Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.     

Propylene Polymerization Behavior of Fluorinated Bis(phenoxy‐imine) Ti Complexes with an MgCl2‐Based Compound (MgCl2‐Supported Ti‐Based Catalysts)

Summary: Fluorinated bis(phenoxy‐imine)Ti complexes 1 – 3 combined with MgCl₂/i‐Bu_nAl(OR)_3−n (MgCl₂‐supported catalysts) were able to polymerize propylene in a living fashion at room temperature to provide slightly to highly syndiotactic poly(propylenes) (PPs) with extremely narrow distributions of molecular weight. These represent the first examples of MAO‐ and borate‐free group 4 metal‐based living catalysts. The supported complexes 2 and 3 formed PPs with higher syndiotacticity and T_m's than the corresponding homogeneous MAO‐activation systems (e.g., 3 : rr 97%, T_m 155 °C; MAO activation: rr 93%, T_m 152 °C). The measured T_m of 155 °C represents the highest known T_m for syndiotactic PPs synthesized at room temperature.

Polymerization of propylene to poly(propylene) with supported Ti‐based catalysts.     

Cloning of a Novel Babesia equi Gene Encoding a 158-Kilodalton Protein Useful for Serological Diagnosis

In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.     

Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.     

Association between thyroid cancer of cribriform variant and familial adenomatous polyposis.

A case of a 20 year old Japanese woman who developed thyroid cancer exhibiting unusual cribriform structures while being followed up for familial adenomatous polyposis/Gardner's syndrome is reported. The patient presented with osteomas, pigmented retinal lesions, and adenomas of the duodenum and the papilla of Vater, in addition to numerous adenomatous polyps in the colorectum. On ultrasonography, the thyroid cancer was localised to the right lobe and was identified as an irregular, internal echo tumour with a peripheral hypoechoic zone, measuring 1.8 cm in diameter. Histological examination of the resected tumour showed a concomitance of papillary proliferation and cribriform structures with follicles of varying sizes. These features can be distinguished from sporadic thyroid cancer.     

Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases.

A new monoclonal antibody (MoAb) HA58 (IgG1) was prepared, which recognizes the binding site on the intercellular adhesion molecule-1 (ICAM-1) antigen to the lymphocyte function-associated antigen-1 (LFA-1). The double-determinant immunoassay (DDIA) was established with use of MoAb HA58 and another anti-ICAM-1, MoAb CL207, to detect the soluble, shedding ICAM-1 antigen. Human recombinant interferon-gamma (IFN-gamma) induced not only the expression of cell surface ICAM-1, but also the shedding ICAM-1 antigen in an IFN-gamma concentration-dependent and incubation-time-dependent manner. DDIA was applied to detect the shedding ICAM-1 antigen in the sera of patients with malignant or benign diseases. The incidence of positivity for ICAM-1 antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM-1 antigen. These findings suggest that serum ICAM-1 antigen may be a useful marker to monitor tumor burden in cancer patients.     

Effect of short physical exercise on the levels of zinc and carbonic anhydrase isoenzyme activities in human erythrocytes

Summary Studies of the influences of physical exercise of short duration (bicycle ergometer, 200 W for 30 min) on the activities of carbonic anhydrase isoenzymes (CA-B and CA-C types) and zinc concentration in erythrocytes were made on 5 untrained healthy male volunteers. The subjects were rested for 30 min after the exercise. There were significant decreases in the levels of zinc, CA-B, total carbonic anhydrase activity and CA-B-dependent activity immediately after exercise, but after 30 min of rest they all returned to their pre-exercise levels. No significant change in CA-C level or CA-C-dependent activity was found after exercise. Immediately after exercise, total carbonic anhydrase activity and CA-B-dependent activity following the addition of Zn²⁺ showed significant increases, compared with their respective activities without Zn²⁺ addition. However, no such effects were observed just before exercise or after rest; the addition of Zn²⁺ had no effect on CA-C-dependent activity at any time. A significant correlation was found between the changes in concentration of zinc and CA-B-dependent activity after exercise (r=0.711). The findings of the present study suggest that active CA-B enzymes are converted in part to inactive enzymes during acute physical exercise, possibly by decreased zinc binding. Moreover, the change in CA-B-dependent activity correlated well with the changes in pH and HCO₃ concentrations in venous blood (r=0.853 and r=0.718, respectively). One may speculate that an adaptive decrease in CA-B-dependent activity in erythrocytes occurs with increased acidification in blood during heavy physical exercise of short duration.The present study was presented to the Fifth International Symposium on the Biochemistry of Exercise, Boston, June 1–5, 1982