Eimeria saudiensis N. Sp. (Apicomplexa: Eimeriidae) from the Arabian Oryx (Oryx leucoryx) in Saudi Arabia

Oocysts of Eimeria saudiensis n. sp. (Apicomplexa: Eimeriidae) are described from the feces of the Arabian oryx, Oryx leucoryx , from the Riyadh Zoo, Saudi Arabia. The oocysts were ellipsoidal or slightly ovoid, 31.2 times 24.5 (24.3–36.5 times 20.0–27.6) μm with a bilayered wall about 1.7 μm thick. The micropyle was covered by a dome-shaped cap. The oocyst residuum was absent, but tiny polar granules were present. The sporocysts were elongate ovoid, 14.3 times 7.2 (11.5–18.5 times 6.0–9.0) μm, had a Stieda body, but lacked a substiedal body. The sporocyst residuum was present, composed of numerous small granules. The sporozoites were elongate club-shaped, and contained two prominent refractile bodies.     

Different sequence environments of cysteines and half cystines in proteins. Application to predict disulfide forming residues.

Protein sequences are often derived by translating genetic information, rather than by classical protein sequencing. At the DNA level cysteines and half cystines are indistinguishable. Here we show that the sequential environments of 'free' cysteine and half cystine are different. A possible origin of this difference is discussed and a simple method to predict cysteines and half cystines from the amino acid sequence is also presented.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Nutrient Leaching and End Product Accumulation in Plastic Composite Supports for L-(+)-Lactic Acid Biofilm Fermentation

Investigations on the leachate bioavailability, leaching rate, and lactic acid accumulation properties of plastic composite supports (PCS) were essential for large-scale or long-term lactic acid fermentation. Leachates from PCS and polypropylene discs (controls) were analyzed by the micro-Kjeldahl method; by absorbances at 260, 275, and 280 nm; and by bioassays with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The amount of leached nitrogen in a 20-ml initial soaking solution had a high correlation with the soaking solution's cell density (r = 0.87) and absorbance at 260 nm (r = 0.95). Leaching rates of various PCS were evaluated by 20 20-ml simulated repeated-batch fermentations (RBF). PCS with only yeast extract as the minor agricultural ingredient had a high leaching rate and leached out 51 to 60% of the total nitrogen during the first RBF. PCS blended with dried bovine albumin, dried bovine erythrocytes, and/or soybean flour had slowed nutrient leaching (20 to 30% of the initial leached nitrogen). Hence, they could still maintain 1 g of lactic acid per liter and measurable cell density (absorbance at 620 nm, 0.4 to 0.6) at the 20th 20-ml RBF. Lactic acid accumulation properties of PCS were evaluated by soaking the supports in a 30% lactic acid solution for 72 h at 45(deg)C. The lactic acid-soaked supports were rinsed three times and then heat treated (121(deg)C, 15 min) in 15 ml of deionized water. The results showed that lactic acid accumulation in PCS was mainly due to absorption and had no correlation with lactic acid production or biofilm formation.     

