Utilization of meat industry by products: protein hydrolysate from sheep visceral mass

Protein hydrolysate was prepared from pre-treated sheep visceral mass (including stomach, large and small intestines) by enzymatic treatment at 43+/-1 degrees C (at the in situ pH 7.1+/-0.2 of the visceral mass) using fungal protease. The enzyme readily solubilized the proteins of the visceral mass as indicated by the degree of hydrolysis (34%) and nitrogen recovery (>64%). Hydrolysis with an enzyme level of 1% (w/w of total solids) at 43+/-1 degrees C with a pH around 7.0 for 45 min was found to be the optimum condition. The yield of protein hydrolysate was about 6% (w/w). The amino acid composition of the protein hydrolysate that was very hygroscopic, was comparable to that of casein.     

Phenotyping of human apolipoprotein E from whole blood plasma by immunoblotting

Human apolipoprotein E (apoE) exists in the population in three common genetically determined isoforms, apoE-2, E-3, and E-4, that are coded for by three alleles epsilon-2, epsilon-3 and epsilon-4 at the apoE structural gene locus resulting in six phenotypes, three homozygotes (E 2/2, E 3/3, and E 4/4) and three heterozygotes (E 2/3, E 2/4, and E 3/4). A new procedure is described that allows identification of apoE isoforms and phenotypes from whole plasma or serum without the need for isolating apoE-containing lipoproteins or two-dimensional gel electrophoresis of serum. This rapid method combines cysteamine treatment of apoE in plasma, separation in parallel of cysteamine-treated and untreated hydrophobic serum proteins by charge-shift electrophoresis, and isoelectric focusing of apolipoproteins with immunoblotting. Compared to phenotyping of apoE after isolation of VLDL, the new procedure agreed in most cases and may be of special value in detecting apoE mutants that differ in their cysteine residues or either are spun off during isolation of lipoproteins or cofocus with other apoproteins and thus escape detection by conventional one-dimensional techniques. The method provides a simple tool to screen apoE isoforms that are known to have a major impact on individual plasma cholesterol levels.     

In-vitro stimulation of TNF-alpha from human whole blood by cell-free supernatants of gram-positive bacteria.

Gram-positive bacteria are being recognized increasingly as the cause of shock-like syndromes, clinically indistinguishable from those seen in association with Gram-negative endotoxic shock. Much clinical and experimental data link tumour necrosis factor-alpha (TNF-alpha) with the pathogenesis of endotoxic shock, and a number of studies of individual Gram-positive species have also implicated TNF-alpha. We report here the first systematic study of the ability of cell-free supernatants of common Gram-positive bacteria to induce TNF-alpha from human peripheral blood monocytes in vitro. Almost all the 63 strains were able to induce TNF-alpha, although the levels were substantially lower than those obtained from supernatants of Gram-negative bacteria, used as controls. Streptococcus pneumoniae, S. pyogenes, viridans streptococci and coagulase-negative staphylococci were consistently more active than group B and D streptococci. TNF-alpha induction did not correlate with conventional markers of pathogenicity; amongst strains of Staphylococcus aureus, commensal and blood culture isolates did not induce significantly different amounts of TNF. We conclude that cell-free supernatants of most Gram-positive bacteria are capable of inducing TNF-alpha from human peripheral blood monocytes in vitro, but the significance of this finding remains to be determined.     

Determination of methanol in whole blood by capillary gas chromatography with direct on-column injection

A gas chromatographic procedure was developed for the determination of methanol in small-volume whole blood samples. Samples (100–200 μl) were prepared by protein precipitation, with direct injection of the supernatant on a wide-bore capillary column. The recovery of methanol and acetonitrile (the internal standard) was approximately 90% and did not vary with sample volume. The assay was linear from 2 μg/ml (the limit of detection) through 1000 μg/ml and was highly reproducible (intra-day coefficient of variation <2.5%). Assay performance was assessed following exposure of rats to methanol. The results indicate that the present procedure is suitable for studies of methanol disposition in small rodent species.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Assimilation of E. coli cells by Daphnia magna on the whole organism level

Consumption of E. coli cells by Daphnia magna was studied. It was found that this organism not only ingested E. coli cells but digested them as demonstrated by the release of ¹⁴CO₂ originating from E. coli grown on ¹⁴C-glucose, and by the transfer of the radioactive label from parental Daphnia to their progenies. In addition the effect of antibiotics on the consumption of E. coli cells by Daphnia magna was studied. In long incubation times, antibiotics inhibited bacterial uptake by Daphnia. The microflora isolated from Daphnia was found to be capable of causing leakage of enzymes out of E. coli cells thus playing at least a partial role in the digestion of E. coli cells by Daphnia.     

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.     

Aqueous two-phase fractionation of biological suspensions for protein recovery from bovine blood

The practical application of a two-stage aqueous two-phase systems (ATPS) for the fractionation and recovery of proteins from biological suspensions is described. A model process for the recovery of proteins from whole bovine blood was selected to study the implementation of an ATPS process. Recycling of used PEG into the initial extraction stage did not significantly influence the partition behaviour of serum albumin in subsequent cycles. © Rapid Science Ltd. 1998