Simple Butyrate Esterase Stain for Monocytes

The esterases used to identify monocytes are best demonstrated using alpha-naphthyl butyrate as substrate. However, the reagents commonly used for this stain are time-consuming to prepare and are unstable. This report describes a quick, easy, and reproducible staining method using stable reagents which are readily available commercially but which may also be prepared in the laboratory.     

Low volume processing of protein blots in rolling drums

We have evaluated an improved method for processing protein blots on nitrocellulose or nylon membranes using cylindrical plastic containers. The method, which is directly analogous to the commonly used method of photographic processing in rolling drums, uses small values of reagents which are constantly washed over the blotting membrane by rotating the drum horizontally on a roller mixer. Volumes of reagents used are typically less than one-10th of those required for conventional methods using plastic bags or trays. The efficiency of probing and washing steps are greatly improved, giving an all-round increase in sensitivity, ease of processing, and economy of reagents.     

Intersectin 2 nucleotide exchange factor regulates Cdc42 activity during Xenopus early development

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than OR6. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the OR6 and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents.     

Electrospray mass spectra of three proprietary detergents

We have determined the major ingredients of the commercially available reagents M-PER, Y-PER, and B-PER from Pierce Chemical Co. using electrospray mass spectrometry. These three proprietary reagents have been widely used in the biochemical community as cell membrane dissolving tools during the initial step of protein purification. However, the identity and mechanism of these reagents remained unknown. In this paper, we identified these reagents as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-octyl-beta-d-thioglucopyranoside, respectively. In addition, we wish to stress here the increasing importance of the role of electrospray mass spectrometry in the analysis of such proprietary biological preparations which are increasingly finding their way into the biochemical literature.     

Anomalously Coupled Nucleosides

Abstract

Polyphosphoric amide reagents are used to prepare purines which are coupled with unprotected 2-deoxy-D-ribose at C-3 of the carbohydrate using tributylammonium polyphosphates in chloroform. Phthalimide can be used instead of purines to produce N-protected 3-amino-2,3-dideoxypentoses.     

High-throughput DNA synthesis in a multichannel format.

We describe an approach to high-throughput parallel DNA synthesis in which a multiwell format is used. The reactions are carried out in open wells using an argon ambient atmosphere to prevent reagent contamination. The controlled-pore glass beads which form the substrate for synthesis are held in individual wells with high-density polyethylene filter bottoms through which reagents are drawn into a vacuum manifold. The synthesis is carried out using direct reagent dispensing into the individual reaction wells. A computer controls the sequence in which reagents are dispensed and the timing of the periodic vacuum pulses required to synthesize the desired sequence. Experiments to date have demonstrated the viability of the approach for a variety of test sequences. Results obtained with HPLC analysis demonstrate coupling efficiencies as high as 99.5% under optimized conditions. Use of the oligomers for DNA sequencing templates and as PCR primers has been demonstrated in production applications. The current instrument design consists of a series of discrete reaction chambers in a 12 channel module which can be multiplexed in a 12 x n format where n can be 1-8, i.e. 96 wells. A projected time interval for 12 parallel syntheses is 2.5 h, with 96 syntheses in 3.5 h. Because of the reduced volume of reagents required in the open well format, significant cost savings are projected.     

A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers

A new procedure for the production of a defined library of random mutants is described. Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three 'wrong' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol. Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents. Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent 'marker' base can be included that allows an early estimate of the mutagenesis efficiency. The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing. This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase. Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase. This approach provides a more complete library than a method using chemical mutagenic reagents.     

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.     

A Rapid lmmunostaining Method for Frozen Sections

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using “Enhanced Polymer One-step Staining” (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.     

Functional residues on the enzyme active site of glyoxalase I from bovine brain

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.     

Evaluation of ready-to-use liquid reagents for clinical chemistry in laboratory animals.

We evaluated the ready-to-use liquid reagents for clinical chemistry (6 tests), to assess their suitability for use in the toxicology laboratory setting. Hitachi 736 automated analyzer was used for the analyses. The evaluation included the following studies: Precision, Linearity, Effects of interference substances such as hemolytic hemoglobin, bilirubin, turbidity to the analytical values and correlation to the solid reagents, which are prepared each time they are needed. The precision and linearity data were within the reagents' specifications. Results of comparison of the liquid reagents and the solid reagents in analyzing plasma samples of rats, dogs and monkeys were generally good except for a bias in results for GOT and GPT, regardless of the animal species tested. It is concluded that these types of liquid reagents can be used in clinical pathology examinations in animal studies.     

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.     

Passive immunization for the treatment and prevention of HIV infection.

