Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

Histamine release from rat mast cells by Vibrio vulnificus protease

Abstract Vibrio vulnificus protease (VVP) stimulated histamine release from isolated mast cells in a dose- and temperature-dependent manner within a range of 0.2–4.0 μ g/0.5 ml. Histamine release was accompanied by degranulation, and no leakage of lactate dehydrogenase from cells was observed, indicating that the histamine release was not due to cytolysis but to exocytosis. This release, completed within 30 s at 37°C, suggested that the mechanism of action of VVP on mast cells is different from that of other proteases, such as trypsin or α-chymotrypsin, which release histamine from the cells slowly.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes

Bovine liver catalase was encapsulated in an aqueous phase of the phospholipid vesicle (liposome) to improve the stability of its tetrameric structure and activity. The catalase-containing liposomes (CALs) prepared were 30, 50 and 100 nm in mean diameters (CAL₃₀, CAL₅₀ and CAL₁₀₀, respectively). The CAL₁₀₀ included the types I, II and III based on the amounts of catalase encapsulated. The CAL₃₀, CAL₅₀ and CAL₁₀₀-I contained one catalase molecule per liposome, and the CAL₁₀₀-II and CAL₁₀₀-III on average 5.2 and 17 molecules, respectively. The storage stability of catalase in either CAL system was significantly increased compared to that of free catalase at 4 °C in a buffer of pH 7.4. At 55 °C, free catalase was much more deactivated especially with decreasing its concentration predominantly due to enhanced dissociation of catalase into subunits while it was so done at excessively high enzyme concentration mainly due to enhanced formation of catalase intermolecular aggregates. Among the three types of CAL₁₀₀, the CAL₁₀₀-II showed the highest thermal stability, indicating that an excess amount of catalase in the CAL₁₀₀-III was also disadvantageous to maintain an active form of the catalase even in liposome. In the CAL₁₀₀-III, however, the stability of catalase was significantly improved compared to that of free catalase at the same concentration. The CAL thermal stability was little affected by the liposome size as observed in the CAL₃₀, CAL₅₀ and CAL₁₀₀-I. An intrinsic tryptophan fluorescence of the catalase recovered from the CAL₁₀₀-II thermally treated at 55 °C revealed that a partially denatured catalase molecule was stabilized through its hydrophobic interaction with liposome membrane. This interaction depressed not only dissociation of catalase into subunits but also formation of an inactive intermolecular aggregate between the catalase molecules in a liposome. Furthermore, either type of CAL₁₀₀ showed a higher stability than free catalase in the successive decompositions of 10 mM H₂O₂ at 25 °C mainly because the H₂O₂ concentration was kept low inside liposomes due to the permeation barrier of the lipid membrane to H₂O₂.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients

A unique feature in inflammatory tissue of rheumatoid arthritis (RA) is the formation of ectopic lymphoid aggregates with germinal center (GC)-like structures that can be considered to contribute to the pathogenesis of RA, because local production of the autoantibody, rheumatoid factor, is thought to be a causative factor in tissue damage. However, the factors governing the formation of GC in RA are presently unknown. To begin to address this, the expression of B cell attracting chemokine (BCA-1) (CXCL13), a potent chemoattractant of B cells, was examined in the synovium of patients with RA or with osteoarthritis (OA). Expression of BCA-1 mRNA was detected in all RA samples, but in only one of five OA samples. Lymphoid follicles were observed in four of seven RA samples and in two of eight OA samples, and in most of them BCA-1 protein was detected in GC. BCA-1 was not detected in tissues lacking lymphoid follicles. Notably, BCA-1 was detected predominantly in follicular dendritic cells in GC. CD20-positive B cells were aggregated in regions of BCA-1 expression, but not T cells or macrophages. These data suggest that BCA-1 produced by follicular dendritic cells may attract B cells and contribute to the formation of GC-like structures in chronic arthritis.