Adjuvant activity of Klebsiella O3 lipopolysaccharide: no contribution of proteins to the expression of adjuvant activity

Previously we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant of strain Kasuya (O3: K1) or its decapsulated mutant strain LEN-1 (O3: K1-) exhibited very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice. The preparation of KO3 LPS after deproteinization by four cycles of treatment with chloroform-butanol (5: 1) usually contained a small percentage of proteins and a definite amount of another antigen which was destroyed by heating at 100 C for 1 hr. This antigen proved to be derived from type 1 fimbriae which are responsible for mannose-sensitive hemagglutination of guinea pig erythrocytes. The preparation of KO3 LPS isolated from culture supernatant of the strains which did not produce type 1 fimbriae exhibited strong adjuvant activity similar to that of the preparation from those which produced them. The preparation of KO3 LPS treated with hot phenol water which is known to remove lipid A-associated proteins exhibited a similar strong adjuvant activity. The preparation of KO3 LPS after extensive deproteinizing, two cycles of pronase treatment followed by ten cycles of treatment with chloroform-butanol, no longer contained detectable amounts of proteins and the fimbrial antigen, but this preparation also exhibited similar strong adjuvant activity. Moreover, there was no difference in strength of the adjuvant activity between the preparation of KO3 LPS isolated from culture supernatant and that isolated by the phenol method from bacterial cells. The present study demonstrates that the strong adjuvant activity of the preparation of KO3 LPS does not depend in any way on proteins contaminating the preparation.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.     

Effects of traditional Chinese medicine on immune responses in abalone, Haliotis discus hannai Ino

A traditional Chinese medicine (TCM) preparation was formulated from orange peel (Pericarpium Citri Reticulatae), hawthorn (Crataegus pinnatifida), astragalus (Astragalus membranaceus (Fisch.) Bunge), pilose asiabell root (Radix codonopsis), indigowoad root (Radix isatidis), taraxacum (Herba taraxaci) and malt (Fructus Hordei Germinatus) at a weight ratio of 1:1:1.5:1.5:1.5:1.5:2. A feeding experiment was conducted to determine the effects of TCM on innate immunity of abalone, Haliotis discus hannai Ino. Artificial diets containing 1%, 3%, 5% TCM preparation, 1% hawthorn or 1% astragalus, respectively, were fed to juvenile abalone (initial weight 10.38+/-2.51 g; initial shell length 44.15+/-4.15 mm) for 80 days. A TCM-free diet was used as a control. Each diet was fed to three replicate groups of abalone using a randomized design. The results indicated that phagocytic activity was significantly higher in abalone fed 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Respiratory burst activity was significantly higher in abalone fed 1%, 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Agglutination titre was significantly higher in abalone fed 5% TCM preparation (P<0.05). Weight gain ratio (WGR), daily increment in shell length (DISL), total haemocyte count (THC), plasma protein concentration, and the activity of acid phosphatase (ACP) were not significantly affected by the TCM preparation (P>0.05). These results indicate that TCM preparation can modulate the immunity of H. discus hannai, and it is very possible that TCM might be used as immunostimulants in abalone farming.     

Chemo-selectivity of the N,O-enzymatic acylation in organic media and in ionic liquids

This paper shows that Lecitase Ultra is an enzyme preparation with a great interest as regioselective biocatalyst in the deprotection of 4 different peracetylated sugars: 1,2,3,4,6-penta-O-acetyl-β-d-galactopyranose (1), 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-d-glucopyranose (4), 1,2,3,4,6-penta-O-acetyl--d-mannopyranose (7) and 2,3,4,6-tetra-O-acetyl-β-d-galacto pyranosyl-(1 → 4)-1,2,3,6-tetra-O-acetyl-β-d-glucopyranoside (9). The enzyme properties (specificity, preference for the per-acetylated sugar and regio-selectivity) were strongly modulated by the immobilization conditions, for example the octyl-LECI preparation was 10 fold more active than the PEI-LECI preparation, while it was more than 40 fold less active against some other substrates. Very interestingly, these changes also affected the regioselectivity, depending on the preparation used it was possible to get free OH groups in anomeric position, position 6 or the mixture of both. Finally, the octyl-LECI preparation did not recognize the -sugars, favouring the β-isomers (in opposition to most commercial lipases or the other LECI preparations). This is potentially useful to obtain pure -peracetylated monosaccharides from a mixture of anomers.     

