ASAP3 is a focal adhesion-associated Arf GAP that functions in cell migration and invasion

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.     

Enzymatic properties of cellobiose 2-epimerase from <Emphasis Type="Italic">Ruminococcus albus</Emphasis> and the synthesis of rare oligosaccharides by the enzyme

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.     

Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.     

Central and peripheral administration of amylin induces energy expenditure in anesthetized rats

Amylin is a peptide hormone that is co-released with insulin from pancreatic β-cells following a meal. Intracerebroventricular (icv) administration of amylin (1–100 pmol), or an amylin agonist, salmon calcitonin, elicited dose-dependent thermogenic, tachycardic, and hyperthermic responses in urethane-anesthetized rats. Intravenous (iv) administration of higher doses of amylin (100 pmol–20 nmol) also induced similar responses, although the amplitudes of these responses were significantly smaller than those elicited by icv administration, suggesting the primary action of amylin to be in the brain. However, the iv administration of amylin induced the responses slightly faster than the icv injection, the former responses occurring <4 min and the latter, at 8–10 min, after the administration. The iv but not the icv injection of amylin increased the respiratory exchange ratio transiently (<20 min), though the thermogenic response lasted for a longer period after both injections, indicating a shift from mixed fuel to predominantly carbohydrate utilization in the initial phase of thermogenesis induced by the iv injection of amylin. The differences in substrate utilization and latency of the responses suggest that the actions of amylin include partly different targets when administered centrally and peripherally. Moreover, pretreatment with a β-adrenergic blocker, propranolol (5 mg kg⁻¹, iv), blocked all responses elicited by either icv or iv administration of amylin, whereas ablation of the area postrema in the hindbrain did not influence the effects of icv-administered amylin. These results suggest the involvement of amylin in postprandial energy expenditure, mediated by peripheral β-adrenoceptors.     

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near −40 °C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

Micro-manipulation system with a two-fingered micro-hand and its potential application in bioscience

A micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces was developed. This micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger. Thus, this hand can grasp, move, rotate, and release micro-objects, such as biological cells. A human operator can operate this hand using a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, two-fingered micro-hands. One hand holds the cell firmly, while the other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the Atomic Force Microscope (AFM) principle. The AFM cantilever is placed within the microscopic field. The micro-finger holds a cell and presses it against the cantilever tip. By measuring the pressing force and the deformation of the cell, the cell's force-deformation curve is obtained.     

Direct observation of fat crystallization in a living fly by X-ray diffraction: fat crystallization does not cause the fly's instantaneous death, but ice formation does

A cool-temperate fly, Drosophila triauraria, stores fat, triacylglycerol (TAG), primarily in the fat storage organ, the fat body, and then diapauses to pass the winter in imago stage. TAG crystallization and ice formation taking place in a living fly by lowering temperatures were studied, in order to clarify the relationship between crystallizations and the fly’s death at lower temperatures. X-ray diffraction, a direct non-invasive method, was used to detect the liquid-to-crystal transformations of TAG and water. During cooling, TAG crystallization preceded ice formation. It was also found that ice formation causes the fly to die instantaneously whereas the TAG crystallization does not.     

Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids

Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.     

IRAK-4-dependent degradation of IRAK-1 is a negative feedback signal for TLR-mediated NF-kappaB activation

The activation of interleukin 1 receptor-associated kinase (IRAK)-1 is a key event in the transmission of signals from Toll-like receptors (TLRs). The catalytic activity of the protein kinase is not essential for its ability to activate nuclear factor (NF) kappaB, because transfection of a kinase-dead mutant of IRAK-1 (IRAK-1KD) is able to activate NF-kappaB in HEK293T cells. In the present study, we observed that the effect of IRAK-1KD was impaired by simultaneous expression of IRAK-4. The effect of IRAK-4 was accompanied by the phosphorylation and degradation of IRAK-1KD. Expression of IRAK-4KD instead of IRAK-4 did not cause these events. In IRAK-4-deficient Raw264.7 macrophages that were prepared by introducing short-hairpin RNA probes, the basal level of IRAK-1 was increased markedly. Stimulation of these cells with TLR ligands did not cause the degradation of IRAK-1, which was clearly observed in the parent cells. These results suggested that the expression of IRAK-4 alone is sufficient to cause the degradation of IRAK-1; the autophosphorylation of IRAK-1 is not necessary to terminate the TLR-induced activation of NF-kappaB. IRAK-4 has an ability to induce the degradation of IRAK-1 in addition to its role as an activator of IRAK-1.