Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

ANALYSIS OF PROLIFERATION AND DIFFERENTIATION OF FOETAL GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN HAEMOPOIETIC TISSUE*

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte-macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea-pig) a period of variable duration was observed when foetal liver CFCs entered a non-cycling G₀ or blocked G₁ phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non-cycling phase again re-entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte-macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.     

Serum is a rich source of ligands for the scavenger receptor of hepatic sinusoidal endothelial cells

The present study was prompted by findings in our laboratory showing that serum effectively inhibits scavenger receptor (SR)-mediated endocytosis in hepatic sinusoidal endothelial cells (SEC). Experiments with SEC in vitro showed that the presence of 20% human serum inhibited endocytosis of SR ligands, ¹²⁵I-formaldehyde treated bovine serum albumin (FSA) and ¹²⁵I-nidogen, by 30 and 50%, respectively, whereas pre-heated foetal bovine serum (10%) inhibited endocytosis of ¹²⁵I-FSA by as much as 56%. Human, bovine and rat serum had similar inhibitory effect on endocytosis in SEC. Fractionation of foetal bovine and human serum on anion exchange chromatography demonstrated that the inhibitory principle co-purified with macromolecules of high negative charge. The serum fraction that most effectively inhibited SR-mediated endocytosis of ¹²⁵I-FSA did not affect mannose receptor-mediated endocytosis of ¹²⁵I-mannan to the same extent. Trap-labelled negatively charged serum fraction administered intravenously to rats was eliminated almost exclusively by liver, with a blood decay of 50% over the first 3 min after injection. Isolation of liver cells showed that the populations of Kupffer cells and SEC contained 39 and 61% of liver radioactivity 30 min after injection of trap-labelled negatively charged fractions prepared from pre-heated (complement inactivated) foetal bovine sera. These findings suggest that the process of serum formation from native blood generates appreciable amounts of macromolecules that compete specifically with the SR for endocytosis in SEC. The inhibitory power of pre-heated serum is particularly great. For this reason pre-heated serum should be used with caution in studies of SR in SEC.     

Comparison of Animal Sera for Suitability in Coagulase Testing

The sera of several animals were examined for suitability in coagulase testing. The assay for coagulase-reacting factor (CRF) activities of the whole sera indicated the following relative concentrations of CRF: human > pig > rabbit > horse > bovine, chicken, and lamb. Human, pig, and rabbit sera had adequate amounts of CRF for coagulase testing. The plasmin activities of the different sera, arranged from the strongest to the weakest, were as follows: rabbit > human > lamb > horse > bovine, chicken, and pig. Fibrinolysis was observed when rabbit, human, lamb, or horse sera were incorporated into coagulase test agars. Pig serum was superior to the other sera for use in the plate test for coagulase since it had adequate amounts of CRF and the plasminogen-plasmin system was not activated by staphylokinase or staphylococcal Müller factor. Heparinized pig plasma was more suitable than citrated pig plasma since citrate interfered with the growth of Staphylococcus aureus, and the use of heparinized plasma prevented false-positive coagulase reactions due to citrate utilization.     

两株聚球藻伴生细菌分离纯化与促生功能鉴定

【背景】蓝藻周围存在伴生细菌,伴生细菌与蓝藻具有复杂的作用关系。【目的】研究淡水聚球藻伴生细菌对聚球藻生长的影响。【方法】采用高通量测序分析聚球藻伴生细菌多样性;平板划线法纯化聚球藻伴生细菌,通过形态观察结合16S rRNA基因序列同源性比对,对其种属关系进行确定;通过聚球藻和不同浓度伴生细菌共培养测定其叶绿素a浓度,分析伴生细菌对聚球藻生长的影响;采用种子发芽试验验证伴生细菌促生功能。【结果】淡水聚球藻伴生细菌优势菌属为产卟啉杆菌属(Porphyrobacter)、根瘤菌属(Rhizobium)、水单胞菌属(Aquimonas)和中慢生根瘤菌属(Mesorhizobium),从聚球藻分离获得了两株伴生细菌JQ1和JQ2,基于16S rRNA基因序列鉴定其分别属于Rhizobium和Peribacillus,通过在聚球藻与不同浓度伴生细菌共培养及水稻发芽试验验证,证明伴生细菌JQ1和JQ2在菌藻比例分别为5:1和15:1时具有促生作用,都对增强秧苗素质和根系发育有一定影响但JQ2与JQ1相比能显著提高水稻种子的发芽率。【结论】淡水聚球藻伴生细菌JQ1和JQ2在适宜的浓度均可显著促进聚球...     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.     