Chemoenzymatic method of 1,2,4-triazole nucleoside synthesis: Possibilities and limitations

Possibilities and limitations of chemoenzymatic synthesis of novel structural analogues of an antiviral preparation of Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) were established. A synthesis of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues—potential substrates of purine nucleoside phosphorylase—has been described. Comparative efficiency of preparation methods of these amides, as well as the methods of introduction of functional groups to the C5 position of heterocyclic system, were investigated. Novel analogues of Ribavirin containing various substitutes in the carboxamide group were synthesized. A biotechnological method was developed for the preparation of 1-β-D-ribofuranozyl-1,2,4-triazole-3-carbonitryl, an intermediate in the synthesis of Viramidine, the modern analogue of Ribavirin.     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

Structure of a novel highly branched α-glucan enzymatically produced from maltodextrin

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility α-glucan containing a large amylase-resistant portion. This α-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility α-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The α-glucan was found to be a novel highly branched α-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.     

Inactivation of follicle-stimulating hormone by a factor in a bovine serum albumin preparation

Bovine serum albumin (BSA) preparations are commonly employed as "carrier" or "protective" proteins in the solutions used to dissolve gonadotropin preparations. The present report describes a BSA preparation that was found to contain a factor that inactivated follicle-stimulating hormone (FSH). Four different BSA preparations (designated BSA1, BSA2a, BSA2b, BSA3) were studied. The FSH preparation (NIH-FSH-S16) was dissolved in 0.15 M NaCl, containing the various BSA preparations. The FSH solutions were injected subcutaneously, twice daily, for 5 days into hypophysectomized immature female rats bearing estrogen capsules. Twenty-four hours after the last injection, the rats were decapitated, and the ovaries were removed, trimmed and weighed. The FSH preparation produced ovarian weight gain when BSA1, BSA2b, or BSA3 was used, but not when BSA2a was used in the vehicle. In animals injected with the FSH dissolved in BSA1 vehicle and injected at a separate site with BSA2a solution, the FSH preparation was fully active, which indicates that contact of the BSA2a preparation with FSH was required for the inactivating factor to be operant. Indeed, after incubation in BSA2a solution, the radiolabeled FSH preparation exhibited a slight decrease in apparent molecular size when chromatographed on a Sephadex G-100 column. This result suggests that the BSA2a preparation contained a factor that may have inhibited FSH by degrading it.     

Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

Characterization of human monocyte activation by a water soluble preparation of Aphanizomenon flos-aquae.

Aphanizomenon flos-aquae (AFA) is a fresh-water microalgae that is consumed as a nutrient-dense food source and for its health-enhancing properties. The current research characterizes the effect of a water soluble preparation from AFA on human monocyte/macrophage function and compares the effect of AFA with responses from known agents that modulate the immune system. At 0.5 μg/ml the AFA extract robustly activated nuclear factor kappa B (NF-kappa B) directed luciferase expression in THP-1 human monocytic cells to levels at 50% of those achieved by maximal concentrations (10 μg/ml) of bacterial lipopolysaccharide (LPS). In addition, the AFA extract substantially increased mRNA levels of interleukin-1β(IL-1β) and tumor necrosis factor-(TNF-), and enhanced the DNA binding activity of NF-kappa B. The effects of AFA water soluble preparation were similar to the responses displayed by LPS, but clearly different from responses exhibited by tetradecanoyl phorbol acetate (TPA) and interferon-gamma (INF-γ). Pretreatment of THP-1 monocytes with factors known to induce hyporesponsiveness suppressed both AFA-dependent and LPS-dependent activation. These results suggest that the macrophage-activating properties of the AFA water soluble preparation are mediated through pathways that are similar to LPS-dependent activation.     

Inducible synthesis of beta-galactosidase in disrupted spheroplast of Escherichia coli

A membrane preparation obtained from osmotic lysate of spheroplasts of Escherichia coli cells showed an activity of synthesizing beta-galactosidase which was dependent upon oxidative phosphorylation. The synthesis was inhibited by the addition of actinomycin D or of chloramphenicol. The beta-galactosidase synthesized in the membrane preparation was completely released into the medium, while that synthesized in the spheroplasts and intact cells remained within the cells. The minimum concentration of the inducer, methyl-beta-d-thiogalactoside, required for the induction of beta-galactosidase was 5 x 10(-5)m for intact cells, 3 x 10(-4)m for spheroplasts and 1 x 10(-3)m for membrane preparation. Incorporation of labeled glucose into insoluble components in membrane preparation was extremely low compared with that in intact cells or in spheroplasts. Based on these and other observations, the nature of this membrane preparation is discussed in relation to the structure of E. coli cells.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Characterization of human monocyte activation by a water soluble preparation of Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (AFA) is a fresh-water microalgae that is consumed as a nutrient-dense food source and for its health-enhancing properties. The current research characterizes the effect of a water soluble preparation from AFA on human monocyte/macrophage function and compares the effect of AFA with responses from known agents that modulate the immune system. At 0.5 μg/ml the AFA extract robustly activated nuclear factor kappa B (NF-kappa B) directed luciferase expression in THP-1 human monocytic cells to levels at 50% of those achieved by maximal concentrations (10 μg/ml) of bacterial lipopolysaccharide (LPS). In addition, the AFA extract substantially increased mRNA levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α), and enhanced the DNA binding activity of NF-kappa B. The effects of AFA water soluble preparation were similar to the responses displayed by LPS, but clearly different from responses exhibited by tetradecanoyl phorbol acetate (TPA) and interferon-gamma (INF-γ). Pretreatment of THP-1 monocytes with factors known to induce hyporesponsiveness suppressed both AFA-dependent and LPS-dependent activation. These results suggest that the macrophage-activating properties of the AFA water soluble preparation are mediated through pathways that are similar to LPS-dependent activation.     

