Biochemical Studies during the Development of the Chick Embryo

The presence and the change of deoxyribosidic compounds in the acid extract of the embryo with the incubation were examined with an aid of the organism, L. leichmannii. The main deoxyribosidic compounds in the extract prepared from the 18 th day embryo were identified as uracil, cytosine and thymine deoxyribosides and deoxyribotides of cytosine and thymine from the behaviour on paper chromatographic and paper electrophoretic separation. A small amount of purine deoxyribosyl compound which was assumed as hypoxanthine deoxyriboside was detected, and the content of which per 1 g of fresh embryo changed with the lapse of the incubated day; especially, the content was minimum at the period from the 10 th to 15 th day incubation. At this period, the total growth promoting compounds contained 50% of deoxyribotide though deoxyribosides was lower than that of the other days. This period is the most significant stage of the embryo growth and the most active time of synthesis of DNA through the embryo growth.     

Purification and Identification of Uracil and 5-Methylcytosine from Chick Embryo DNA

It was found that minor components were approximately 1% of the total deoxyribosides in the analysis for base composition of chick embryo DNA. In order to identify the minor components, this work was done. The separation of the minor components from the major components in a DNA hydrolysate was carried out by celite column chromatography and the final purification was accomplished by paper chromatography. For detection of minor components at the deoxyriboside level, the microbial assay method with Lactobacillus leichmannii ATCC 7830 was applied. Thus, two minor deoxyribosides of the DNA were identical with deoxyuridine and 5-methyldeoxycytidine in spectral, electrophoretic and chromatographic properties, respectively. Furthermore, uracil and 5-methylcytosine were purified and identified from perchloric acid hydrolysate of the DNA.     

Thymineless death inLactobacillus acidophilus R-26

Thymineless death (TLD) as well as deoxyribosideless death (DRLD) can be observed inLactobacillus acidophilus R-26 during growth in media lacking thymine or deoxyriboside respectively. Both phenomena exhibit the same interval of lage period (2–3 h) but the rate of inactivation is 2–3 times faster in TLD. Transfer experiments show that inactivation of bacterial reproduction is accelerated immediately if—DR medium is replaced by—T one. In the opposite case the deceleration of the inactivation rate does not appear immediately but after a 1–2 h lag period, in which no changes in the number of viable bacteria can be observed. Our results suggested that the accumulation of deoxyriboside compounds has no causal role in the inactivation of bacterial reproduction. However, the presence of deoxyribosides can accelerate the process of inactivation.     

Profiling the Lipophilic Fractions of Pithecellobium dulce Bark and Leaves Using GC/MS and Evaluation of Their Antioxidant,Antimicrobial and Cytotoxic Activities

Pithecellobium dulce has been used in traditional medicine to treat various ailments owing to its restorative properties. The biological activities and chemical profiles of the lipophilic fraction of P. dulce bark and leaves were assessed herein. Fatty acid methyl esters (FAME) and unsaponifiable matter (USM) were prepared and analyzed by GC/MS. A total of 40 compounds were identified in the bark saponifiable fraction, whereas 9 compounds were annotated in the leaves. Palmitic acid methyl ester was the major compound identified accounting for 41.48 % of the bark and 19.03 % of the leaves composition. Besides, linolenic acid methyl ester (22.40 %) and linoleic acid (12.69 %) were annotated in the leaves saponifiable fraction. A total of 63 compounds were detected in the bark USM and 4 compounds were identified in the leaves. Phytol represented the major component in the leaves (52.57 %) followed by lupeol (20.68 %) and lupenone (8.60 %). Meanwhile, n‐dodecane dominated in the bark USM accounting for 24.69 % of the total composition. The leaves and bark lipophilic fractions revealed moderate antioxidant and antibacterial activities. Both extracts showed no antifungal activity. No cytotoxicity was observed for both lipophilic fractions. P. dulce offers a good source of antioxidant compounds that can be introduced to food and pharmaceutical industry.     

