that the clustering of phosphate residues which characterizes alphas- and beta-caseins can be rationalized on the basis that it facilitates co-operative interactions between these phosphates and the Ca phosphate of the casein micelle and/or basic residues within casein polypeptide chains.

Multicentre general practitioner assessment of 'Tenormin' and methyldopa.

A large scale study in general practice was set up to investigate the effects of transferring hypertensive patients from treatment with usually less than 1 g daily of methyldopa to atenolol ('Tenormin') 100 mg daily. The results demonstrate an improvement in blood pressure control with atenolol treatment and a reduction in the incidence of side-effects. The simple dosage regime, combined with proven effectiveness and a relative lack of side-effects makes atenolol a useful treatment for the hypertensive patient.

A study of cow's milk containing high levels of linoleic acid: isolation and properties of the fat-globule membrane.

Properties of whole milk and milk fractions from cows fed a diet that gave a greatly increased proportion of unsaturated fatty acid residues (especially of linoleic acid) in the milk lipids were studied, and this milk (high-linoleic milk) was compared with milk from cows on a control diet (control milk). The milk fractions were isolated by high-speed centrifugation of whole milk or cream and were examined by chemical analysis and electron microscopy. During centrifugation the globules of milk fat were disrupted and the membranes (fat-globule 'ghosts') floated as a layer beneath the free lipid. Membrane proteins from the 2 sorts of milk gave the same electrophoretic pattern and the amino acid compositions were the same. Lipid analysis of the membrane fraction from high-linoleic milk showed the expected increase in the proportion of unsaturated fatty acid residues in the neutral lipids, but there was an unexpected decrease in the proportion of unsaturated residues in the membrane phospholipids. No differences were found between high-linoleic and control milk in the ultrastructure of the milk-fat globules or the isolated membranes.