The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

Characterization of β-amylase and its deficiency in various rice cultivars

 β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed. Received: 8 May 1998 / Accepted: 5 June 1998     

Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Settling-site selection and survival of two scale insects,Ceroplastes rubens andC. ceriferus,on citrus trees

We studied settling-site selection and the resulting survival of two sessile scale insects, Ceroplastes rubens and C. ceriferus, in the citrus tree, Citrus unshiu, in central Japan. C. rubens preferred 0-year-old twigs most as a settling-site; the density of nymphs settling on 0-year-old twigs was significantly higher than those on ≥1-year-old twigs, and few nymphs settled on ≥3-year-old twigs. The mean survival rates from settling until reproduction in the next year were significantly higher on more preferred twigs than on less preferred ones. In C. ceriferus, nymphs significantly preferred 1- and 2-year-old twigs to 0- and ≥3-year-old ones, and the mean survival rates on the more preferred 1- and 2-year-old twigs were significantly higher than those on less preferred ≥3-year-old twigs. However, the survival rate on less preferred 0-year-old twigs was slightly higher than those on 1- and 2-year-old ones. Thus, in both species of scale, it was the preferred twigs which were more profitable sites for survival after settling, except for less preferred 0-year-old twigs for C. ceriferus. In both scale species, most mortality was due to growth cessation, which is believed to be related to the twig quality as a food source. Predators and parasitoids were minor mortality factors. Both species showed constant survival rates until the density of settled nymphs exceeded double the “upper-limit” density, whereupon they decreased drastically. Nymphs of C. rubens settling on twigs of high scale density showed a spacing-out distribution, those of C. ceriferus did not. In C. rubens, an increase in preference for originally less profitable twigs at the later stage of the settling season was observed, but not in C. ceriferus. Accordingly, individuals of C. rubens showed a stronger tendency to avoid conspecifics than did C. ceriferus. Although nymphs of the two scales clearly preferred more profitable sites, their settling-site selection did not agree with the predictions from the ideal free distribution theory (Fretwell and Lucas, 1970). The discrepancies were (1) frequent settling on less profitable sites at the early stage of the settling season, (2) insufficient utilization of the most profitable twigs, and (3) virtually 100% mortality on overcrowded twigs under conditions where unoccupied profitable twigs still remained. These discrepancies are thought due to the limited dispersal time of nymphs.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

High performance liquid chromatography of nucleic acids and the related compounds on a fluorinated silica gel column.

Separations of nucleic acids and the related compounds were investigated by HPLC on a new fluorinated bonded silica gel column. Polyadenylate (Poly (A)) enzymatic partial hydrolysate sample and the mixture of various polynucleotide samples were sufficiently separated by the reversed-phase mode using a gradient elution with aqueous ammonium acetate/acetonitrile system. Mixed-mode separation on the fluorinated bonded phase coated with a tert-alkylammonium salt was also examined for the separation of the various polynucleotides including tRNAs.     

How to discriminate between vibration syndrome and local fatigue of motorcyclists

Motorcyclists who work in some offices sometimes complained of coldness, pain and numbness of upper limbs. We studied how to discriminate between vibration syndrome and local fatigue of the motorcyclists. Subjects are 42 motorcyclists of an office in Aichi prefecture. 25 of them held several letters in their left hand when they delivered the letters. They complained of coldness, pain and numbness in the left upper limbs more than in the right limbs (p less than 0.01). We think that it is the local fatigue rather than the disorder of vibration syndrome that causes such symptoms. So it is very important to recognize the existence of local fatigue in order to know how to discriminate between vibration syndrome and local fatigue of the motorcyclists.     

Fractal property of eye movements in schizophrenia

 On the basis of a temporal model of animal behavior we conducted temporal analysis of eye movements in schizophrenic subjects (n=10) and normal controls (n=10). We found a fractal property in schizophrenic subjects, the fixation time of eye movement during reading ambiguous and difficult sentences showing a clear inverse power law distribution. An exponential distribution of a nonfractal nature was found in normal controls. Received: 21 July 1995/Accepted in revised form: 30 April 1996     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.