Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein

A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.Revisions requested/16 November 2004; Revisions received 12 November 2004/14 December 2004     

适合于咸宁基地生产的螺旋藻品系的选育研究

1　引　　言　　为综合利用螺旋藻资源,我国第一座利用高原碱水湖(ｐＨ8.6～9.3)、光、温、地热等自然资源[2]的中试基地在云南程海湖畔建成,并通过改造温棚和利用当地的地热在武汉咸宁建成第一个利用地热控温、半封闭、全循环、高洁净的螺旋藻生产基地.本项研究旨在选育一株适合于该基地特有自然条件生长的优良品系.2　自然概况与研究方法2.1　基地概况　　咸宁基地是由4个玻璃温室改建成的螺旋藻生产工厂,面积为1×104ｍ2.该基地结合当地自然条件和自然资源有如下特征:1)温度控制系统:热水资源(来自地热水)、冷水资源(来自流过工厂的小河)…     

The selection of suitable indices for the measurement and analysis of fish condition

A survey of the studies published in two leading fisheries journals revealed that the analysis and measurement of condition, based on length-weight data, has been performed using a wide variety of indices and statistical procedures. Eight forms of index were identified which can be categorized into those which measure the condition of individual fish, i.e. condition factors', and those which measure the condition of subpopulations as a whole, i.e. regressions of log, 10 weight on log, 10 length and the parameters of such regressions. Analysis of a test data set indicated that both the form of index and properties of the data set size can dictate the patterns of condition observed. The various indices were reviewed in terms of appropriateness, simplicity and statistical correctness. It was concluded that an index should be selected only after a detailed examination of both the underlying assumptions of the index and the properties of the data set.     

Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

The uptake of noxythiolin by a urinary isolate of Escherichia coli was examined initially at 37°C but the adsorption isotherm was complicated by the concomitant degradation of the compound. When drug adsorption was investigated at 4°C, to reduce the degradation rate of the compound, it was observed that noxythiolin was taken up by the urinary isolate in a linear fashion. The resulting adsorption patterns are discussed in relation to their possible classification. The implications of this uptake are considered with respect to the antimicrobial activity of noxythiolin.     

Methods for the preparation of protein-oligonucleotide-lipid constructs

A mixture of ionizable cationic lipids, steric barrier lipids, and colipids is used to encapsulate oligonucleotide DNA in lipidic particles called SALP. This material is under development as an adjuvant for vaccines. Previously we have shown that coupling the antigen directly to the surface of SALP can lead to enhanced immunological responses in vivo. Two different methods for preparing ovalbumin-SALP were assessed in this work. Originally the conjugates were prepared by treating SALP containing a maleimide-derivatized lipid with thiolated ovalbumin, a method we refer to as active coupling. This reaction was found to be difficult to control and generally resulted in low coupling efficiencies. The issues relating to this approach were characterized. We have recently developed alternative techniques based on first coupling ovalbumin to a micelle and then incubating the resultant product with SALP, methods we refer to as passive coupling. We have shown that this method allows accurate control of the levels of protein associated SALP and does not suffer from surface saturation effects seen with the active coupling method that places maximum limits on the amount of protein that can be coupled to the SALP surface. The products from the passive coupling protocol are shown to have activity comparable to those derived from the active coupling protocol in investigations of in vivo immune responses.     

Methods for assaying viability of frozen-thawed granulocytes.

