Unfractionated Preparations Research Articles

A multi-approach peptidomic analysis of hen egg white reveals novel putative bioactive molecules

Chicken egg white is a raw material broadly used as additive for the preparation of food and cosmetoceutical products. To describe its molecular properties, various proteomic investigations were performed in the last decade characterizing highly abundant components. No peptidomic counterparts were accomplished so far; scientific literature only reports on the characterization of specific bioactive peptides or preparations from egg white and its hydrolysates, which was performed through dedicated functional assays. In this study, a broad description of the egg white peptidome at 24, 336 and 672 h after laying was achieved using three peptide extraction procedures, which were combined with MALDI-TOF-TOF-MS and nanoLC-ESI-Q-Orbitrap-MS/MS analyses. In the whole, 506 peptides were characterized; they mostly resulted from the physiological degradation of intact proteins following the activity of endoprotease ArgC-, trypsin- and plasmin-like enzymes. Eventual detection of peptide post-translational modifications also provided structural information on parental proteins. When analyzed by bioinformatics and/or compared with literature data, identified peptides allowed recognizing a number of protein fragments associated with different hypothetical biological activities. These results confirmed previous observations regarding functional characteristics of egg white unfractionated preparations or purified molecules, emphasizing the useful application of this raw material in human nutrition and cosmetics. Finally, a comparative label-free peptidomic evaluation of samples stored for different times under refrigeration identified 31 peptides showing significant quantitative changes during storage. Biological significanceThis study provided the largest inventory of peptides described in chicken egg while so far. In addition, it identified a number of protein fragments associated with hypothetical antihypertensive, antioxidant, antiinflammatory, antimicrobial, anticancer, antiviral, antibiofilm, calcium-binding, antidiabetic, antithrombotic, adipogenic differentiating, stimulating/immunostimulating, hormonal, lipid-binding and cell adhesion-affecting activities. These results confirmed previous observations regarding functional characteristics of egg white unfractionated preparations or purified molecules, emphasizing the useful application of this raw material in human nutrition and cosmetics.

An Extensive Description of the Peptidomic Repertoire of the Hen Egg Yolk Plasma.

Hen egg is a raw material widely used for the preparation of food, pharmaceutical and cosmetoceutical products. Dedicated proteomic studies were accomplished on eggshell membrane, egg white, and yolk, identifying the most abundant proteins. No similar peptidomic studies have been performed so far. Only preliminary investigations on bioactive peptides in egg fractions and digestates were accomplished through functional screening assays, characterizing antioxidant, antibacterial, antiviral, immunomodulatory, and antihypertensive preparations and isolated components. This study fills this gap and provides a comprehensive picture of the peptides present in the yolk plasma of different hen egg types after 24 and 264 h of laying, taking advantage of a procedure based on a two-step fractionation followed by combined MALDI-TOF-TOF-MS- and nanoLC-ESI-Q-Orbitrap-MS/MS-based analysis. Six hundred and twenty-eight peptides were characterized as deriving from the proteolytic processing of larger protein components after the physiological action of chicken chymotrypsin-like and pepsin-like enzymes. Structural details on their post-translational modifications were also provided. Identified peptides were subjected to bioinformatic analysis and further compared with available data from the literature, ascertaining 198 peptides associable with putative antihypertensive, antimicrobial, anticancer, antiviral, antibiofilm, anorectic, calcium-binding, and anti-inflammatory activities. This analysis was often confirmative of previous experimental evidence on functional properties of unfractionated preparations or isolated molecules. These results further emphasize the bioactive action of yolk-derived peptides as related to egg consumption, and the potential use of these molecules as additive ingredients in the preparation of functional foods and cosmetics.