Antigenic Relationship Between Plasmodium falciparum and Babesia bovis: Reactivity with Antibodies to Culture-Derived Soluble Exoantigens1

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey—P. falciparum; bovine—B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.     

Serological Studies of an Acid-Labile O-Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.     

Antigenic relatedness of immunoglobulins toLegionella pneumophila serogroups 1–6 from humans and the nonhuman primateMacaca fascicularis

The purpose of this study was to determine whether cynomolgus monkey antisera toLegionella pneumophila serogroups 1–6 antigens could be used as positive controls in the indirect immunofluorescence assay (IFA) for legionellosis. Immunoelectrophoretic mobilities and Ouchterlony analyses with heavy chain-specific antisera and IFA titers with immunoglobulin class-specific conjugates were used to show antigenic relatedness of immunized monkey immunoglobulins to those produced as a result of infection in humans. Identical immunoelectrophoretic precipitation patterns were obtained for human and monkey sera with antihuman gamma, mu, and alpha heavy-chain-specific antisera. Ouchterlony analyses showed precipitin bands of partial identity between human and monkey IgG, IgM, and IgA classes. IFA titers in the monkey hyperimmune antisera were >16,000 with antihuman conjugate. These data suggest that hyperimmune cynomolgus monkey antisera are suitable alternatives to human sera for IFA-positive controls.     

幽门螺杆菌黏附素保守区蛋白的免疫原性、安全性和黏附作用的体外评价

探讨幽门螺杆菌（Hp）保守区（AB）蛋白的体外安全性、免疫原性和黏附作用,以确定AB在Hp疫苗研制中的应用价值.ELISA法测定Hp感染者血清中抗AB抗体,四唑盐比色法（MTT）测定T细胞对AB的增殖反应,流式细胞术检测AB致T细胞表达FasL的作用,二苯胺（DPA）法测定AB致T细胞凋亡率,光镜计数法研究AB抗体对Hp与胃癌细胞黏附的影响.ELISA法共检测了55份血清,以快速尿素酶实验(RUT)作为平行对照,两法的评价判断一致性程度的指标卡帕系数为0.76. 同时,低剂量AB即可刺激Hp⁺T细胞的增殖.体外安全性实验表明,AB无明显调节T细胞表达FasL的作用以及无明显致Hp^－T细胞凋亡的作用. AB抗血清能部分阻断Hp与胃癌细胞系的黏附,在光镜下表现为经抗AB兔血清预处理后,每细胞周围黏附的细菌数较免疫前兔血清预处理组显著减少（P＜0.05）.研究表明AB是安全的、具有免疫原性的Hp菌体成分,既可以刺激体液免疫,又能够提高细胞免疫,并且其抗体还可防止Hp与胃上皮细胞的黏附.     

EFFECT OF SOIL SURFACE TOPOGRAPHY AND LITTER COVER ON THE GERMINATION,SURVIVAL, AND GROWTH OF MUSK THISTLE (CARDUUS NUTANS)

In natural populations of musk thistle (Carduus nutans L.) germination occurs over several months in the fall and spring. To better understand the physical environmental mechanisms that influence this pattern of germination, a greenhouse experiment was established consisting of ten treatments in which soil surface topography and the amount and consistency of litter cover was varied. Significant differences occurred among the treatments for germination percentage, levels of mortality and individual growth rates. Optimum levels of germination, survival and growth occurred in treatments that provided microhabitats with reduced evaporation. Treatments with cracks or moderately irregular surface topographies provided the best conditions. A light covering of litter benefited establishment and growth but thick layers of litter prevented seeds from reaching the soil surface and reduced germination and establishment. These results were consistent with what is known about the germination ecology of musk thistle in natural populations.     

The Acidophilia of Elastin

Conditions relevant to the acceptance of acid dyes by the elastin of the aorta are as follows: (a) Fixation. Cold alcohol is poor, formol-sublimate, hest; others are intermediate. (h) Acid dyes used. Bromphenol blue is best, light green is poor, acid fuchsin is unstable in alkaline solution. Ponceau, aniline blue, fast green, chromotrope, erythrosin, and eosin are only moderately useful. (c) Staining pH. Elastica shares with eosinophil granules the property of retaining acid dye up to pH 11. (d) Autolysis. There is no effect even after 24-36 hr post mortem on acid dye affinity. (e) Speed of staining. Minor variations will appear if staining is less than 30 min. (f) Species of animal. Rat, mouse, cat, sheep, rabbit, guinea pig, opossum, ferret, rhesus monkey, horse, cow, and pig all possess acidophilic aortic elastin. A special feature has been the effect of age on the staining of human aortic elastin; bromphenol blue washes out quickly from the aortic elastin of old subjects, slowly from that of infants.     