Quantitative and qualitative variations in the glycoproteins in the chick embryo brain

Sugar content was examined in soluble and insoluble glycoproteins extracted from the chick embryo brain at different developmental stages. The content of hexosamines and uronic acids in the soluble fraction is higher during the whole period examined. The difference between the two fractions reaches a maximum at the 15th day. The insoluble fraction shows the highest content of sialic acid and fucose in comparison with the soluble one, especially toward hatching. The sialic acid/fucose ratio shows a different pattern in the two fractions examined, particularly in the soluble glycoproteins. The patterns of sialic acid and fucose indicate that quantitative and qualitative developmental changes occur in the soluble and insoluble glycoproteins. All sugars examined show significant changes on the 15th day, suggesting that this stage may represent a critical period in the development of the chick embryo brain.     

Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Investigation of the mechanisms affecting Cu and Fe bioavailability from legumes

Chemical composition and content in polyphenols, phytic acid, and dietary fiber of whole cooked common bean (Phaseolus vulgaris L.) and faba bean (Vicia faba L.) and of soluble and insoluble fractions separated from them were determined. Simultaneous determination of Cu, Fe, and protein bioavailability in the small intestine of rat was carried out in single-dose, short-term (1 h) experiments. After cooking, about 80% of seed components (on a weight basis) of either legume was recovered in the precipitate (insoluble fraction) after extraction with water. Protein, lipid, starch, dietary fiber, and polyphenols underwent the most severe insolubilization, together with more than 70% of total Cu and Fe. Cu, Fe, and protein bioavailability showed a similar trend (i.e., the lower the protein, the lower the Cu and Fe availability). Availability of proteins, Cu, and Fe in the insoluble fractions were the lowest, but Cu bioavailability was higher than that of Fe in all fractions. The results provide evidence that the heat-induced insolubilization process adversely affects not only protein but also Cu and Fe bioavailability from legumes and that polyphenols are likely to be a major inhibitor on absorption.     

Growth of Lactobacillus acidophilus in the absence of folic acid

Soska, Jirí (Kansas State University, Manhattan). Growth of Lactobacillus acidophilus in the absence of folic acid. J. Bacteriol. 91:1840-1847. 1966.-A chemically defined medium, containing no folic acid, was used for the cultivation of Lactobacillus acidophilus R-26. In such a medium, the organism required thymine in addition to a deoxyriboside, purines, pyrimidines, and most amino acids. If thymine was present in this medium, an unlimited exponential growth was possible. The influence of the components of this medium on the growth is described. The concentration and type of adenine compounds in this medium were most important. Adenine and adenosine inhibited utilization of thymine, but not of thymidine, whereas adenylic acid inhibited recovery from amino acid starvation. In the absence of thymine or deoxyribosides, cells continued to grow in length, and after 3 hr a slow decline in viable count ensued.     

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Dynamic response of UV-absorbing compounds, quantum yield and the xanthophyll cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort

We studied the diel responses of the liverwort Jungermannia exsertifolia subsp. cordifolia to radiation changes under laboratory conditions. The samples were exposed to three radiation regimes: P (only PAR), PA (PAR + UV-A), and PAB (PAR + UV-A + UV-B). The day was divided in four periods: darkness, a first low-PAR period, the high-PAR plus UV period, and a second low-PAR period. After 15 days of culture, we measured photosynthetic pigments, chlorophyll fluorescence and UV-absorbing compounds in the four periods of the day on two consecutive days. With respect to UV-absorbing compounds, we analyzed their global amount (as the bulk UV absorbance of methanolic extracts) and the concentration of seven hydroxycinnamic acid derivatives, both in the soluble (mainly vacuolar) and insoluble (cell wall-bound) fractions of the plant extracts. PAB samples increased the bulk UV absorbance of the soluble and insoluble fractions, and the concentrations of p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall. Most of these variables showed significant diel changes and responded within a few hours to radiation changes (more strongly to UV-B), increasing at the end of the period of high-PAR plus UV. F_v/F_m, Φ_PSII, NPQ and the components of the xanthophyll cycle showed significant and quick diel changes in response to high PAR, UV-A and UV-B radiation, indicating dynamic photoinhibition and protection of PSII from excess radiation through the xanthophyll cycle. Thus, the liverwort showed a dynamic protection and acclimation capacity to the irradiance level and spectral characteristics of the radiation received.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Characterization of Wolfgram proteolipid protein of bovine white matter and fractionation of molecular weight heterogeneity

