Specific non-histone protein fractions associated with ‘active’ chromatin in immunized rats

Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [³H]tryptophan, [³H]methionine and [³H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (M_r 12 000) and H (M_r 3 000) highly labelled with [³H]tryptophan, lower with [³H]methionine but not with [³H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg^2– soluble fractions which suggests that, similar to the fractions I (M_r below 3 000) and B (M_r 120 000) described previously (5), these fractions may be associated with active transcribed genes.     

GLUT4 translocation in C2C12 myoblasts and primary mouse hepatocytes by an antihyperglycemic flavone from Tillandsia usneoides

BackgroundType 2 Diabetes (T2D) is characterized by deregulation in carbohydrate and lipid metabolism, with a very high mortality rate. Glucose Transporter type 4 (GLUT4) plays a crucial role in T2D and represents a therapeutic target of interest. Tillandsia usneoides (T. usneoides) is a plant used as a remedy for diabetes. T. usneoides decreased blood glucose in different experimental models. However, the involvement of GLUT4 in this effect has not yet been explored.PurposeThis study aimed to investigate whether any component in T. usneoides might participate in the effect on blood glucose through a bioassay-guided fractionation, testing its potential antihyperglycemic effect in mice, as well as its influence on GLUT4 translocation in C2C12 myoblasts and primary hepatocytes.MethodsThe aqueous extract and the Ethyl Acetate fraction (TU-AcOEt) of T. usneoides were evaluated in a hypoglycemic activity bioassay and in the glucose tolerance test in CD-1 mice. TU-AcOEt was fractionated, obtaining five fractions that were studied in an additional glucose tolerance test. C1F3 was fractioned again, and its fractions (C2F9-12, C2F22-25, and C2F38-44) were examined by HPLC. The C2F38-44 fraction was analyzed by Mass Spectrometry (MS) and subjected to additional fractionation. The fraction C3F6-9 was explored by Nuclear Magnetic Resonance (NMR), resulting in 5,7,4´-trihydroxy-3,6,3´,5´-tetramethoxyflavone (Flav1). Subsequently, a viability test was performed to evaluate the cytotoxic effect of Flav1 and fractions C2F9-12, C2F22-25. C2F38-44, and C3F30-41 in C2C12 myoblasts and primary mouse hepatocytes. Confocal microscopy was also performed to assess the effect of Flav1 and fractions on GLUT4 translocation.ResultsThe TU-AcOEt fraction exhibited a hypoglycemic and antihyperglycemic effect in mice, and its fractionation resulted in five fractions, among which fraction C1F3 decreased blood glucose. MS and NMR analysis revealed the presence of Flav1. Finally, Flav1 significantly promoted the translocation of GLUT4 in C2C12 myoblasts and primary hepatocytes.ConclusionTo date, Flav1 has not been reported to have activity in GLUT4; this study provides evidence that T. usneoides is a plant with the potential to develop novel therapeutic agents for the control of T2D.     

Enhancement Of Immunogenicity of an Immunoprotective Glycophospholipid Antigen of Mycobacteria Using Liposomes Containing Lipid A

Abstract

Role of liposomes as the carriers of mannophosphoino-sitides (PIMs) antigen of mycobacteria have been investigated. PIMs when incorporated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH)/ in a molar ratio of 2: 1. 5 respectively/ elicited both humoral (anti-PIMs IgG) and cell-mediated (DTH) immune responses in mice. Further/ lipid A containing liposomes as carriers of PIMs were observed to induce almost two times more anti-PIMs immune responses as compared to those without lipid A. These findings clearly establish that the lipid A containing liposomes not only replace FIA, but are even better adjuvants for PIMs.     

Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics

Abstract

The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is – at least to some extent – influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.     