Purification of the Endospores and Sporangia of Rhinosporidium Seeberi on Percoll Columns

Human rhinosporidial tissue was used as the source of the various developmental stages of Rhinosporidium seeberi - endospores with electron dense bodies, juvenile, and immature sporangia. After homogenisation in phosphate buffered saline (PBS) and removal of tissue fragments by centrifugation, the rhinosporidial bodies were isolated on centrifuged Percoll columns with gradients of densities or on triple-layered columns of varying density. The separated bands, after repeated washing in PBS gave bodies free from human tissue as shown on Leishman and PAS staining and indirect immunofluorescence with rabbit and human patients' anti-rhinosporidial sera. Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining. Percoll columns were shown to be capable of isolating these stages of R. seeberi, free from human tissue and contaminating bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

TAURINE AND HYPOTAURINE: THEIR EFFECTS ON MOTILITY,CAPACITATION AND THE ACROSOME REACTION OF HAMSTER SPERM IN VITRO AND THEIR PRESENCE IN SPERM AND REPRODUCTIVE TRACT FLUIDS OF SEVERAL MAMMALS*

Previous work from this laboratory has shown that the β-amino acid taurine can support and stimulate hamster sperm motility during in vitro capacitation in the presence or absence of epinephrine. The present report describes in vitro results which demonstrate that hypotaurine, a precursor of taurine, can also support and stimulate motility under these conditions and that a higher number of acrosome reactions occur in the presence of taurine as compared to hypotaurine (both in the presence and absence of epinephrine). In all cases, the greates percentage of acrosome reactions occurs in the presence of epinephrine. Whether these β-amino acids act independently of epinephrine of in a synergistic manner with it remains to be determined. In addition to these in vitro studies, we report that hypotaurine and taurine are present at high levels in bovine follicular fluid, rabbit uterine and ampullar oviductal fluid (11 hr after mating, i.e., 1 hr after ovulation), monkey oviductal fluid, bovine adrenal cortex “motility factor” preparation and human, guinea pig and hamster sperm preparations. Based on these results, we suggest the possibility that taurine and hypotaurine may have roles in vivo in the maintenance and stimulation of sperm motility and stimulation of capacitation and/or acrosome reactions.     

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat

Aims: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. Methods and Results: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. Conclusions: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. Significance and Impact of the Study: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth‐promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.     

Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance

• There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity.
• Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant‐growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied.
• Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na⁺ in its shoots, thus improving sodium phytoextraction capacity.
• Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils.

     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Presence and comparison of angiotensin converting enzyme in commercial cell culture sera

This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide. The heat inactivation of commercial sera at 56 degrees C for 30 min showed a reduction of ACE activity of about 35-80%. Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured "in vitro."     

Partial Characterization of the Antimicrobial Activity of Streptomyces antimycoticus FZB53

The paper describes experiments aimed at evaluating the sensitivity of different fungi, most of them plant pathogens and bacteria towards Streptomyces antimycoticus FZB53, a biocontrol agent that, when applied as a seed treatment, in previous studies has shown good activity against different seed‐borne fungal diseases. When incorporated into agar media, the filtrate from shake cultures of S. antimycoticus FZB53 inhibited the mycelial growth or spore germination, respectively, of a broad spectrum of fungi. The most sensitive of the fungi tested was Fusarium culmorum. The inhibitory activity could be removed from the culture filtrate by extraction with ethyl acetate. When ethyl acetate extracts of the pellet and supernatant obtained by centrifugation of the shake culture were added to the agar medium, inhibition of mycelial growth of F. culmorum was restored, especially with the extracts of the pelleted biomass. Autoclaving of the culture filtrate reduced the inhibition of F. culmorum but completely eliminated the inhibitory activity against Fusarium graminearum. Among the bunt fungi tested, spore germination of Tilletia tritici was more sensitive to the culture filtrate of S. antimycoticus FZB53 than spore germination of Ustilago avenae and U. tritici. Separation by thin layer chromatography (tlc) and spraying with different reagents showed that ethyl acetate extracts from shake cultures or biomass scraped from agar media contained several hydrophobic metabolites. When eluted from the tlc‐plates, the material from one of the spots had strong antifungal activity against spore germination of T. tritici and mycelial growth of F. culmorum, respectively. Ethyl acetate extracts from biomass of S. antimycoticus FZB53 prevented the growth of the tested Gram‐positive bacteria, namely Clavibacter michiganensis and different species of Bacillus. The results indicated that these bacteria were at least as sensitive towards the metabolites of S. antimycoticus FZB53 as F. culmorum. The tested Gram‐negative bacteria were not affected.