Comprehensive Discovery and Quantitation of Protein Heterogeneity via LC-MS/MS Peptide Mapping for Clone Selection of a Therapeutic Protein.

Development of biopharmaceutical production cell lines requires efficient screening methods to select the host cell line and final production clone. This is often complicated by an incomplete understanding of the relationship between protein heterogeneity and function at early stages of product development. LC-MS/MS peptide mapping is well suited to the discovery and quantitation of protein heterogeneity; however, the intense hands-on time required to generate and analyze LC-MS/MS data typically accommodates only smaller sample sets at later stages of clone selection. Here we describe a simple approach to peptide mapping designed for large sample sets that includes higher-throughput sample preparation and automated data analysis. This approach allows for the inclusion of orthogonal protease digestions and multiple replicates of an assay control that encode an assessment of accuracy and precision into the data, significantly simplifying the identification of true-positive annotations in the LC-MS/MS results. This methodology was used to comprehensively identify and quantify glycosylation, degradation, unexpected post-translational modifications, and three types of sequence variants in a previously uncharacterized non-mAb protein therapeutic expressed in approximately 100 clones from three host cell lines. Several product quality risks were identified allowing for a more informed selection of the production clone. Moreover, the variability inherent in this unique sample set provides important structure/function information to support quality attribute identification and criticality assessments, two key components of Quality by Design.

Antizyme mRNA distribution and regulation in rat small intestinal enterocytes.

The protein ornithine decarboxylase antizyme (AZ) is inhibitory to both polyamine transport and synthesis. Experiments were performed to examine the distribution and regulation of AZ mRNA in cells of the small intestinal epithelium, a tissue exposed to high concentrations of extracellular polyamines and high levels of ornithine decarboxylase (ODC) activity. AZ mRNA was expressed in acutely isolated epithelial cells of rat jejunum and ileum; expression was higher in proximal than distal small intestine. In cells isolated from jejunal crypt-villus axis, AZ was expressed to high levels in cells from the small intestinal crypts but the message fell to near undetectable levels in cells of the villus tip. Western blot analysis demonstrated that distribution of AZ protein followed the distribution of AZ message. The distribution of ornithine decarboxylase activity along the crypt-villus axis was also determined. ODC activity and ODC protein were higher in cells from the upper villus than in cells isolated from the crypt. The intestinal lumen contains extremely high concentrations of free polyamines. The effect of depletion of endogenous polyamines or the addition of exogenous polyamines on AZ mRNA was evaluated in IEC-6 cells. Cells were depleted of intracellular polyamines by 72 hr of incubation in difluoromethylornithine. The fall in intracellular polyamine content was accompanied by a corresponding fivefold fall in AZ mRNA. When polyamine-depleted cells were treated with putrescine, the level of the AZ mRNA transcript was increased ninefold. These data demonstrate the expression of AZ gene in the longitudinal and crypt-villus axes of rat small intestine and show that AZ gene transcription is modulated by polyamines, an effect which may be involved in product suppression of polyamine synthesis.

Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana.

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.

A rapid and accurate closed-tube immunoassay for platelets on an automated hematology analyzer.

Accurate and precise platelet counts are important for patients with severe thrombocytopenia or who are receiving chemotherapy. We developed a novel flow cytometric analysis of platelets that may be particularly valuable for assessing the necessity for platelet transfusions. This ImmunoPlt (CD61) assay is based in part on CD61 monoclonal antibody labeling and has been automated and implemented on the CELL-DYN 4000 hematology analyzer. It is well suited for thrombocytopenic specimens, since it reduces interference by nonplatelet particles. It takes less than 5 minutes from closed-tube aspiration to report. Data for more than 350 thrombocytopenic specimens demonstrate that the ImmunoPlt (CD61) assay is more accurate than the optical scatter or the impedance count for specimens with platelet counts between 1 and 60 x 10(3)/microL (1 and 60 x 10(9)/L). The ImmunoPlt (CD61) assay is more precise than the optical scatter or the impedance count for specimens with platelet counts between 1 and 50 x 10(3)/microL (1 and 50 x 10(9)/L).

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Resistance to osmotic stress of horse spermatozoa: the role of ionic pumps and their relationship to cryopreservation success.