Mass separation and in vitro immunological activity of membrane-fractionated polysaccharides from fruiting body and mycelium of Agaricus subrufescens

Membrane technology has been applied to separate polysaccharides from Agaricus subrufescens (ASPs). The membrane-retained fractions and unfractionated preparations have been tested for in vitro immunological activity. Both the microfiltration (MF) and ultrafiltration (UF1) membranes were able to separate high-molecular weight polysaccharides from fruiting body (ASP-FB) and submerge-fermented mycelium (ASP-SmF) extracts. All fractions showed immunostimulatory effects on RAW 264.7 macrophages, measured by TNF-α, iNOs gene expression, and NO production. In contrast, antibody and proliferation levels in B lymphoblastoid SKW 6.4 cells were significantly increased after treatment with ASP-FB, but did not with ASP-SmF preparations. The ASPs- and LPS-induced stimulation could be differentiated by the finding that polymyxin B, a specific inhibitor of LPS, did not significantly affect the immunoactivating response and proliferation activity of ASPs on macrophages and B cells, respectively. Furthermore, the ASP-FB treatment was unable to induce IL-6 production by B cells unlike LPS activation, sustaining distinct signaling pathways for ASP-FB and LPS. The overall results provided additional information about the action of ASPs on the immune system and support the membrane method to separate and concentrate high- molecular weight ASPs for immunopharmacological and biotechnological applications.

未分画ヘパリン中のムコ多糖類の確認方法およびその工業的除去方法

未分画ヘパリン中のムコ多糖類の確認方法およびその工業的除去方法

Heparin-associated anti-Xa activity and platelet-derived prothrombotic and proinflammatory biomarkers in moderate to high-risk patients with acute coronary syndrome.

Heparin compounds, to include fractionated and unfractionated preparations, exert both antithrombotic and antiinflammatory effects through combined inhibition of factor Xa and thrombin. The contribution of modulated platelet activity in vivo is less clearly defined. The SYNERGY library was a prospectively designed repository for candidate clinical, hemostatic, platelet, and molecular biomarkers from patients participating in SYNERGY--a large-scale, randomized clinical trial evaluating the comparative benefits of unfractionated heparin (UFH) and enoxaparin in high-risk patients with acute coronary syndrome (ACS). Samples were collected from 201 patients enrolled at 26 experienced, participating sites and shipped to established core laboratories for analysis of platelet, endothelium-derived, inflammatory and coagulation activity biomarkers. Tissue factor pathway inhibitor (TFPI)--a vascular endothelial cell-derived factor Xa regulatory protein-correlated directly with plasma anti-Xa activity (unadjusted: r = 0.23, P < 0.0001; adjusted: β = 0.10; P = 0.001), as did TFPI-fXa complexes (unadjusted: r = 0.34, P < 0.0001; adjusted: β = 0.38; P = < 0.0001). In contrast, there was a direct and inverse relationship between anti-Xa activity and two platelet-derived biomarkers-plasminogen activator inhibitor-1 (unadjusted: r = -0.18, P = 0.0012; adjusted: β = -0.10; P = 0.021) and soluble CD40 ligand (unadjusted: r = -0.11, P = 0.05; adjusted: β = -0.13; P = 0.049). All measured analyte relationships persisted after adjustment for age, creatinine clearance, weight, sex, and duration of treatment. Differences in biomarkers between patients receiving UFH and those randomized to enoxaparin were not observed. The ability of heparin compounds to affect the prothrombotic and proinflammatory states which characterize ACS may involve factor Xa-related modulation of platelet activation and expression. Whether this potentially beneficial effect is direct or indirect and achieved, at least in part, through the release of endothelial cell-derived coagulation regulatory proteins will require further investigation.

Characterization of putative stem cells in isolated human colonic crypt epithelial cells and their interactions with myofibroblasts

Colonic epithelial stem cells are believed to be located at the crypt base where they have previously been shown to express musashi-1. The colonic stem cell niche, which includes extracellular matrix and myofibroblasts (together with other cell types), is likely to be important in maintaining the function of the progenitor cells. The aims of our studies were to characterize stem cells in isolated and disaggregated human colonic crypt epithelial cells and investigate their interactions with monolayers of primary human colonic myofibroblasts. In unfractionated preparations of disaggregated colonic crypts, musashi-1 positive cells preferentially adhered to colonic myofibroblasts, despite the presence of excess blocking anti-beta(1)-integrin antibody. These adherent epithelial cells remained viable for a number of days and developed slender processes. Cells with side population characteristics (as demonstrated by ability to expel the dye Hoechst 33342) were consistently seen in the isolated colonic crypt epithelial cells. These side population cells expressed musashi-1, beta(1)-integrin, BerEP4, and CD133. Sorted side population crypt epithelial cells also rapidly adhered to primary colonic myofibroblasts. In conclusion, in preparation of isolated and disaggregated human colonic crypts, cells with stem cell characteristics preferentially adhere to primary human colonic myofibroblasts in a beta(1)-integrin-independent fashion.

