Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin betaE subunit

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin beta subunit genes, betaC and betaE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin betaE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells.     

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes

Background

The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.

Methods

Subcellular localization of Lyn and its mutants was determined by confocal microscopy.

Results

We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.

General significance

Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Modification of small molecules by using cytochrome P450 expressed in Escherichia coli

We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening.     

Can an arbitrary sequence evolve towards acquiring a biological function?

Abstract To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution.     

Study of chiral recognition of bilayered phosphatidylcholine vesicles using a helicene probe: characteristic function of cholesterol

Incorporated into bilayered chiral phosphatidylcholine (PC) vesicles, 2-hydroxymethyl[5]thiaheterohelicene (5HM) having a labile helix that functioned as a chirality probe, exhibited induced CD absorptions. The Cotton effects of these absorptions demonstrated opposite signs according to the difference in chirality of PC applied, indicating the chiral recognition of the vesicles. The vesicles formed by PCs with unsaturation or acyl chains shorter than dipalmitoyl-PC (DPPC) exhibited a slightly stronger CD absorptions of 5HM, presumably because of an increase in the constraint by the vesicles. The phenomenon that an increase in fluidity results in a decrease in the CD intensity was examined by CD measurements at various temperatures. The vesicles formed with egg lecithin and bovine heart lecithin also induced CD absorptions of 5HM similar to those of (L)PC vesicles. The influence of cholesterol (Cho) and four kinds of analogs with different structures at the 3- or 5-position of a Cho molecule on the CD intensities was investigated. Following addition of Cho, the CD intensities of 5HM decreased slightly in the (L)DPPC vesicles and increased moderately in the (D)DPPC vesicles. On the other hand, by addition of Cho analogs the CD intensities of 5HM were nearly unchanged in (L)DPPC vesicles and weakened moderately in the (D)DPPC vesicles.     

Structure-activity relationship of benzo[b]thiophene-2-sulfonamide derivatives as novel human chymase inhibitors

We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously discovered by in silico screening. TY-51076 (7) showed high potency (IC(50)=56 nM) and excellent selectivity for chymase compared to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure-activity relationship of this class are described.     

Design,synthesis and photochemical properties of caged bile acids

Photolabile derivatives of bile acids (8-10 and 13) were synthesized via silver (I) oxide promoted selective etherification of 3alpha-hydroxyls. Quantitative production of the parent cholic acid was detected from the photolytic mixture of 3-NB-CA (8) in Tris buffered solution. Interestingly, the unexpectedly stable nitroso-hemiacetal intermediate (14) was detected when the photolysis was conducted in methanol. The enzymatic analysis using 7alpha-HSDH showed 8 and 9 could serve as caged bile acids that might be able to regulate certain biological processes upon UV irradiation.