Phagocytosis and microbial killing by granulocytes is a complicated process which is not yet completely understood. Innumerable in vitro and in vivo tests have been outlined for the several stages of granulocyte activity leading to microbial killing. No single simple test is sufficient to determine the nature of the lesion observed in abnormal frozen and thawed granulocytes, Several procedures are required to define such lesions before attempts can be made to inhibit or reverse this damage. The tests most commonly in use measure production, mobilization, chemotaxis, opsonization, phagocytosis, degranulation, peroxidation, and microbial killing. A test of microbial killing, either in vitro or in vivo, should always be used as the definitive assay.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Cytotoxicity testing of scaffolds potentially suitable for the preparation of three-dimensional skin substitutes

The preparation and study of three-dimensional functional skin substitutes has been the focus of intense research for several decades. Dermal substitutes are now commonly used in medical practice for a variety of applications. Here, we assess the toxicity of seven selected acellular dermal matrix materials to establish their potential for use in future three-dimensional skin substitute studies. The cytotoxicity of acellular dermis (of Allo- and Xenograft origin) prepared in our lab and biomaterials based on collagen and hyaluronic acid (Coladerm H and Coladerm H–L) were compared to that seen in three commercially available products (Xe-Derma, AlloDerm and Xeno-Impl). Murine fibroblasts NIH-3T3 and human dermal fibroblasts were used in cytotoxicity tests, with any resultant cytotoxic effects caused by the seven tested dermal scaffolds visualised using an inverted microscope system and confirmed in parallel using colorimetric MTT cell proliferation assays. While most of the dermal substitutes did not demonstrate a cytotoxic effect on our two cell types, Xeno and Xeno-Impl scaffolds clearly did. The cytotoxic effect of acellular Xeno dermal matrix could essentially be removed through a regime of multiple washes, but we were unable to remove the cytotoxic effect of Xeno-Impl. Thus, Xeno-Impl alone has been excluded from our future work on preparation of 3D skin substitutes.     

Production of purified alginates suitable for use in immunoisolated transplantation

Alginate is used as a matrix for immunoisolation of cells and tissues in vivo. We have demonstrated previously that commercial alginates contain various fractions of mitogenic impurities and that they can be removed by free flow electrophoresis. The use of purified material is a necessity in order to reveal the parameters that control biocompatibility of the implanted material (such as stability, size, surface charge and curvature, etc.). In this study, we present a protocol for the chemical purification of alginates on a large-scale. Beads made from alginates purified by this multi-step chemical extraction procedure did not induce a significant foreign body reaction when implanted for 3 weeks either intraperitoneally or beneath the kidney capsule of Lewis or non-diabetic BB/Gi rats. Correspondence to: U. Zimmermann     

The preparation of protected fragments of lysozyme for semisynthesis.

This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain...     

Parameters for evaluation of viability assays: accuracy, precision, specificity, sensitivity, and standardization

The selection of appropriate viability assays is critical in evaluating the efficacy of any cryopreservation procedure. The appropriateness of a given assay depends on the specific tissue and the function which is being optimized. Although a broad range of "viability" assays have been used, these assays can be classified in seven principle groups: (i) Morphological procedures, including routine histology, surface antigen localization, and transmission electron or scanning microscopy; (ii) proliferation studies; (iii) metabolic assays; (iv) implantation; (v) mechanical assays; (vi) motility; and (vii) DNA or RNA synthetic assays. Regardless of the class of assay, each assay may be further characterized as qualitative, quantitative, or quantal and each type may vary in the degree of subjectivity. In selecting a specific viability assay, biological variability, assay bias, and the statistical probability of both Type I and Type II errors should be considered crucial. Here we discuss a number of critical factors involved in validating viability assays, including accuracy, precision, standardization, specificity, sensitivity, selection of statistical methodology, and range of the assay.     

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

Background  

Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.     

Use of solubilizable acrylamide disulfide gels for isolation of DNA fragments suitable for sequence analysis

Acrylamide gels which are crosslinked by disulfide bridges are found to be an excellent electrophoretic medium for DNA restriction fragments over a wide size range. The gels are solubilized by mercaptoethanol and the fragments are rapidly and conveniently recovered on DEAE-cellulose columns. Fragment recovery appears nearly quantitative. The gel formulation described here requires polymerization at a temperature of about 40°C. Recovered fragments are active in all systems tested, including subsequent restriction, kinasing, ligation, nick translation, and sequencing by the method of A. M. Maxam and W. Gilbert (1980, in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 499–560, Academic Press, New York).