To study the resistance of horse spermatozoa against hyperosmotic stress, cells were incubated in solutions of 600 to 4000 mOsm(undisturbed media). Then, semen was immediately placed into an iso-osmotic solution (disrupted media). Incubation in undisturbed media decreased sperm viability in an osmolarity- and temperature-dependent manner. Viability was further decreased in disrupted media, with the effect dependent upon the initial osmolarity of the media and on the temperature. Treatment with ouabain or amiloride impaired the resistance of horse spermatozoa to hyperosmotic stress. Very few correlations were strong between viability after hyperosomotic stress and quality parameters of fresh and frozen-thawed horse semen. The results indicate that the usefulness of resistance to hyperosmotic stress in assessing frozen-thawed semen quality is compromised, since other factors are involved in the resistance to freezing-thawing. Both Na (+)K (+) ATP-ase and the Na (+)H (+) antiporter act in the resistance to hyperosmotic stress in horse spermatozoa.

The development of a blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats (Rattus norvegicus, Berk.).

1. A new test for identifying levels of difenacoum resistance in the Norway rat is described, based upon the differential physiological response to difenacoum administration. 2. This test is based on changes in blood clotting activity over 4 days, following administration of the rodenticide difenacoum in conjunction with menadione (vitamin K3). 3. The anticoagulant effect is reduced only in rats that are resistant or tolerant to difenacoum. 4. This test procedure is quicker than traditional feeding tests, and identifies the degree of resistance in both laboratory and wild rats that have difenacoum resistance genes.

Laboratory trials of five rodenticides for the control of Mesocricetus auratus Waterhouse.

The efficacy of five rodenticides for use in bait against the golden hamster (Mesocricetus auratus Waterhouse) was investigated in the laboratory. The species proved to be resistant to warfarin (up to 0.5%) and difenacoum (0.005%), but brodifacoum (0.005%) gave complete mortality after three days' feeding. Calciferol (0.1%), though toxic, was significantly unpalatable. Zinc phosphide (5.0%) presented in a choice test for two days against unpoisoned feed gave 100% mortality, and appears to be the most suitable of these compounds for the control of M. auratus in the field.

Laboratory tests of seven rodenticides for the control of Meriones shawi.

The response of Meriones shawi to seven rodenticides was investigated in laboratory feeding tests. The species proved to be much less susceptible to anticoagulants than most other species of rodent pests. Brodifacoum (at 0.005%), although giving complete mortality after only 8 days' continuous feeding, was more toxic than warfarin (0.025%), coumatetralyl (0.0375%), difenacoum (0.005%) and bromadiolone (0.005%). Calciferol (0.1%), though toxic, was significantly unpalatable. Zinc phosphide (5.0%) presented for 2 days in a choice test against unpoisoned food gave 80% mortality and appears to be the most suitable of these compounds for the control of M. shawi in the field.

A cyanine dye distinguishes between cycling and non-cycling fibroblasts.

Cellular proliferative activity has previously been determined by measuring the incorporation of radiolabelled nucleotides or by visual inspection of cellular morphology. Although two flow cytometric methods have recently been developed which can distinguish cycling from non-cycling cells, both have serious disadvantages. One method requires uptake of a substantial amount of BUdR, limiting its usefulness for in vitro systems. The other method utilizes RNA/DNA content differences but its successful application has proved cell-type dependent. We have now used the findings that the cell membrane is more highly polarized in resting than in proliferating cells and that cyanine dyes carrying a delocalized positive charge enter live cells to an extent that depends on the cell membrane potential, to develop a method of distinguishing between cycling and non-cycling cells. The greater the membrane polarization, the greater is the concentration of dye within the cell. At high concentrations, the dye molecules aggregate and their fluorescence is quenched. Thus, for a given external dye concentration, cells of different membrane potential would accumulate different amounts of fluorescent (non-aggregated) dye. Using fibroblasts in culture conditions chosen to provide various models of cycling and non-cycling cells, we found that fluorescence intensity with the dye, 3,3'-diheptyloxycarbocyanine (Di-O-C,(3)) was consistently greater in the former than the latter.

Laboratory trials of seven rodenticides for use against the cotton rat (Sigmodon hispidus).

