Further Investigation on Naturally Occurring Tocopherols and Tocotrienols in Bovine Milk Fat

The polar lipid material which contains most of unsaponiriable matter of milk fat was collected by means of neutral alumina column chromatography. After saponification of the polar lipid material, the unsaponiriable matter was purified by repeated Florisil and neutral alumina column chromatography and the total tocopherol fraction was obtained. It was found that the total tocopherol fraction isolated from milk fat contained 6 of the known naturally occurring tocopherols, that is, α-, β-, γ-, and δ-tocopherols and α- and γ-tocotrienols. These were identified by two-dimensional thin-layer and gas-liquid chromatography before and after hydrogenation.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

N-glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: a comparison

N-glycans of the mouse glycoprotein HSA and its human analogue CD24 from lymphoblastoma, neuroblastoma and astrocytoma cell lines as well as from mouse brain homogenate were analysed and compared to each other and to the N-glycosylation pattern of total glycoproteins from mouse and human brain. The N-glycans were released from PVDF-blotted HSA or CD24 and separated on Carbograph SPE into neutral and acid glycans. The naturally neutral glycan fraction and the fraction of glycans rendered neutral after neuraminidase treatment were analysed without further purification by MALDI-MS. In each fraction, about 25 molecular ions with an intensity >10% of the base peak were identified which corresponded to glycans with distinct isobaric monosaccharide compositions. Comparison of the neutral and desialylated glycans revealed some similarities between the samples analysed, but also clear differences. HSA and CD24 from all cell lines express almost no neutral N-glycans with two or more fucose in contrast to brain HSA and glycoproteins from mouse and human brain. The lack of extensive fucosylation was also observed for desialylated glycans of HSA and CD24 from all cell lines analysed except for CD24 from a human neuroblastoma cell line which exhibits like total human and mouse brain glycoproteins a large variety of highly fucosylated, higher branched N-glycans. HSA from mouse brain carries in addition desialylated non-fucosylated glycans of high abundance which were detected, if at all, only at low intensity in all other samples analysed suggesting that they may be implicated in specific functions of mouse brain HSA. Therefore, a rapid assessment of similarities or differences between glycosylation patterns of a glycoprotein isolated from different sources is possible using methods as described here.     

Phase properties of senescing plant membranes. Role of the neutral lipids

Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes.     

Cytoplasmic origin of the so-called nuclear neutral histone protease.

1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.     

Comparison of DNA and protein polymorphisms between humans and chimpanzees

To examine the nucleotide diversity at silent (synonymous + intron + untranslated) and non-silent (nonsynonymous) sites in chimpanzees and humans, genes at six nuclear loci from two chimpanzees were sequenced. The average silent diversity was 0.19%, which was significantly higher than that in humans (0.05%). This observation suggests a significantly larger effective population size and a higher extent of neutral polymorphism in chimpanzees than in humans. On the other hand, the non-silent nucleotide diversity is similar in both species, resulting in a larger fraction of neutral mutations at non-silent sites in humans than in chimpanzees. Other types of polymorphism data were collected from the literature or databases to examine whether or not they are consistent with the nuclear DNA sequence polymorphism observed here. The nucleotide diversity at both silent and non-silent sites in mitochondrial (mt) DNA genes was compatible with that of the nuclear genes. Microsatellite loci showed a similar high extent of heterozygosity in both species, perhaps due to the combined effect of a high mutation rate and a recent population expansion in humans. At protein loci, humans are more heterozygous than chimpanzees, and the estimated fraction of neutral alleles in humans (0.84) is much larger than that in chimpanzees (0.26). These data show that the neutral fraction in non-silent changes is relatively large in the human population. This difference may be due to a relaxation of the functional constraint against proteins in the human lineage. To evaluate this possibility, it will be necessary to examine nucleotide sequences in relation to the physiological or biochemical properties of proteins.     

Ethanol extraction of free amino acids from soil

Summary A technique was developed for extracting and analyzing the free amino acid fraction of soil. Ethanol was used as an extracting agent. Ethanolextraction curves showed 20 per cent ethanol was the optimum percentage for extraction. Extraction-time curves indicated 18 to 20 hours of extraction with 20 per cent ethanol produced satisfactory results.The free amino acid fraction of soil was characterized and the limitations of the technique were determined. The naturally occurring amino acids extracted with 20 per cent ethanol were limited to acidic and neutral amino acids; basic amino acids were not extracted in sufficient quantities to permit detection. Based on the percent recovery of amino acids incorporated into soil and extracted with 20 per cent ethanol 90 to 95 per cent of the acidic, 80 to 85 per cent of the neutral and 1 to 5 per cent of the basic amino acids used were recovered with the technique.     

The lipid composition of flight muscle mitochondria isolated from the blowfly, Phormia regina.

