Preparation of Australia Antigen Free Human Albumin with Polyethylene Glycol

Albumin was prepared from human plasma diluted In 0.05M phosphate buffer pH 8.6, by the precipitation of approximately 95% of the associated plasma proteins with 357. polyethylene glycol. More than 70% of the albumin In the original plasma was recoverable as a viscous liquid on lowering the pH to 5.5. The albumin prepared by this technique is associated with 5 to 10% or and B globulin. Plasma, positive for Australia antigen (Au(SH)ag) yields an albumin preparation negative for the antigen.     

Purification and Characterization of Novel Antifungal Compounds from the Sourdough Lactobacillus plantarum Strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml⁻¹ was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml⁻¹ except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml⁻¹). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.     

Preparation and Characterization of Nanoliposomes Loaded with the Antimicrobial Peptide CGA-N9

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) are considered ideal antibiotic candidates, but their therapeutic ability and application are restricted...     

洋葱伯克霍尔德菌CF-66环二肽的分离纯化及抑菌活性研究

植物真菌病害给农业生产带来了巨大损失,因此对高效、低毒、低残留的生物农药的开发迫在眉睫。洋葱伯克霍尔德菌CF-66（Burkholderia cepacia CF-66）对真菌类病原菌具有强烈的抑制作用。其发酵液经减压浓缩和乙酸乙酯萃取得到粗提液,粗提液经反复硅胶柱层析和反相高效液相色谱（RP-HPLC）多步柱层析,首次分离纯化得到一种环二肽——cyclo(Phe-Pro)(cFP)。利用气质联用（GC-MS）系统和HPLC进行定性和定量,结果表明分离纯化物质呈单峰,纯度较高且经标准曲线算出其浓度约为15 mg/ml。MIC值的测定结果表明该物质对立枯丝核菌、黄瓜菌核、玉米弯孢病菌等植物病原菌及冻土毛霉、黄曲霉、米根霉等食品腐败菌均具有较强的抑制作用。经显微镜观察发现,该物质可使丝状真菌菌丝生长异常,菌丝由光滑细长变得粗糙、弯曲、短粗且顶端膨大呈泡状。     

Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.     

深海放线菌08A4的鉴定及其抗真菌活性产物研究

从南海深海分离得到1株放线菌08A4,其发酵产物具有抗植物病原真菌活性,分离纯化得到3个化合物,通过1H-NMR初步鉴定为抗霉素类物质。结合形态学鉴定方法与16S rDNA序列分析方法,鉴定该菌株为微白黄链霉菌(Streptomyces albidoflavus)。     

Characterization of a Phenazine-Producing Strain Pseudomonas chlororaphis GP72 with Broad-Spectrum Antifungal Activity from Green Pepper Rhizosphere

A new Pseudomonas strain, designated GP72, was isolated from green pepper rhizosphere and identified as a member of species Pseudomonas chlororaphis based on morphology; conventional biochemical and physiologic tests; Biolog GN system (Biolog Inc., Hayward, CA); and 16S rDNA sequence analysis. The secondary metabolites produced by this strain have shown broad-spectrum antifungal activity against various phytopathogens of agricultural importance in vitro. Two main antifungal substances produced by this strain proved to be phenazine-1-carboxylic acid and 2-hydroxyphenazine with further purification and structure elucidation based on ultraviolet-absorbent spectrum scanning, atmospheric pressure chemical ionization–mass spectrometry (APCI-MS) spectrum, and ¹H,¹³C nuclear magnetic resonance spectrums. Strain GP72 could produce quorum-sensing signaling molecules of N-butanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone, which were found to accumulate with different quantities in King’s medium B and pigment producing medium, respectively.     

Identification and Characterization of a Chitinase-Produced Bacillus Showing Significant Antifungal Activity

Strain MY75 is a gram-positive, aerobic, endospore-forming bacterium that can secrete high levels of extracellular chitinase (4.645 U/ml) when chitin powder exists as an inducer. This strain was identified as Bacillus licheniformis using the Biolog MicroLog microbial identification system and sequence analysis of 16S rDNA, gyrA and rpoB genes. Strain MY75 has the ability to inhibit the growth of Gibberella saubinetii and Aspergillus niger, two major pathogenic fungi in agriculture, and to restrain their spore germination completely. The chitinase was proved to play an important role in the strain’s antifungal activity.     

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m⁻² day⁻¹, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]⁻¹ day⁻¹, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS⁻¹ day⁻¹). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.     

Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma–Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4′-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.     

Novel Techniques for Identification and Characterization of Proteins Loaded on Gels in Femtomole Amounts

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H₂ ¹⁸O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the ¹⁸O/¹⁶O-labeled fragmentation ions in the PSD spectra.     

Characterization of a human B lymphocyte-specific antigen

A human B lymphocyte-specific antigen (B1) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence, cytotoxicity, and quantitative absorption, B1 was present on approximately 9% of the peripheral blood mononuclear cell fraction and >95% of B cells from blood and lymphoid organs in all individuals tested. Monocytes, resting and activated T cells, null cells, and tumors of T cell and myeloid origin were B1 negative. B1 was distinct from standard B cell phenotypic markers, including Ig and Ia antigen. Removal of the B1 positive population in peripheral blood eliminated all B cells capable or responding to pokeweed mitogen by maturation to Ig-producing cells.     