Abstract— Wolfgram proteolipid protein fraction (WPF) was prepared as the insoluble pellet resulting from the extraction of myelin three times with chloroform-methanol (CM, 2:1, v/v). Amino acid composition analysis showed that the WPF described here is comparable to that described by Wolfgram (1966) and by Eng et al. (1968). Disc gel electrophoresis in two different buffer systems revealed three major protein bands, W1, W2 and W3, having apparent mol. wt of 23,500, 54,000 and 62.000 daltons respectively. The 54,000–62,000 doublet is stable to performic acid oxidation and to reduction with β-mercaptoethanol. Characterization of WPF by sedimentation velocity revealed two peaks having S_{20, w} values of 1 96 and 0 84. In comparison, water soluble Folch-Lees proteolipid apoprotein (APL) prepared in this laboratory (Hendrickson et al., 1972) differs from WPF in its amino acid composition and in its behavior on disc gels and in the ultracentrifuge. We employed preparative electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate (SDS) in order to separate and purify heterogeneous components observed in WPF. We obtained a fraction containing essentially pure W1 protein and determined that it has a unique amino acid composition. Various fractions containing partly purified high molecular weight components were also recovered. Gel filtration chromatography on columns of Sephadex G-200 was also successfully employed in this study of WPF.     

The invariance of macromolecular composition with altered light limited growth rate of Amphidinium carteri (dinophyceae)

The effect of irradiance on the growth rate, macromolecular composition and photosynthetic carbon metabolism of Amphidinium carteri was studied in batch culture. Growth rate increased linearly with increasing irradiance up to a maximum growth rate of 0.04 h^-1 at an irradiance of 80 Em^-2s^-1. In contrast to a number of other studies on both prokaryotic and eukaryotic microorganisms, ours showed that cellular content of RNA, DNA, protein and carbohydrate of A. carteri were invariant with growth rate over the range =0.04 to 0.007 h^-1. The invariant macromolecular composition was correlated with a constant modal cell volume. Chlorophyll and lipid per cell increased with decreasing irradiance. The distribution of [¹⁴C]-bicarbonate in the major end products of photosynthesis after incubation with isotope for 14% of a doubling time showed that the percentage carbon in the chloroform (lipids and pigments) fraction increased with decreasing irradiance while that of the trichloroacetic acid soluble (carbohydrate) fractions decreased. The percentage of isotope in the trichloroacetic acid insoluble (protein) fraction and methanol: water fraction (metabolites) remained constant. Because this species, under light-limited growth, differs from other organisms so far studied, more species must be similarly examined before nucleic acid content is used as an index growth rate in the field.This paper is presented with our best wishes on the occasion of Professor G. Drews 60th birthday     

Seed dispersal by ants: behaviour-releasing compounds in elaiosomes

Summary In a study of the biochemical basis of seed dispersal by ants, elaiosomes of Acacia myrtifolia and Tetratheca stenocarpa induced seed collection: intact diaspores and elaiosomes were taken rapidly by ants while most seeds remained on the forest floor. Extracts of elaiosomes (non-polar lipids, polar lipids, and aqueous fractions) were differentially collected by ants. Small pieces of pith impregnated with the polar lipid fraction from elaiosomes of either species elicited a removal rate by ants equivalent to that of intact elaiosomes and significantly higher than that of untreated pith. The non-polar lipid fraction, highest in concentration in elaiosomes of both species, elicited removal that did not differ from that of untreated pith. In T. stenocarpa, however, the aqueous fraction also induced removal equivalent to the polar lipid fraction. 1,2-Diglycerides with unsaturated groups are present in the active polar fractions of both species and unsaturated oleate is the major acid group of the glycerides in elaiosomes. Most oleate-containing compounds tested were taken more rapidly by ants than saturated compounds, and oleic acid, associated with corpse-carrying in ants, induced rapid removal. 1,2-Diolein, but not 1,3-diolein, was taken suggesting that the specific configuration of fatty acid moieties influences collection by ants. We hypothesize that a small suite of oleyl-containing compounds in elaiosomes elicit a stereotyped carrying response by a variety of ants. While the nutrient composition of elaiosomes may provide the underlying selective advantage for ants in seed dispersal, specific compounds may manipulate their behaviour and maximize seed dispersal.     