The role of lipids in membrane structure and function was studied by measuring the major lipid classes in mitochondria isolated from flight muscle of the blowfly, Phormia regina. Approximately 98% of the total lipid is phospholipid. Neutral lipid constitutes the remaining 2% of the total. Phosphatidylethanolamine accounts for 55–60% of the phospholipid. A molecular ratio of 4:1:1 is found for phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (diphosphatidylglycerol). The neutral lipids include cholesterol, about 20%, and quinone, 40–45% of the total. The free fatty acid content of the neutral lipid fraction is variable, apparently being generated by endogenous phospholipase activity. The fatty acids of the neutral and phospholipid classes are predominantly 14–18 carbon acids; long-chain fatty acids of 20 and 22 carbons are essentially absent. The neutral lipid fraction contains 43% saturated and 51% monoenoic fatty acids. More than 65% of the phospholipid fatty acids are unsaturated. The principal fatty acids are palmitic, palmitoleic, oleic, linoleic, and linolenic. No trace of α- or β-tocopherol is detected. As vitamin E is considered an important naturally occuring antioxidant that prevents lipid peroxidation, the apparent absence of α- and β-tocopherol in these mitochondria coupled with intense oxidative activity of the mitochondria leads to the suggestion that blowfly flight muscle mitochondria may be particularly susceptible to peroxidative damage.     

Proteolysis in mitochondrial preparations and in lysosomal preparations derived from rat liver

A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker beta-glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.     

Detection of 1-nitropyrene in yakitori (grilled chicken)

Pieces of raw chicken with or without a marinating sauce were grilled over a city gas flame, extracted with benzene-ethanol (4:1) by ultrasonication and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor 1254-treated Sprague-Dawley rat liver (S9 mix). The basic fraction of yakitori without the sauce was more mutagenic than the other fractions for S. typhimurium strain TA98 in the presence of S9 mix. This is probably due to the presence of amino acid or protein pyrolysates. However, when the chicken was grilled with the sauce, the basic fraction showed lower mutagenicity for strain TA98 in the presence of S9 mix than did the same fraction without the sauce. The neutral fraction of yakitori with sauce showed high mutagenicity for strain TA98 in the absence of S9 mix, but low mutagenicity for strains TA98NR and TA98/1,8-DNP6, suggesting that this fraction might contain nitropyrenes (NPs). The neutral fraction of yakitori was analyzed by high-performance liquid chromatography (HPLC). The neutral fraction of the chicken grilled with the sauce for 3, 5 and 7 min contained 3.8, 19 and 43 ng, respectively, of 1-NP per gram of yakitori accounting for 3.0, 2.7 and 1.3%, respectively, of the total mutagenicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study on the suppressive effect of iodine-enriched egg on LT-C4 production in arachidonic acid-induced ear inflammation

The anti-inflammatory mechanism of iodine-enriched egg was investigated in mice by means of arachidonic acid-induced ear inflammation. The lipid fraction of iodine-enriched egg was capable of suppressing the increase in ear weight induced by arachidonic acid in a dose-dependent manner. The lipid fraction was further separated into neutral and polar lipid fractions. Of these two fractions, only the neutral lipid fraction was capable of suppressing LT-C₄ production in arachidonic acid inflammation. Neither the neutral nor polar lipid fractions of ordinary egg, however, showed any anti-inflammatory effect. These results suggest that the anti-inflammatory activity of iodine-enriched egg is present in the neutral lipid fraction, and its mechanism is assumed to be inhibition of LT-C₄ production.     

Possible association of Neu2 with plasma membrane fraction from mouse thymus exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU‐Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP‐40 or DOC/NP‐40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris‐buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane‐rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2‐transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.     

Biosynthesis of Triglyceride and Other Fatty Acyl Esters by Developing Rat Brain

Abstract: The biosynthesis of triglyceride from 1,2-diglyceride and long-chain acyl coenzyme A (CoA) was studied in developing rat brain. Diglyceride acyltransferase activity was highest in a microsomal fraction, had a neutral pH optimum, and was stimulated by MgCl₂. Palmitoyl CoA and oleoyl CoA served equally well as acyl donors. The enzyme catalyzed the acylation of both endogenous diglyceride and several naturally occurring and synthetic exogenous diglycerides. In addition, short-chain primary and secondary alcohols were found to be acylated under these conditions. A second acylation system, active at low pH, was found to catalyze esterification of ethanol and cholesterol, but not diglyceride, with free fatty acid. These results demonstrate that brain has the capacity to acylate a wide variety of physiological and nonphysiological hydroxyl compounds.     