Contaminant in the Recalled Unfractionated Heparin Preparations: Where is the Problem?

In a recently held press conference, the USFDA (United States Food and Drug Administration) briefed the press that the potential contaminant in the recalled Baxter product is a heparin-like substance. The USFDA commented that heparin-like molecules have been identified using high-tech methods. No specifics regarding these contaminants were given. The manufacturing process of heparin is such that other heparin-like contaminants, such as the dermatan sulfate, heparan sulfate, and chondroitin sulfate are removed effectively. These represent the main contaminants in heparin. However, these contaminants are unlikely to produce any allergic effects, nor the reported adverse reactions, because some of the antithrombotic drugs contain significant amounts of these heparin-like substances. Because of the carbohydrate nature of the contaminants identified by a special method of nuclear magnetic resonance (NMR), it is quite disturbing to note that such contaminants may have inadvertently been added to the recalled heparin. Heparin-like materials have been isolated from the shellfish, marine plants, bones, or skin of mammalian origin for several therapeutic purposes. Although structurally similar, these heparin-like substances exhibit different biochemical and pharmacologic effects. There are also various protein contaminants that can be expected along with the carbohydrates. Thus, it is likely that besides the carbohydrate contaminants, some unknown protein contaminants may also be present in these products. The statement that a heparin-like material was identified using high-tech methods is ambiguous and misleading. Such statements should be specific and clear regarding the methods used and the nature In late January, Baxter Healthcare Corporation voluntarily recalled the following lots of heparin: 107054, 117085, 047056, 097081, 107024, 107064, 107066, 107074, and 107111. These products were labeled as heparin 1000 U/mL in 10 mL vials and heparin 1000 U/mL in 30 mL vials. The reason for this recall was the reported adverse reactions, which were associated with the use of these heparin batches. These included abdominal pain, hypotension, burning, chest pain, diarrhea, dizziness, dyspepsia, dyspnea, arrhythmia, flushing, headache, hyperthyroidism, hypoesthesia, increased lacrimation, loss of consciousness, malaise, nausea, pallor, palpitation, paresthesia, pharyngeal edema, restlessness, vomiting, stomach discomfort, tachycardia, thirst, trismus, unresponsiveness to stimuli, and drug ineffectiveness. These adverse reactions were reported in hundreds of patients treated with these heparins in the USA. Initially 19 deaths were linked with the use of the recalled heparins. However, this figure has now reached 81, and it is likely that it will go even higher. These multiple adverse reactions were later attributed to heparin manufactured by Baxter’s [Deerfield, IL] suppliers using Chinese raw material from SPL Changzhou, China. Subsequently, Baxter recalled all of the remaining lots and doses of its heparin sodium injection multidose, single dose vials, and Hep-lock heparin flush product.

Soluble factors from rabbit spleen cells kill and lyse Treponema pallidum in vitro

Antibody and complement immobilize (kill) Treponema pallidum in vitro. Recent evidence also documents immobilization by soluble factors released by activated macrophages and lymphocytes. Immune-mediated lysis of treponemes, however, has not been reported. The findings in this paper focus on apparent treponemal lysis by rabbit splenic cell preparations. Using cells from animals infected testicularly for 9 to 12 days, unfractionated splenic preparations, as well as adherent and nonadherent preparations, killed and lysed T. pallidum. Phagocytosis alone could not explain the detrimental effects of adherent cells. When cytochalasin B was used to block phagocytosis, decreases in treponemal numbers were still detected. In related studies, immune rabbit sera did not enhance treponemicidal activity of the adherent cells. To assess the specificity of these reactions, T. pallidum was incubated with two monocyte-like cell lines (human U937 and mouse P388D1). Neither cell line was detrimental, and treponemal numbers were not lowered. The soluble nature of the treponemicidal factors from adherent and nonadherent preparations was shown by physically separating these cells from the organisms and demonstrating treponemal killing and lysis. In summary, clearance of T. pallidum from infected tissues is probably at least partially attributed to macrophage phagocytosis. Our findings suggest another mechanism involving lytic factors secreted by activated adherent and nonadherent cells.

A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.