Passive immunization using serum or immunoglobulin preparations has been used in the prophylaxis and treatment of many bacterial and viral diseases. Preliminary attempts to use these methods to prevent HIV infection in chimpanzees have been promising. With the identification of polyclonal and monoclonal antibodies with protective activity against HIV in in vitro systems, the possibility of using these reagents in vivo takes on new relevance. The potential and problems of using passively administered anti-HIV antibodies for HIV prophylaxis and treatment are discussed, as well as the relative merits of polyclonal versus monoclonal reagents.     

Proteomic analyser with applications to diagnostics and vaccines

This paper describes a method for proteomic analysis with applications to diagnostics and vaccines. A panel of N (> or = 1) reagents called X(j), with j = 1 to N, is used. The binding strength of each of the X(j) reagents to each other is measured, for example by an ELISA assay, giving an N x N matrix K. The matrix K is used to define another set of N reagents called Y(j), with j = 1 to N, each of which is a linear combination of the X(j) reagents and each of which is tailored to be complementary to one of the X(j) reagents. Each of the N pairs of reagents X(j) and Y(j) defines an axis in an N-dimensional shape space. The definition of these axes facilitates proteomic analysis of diverse biological samples, for example, mixtures of proteins such as serum samples or T cell extracts. A method for defining and measuring similarity between pairs of biological samples and between sets of biological samples in the context of the set of N reagent pairs is described. This leads to methods for using the N reagent pairs in the diagnosis of diseases and in the formulation of preventive and therapeutic vaccines. The relationship of this work to previous research on shape space is discussed.     

Biochemical engineering: cues from cells

Engineering principles are used in the exploitation of biocatalysts derived from cells. The purity of reagents, catalysts and maintenance of operation variables are extremely important for bioengineering systems. Any change in the purity of reagents or in operation variables usually leads to a dramatic decrease in productivity. Cellular systems, however, are able to work with relatively high impure conditions and increase their productivity in response to external signals. Thus the seemingly disordered 'bag of juice' or cytoplasm has more order and much higher order of integration than first appears. Learning the semantics of this paradoxical ability of order and integration would help bioengineers to understand and enhance productivity even using impure reagents.     

RNAi Transfection Results in Lipidome Changes

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock‐down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non‐targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane‐associated phenomena.     

Preparation of arylzinc reagents and their use in the rhodium-catalyzed asymmetric 1,4-addition for the synthesis of 2-aryl-4-piperidones

A protocol for the catalytic asymmetric synthesis of 2-aryl-4-piperidones with high enantiomeric excess (ee) (typically > or = 99% ee) has been described here. The preparation of arylzinc reagents, which are used as nucleophiles in catalysis, is also included. The whole protocol can be completed in 10-20 h, starting from the preparation of the arylzinc reagents, depending on the reaction time of the rhodium-catalyzed process. A detailed protocol is described using the preparation of 4-fluorophenylzinc chloride and its addition to benzyl 3,4-dihydro-4-oxo-1(2H)-pyridinecarboxylate in the presence of [RhCl((R)-binap)]2 as an example.     

A novel, mild, specific and indirect maleimido-based radioiodolabeling method. Radiolabeling of analogs derived from parathyroid hormone (PTH) and PTH-related protein (PTHrP).

In an effort to design a mild, non-oxidative and site-specific means of radiolabeling bioactive molecules we have employed maleimido-sulfhydryl chemistry to produce bioactive hormone radioligands. We have prepared two novel radioiodolabeled reagents, 3'-maleimidopropanoyl-3-125I-tyramide and its retro analog, N-maleoyl-N'-3-(4-hydroxy-3-125I-phenyl)propanoyl ethylenediamide, by either oxidative radioiodination of the precursors or radiolabeling of the phenolic component prior to its incorporation into the radiolabeling reagents. These reagents were then used to radiolabel analogs of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) in an efficient way, yielding reaction mixtures which were easily purified. The radioligands obtained are stable upon storage and bind in a reversible manner to a single population of binding sites displaying affinity in the low nanomolar range. The potencies of these analogs are comparable to the non-modified PTH and PTHrP analogs. This study demonstrates the utility of the novel maleimido-based indirect radioiodination approach and highlights some of its advantages over either direct oxidative procedures or acylation using the Bolton-Hunter reagent.     

Guanidination of a peptide side chain amino group on a solid support

A novel guanidination method of converting a peptide side chain amino group to a guanidino group on a solid support is described. Four guanidinating reagents were evaluated using a model tetrapeptide attached to a polystyrene resin. Experimental data indicate that the two nitroguanidinating reagents, but not the two tosylguanidinating reagents, can be used effectively in solid phase peptide synthesis.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.