A Conjugate Gradient Algorithm with a Trust Region for Molecular Geometry Optimization

An algorithm is presented for the optimization of molecular geometries and general nonquadratic functions using the nonlinear conjugate gradient method with a restricted step and restart procedure. The algorithm only requires the evaluation of the energy function and its gradient, therefor less memory storage is needed than for other conjugate gradient algorithms. Some numerical results are also presented and the efficiency and behaviour of the algorithm is compared with the standard conjugate gradient method. We also present comparisons of both conjugate gradient and variable metric methods with and without the trust region technique. One of the main conclusions of the present work is that a trust region always improves the converge of any optimization method. A sketch of the algorithm is also given.     

加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定

细胞毒T淋巴细胞(CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用,因而特异性CTL的检测相当重要;而过去检测CTL的方法都是间接的,最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法,成为目前研究T细胞免疫应答的关键技术。报道一种简化的四聚体制备程序,利用该程序成功制备加载人巨细胞病毒(HCMV)抗原肽的HLA-A2四聚体,具有特异性结合CTL活性。HLA-A*0201重链基因是通过RT-PCR方法从HLA-A2⁺供者白细胞中克隆,进而以PCR方法构建在羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*0201(A2)重链胞外区原核表达载体, A2重组蛋白在大肠杆菌中得到高表达,主要以包涵体形式存在。加载抗原肽的可溶性A2单体是A2胞外区在轻链β2微球蛋白和HLA-A2限制性HCMV pp65_495-503抗原肽(NLVPMVATV,NLV)存在时通过稀释法复性获得,以BirA对其进行生物素化,然后以阴离子交换树脂纯化,得到的纯化A2-NLV单体与Streptavidin_PE按4:08比例混合形成四聚体,结合程度在85％以上,流式细胞仪分析显示该四聚体具有与HLA-A2+供者的特异性CTL结合活性。总之,这种简化的四聚体制备程序,不仅有利于该技术的推广,为特异性T细胞免疫研究建立必要的技术平台,而且A2-NLV四聚体在临床监测CMV特异性CTL水平等方面也有应用价值。     

Getting the Jump on the Development of Bullfrog Oil Microemulsions: a Nanocarrier for Amphotericin B Intended for Antifungal Treatment

Amphotericin B (AmB), a potent antifungal drug, presents physicochemical characteristics that impair the development of suitable dosage forms. In order to overcome the AmB insolubility, several lipid carriers such as microemulsions have been developed. In this context, the bullfrog oil stands out as an eligible oily phase component, since its cholesterol composition may favor the AmB incorporation. Thus, the aim of this study was to develop a microemulsion based on bullfrog oil containing AmB. Moreover, its thermal stability, antifungal activity, and cytotoxicity in vitro were evaluated. The microemulsion formulation was produced using the pseudo-ternary phase diagram (PTPD) approach and the AmB was incorporated based on the pH variation technique. The antifungal activity was evaluated by determination of minimal inhibitory concentration (MIC) against different species of Candida spp. and Trichosporon asahii. The bullfrog oil microemulsion, stabilized with 16.8% of a surfactant blend, presented an average droplet size of 26.50?±?0.14 nm and a polydispersity index of 0.167?±?0.006. This system was able to entrap AmB up to 2 mg mL^?1. The use of bullfrog oil as oily phase allowed an improvement of the thermal stability of the system. The MIC assay results revealed a growth inhibition for different strains of Candida spp. and were able to enhance the activity of AmB against T. asahii. The microemulsion was also able to reduce the AmB toxicity. Finally, the developed microemulsion showed to be a suitable system to incorporate AmB, improving the system’s thermal stability, increasing the antifungal activity, and reducing the toxicity of this drug.     

Molecular Characterization of a Variant of Bacillus anthracis-Specific Phage AP50 with Improved Bacteriolytic Activity

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.     

Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile¹⁷⁰ of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.     

Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of Sucrose

A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical β-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase. Received: 23 May 2000 / Accepted: 26 June 2000     

Characterization of a subset of human B lymphocytes interacting with natural killer cells

Tonsil B cells were analyzed for their capacity to interact directly with NK cells in vitro. A specific, direct interaction between NK cells and B cells could be detected by direct conjugation and by cold target inhibition using the B lymphoblastoid cell line BJA.B as a labeled target. The data further suggest that the B cell interaction with NK cells specifically activates the NK effectors and induces their production of IFN-gamma. The NK-interactive population of tonsil B cells were characterized as low-buoyant density cells (by Percoll gradient fractionation) that stained more brightly with Hoechst 33342, both characteristics of activated B cells. Immunofluorescent staining of NK cell-B cell conjugates allowed determination of the cell-surface antigenic phenotype of conjugate-forming B cells. B cell targets were ICAM-1bri, 4F2+, TfR+, CD32+, BB1+, and CD77-. They tended to be CD38-, but overlapped the CD38+ population. No correlation was seen with CD37, CD44, CD75, CD76, HC2, or Ig kappa. This phenotype is most consistent with a late activation stage of differentiation, just before and overlapping the expression of CD38. These B cells do not appear significantly sensitive to NK-mediated cytolysis, suggesting that NK cell cytokine synthesis and secretion (e.g., IFN-gamma) may be more important in the NK cell regulation of the humoral response.