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) isoforms (2b and 3) as well as four Ca ²⁺ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP₃ binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca ²⁺ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P₃R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP₃R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP₃R-1 and InsP₃R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP₃R-1 and InsP₃R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP₃R isoforms, the two SERCA isoforms and the four Ca²⁺ binding proteins investigated. This heterogeneity may underlie specialization of the Ca²⁺ stores and the subsequent initiation of intracellular Ca²⁺ signals.     

Performance of the irregular surface compensator compared with four-field box and intensity modulated radiation therapy for gynecologic cancer

PurposeA retrospective planning study was undertaken to evaluate the dosimetric advantages of the irregular surface compensator (ISC) technique, a forward planning technique with electronic compensation algorithm available on Varian Eclipse treatment planning system. This was extensively compared to the conventional four-field box (4FB) and intensity modulated radiation therapy using 5 fields (IMRT5F) on gynecologic cancer patients.MethodsTwenty-two patients were enrolled. The prescribed dose was 50.4 Gy in 28 fractions to the primary target including pelvic lymph nodes. 4FB treatment plans were generated, then fluence of anterior and posterior fields were modified to generate ISC plans. IMRT5F were inversely optimized with equally spaced five coplanar fields. Dose-volume parameters were evaluated for the comparison of three planning techniques. The MU and delivery time were also estimated.ResultsIn terms of target coverage, the conformity and homogeneity index of ISC (1.67 and 1.03, respectively) were superior to those of 4FB (2.43 and 1.06, respectively) but slightly inferior to those of IMRT5F (1.10 and 1.02, respectively). ISC also illustrated an overall improvement in normal organ saving. Compared to 4FB, the mean dose of the rectum was reduced by about 4.0–5.0 Gy with ISC and IMRT5F. The volume receiving large doses was reduced for bladder with statistical significance with ISC and more with IMRT5F relative to 4FB. The mean number of MU per fraction were 200.86 (4FB), 446.09 (ISC) and 895.59 (IMRT5F).ConclusionThe ISC technique has the superior target coverage and healthy tissue sparing in comparison with conventional 4FB and comparable normal organ saving compared to IMRT5F. The ISC can be an available option for gynecologic radiotherapy.     

Investigations of Antibacterial,Antioxidant, and Antidiabetic Potential of Extract and Its Active Fractions from the Leaves of Horsfieldia spicata (Roxb.) J. Sinclair

This study was undertaken to analyse the potential bioactivities including antibacterial, antioxidant and antidiabetic derived from the methanolic extract and the column chromatography ethyl acetate fraction (AcOEt Fr) of Horsfieldia spicata leaves. Methanolic extract and 4 other fractions was calculated for total phenol and flavonoid contents along with tested for antibacterial, antioxidant and antidiabetic properties. Interestingly, the AcOEt Fr had the highest value for total flavonoid content and the best antioxidant, and antidiabetic activities. Therefore, the AcOEt Fr was further separated using column chromatography technique for obtaining 9 selected fractions namely fraction 1 (F1) - fraction 9 (F9) which were further tested. The results showed that the AcOEt column chromatography fractions namely F2, F3, F4 and F6 had the best clear inhibition antibacterial value against all bacterial tested. In addition, these fractions also exhibited better Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC) values than others. Antioxidant, 2,2-diphenylpicrylhydrazyl (DPPH) assayed indicated that AcOEt Fr had the strongest IC₅₀ value of 47.30 μg/mL. Further, F4 column chromatography fraction showed the best inhibition against α-Glucosidase enzyme related to antidiabetic activity with an IC₅₀ value of 6.11 μg/mL. Liquid chromatography tandem-mass spectrometry (LC/MS/MS) analysis identified that F4 derived from AcOEt fraction had several compounds belonging to the flavonoid and phenolics such as 3′,5-dihydroxy-7,4′-dimethoxyflavone, 5,7-dihydroxy-3-(4′-hydroxybenzyl)chromone, and Kadsurenin I.     

