Effect of addition of polyethyleneimine on thermal stability and activity of glucose dehydrogenase

When polyethyleneimine (PEI), a water-soluble cationic polymer, was added to solutions of glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 at a molar ratio of PEI to GDH greater than 10, the thermal stability of GDH markedly increased. By addition of PEI, the rate of GDH-catalysed oxidation of -d-glucose increased in a low concentration range of NAD⁺ and NADP⁺ and the Michaelis constants and inhibition constants for both NAD⁺ and NADP⁺ decreased. These results suggested that negatively charged GDH interacts with cationic water-soluble polymers to form conjugates by electrostatic attraction, and also that negatively charged coenzymes are adsorbed by the polymers, resulting in enrichment of the coenzymes in the vicinity of GDH. Addition of PEI was also found to be effective for preventing the denaturation of GDH by acrylamide.Correspondence to: M. Teramoto     

The fine structure of ventral prostatic secretory epithelial cells in older rats

Summary The ventral prostatic secretory epithelial cells in older rats were studied by light and electron microscopy. The cells vary in height in different parts of the same organ, and ultrastructurally they show the presence of a developed secretory apparatus such as well-developed Golgi body and abundant rough endoplasmic reticulum. They also show signs of a depressed secretory activity, involving occasional emiocytosis of apical secretory vacuoles and a paucity of condensing vacuoles in the Golgi region and above it. Further, they are characterized by the frequent occurrence of supra and paranuclear pleomorphic lysosomes.     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )     

Effect of photoperiod on winter and summer diapause of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae)

To understand the geographical differences between diapause systems and synchronization of adult occurrence in the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae), we examined the timing of winter diapause termination and intensity of summer diapause using univoltine and potentially bivoltine individuals in Iwate, Japan. In laboratory rearing experiments of mature larvae maintained at constant temperature (20 °C), winter diapause intensity weakened by January without photoperiodic responses. Meanwhile, summer diapause was maintained by the long day length and presumably terminated with the photoperiodic transition from long to short day length. The intensity of summer diapause was stronger for cocoons that transitioned from a 16 h light to 8 h dark (LD 16:8) to a LD 15:9 photoperiod than for those that transitioned from LD 15:9 to LD 14:10. These results suggest that populations distributed in relatively low-latitude areas, with partly or potentially bivoltine individuals, would have a weaker summer diapause or none at all. Moreover, sexual differences in the number of days to emergence were not detected when individuals experienced a photoperiodic transition from long to short day length, suggesting that the summer diapause system may function to synchronize the emergence of males and females in the population examined.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFbeta-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.     

Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)--BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)--for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.     

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.