Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Medium‐Chain Oil Reduces Fat Mass and Down‐regulates Expression of Adipogenic Genes in Rats

Objective: To test the hypothesis that adipose tissue could be one of the primary targets through which medium‐chain fatty acids (MCFAs) exert their metabolic influence. Research Methods and Procedures: Sprague‐Dawley rats were fed a control high‐fat diet compared with an isocaloric diet rich in medium‐chain triglycerides (MCTs). We determined the effects of MCTs on body fat mass, plasma leptin and lipid levels, acyl chain composition of adipose triglycerides and phospholipids, adipose tissue lipoprotein lipase activity, and the expression of key adipogenic genes. Tissue triglyceride content was measured in heart and gastrocnemius muscle, and whole body insulin sensitivity and glucose tolerance were also measured. The effects of MCFAs on lipoprotein lipase activity and adipogenic gene expression were also assessed in vitro using cultured adipose tissue explants or 3T3‐L1 adipocytes. Results: MCT‐fed animals had smaller fat pads, and they contained a considerable amount of MCFAs in both triglycerides and phospholipids. A number of key adipogenic genes were down‐regulated, including peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein α and their downstream metabolic target genes. We also found reduced adipose tissue lipoprotein lipase activity and improved insulin sensitivity and glucose tolerance in MCT‐fed animals. Analogous effects of MCFAs on adipogenic genes were found in cultured rat adipose tissue explants and 3T3‐L1 adipocytes. Discussion: These results suggest that direct inhibitory effects of MCFAs on adiposity may play an important role in the regulation of body fat development.     

Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251bp with an open reading frame (ORF) of 636bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.     

Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Synthesis and antifungal activity of noble 5-arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles

5-Arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles 4 and 5 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 5-Arylamino-4,7-dioxobenzoselenazoles 4 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzoselenazoles 5. The results suggest that 5-arylamino-4,7-dioxobenzoselenazoles 4 would be potent antifungal agents.     

NELIN, a new F-actin associated protein, stimulates HeLa cell migration and adhesion

A new gene (GenBank Accession No. AF114264) was cloned from umbilical vein wall tissue by using RT-PCR. The gene shares high similarity to the gene encoding F-actin binding protein nexilin, so named as NELIN. A clone of 2737bp contains open reading frame of 1344bp extending from 412 to 1755. NELIN was expressed primarily in the heart and skeletal muscle among eight tested normal tissues. Immunofluorescence and immunoprecipitation demonstrated that NELIN product was associated with F-actin. Stable transfection of NELIN into HeLa cells increased the cell migration by 2.17-fold and the adhesion by 1.67-fold, respectively, compared to cells with the empty vector (P<0.05). The results support that NELIN product is an F-actin associated protein and mediates cell motility.