The effect of the preservative sorbic acid on the lipid composition of the ascomycete fungus <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> Thom

The mechanism of action of potassium sorbate, a widely used food preservative on the lipid composition of the Ascomycete fungus Penicillium roqueforti, the main contaminant of cheese, was investigated. The inhibition of fungal growth by potassium sorbate was found to be associated with a change in the composition of phospholipids (a decrease in phosphatidylcholine content and an increase in phosphatidylethanolamine and phosphatidic acid content) and of neutral lipids (a decrease in the triacylglycerol and sterol content and an increase in the free fatty acid content). The fatty acid composition of fungal lipids also changed. A drastic decrease in the linoleic acid content occurred both in the total lipid fraction and in the triacylglycerol and total phospholipid fractions, whereas the oleic acid content increased correspondingly. This suggests that sorbic acid (SA) affects Δ12 desaturase activity, which controls the adaptive response of mycelial fungi to deleterious environmental factors.     

Anti-microfouling Activity of Lipidic Metabolites from the Invasive Brown Alga Sargassum muticum (Yendo) Fensholt

The purification of the chloroform extract from the brown invasive macroalga Sargassum muticum, through a series of chromatographic separations, yielded 12 fractions that were tested against strains of bacteria, microalgae, and fungi involved in marine biofilm formation. The chemical composition of four (a, c, g, and k) out of the six fractions that exhibited anti-microfouling activity was investigated. Fraction a contained saturated and unsaturated linear hydrocarbons (C₁₂–C₂₇). Arachidonic acid was identified as the major metabolite in fraction c whereas fraction g contained mainly palmitic, linolenic, and palmitoleic acids. Fraction k was submitted to further purification yielding the fraction kAcaF1e that was composed of galactoglycerolipids, active against the growth of two of the four bacterial strains (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi. These promising results, in particular the isolation and the activity of galactoglycerolipids, attest the potential of the huge biomass of S. muticum as a source of new environmentally friendly antifouling compounds.     

不溶性基质中天冬氨酸丰富的蛋白在珊瑚的钙质骨片形成中的重要作用

一般认为, 酸性蛋白在控制矿物的形成和发展中发挥重要作用。因此, 在不溶性有机基质中鉴定酸性蛋白对于理解珊瑚中个体蛋白的功能是非常重要的一步。在短指多型软珊瑚(Sinularia polydactyla)的可溶性和不溶性基质层中分析蛋白组分表明, 在不溶性基质和可溶性基质层中天冬氨酸的含量分别是61%和29%。利用体外分析法发现, 基质蛋白诱导碳酸钙形成非晶态析出相先于其形成钙质的结晶态。利用X-射线衍射来鉴定骨片上结晶态的碳酸钙, 结果表明钙质的多晶态呈现强反射。傅利叶变换红外光谱分析表明珊瑚基质中富含天冬氨酸的蛋白和多醣的结构。在不溶性基质组分中用钙离子结合分析显示一个分子量为109 kD的蛋白质可以与形成骨片的钙离子结合, 这一过程对骨片形成非常重要。在对生物钙化过程中起重要作用的碳酸酐酶的分析中显示了此酶的新颖的活性。以上结果显示珊瑚中不溶性基质内的富含天冬氨酸的蛋白在生物矿化调控过程中起重要作用。     

Shoyu (Soy Sauce) Flavor Components: Neutral Fraction

From the vacuum distilled volatiles of shoyu, a neutral fraction was obtained. Shoyu was also directly extracted with dichloromethane and then the extract was separated into ten (A~J) fractions. The J fraction was a neutral one. The J fraction was further separated into twelve fractions by liquid column chromatography. All fractions obtained were analyzed by gas chromatography and combined gas chromatography-mass spectrometry. Consequently, 142 components were identified, 82 of which have not been reported previously as volatile constituents of shoyu. The identified compounds were 37 hydrocarbons, 22 alcohols, 22 carbonyls, 22 esters, 12 furans, 6 sulfurous compounds, 1 pyrone, 5 phenols, 1 furanone, 1 acid, 1 lactone and 12 other compounds. From the results of quantitative analysis and organoleptic evaluation, phenylacetal-dehyde is considered to be most important in the neutral fraction.