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients.To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation.In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study.Acute leukemia (AL)¹ is the most common childhood cancer, accounting for a quarter of all pediatric malignancies (1). Accumulated chromosomal translocations and mutations in proto-oncogenes alter proliferation, differentiation, apoptosis and death in developing hematogones, ultimately leading to the development of leukemia (2, 3). The most recent understanding of these cancer-related changes is based on molecular genetic studies that focused primarily on DNA and mRNA alterations. High-throughput molecular genetic technologies, such as mRNA expression profiling and next generation sequencing, are widely used in clinical research. These techniques can provide new classification schemes, define new prognostic subgroups and outline the background of some pathological mechanisms (2, 4, 5, 6, 7) but they cannot easily elucidate the functional consequences at the cellular level. Proteins are the principal carriers of cellular functions. Thus, the analysis of proteins and protein modifications can elucidate the pathological mechanisms of leukemia or clarify the response mechanisms to current and emerging therapies. Currently, flow cytometry is used in clinical laboratories to analyze dozens of proteins that are expressed by leukemic cells (8, 9). These proteins, which are mostly surface CD markers, can reflect lineage commitment, developmental status and even the underlying genetic lesion (10, 11) but they do not carry information about the intracellular processes that control malignant transformation. Moreover, many cancer alterations are manifested only at the functional level, including changes in subcellular localization, post-translational modification (e.g. phosphorylation), protein cleavage, or protein–protein interactions (12). Proteomic techniques that can capture disease-associated changes are needed. Mass spectrometry (MS) is presently the technique of choice for large-scale proteomic analysis. MS can uncover thousands of molecules without an a priori probe selection, e.g. new disease-associated features in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (13, 14). Despite its tremendous analytical power, MS is complex and not widely accessible. Unlike MS, affinity proteomics is a simple technology suitable for large-scale protein analysis in primary cancer samples in the clinical laboratories. Recently, a technique linking size exclusion chromatography (SEC) to microsphere-based antibody arrays (microsphere-based affinity proteomics (MAP)) has been developed (15, 16). SEC-MAP enables the detection of hundreds of proteins in a single sample and provides essential information about protein size. Because only five to ten million cells are necessary, SEC-MAP can serve as a sensitive, sample-sparing and high-content tool for protein profiling in leukemia samples probing the relative amounts of different proteins, as well as protein size and cleavage (17). Our in-house-assembled MAP array is a set of 1152 populations of fluorescent-labeled microbeads, each carrying an antibody against a single human antigen. Native cellular proteins (and their complexes) are isolated from cellular compartments using detergents, labeled with biotin (biotin-PEO4-NHS) and subjected to SEC to obtain 24 size fractions. The SEC fractions are incubated with MAP microbeads, and antibody-protein binding is detected using phycoerythrin (PE)-labeled streptavidin with flow cytometry. The flow cytometer resolves the color code of each microbead population and reads the amount of bound protein. The data from 24 SEC fractions are combined, and a protein''s binding relative to its size is detected as a “protein entity.” Data are analyzed with in-house R-based software. This approach permits automatic batch processing of raw flow cytometry standard (FCS) files in addition to advanced analyses including quality control steps (the minimal number of microspheres required in a population and the unimodality of the signal in the PE channel is checked) (17). We wanted to find out whether SEC-MAP can be used in the clinical laboratory to bring a biologically important information, e.g. to classify acute leukemias or to find the marker with a prognostic relevance. We assembled MAP arrays to carry antibodies against proteins that are known to be important for leukemia diagnostics (18, 9) and against components of intracellular signaling networks (16). Through extensive testing on leukemia samples, we have identified antibodies that are suitable for immunoprecipitation-based techniques. Furthermore, we have improved the software tools to allow for large-scale data normalization, fast automatic protein entity detection with manual correction, and the discovery of differentially expressed entities in multiple samples. Using innovative software tools, we have identified entities that were differentially expressed between particular AL subgroups. To ensure the specificity we have validated the data collected by SEC-MAP with classical flow cytometry-based immunophenotyping (FACS), Western blot (WB) and quantitative real-time PCR (qRT-PCR). Moreover, we have addressed practical sample processing issues related to patient material handling and logistics. Based on the protein size profile, we were able to discriminate proteolytically degraded samples from those with an uncleaved proteome. Importantly, proteolysis would be missed by conventional protein load controls in Western blots. Thus, the SEC-MAP array was demonstrated to be a useful, reproducible and accurate high-content proteomic tool for the assessment of primary leukemia samples.     

Characterization of Plant Alcohol Dehydrogenase

The final activity of the alcohol dehydrogenase (E.C.1.1.1.1, abbreviated ADH) from germinating pea, isolated by fractionating with ammonium sulphate, chromatography on DEAE cellulose and gel filtration, was 80,000, from bean 25,000 and from lentil 13,500 units per mg protein. Molecular weights of the ADHs are close to each other: pea and bean ADH 60,000, lentil ADH 70,000. The K_m values are mutually similar with three enzymes, i.e. of the order of 10⁻⁴M for NAD and 10⁻²M for ethanol. The pH optima lie in the alkaline region. These enzymes catalyse oxidation of a number of monovalent alcohols. At temperatures above 60°C the enzymes are thermally unstable. Stability is enhanced slowly by ethanol but not by NAD. Pyrazol, imidazol and pyridine inhibit plant ADH similarly to the enzyme from horse liver. There is a similarity between plant alcohol dehydrogenases and animal and yeast enzymes.