Multiple forms of syringomycin

Preparations of syringomycin purified from three isolates of Pseudomonas syringae pv. syringae according to published procedures have been shown to contain a group of structurally related peptides which can be resolved by HPLC on a reverse phase column. In the acid hydrolysate of all components serine, phenylalanine, 2,4-diaminobutyric acid and arginine in the ratio 2:1:2:1 have been found. These products account for nearly 50% of the molecular weights determined by FAB mass spectrometry. Most of the antibiotic activity of the unfractionated preparations is recovered in a limited number of peaks.

The role of L3T4+ cells in the pathogenesis of lupus in lpr-bearing mice. I. Defects in the production of interleukins 2 and 3.

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy-1+, L3T4-, Lyt-2-, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A-induced interleukin 2 and 3 production in the spleens of MRL-lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4- T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt-2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr-bearing mice.

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49–64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118–123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the p K a of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (p K a = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine p K a shift in the colipase micelle complex may explain why nitrated colipase can reactive lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.

In-vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus

Several types of membrane-localized flavin binding sites were investigated in sporangiophores (spph) and mycelia of Phycomyces blakesleeanus. In-vitro binding of riboflavin, riboflavin-5'-phosphate, and flavin-adenine-dinucleotide was demonstrated with unfractionated membrane preparations by means of competition of [(14)C]riboflavin binding. Saturation of binding was only obtained with the highly water-soluble riboflavin-5'-phosphate, but by extrapolation it was shown that riboflavin showed the highest affinity towards the binding sites (KD about 4·10(-6)M). The number of binding sites was estimated to be 0.7 nmol g(-1) fresh-weight equivalent. Analysis of endogenous soluble flavin revealed that only riboflavin, riboflavin-5'-phosphate, and flavin-adenine-dinucleotide occurred in Phycomyces, and at a concentration of at least 1 nmol g(-1) fresh-weight equivalent in entire spph. Thus, the measured binding sites could reach saturation in-vivo. In the apical part of spph to which blue-light sensitivity is restricted, the amount of soluble flavin was three-fold higher. Exclusively in this zone, heat-labile riboflavin proteins were measured at a concentration of about 3 nmol g(-1) fresh-weight equivalent. The amount of covalently bound flavin was higher in spph tips than in intact spph (8 nmol and 3 nmol g(-1) fresh-weight equivalent, respectively). In either case, the concentrations of the flavin-membrane complexes were higher than the theoretical calculated concentration of (anisotropic) blue-light photoreceptor in Phycomyces (Bergman et al. 1969), and their involvement in blue-light photoreception is considered.

Effects of Different Fractions of &lt;italic&gt;Corynebacterium parvum&lt;/italic&gt; on the Cytotoxic T-Cell Response to Alloantigens in Mice&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;

The effects of several biochemically derived fractions of Corynebacterium parvum and chemically treated intact organisms on the generation of cytotoxic T-lymphocytes (CTL) were assessed in a tumor allograft model with the use of C57BL/6 and DBA/2 mice. The acid-modified, active and inactive fragmented preparations and pyridine extract and residue were all capable of inhibiting primary spleen cell allocytotoxicity. Only the active fragmented, pyridine residue and unfractionated preparations caused splenomegaly and prevented the secondary in vitro generation of CTL. Periodate treatment, acid modification, and pyridine extraction abrogated the ability of C. parvum to activate suppressor macrophages. Although incapable of activating suppressor macrophages, the phenol and pyridine extracts significantly enhanced suppressor T-cell activity. The effect of whole C. parvum organisms on T-cell-mediated cytotoxicity is thus extremely complex and due to the direct activation or inhibition of various cell types that interact with each other.

Heterogeneity in the composition of commercial heparins: Comparison of anticoagulant activities and biochemical compositions of anionic density-fractionated heparins

In this study the nature of compositional heterogeneity in commercial heparins was investigated. Five hog-mucosal (HM) and one beef-lung (BL) heparin were subjected to partition fraction in a two-phase system of 1-butanol containing hexadecylpyridinium chloride/aqueous NaCl, a system which fractionates heterogeneous heparins larger than 10,000 daltons according to anionic density. The heparins differed markedly in purity as determined by both the galactosamine content of the unfractionated preparations and the amount of uronate extracted at lower NaCl concentrations in the fractionation scheme. All fractions afforded linear plots of the logarithm of clotting time vs heparins concentration in the APTT assay with human plasma, thereby permitting measurement of concentration - independent specific activities. Equivalent fractions from different HM heparins had similar compositions, and with the exception of an unbleached sample, had equivalent specific APTT anticoagulant activities. These latter were linearly related to the square of anionic density. In contrast, the BL heparin behaved quite differently. Its most abundant fractions were extracted at higher NaCl concentrations than was the case for HM heparins, little systematic variation in anionic density was observed, and its fractions had much lower specific activities than equivalent HM fractions.