Distribution and mobility of scandium and yttrium in selected types of soils in Poland

Abstract

This study was conducted to investigate the chemical speciation of yttrium and scandium in selected types of soils and also determined the total content and profile distribution of those elements. The research was performed on soil samples from forest areas in Puszcza Borecka (Poland) known as a non-contaminated site. Soil samples were collected from natural brown, lessive and rusty soil profiles.

The sequential extraction procedure was applied to separate fractions of scandium and yttrium according to the Tessier method. The range of total content of scandium and yttrium was respectively: 0.63-6.48 mg kg^-1 and 2.25 to 27.93 mg kg^-1. Scandium and yttrium occurred predominantly in residual fraction (F5) in each genetic horizon. Also both elements occurred mainly in fraction bound to organic matter (F4). The mean percentage content of scandium measured in mobile fractions (∑F1-F2) reached about 3% (in Bbr, Bt horizons) to 14% (in Bv horizons) in relation to the total content of the element in the analysed soils. The percentage content of yttrium measured in mobile fractions reached about 4% (in A, Bv horizons) to 28% in Bt horizons in relation to the total content of the element in forest soils of Puszcza Borecka.     

Preparation,structural characterization and <Emphasis Type="Italic">in vitro</Emphasis> antitumor activity of <Emphasis Type="Italic">kappa</Emphasis>-carrageenan oligosaccharide fraction from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum to compare the antitumor activity with carrageenan polysaccharides. Oligosaccharide fractions were isolated by gel permeation chromatography and the structure of fraction 1 (F1) was studied by using negative-ion electrospray ionization-mass spectrometry (ESI-MS), and ¹H and ¹³C-NMR spectrometry. The in vitro antitumor effects in three human neoplastic cell lines (KB, BGC, and Hela) of polysaccharides and F1 were investigated. The bioassay results showed that F1 exhibited relatively higher antitumor activity against the three cancer cells than polysaccharides.     

Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

Normal Range of Hepatic Fat Fraction on Dual- and Triple-Echo Fat Quantification MR in Children

ObjectivesTo evaluate hepatic fat fraction on dual- and triple-echo gradient-recalled echo MRI sequences in healthy children.ResultsIn 54 children (M:F = 26:28; 5–15 years; mean 9 years), the dual fat fraction (0.1–8.0%; median 1.6%) was not different from the triple fat fraction (0.4–6.5%; median 2.7%) (p = 0.010). The dual- and triple-echo fat fractions showed good agreement using a Bland-Altman plot (-0.6 ± 2.8%). Eight children (14.8%) on dual-echo sequences and six (11.1%) on triple-echo sequences had greater than 5% fat fraction. From these children, six out of eight children on dual-echo sequences and four out of six children on triple-echo sequences had a 5–6% hepatic fat fraction. When using a cut-off value of a 6% fat fraction derived from a reference interval, only 3.7% of children were diagnosed with fatty liver. There was no significant correlation between clinical and laboratory findings with dual and triple-echo fat fractions.ConclusionsDual fat fraction was not different from triple fat fraction. We suggest a cut-off value of a 6% fat fraction is more appropriate for diagnosing fatty liver on both dual- and triple-echo sequences in children.     

Gene Transfer to Lentil Protoplasts by Lipofection and Electroporation

Abstract

Cationic liposomes made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were prepared by reverse-phase evaporation and were able to interact spontaneously with plasmid DNA. The loaded vesicles delivered a β-glucuronidase (GUS)-carrying plasmid to lentil Lens culinaris) protoplasts, leading to transient expression of the GUS reporter gene. The transfection efficiency achieved by using stearylamine-containing liposomes (lipofection) was comparable to the one obtained by electroporating the protoplasts at 500 μF and different field strengths. Furthermore, the combination of electroporation and lipofection, though reducing cell survival, increased the activity of the reporter enzyme detected in the cell lysates, yielding transient expression levels higher than those recorded after lipofection or electroporation alone.     