The efficacy of seven rodenticides for use against Sigmodon hispidus was investigated in the laboratory. The poisons (warfarin, coumatetralyl, difenacoum, brodifacoum, bromadiolone, calciferol and zinc phosphide) were all toxic at the concentrations normally used against Rattus rattus and R. norvegicus and all were palatable. Trials are now needed to confirm the efficacy of these poisons in the field, but it seems likely that, if used in suitable bait formulations, they would all be useful for the practical control of S. hispidus.

Laboratory and field evaluation of pyriminyl as a poison for Rattus norvegicus.

Laboratory tests indicated that the optimum concentration for pyriminyl in rat baits was between 1% and 3%. In field trials in which 0.5% pyriminyl (the concentration in commercial use) was compared with 2.5% zinc phosphide for the control of rats on farms, the pyriminyl treatments were significantly less effective than the zinc phosphide even when the poisoned baits were left down for 7 days instead of 1 day after prebaiting. Both poisons were as effective in medium oatmeal bait as they were in medium oatmeal containing 5% corn oil and 5% sugar.

Laboratory evaluation of bromadiolone as a rodenticide for use against warfarin-resistant and non-resistant rats and mice.

Laboratory feeding tests were carried out to determine the efficacy of the anticoagulant rodenticide bromadiolone against Rattus norvegicus, R. rattus and Mus musculus. Using 0.005% bromadiolone, complete kills of R. norvegicus and R. rattus not resistant to warfarin were obtained after exposure to the poison for 1 and 5 days respectively. Warfarin-resistant R. norvegicus were all killed in 4 days, and resistant M. musculus in 12 days. In general, the results resembled those obtained with difenacoum. Acceptance of bromadiolone was very good.

Laboratory test of seven rodenticides for the control of Mastomys natalensis.

Laboratory feeding tests were carried out to assess the efficacy of seven rodenticides against Mastomys natalensis. The poisons (warfarin, coumatetralyl, difenacoum, brodifacoum, bromadiolone, calciferol and zinc phosphide) were all toxic at the concentrations normally used against Rattus norvegicus (Berk.), although several were unpalatable. Trials are now needed to demonstrate the relative efficacy of these poisons in the field, but it is likely that, given suitable bait formulations, they would all be useful as practical control agents.

Warfarin-resistance genotype determination in the Norway rat, Rattus norvegicus.

The 2-stage determination is based on changes in blood coaggulation activity brought about both by the administration of warfarin in conjunction with vitamin K1 epoxide and by feeding a vitamin K-free diet for 4 days. When it was applied to laboratory-bred rats of known warfarin-resistance genotype, 35/35 homozygous susceptible, 44/44 homozygous resistant and 131/133 heterozygous rats were correctly classified. This method was equally effective in identifying the genotype of wild rats carrying the warfarin-resistance gene, Rw2. The procedure is rapid and accurate.

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.

The development and use of a test to identify resistance to the anticoagulant difenacoum in the Norway rat (Rattus norvegicus).

Feeding tests were carried out in the laboratory to obtain basic data on the susceptibility of wild Norway rats to difenacoum. The results were used to derive a standard test procedure for the identification of difenacoum resistance in warfarin-susceptible and resistant rats. Details are given of tests on rats from suspected difenacoum-resistant infestations on farms.

A comparison of acridine orange and Feulgen cytochemistry of human tumor cell nuclei.

Specimens of cells derived from tumors of the human female genital tract plus normal cells as standards have been divided into aliquots and stained according to acridine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nm nuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor cell:normal cell ratio of mean nuclear fluorescence following acridine orange staining was greater than the tumor cell:normal cell ratio of mean nuclear absorbance following Feulgen staining. The tumor cell:normal cell ratio of mean nuclear fluorescence ranged from 2.3 for a nonkeratinizing squamous cell carcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor cell:normal cell ratio of mean nuclear absorbance ranged from 1.4 for a mixed mesodermal sarcoma to 2.3 for a small cell squamous cell carcinoma. These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cells cannot be explained solely on the basis of elevated Feulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal binding substrate for intranuclear acridine orange and that the DNA of certain tumor cells is more accessible to acridine orange than is the DNA of normal cells.