Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH₂-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Cold exposure suppresses serum adiponectin levels through sympathetic nerve activation in mice

Objective: Several lines of evidence suggest important roles for adiponectin in glucose and lipid metabolism and atherosclerosis. However, the mechanisms regulating serum adiponectin levels and adiponectin production are still not completely understood. Our aim was to determine whether adiponectin synthesis is physiologically regulated by the sympathetic nervous system (SNS). Research Methods and Procedures: Mice were exposed to cold (4 °C) for 12 hours and for 24 hours with or without inhibition of noradrenaline synthesis or pan‐β adrenergic function, followed by measurement of serum adiponectin concentrations and levels of adiponectin and uncoupling protein (UCP) 1 expressions in various white adipose tissues (WATs). Results: Cold exposure significantly reduced serum adiponectin concentrations without changing body weights or WAT sizes in either subcutaneous or intra‐abdominal fat tissues. The serum adiponectin reduction was associated with a decrease in adiponectin mRNA expression in subcutaneous, epididymal, and mesenteric fat tissues. In these adipose tissues, UCP1 expression was markedly enhanced, suggesting SNS activation in these tissues. Administration of α‐methyl‐p‐tyrosine or a combination of SR59230A and propranolol reversed the cold‐exposure‐induced decreases in serum adiponectin concentrations and adiponectin mRNA expression in these tissues. In contrast, in retroperitoneal fat, the effects of cold exposure on adiponectin and UCP1 expressions were strikingly weak but were not reversed by SNS inhibitors. Discussion: SNS physiologically regulates serum adiponectin levels and adiponectin synthesis in WATs in vivo, although responsiveness to SNS stimulation differs markedly among WATs. Sympathetic activation might be involved in development of the metabolic syndrome by modulation of serum adiponectin concentrations.     

Expression of cys-cys chemokine ligand 21 on human gingival lymphatic vessels

The cys-cys (C-C) chemokine ligand 21 is a member of the C-C chemokines that constitute a group of heparin-binding cytokines with a pattern of four or six conserved cysteines. The CCL21 is known to be expressed in secondary lymphoid tissues, however it has rarely been reported for the expression on peripheral lymphatic vessels in somatic tissue. Here we investigated the expression of CCL21 on lymphatic vessels identified by anti-desmoplakin in uninflamed and inflamed human gingiva. In uninflamed tissue the expression of CCL21 was detected on lymphatic vessels in gingiva. In uninflamed gingiva the expression of CCL21 was detected on all lymphatic capillaries of the mucosal connective tissue papillae. There were two types of collecting lymphatic vessels in the lamina propria mucosae expressing CCL21 strongly or very weakly. In inflamed gingiva no expression of CCL21 was detected on lymphatic vessels. In all tissue sections no blood vessels expressing CCL21 were observed. These results may suggest that the expression of CCL21 is predominantly induced in the peripheral lymphatic endothelium of the uninflamed mucosal microcirculation, and that under inflamed conditions a reduction of CCL21 occurs in lymphatic endothelium.     

Treatment with leucine stimulates the production of hepatocyte growth factor in vivo

Hepatocyte growth factor (HGF) has pleiotropic effects. Up-regulation of HGF activity in vivo may be beneficial. Branched-chain amino acids (BCAAs) are known to modulate various cellular functions. When starved rats received intraperitoneal injections of valine, leucine or isoleucine, only leucine treatment increased both hepatic and circulating levels of HGF in a dose-dependent manner, up to 1.5 and 2.3 times higher, respectively, than in controls. When young growing rats with free access to food were injected with leucine once a day for a week, HGF levels and liver weights were significantly higher than those of control rats. Furthermore, 1 week of leucine treatment of adult rats resulted in elevated serum albumin levels with an increase in HGF levels. Taken together with our previous report showing that leucine stimulates HGF production by hepatic stellate cells in culture, leucine, among BCAAs, may induce an increase in HGF production by the liver in vivo.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

The SNP in the promoter region of the bovine ELOVL5 gene influences economic traits including subcutaneous fat thickness

Genetic analyses have contributed to improvements of economically important traits derived from adipose tissue such as fatty acid composition in beef. Elongation of very long chain fatty acids (ELOVL) genes encode for the enzymes that play an important role in elongation of long-chain fatty acids. In this study, we aimed to discover genetic polymorphisms of ELOVL gene family in cattle populations to develop genetic markers. As a result, five synonymous mutations were detected in the coding regions of the ELOVL1, ELOVL2, ELOVL3 and ELOVL5 genes. In addition, six mutations were identified in promoter region of the ELOVL5. Two of five mutations in the promoter region of ELOVL5 were expected to alter the ELOVL5 expression and influence the economic traits, because of the high synteny of the region which was essential for activation of Elovl5 in mouse. Therefore, we performed association analysis between the genotypes and traits and our result revealed that T allele of g.-110T>C in ELOVL5 gene promoter indicated significantly thinner subcutaneous fat thickness (TT, 2.39 cm; CT, 2.35 cm) than that of C allele (CC, 2.68 cm) in a Japanese Black population. Our results suggest that the g.-110T>C is a useful genetic marker for the breeding in beef cattle.     

Structure–activity-relationship studies on dihydrofuran-fused perhydrophenanthrenes as an anti-Alzheimer’s disease agent

As an extended study on development of anti-Alzheimer’s disease agent, we newly synthesized various dihydrofuran-fused perhydrophenanthrenes via o-quinodimethane chemistry. This study revealed that the introduction of carbon side-chain on 8-position or removal of the acetal moiety on 3-position arose a cytotoxicity on rat cortical neurons. On the other hand, the ethereal or thio-ethereal substituent on 8-position enhanced the elongation effect on Aβ-damaged neurons. The necessity of the cyano group on 10b position was also proved in this structure–activity-relationship study.     

Formation of fibrin gel in fibrinogen-thrombin system: static and dynamic light scattering study

The dynamics of thrombin-induced fibrin gel formation was investigated by means of static and dynamic light scattering. The decay time distribution function, obtained by the dynamic light scattering, clearly revealed a stepwise gelation process: the formation of fibrin and protofibril from fibrinogen followed by the lateral aggregation of protofibrils to form fibrin fibers and the formation of a three-dimensional network consisting of fibers. This conversion process was correlated with the angular dependence of the scattered light intensity (static light scattering). The correlation function of dynamic light scattering was analyzed in terms of sol-gel transition and gel structure. The correlation function showed a stretched exponential type behavior before the sol to gel transition point, and it showed a power law behavior at the gelation point.