Molecular characterization of total organic matter and carbohydrates in peat samples from a cypress swamp by pyrolysis-mass spectrometry and wet-chemical methods

The organic matter present in coarse- and fine-grained sample preparations of a peat core from Minnie's Lake, Okefenokee Swamp (Georgia, USA) was analysed on a molecular level by gas chromatography-mass spectrometry of alditol acetates obtained from polysaccharides after acid hydrolysis and derivatization and by flash pyrolysis-mass spectrometry. We found that the coarse-grained fraction is dominated by neutral monosaccharides derived from vascular plants whilst the fine-grained fraction is dominated by neutral monosaccharides derived from microorganisms. Relatively high contributions of xylose discriminate the Taxodium derived peat from the underlying peats derived from Carex and Nymphaea which show relatively low contributions of this sugar. Factor-discriminant analysis of the Py-MS data indicates that most carbohydrate degradation is restricted to the upper peat levels and that guaiacyl and syringyl type lignins are better preserved than carbohydrates. Resinous organic matter - presumably derived from Taxodium — is selectively preserved in the fine-grained fraction. Deviant monosaccharide concentrations and Py-MS results observed for the top sample of the Carex peat were explained by oxygenation of the peat, a concomitantly high microbial activity and subsequent preservation of the organic material due to the action of phytotoxins derived from Taxodium.     

The influence of experimental diabetes on the serum and urinary glycopeptide compounds in rats.

In alloxan-diabetic rats the protein level in the secomucoid and the levels of neutral sugars and peptides in the serum and urinary non-diffusible sugar-peptide (serum and urinary NSP) fractions increased. The proportion of neutral sugars in seromucoid and serum NSP fraction decreased, while in the urinary NSP fraction this ratio increased. In streptozotocin-diabetic rats the ratio of neutral sugars to the peptides did not change in either fraction, while the ratio of these sugars to the total nitrogen in the serum and urinary NSP fractions increased. The total level of seromucoid as well as the levels of neutral sugars in the serum and urinary NSP fraction were raised in these rats. The comparison of the results with those obtained previously by Sarnecka-Keller et al. (1968, 1972, 1972 a) in natural diabetes showed that the alloxan-diabetic model is more suitable for the study of glycopeptide compound metabolism in this disease.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

The interaction of a trypsin-dependent neutral protease and its inhibitor found in tumour cells. Analysis of complex kinetic data involved in a thiol-disulphide exchange mechanism

Ehrlich ascites tumour cells contain a granule-derived zymogen which on trypsin activation yields a collegenolytic neutral protease. The preparation of the granule fraction by subcellular fractionation procedure results in the preparation of a second fraction referred to as the post-granule supernatant fraction. The post-granule supernatant fraction contains a latent form of the granule-derived neutral protease and an excess of cytoplasmic inhibitor for this enzyme. The inhibitor of neutral protease is also capable of inhibiting trypsin and in each case the chemical mechanism of enzyme.inhibitor complex formation has been shown to be a reversible thiol-disulphide exchange. The post-granule supernatant fraction exhibited complex kinetic data when the interactions between the inhibitor, the latent enzymes and trypsin were examined simultaneously by incremental analysis. The data were interpreted and quantitatively analysed by computer analysis. It was demonstrated that the conventional types of analysis could not have provided meaningful interpretations of the experimental data provided by these complex-interacting systems.     

Decomposition process of organic matter derived from freshwater phytoplankton

We investigated the biodegradation process of freshwater phytoplanktonic organic matter using incubation experiments, with special reference to changes in three major biomolecules: neutral aldoses, amino acids and fatty acids. The concentration of neutral aldoses decreased drastically relative to amino acids and fatty acids during the early decomposition phase (days 0–7), owing largely to the rapid decomposition of storage carbohydrate. This resulted in a temporary decrease in the C/N ratio of organic matter. During the late phase (days 7–60), however, the rate of decrease in neutral aldoses slowed, while the considerable decrease in amino acids and fatty acids continued. The inconspicuous change in amino acid composition was probably due to the fact that no protein and/or peptide is composed of a limited species of amino acid. Although the compositional variety of organic matter among the phytoplankton was clearly observed at the start of decomposition, it became obscure in the course of 60 days. This indicates that while the organic composition of the labile fraction of phytoplanktonic organic matter varies depending on the phytoplankton groups, the refractory fraction has similar composition. The addition of bacterial organic matter is likely another reason for the similar composition of the remaining organic matter at day 60.     

Production of chemotactic factor for lymphocytes by neutral SH-dependent protease of rabbit PMN leukocytes from immunoglobulins,especially IgM

In inflammation, PMN leukocyte emigration is followed by lymphocyte emigration. Two neutral proteases were isolated from lysosomal fraction of rabbit PMN leukocytes and purified by chromatography. The SH-dependent protease converted in vitro a naturally occurring IgM and specific antibody IgM to a chemotactic factor for lymphocytes; its molecular size was assumed to be around 14,000. Lymphocytes were collected from the thoracic duct lymph of rats. The chemotactic generation was induced by a short treatment with small amount of the enzyme, but the chemotactic factor produced was inactivated by a prolonged digestion with the enzyme. The chemotactic generation by the enzyme of rabbit IgG was apparently less marked. On the other hand, the SH-independent protease was ineffective for such chemotactic generation, suggesting different enzymatic characteristics of these proteases.