In Vivo Screening for Anti-Osteoporotic Fraction from Extract of Herbal Formula Xianlinggubao in Ovariectomized Mice

MethodsThe extract of XLGB formula was separated into three fractions using chromatography, i.e., XLGB-A, XLGB-B and XLGB-C. They were administrated to 4-month old ovariectomized (OVX) mice for 6 weeks to determine which bioactive fraction(s) were more effective for preventing OVX-induced bone loss evaluated by microCT, biomechanical testing and biochemical markers. The main peaks of the key fraction were identified using reference compounds isolated from the fraction. In addition, the effects of the composite compounds in XLGB-B on osteoblasts’ proliferation and mineralization were evaluated in UMR 106 cells.ResultsXLGB-B with a yield of 13.0% from herbal Fufang XLGB was identified as the most potential one among the three fractions for prevention of OVX-induced bone loss confirmed with bone mass, bone microarchitecture, bone strength and bone turnover markers. Nine compounds in HPLC fingerprint were identified in the XLGB-B fraction, including phenylpropanoids from Herba Epimedii, terpenes from Radix Dipsaci and coumarins from Fructus Psoraleae. In addition, the identified compounds effectively promoted proliferation and/or mineralization of osteoblast-like UMR 106 cells in vitro.ConclusionXLGB-B with defined phytochemical structures was screened as the key fraction that demonstrated preventive effects on OVX-induced bone loss in mice. The present study laid down a foundation towards a new generation of herbal Fufang characterized with “less herbal materials for achieving equal treatment efficacy” in development strategy of TCM for prevention of OVX-induced osteoporosis.     

The histones of rat testis

Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X₁, X₂, and X₃) were observed. Fraction X₁ had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X₁ were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X₁ had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels.Fraction X₂ migrated between histone fractions F1 and F3 on electrophoresis while X₃ migrated between fractions F2b and F3. Fraction X₃ was isolated with F2b during fractionation by the Johns procedure. Fraction X₂ has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X₂ can be seen in electrophoretic patterns of rat liver histones.The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X₁ and F1, or X₃ and F2b are not attributable to phosphorylation.     

Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

     

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones)

Partially purified lymphocytic factors were obtained from bovine spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo:[³H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited: similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked.[³H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced.The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in culture as far as thymidine incorporation into DNA is concerned.The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F′.Both fractions F and F′ were further fractioned by chromatography on a Sephadex G75 column: 7 peaks were obtained (F₁–F₇). Biological activity was found in fraction F₁, corresponding to high molecular weight material, and in fraction F₁, corresponding to low molecular weight substances.By rechromatography on Sephadex G 75, it is easy to dissociate from F₁ a small molecular weight fraction which might be similar to F₆ as far as elution volume and biological properties are concerned.     

Cell-mediated responses and protection elicited by a carbohydrate-lipid-containing fraction extracted from Leishmania major promastigotes

Carbohydrate-lipid-containing fractions (CLF) extracted from Leishmania major promastigotes and recognized by sera from immune but not from normal human donors were evaluated for their capacity to elicit cell-mediated responses. It was found that one of these fractions, CLF-1, stimulated the in vitro response of lymphocytes from immune but not from normal human donors. A similarly extracted fraction from L. donovani parasites also elicited an in vitro response by cells from donors immune to L. major. The response was mediated by antigen-presenting cells, and specific Leu 3+ Leu 2- T cells from a human T-cell line responded to the antigen. In vivo, the CLF-1 elicited delayed-type hypersensitivity (DTH) response in L. major-immunized C3H mice, which was comparable to the DTH response elicited by freeze-thawed and sonicated L. major promastigotes. C3H mice were vaccinated with CLF-1 prior to challenge with live L. major promastigotes. Mice vaccinated with CLF-1-containing liposomes showed a significant degree of protection to challenge. These results suggest that the carbohydrate-lipid-containing fraction described here may represent a functional antigenic entity from Leishmania parasites.