Purification and properties of a major isoform of β-1,3-glucanase from pearl millet seedlings

A major isoform of β-1,3-glucanase from pearl millet seedlings was purified following ammonium sulfate precipitation, ion-exchange chromatography and gel filtration techniques. The enzyme had a molecular weight of 20.5 kDa on SDS–PAGE and was highly basic with a pI of 9.6. It was thermostable with a broad temperature optima for activity ranging from 37 to 70°C and had an optimum pH of 5.2. Mercuric chloride and para-chloromercuric benzoate inhibited completely the enzyme while manganese chloride activated it. Antibodies raised against the purified β-1,3-glucanase identified another protein with an apparent molecular weight of 30 kDa in western reactions. Significance of this enzyme in pearl millet–downy mildew host–pathogen interaction is discussed.     

DNA binding mediated by the wheat HMGa protein: a novel instance of selectivity against alternating GC sequence

The high-mobility-group (HMG) chromosomal protein wheat HMGa was purified to homogeneity and tested for its binding characteristics to double-stranded DNA. Wheat HMGa was able to bind to P268, an A/T-rich fragment derived from the pea plastocyanin gene promoter, producing a small mobility shift in gel retardation assays where the bound complex was sensitive to addition of proteinase K but resistant to heat treatment of the protein, consistent with the identity of wheat HMGa as a putative HMG-I/Y protein. Gel retardation assays and southwestern hybridization analysis revealed that wheat HMGa could selectively interact with the DNA polynucleotides poly(dA).poly(dT), poly(dAdT).poly(dAdT), and poly(dG).poly(dC), but not with poly(dGdC).poly(dGdC). Surface plasmon resonance analysis determined the kinetic and affinity constants of sensor chip-immobilized wheat HMGa for double-stranded DNA 10-mers, revealing a good affinity of the protein for various dinucleotide combinations, except that of alternating GC sequence. Thus contrary to prior reports of a selectivity of wheat HMGa for A/T-rich DNA, the protein appears to be able to interact with sequences containing guanine and cytosine residues as well, except where G/C residues alternate directly in the primary sequence.     

Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study

To understand the mechanism of quail eggshell biomineralization, we have performed two CaCO(3) precipitation experiments. In the reprecipitation experiments, supersaturated Ca(HCO(3))(2) was prepared by bubbling CO(2) through a slurry of biogenic CaCO(3) obtained from bleach-treated eggshell followed by filtration to obtain a clear solution for crystallization experiments. The nucleated crystals were collected at various time intervals and analyzed. In the second experiment, the extracted SOM from the bleach-treated eggshell was added to the supersaturated clear solution of Ca(HCO(3))(2) solution obtained by bubbling CO(2) gas through a slurry of synthetic CaCO(3) followed by filtration. The crystals/precipitates collected at various time intervals were analyzed. Both experiments showed that amorphous CaCO(3) (ACC) was precipitated in the early stages, which then transformed to the most stable crystalline calcite phase. Amino acid analysis of the soluble organic matrixes (SOM) indicated the presence of high amounts of Glx and Asx amino acids. Ovomucoid--an acidic glycoprotein, and lysozyme--a basic protein, are the two major components along with a few low molecular weight peptides present in the SOM of quail eggshell matrix. Both ovomucoid and lysozyme did not induce precipitation of the ACC phase in in vitro conditions, while the fraction containing low molecular weight peptides induced the precipitation of ACC, suggesting that the latter play an important role in the eggshell biomineralization. Thus, organisms can produce inorganic minerals which assume nonequilibrium morphologies and intricate architecture by precipitating transient ACC, which then transformed into the crystalline phase. Altogether, these observations further demonstrate that this strategy may be common in both vertebrate and invertebrate mineralized structures.     

Interobserver variation in breast cancer grading: a statistical modeling approach

OBJECTIVE: To study random and the systematic error in breast cancer grading, to find the source of disagreements and measure the reliability of graders so that appropriate corrective action can be taken. STUDY DESIGN: Five independent observers graded 50 breast carcinoma slides from 50 consecutive breast cancer specimens according to the Nottingham criteria. The polychoric correlation was used to measure association. Stuart-Maxwell and McNemar tests were used to measure equality of thresholds. RESULTS: The polychoric correlation among observers was high (mean = 0.803, 0.712, 0.797 and 0.602 for the final grade, tubule formation, nuclear pleomorphism and mitotic figures, respectively). However, there were significant differences in thresholds (6, 7, 7 and 9 pairs of 10 showing significant differences in classification of grades/scores for final grade, tubule formation, nuclear pleomorphism and mitotic counts, respectively). CONCLUSION: The high polychoric correlations suggest that random error in grading breast cancers in this study was low, confirming the underlying reliability of grading and graders. However, significant differences in the thresholds lowers raw agreement. Such a scenario may be rectified by increased intradepartmental discussion.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.