Unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs

The effects of the absence or presence of prostatic fluid (PF) during cauda epididymal sperm retrieval were assessed as regards semen quality after freezing semen in egg yolk Tris-fructose citrate solution (EYT-FC). Epididymal spermatozoa from the left testis of each of 10 dogs was retrieved into PF, whereas that from the right testis into EYT-FC only. At sperm recovery, the only difference between the two groups was that the incidence of spermatozoa with cytoplasmic droplets (immature sperm) was lower in the PF group (P < 0.01). In contrast, after freezing-thawing, (mean +/- S.E.) sperm motility (32.0 +/- 1.4 versus 12.5 +/- 2.0%, P < 0.01) and viability (58.2 +/- 3.5 versus 41.8 +/- 5.6%, P < 0.05) were higher in the PF group than in the EYT-FC group, respectively. Furthermore, 25.6 +/- 2.7% spermatozoa in the PF group were still motile after being maintained at 20 degrees C for 6 h. The incidence of immature spermatozoa post-thaw was lower compared to that after recovery in the EYT-FC group (P < 0.01), but was still higher than that in the PF group (P < 0.05). Frozen-thawed spermatozoa (2 x 10(8)) were used for unilateral intrauterine AI. The conception rate of PF-unsensitized sperm was 20% (2/10), but that of PF-sensitized sperm was 80% (8/10; P < 0.01). Therefore, sperm recovered in PF and frozen-thawed were of good quality. Sensitization of epididymal sperm with PF before freezing clearly improved the conception rate to AI of spermatozoa derived from the cauda epididymus.     

Modulation of hepatocyte growth factor induction in human skin fibroblasts by retinoic acid

Topical treatment of skin with all-trans-retinoic acid (ATRA), the major biologically active form of vitamin A, results in hyperproliferation of basal keratinocytes, leading to an accelerated turnover of epidermis cells and thickening of the epidermis, probably via induction of production of paracrine growth factors for keratinocytes in epidermal suprabasal keratinocytes and/or dermal fibroblasts. Since hepatocyte growth factor (HGF) is a factor mitogenic to epidermal keratinocytes secreted from dermal fibroblasts, the effect of ATRA on basal and induced HGF production in human dermal fibroblasts in culture was examined. ATRA alone did not induce HGF production, but it significantly enhanced HGF production induced by the cAMP-elevating agent cholera toxin or the membrane-permeable cAMP analog 8-bromo-cAMP. Cholera toxin-induced activation of cAMP responsive element (CRE)-binding protein (CREB) was enhanced by pretreating cells with ATRA for 24 h. In contrast, HGF production induced by epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) was potently inhibited by ATRA. These modulatory effects of ATRA were different from the effects of transforming growth factor-beta1 (TGF-beta) and dexamethasone, both of which inhibited HGF production induced by all of the four inducers. Up-regulation of HGF gene expression by cholera toxin and EGF was also enhanced and inhibited, respectively, by ATRA. Both 9-cis-retinoic acid (9-cis-RA) and 13-cis-retinoic acid (13-cis-RA), which are stereo-isomers of ATRA, showed a modulatory effect on HGF induction similar to that of ATRA. These results suggest that ATRA augments the induction of HGF production caused by increased intracellular cAMP.     

Parasporin-1, a novel cytotoxic protein to human cells from non-insecticidal parasporal inclusions of Bacillus thuringiensis

Pro-parasporin-1 is a parasporal inclusion protein of the non-insecticidal Bacillus thuringiensis strain A1190. Cytotoxic fragments, named parasporin-1, were generated from pro-parasporin-1 by trypsin digestion. Parasporin-1 was purified by a combination of chromatography procedures based on the cytotoxic activity to HeLa cells. Two different fragments of 15-kDa and 56-kDa were detected in the purified parasporin-1 fraction. These fragments were tightly associated with each other and could not be separated by chromatography under conditions that preserve cytotoxic activity, indicating that the active form of parasporin-1 is a heterodimer of the 15- and 56-kDa fragments. Amino acid sequencing and MALDI-TOF mass spectrometric analysis revealed that parasporin-1 is generated from pro-parasporin-1 by trypsin digestion at Arg 93 and Arg 231. Of 12 human cell lines tested, parasporin-1 showed strong cytotoxicity to four cell lines derived from cancer tissues, but low to no cytotoxicity to the other cell lines. The time-courses of cytotoxicity indicated that the mode of action of parasporin-1 to sensitive cells differs from that shown for previously isolated cytotoxic proteins from Bacillus thuringiensis, Cyt proteins, and other bacterial pore-forming toxins. Thus, parasporin-1 is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis, and may be useful as a tool to recognize and destroy specific cancer cells.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

Identification of a novel endocytic recycling signal in the D1 dopamine receptor

A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. We have identified a sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking.     

Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells

The NADPH oxidase 1 (Nox1) is a gp91(phox) homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O(2)(-)) of 160 nmol/mg protein/h and expressed the Nox1, p22(phox), p67(phox), and Rac1 mRNAs, but not the gp91(phox), Nox4, p47(phox), p40(phox), and Rac2 mRNAs. They also expressed novel homologues of p47(phox) and p67(phox) (p41(nox) and p51(nox), respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22(phox), p51(nox), and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O(2)(-) (<2 nmol/mg protein/h). Cotransfection of p41(nox) and p51(nox) cDNAs in T84 cells enhanced PMA-stimulated O(2)(-) release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O(2)(-) release in association with the induction of Nox1 protein. The enhanced O(2)(-) production by cotransfection of p41(nox) and p51(nox) vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-beta-activated kinase 1 and TGF-beta-activated kinase 1-binding protein 1. A potent inhibitor for NF-kappaB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O(2)(-) production and induction of Nox1 protein. These results suggest that p41(nox) and p51(nox) are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.     

Screening and evaluation of resistance to downy mildew (Peronospora parasitica) and clubroot (Plasmodiophora brassicae) in genetic resources of Brassica oleracea

52 entries including landraces, old cultivars and wild accessions of B. oleracea and closely related Brassica species were screened for resistance against downy mildew and clubroot. Several accessions resistant to downy mildew and a few to clubroot were found. Genetic inheritance of the resistance in downy mildew was investigated by screening F1 and BC1F1 offspring from three resistant landrace accessions crossed with both a resistant and a susceptible father. The seedling resistance against downy mildew was found to be inherited recessively. This is a bit surprising as earlier papers mostly report of inheritance controlled by a single dominant gene. Previous screenings of B. oleracea resistance against downy mildew at the cotyledon stage have been done with P. parasitica isolated from B. oleracea as the original host plant. The recessive nature of the cotyledon resistance found in this screening might be due to the fact that the P. parasitica isolate was collected from B. napus fields. The clubroot seedling resistance was found to be controlled by recessive inheritance after screening the F1 offspring, this in agreement with earlier results/reports.     

N,beta-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa

From leaves of Psychotria leiocarpa, an indole alkaloid was isolated to which the structure N,beta-D-glucopyranosyl vincosamide (1) was assigned. This represents the first report of an N-glycosylated monoterpenoid indole alkaloid. In field-grown plants highest amounts of 1 were found in the leaves (2.5% of dry wt) and fruit pulp (1.5% dry wt). Lower amounts were found in the stems (0.2% dry wt) and the seeds (0.1% of dry wt), whereas the alkaloid was not detected in the roots. The accumulation of 1 in aseptic seedlings was also restricted to the shoots and increased with plant age and light exposure, independent of the supply of sucrose in the culture medium.