Plasmid transformation in Bacillus subtilis: fate of plasmid DNA.

Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA. Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA. We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.

Characterization of antigens from Aspergillus fumigatus. IV. Evaluation of commercial and experimental preparations and fractions in the detection of antibody in aspergillosis.

Commercially available preparations of Aspergillus fumigatus, experimental extracts and fractions of A. fumigatus, and extracts of 5 other aspergilli were evaluated in a blind manner for their abilities to detect antibody in 66 serum specimens from patients with aspergilloma, allergic bronchopulmonary and invasive aspergillosis, and unconfirmed aspergillosis. Rocket immunoelectrophoresis was more sensitive than immunodiffusion in 2 dimensions. Although qualitative differences in antigen composition could be detected, commercial preparations compared favorably with the best experimental extracts in detecting positive specimens. Extracts from young, actively-growing myceliums were most effective and produced the largest numbers of precipitin bands. In contrast to all unfractionated preparations, several fractions of A. fumigatus were devoid of substances that react with C-reactive protein of serums, yet were as effective as the best preparations in detecting serums positive for Aspergillus.

Action of bradykinin on the mesenteric microcirculation in rats

It was shown that treatment by 2-3 M KCl solutions of complex unfractionated preparations of heparin with hexamminecobalt (III) resulted in incorporation into the solution of a heparin fraction containing 3 residues of sulphuric acid per 1 residue in glucosamine. In the precipitate released of hexamminecobalt (III) there remains a heparin fraction containing 4 residues of sulphuric acid per 1 residue of glucosamine. Both fractions were isolated in a chemically individual form.

Major mRNA species from spinach chloroplasts do not contain poly(A).

POLY(A) is a constituent of most animal cytoplasmic mRNAs1,2 and has been demonstrated in rapidly-labelled, polysomal RNA in plants3,4. Verma et al.5 have shown that cellulase mRNA, isolated from auxin-treated pea epicotyls, contains poly(A). Although a significant proportion of eukaryotic polysomal mRNAs are known to lack poly(A)6–8, the presence of poly(A) is still regarded as a characteristic feature of unfractionated preparations of eukaryotic mRNA. By contrast, poly(A) has not been detected in mRNA from prokaryotic cells8. Several reports9–11 have indicated that poly(A) is present in HeLa cell mitochondrial RNA, although Groot et al.12 were unable to detect poly(A) in yeast mitochondrial RNA. Here we describe experiments in which we have assayed for the presence of poly(A) in spinach chloroplast RNA by two methods; (1) hybridisation of chloroplast RNA with 3H-poly(U) and (2) binding of chloroplast RNA, pulse-labelled in vivo, to oligo(dT) cellulose. Additionally, the mRNA activities of chloroplast poly(A)-containing RNA and non-poly(A)-containing RNA (hereafter referred to as poly(A)+ RNA and poly(A)− RNA respectively) have been investigated by measuring their translation into specific polypeptides in cell-free extracts from Escherichia coli. From the results, we conclude that chloroplast mRNA lacks poly (A).

Comparative antileukemic action of RNA and histones from intact and regenerating homologous tissue

The effect of unfractionated preparations of RNA and histones isolated from intact and regenerating lymphoid tissue on the survival and growth of leukemic lymphocytes in syngeneic recipients was studied. The inhibitory action of histones on leukemic growth was well-marked but was independent of the morphological and functional state of the tissue at the time of their extraction from it. Electrophoresis in polyacrylamide gel showed no appreciable differences in the fractional composition of histones from intact and regenerating tissues. Preliminary treatment of the leukemic cells with RNA from regenerating tissue reduced the survival rate and increased the life span of the mice compared with animals receiving cells incubated with RNA from intact tissue. A spectrophotometric study of the Melting profile of the RNA preparations revealed differences in the ratio between the thermostable and unstable components of RNA from intact and regenerating tissues.