蔗糖酯类超声造影剂的制备及兔肝脏超声造影的实验初步观察

目的:探索蔗糖酯类超声造影剂的制备方法,并研究其造影效果。方法:采用声振法制备蔗糖酯类造影剂,将F68和SE-5按一定质量比称取配置微泡包膜材料,以微泡浓度为指标进行制备优化,将F68和SE-5按1:1质量比称取,将其配置成乳状液置入声振仪,以不同超声功率(200 W、400 W、600 W及800 W),不同声振时间(30 s、60 s、90 s、120 s、150 s、180 s、210 s、240 s、270 s及300 s)进行声振处理。将F68和SE-5分别按6:1、5:1、4:1、3:1、2:1、1:1、2:1、3:1及4:1的质量比称取制成乳状液,选择最佳声振功率与声振时间,筛选最佳溶液配比。选用优化条件下制备的微泡,观察兔肝脏造影效果。结果:蔗糖酯类造影剂的最佳制备条件为:泊洛沙姆188(Pluronic F68)和蔗糖酯-5(SE-5)的配比为2:1,声振仪功率为600 W,声振持续时间为240 s。注射造影剂后兔肝脏超声造影图像清晰,回声强度明显增强。结论:通过优化制备工艺,制备出的蔗糖酯类微泡浓度及直径均符合超声造影要求,具有良好显影效果。     

The effect of myelopid on antibody formation in gamma-irradiated animals

The effect of a new immunocorrecting preparation, Myelopid, on the antibody-forming cell content of mouse spleen after gamma-irradiation (1-3 Gy) has been investigated. The preparation administered after immunization of mice with sheep erythrocytes increases the number of antibody-forming cells in the spleen, the effect being a function of radiation dose and time interval between the exposure and immunization. The preparation is ineffective when delivered after irradiation, but prior to immunization.     

Binding of prostaglandin E1 to human erythrocyte membrane

Prostaglandin E1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E1 binding sites which differed in their affinity for the autacoid. The high-affinity class had Kd = 3.6 X 10(-9) M and the low-affinity class had Kd = 5.6 X 10(-5) M. The optimum pH for the binding of [3H]prostaglandin E1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occurred at a concentration of 5 mM Mg2+ in the incubation mixture. [3H]Prostaglandin E1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC50 (concentration of the nucleotide displacing 50% bound [3H]prostaglandin E1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [3H]prostaglandin E1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.     

The biosynthesis of raffinose

1. Reaction of UTP and alpha-d-galactose 1-phosphate with [U-(14)C]sucrose in the presence of a Vicia faba dormant-seed preparation yielded the trisaccharide raffinose. 2. UTP-alpha-d-galactose 1-phosphate-uridylyltransferase activity has been demonstrated in the bean preparation and evidence for the participation of UDP-galactose in the trisaccharide synthesis is presented. 3. UDP-galactose 4-epimerase is present in the dormant seed. 4. The biosynthesis of raffinose in relation to the metabolism of other carbohydrates in plants is discussed.     

复合微生态制剂对雏鸡实验性感染鸡白痢沙门菌病的预防实验

实验用200羽伊莎B-380雏鸡,随机分为5组,在1~3组雏鸡日粮中分别添加0.5%、1.0%、1.5%HM-强效复合微生态制剂,第4组日粮中添加0.2%氟哌酸,第5组饲料中不添加任何药物作为空白对照,用鸡白痢沙门菌进行人工感染,以观其药物的保护作用。结果显示添加0.5%HM-强效复合微生态制剂有98%的保护率,添加1.0%、1.5%HM-强效复合微生态制剂和添加0.2%氟哌酸有100%的保护率,检查发现添加HM-强效复合微生态制剂组雏鸡肠道中大肠埃希菌明显减少,而乳酸杆菌和双歧杆菌有益微生物明显增多。