The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

A reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A variety of insects accumulate high contents of riboflavin (vitamin B₂) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3^oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.     

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets

Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.     

Mating-type differentiation in ascosporogenous yeasts on the basis of mating-type-specific substances responsible for sexual cell-cell recognition

Summary Sexual agglutination occurred only between cells of opposite mating types of the same species in all the Sacharomyces, Hansenula, Saccharomycodes, and Pichia yeasts tested. We succeeded in solubilizing the sex-specific glycoprotein, cell wall agglutination substance responsible for sexual agglutinability by briefly autoclaving these yeasts. The agglutination substances of all the above yeasts were univalent and sensitive to the enzyme pronase. The formation of complementary complexes was observed only between agglutination substances of opposite mating types of the same species. In general, the agglutination substance of one mating type was more resistant to heat treatment at 100°C in 3% acetic acid and more sensitive to 5% 2-mercaptoethanol treatment than the agglutination substance of the other mating type in these yeasts. On the basis of these results together with the pheromone response and production, we expect that almost all ascosporogenous yeasts can be classified into the two mating types corresponding to a and mating types in Saccharomyces cerevisiae, respectively.     

The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation.

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.     

A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm,Bombyx mori

Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV–silkworm baculovirus expression vector system to produce proteins of interest.     

Silkworm strains that can be clearly destined towards either embryonic diapause or direct development by adjusting a single ambient parameter during the preceding generation

In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), embryonic diapause is under maternal control and the decision between diapause and direct development in bivoltine strains depends on environmental factors such as temperature and photoperiod experienced by the preceding generation. We reared ten bivoltine silkworm strains (c10, g32, k06, n25, p21, p22, p24, p44, p50 and p63) under various thermal and light conditions and examined the incidence of embryonic diapause in the next generation to identify strains in which the incidence of diapause can be controlled within the range of 0 to 100% by adjusting a single ambient parameter. Some strains were clearly destined towards either diapause or direct development. The diapause incidence in the c10, p22 and p50 strains was controlled by temperature during the egg stage (0% at 18°C and 100% at 25°C), that in the p50 strain was also determined to be dependent on illumination during the egg stage (0% under continuous darkness and 100% under continuous illumination), and photoperiod during the larval stage regulated diapause in p44 and p50 (0% and 100% under long‐day and short‐day photoperiod, respectively), when all other external parameters remained constant under each experimental condition. These diapause‐controllable silkworm strains might serve as model systems for studies of insect diapause, especially for the differential screening of related factors.     

Effect of temporary changes in light intensity on carbon transport, partitioning and respiratory loss in young tomato seedlings raised under different light intensities

Tomato plants were grown under light intensities of 36 or 90 W m⁻² [photosynthetically active radiation (PAR)], and then the light intensity was changed to 36, 90 or 180 W m⁻² for 8 h to investigate the effect of temporary changes in light intensity on the carbon budget of photoassimilates from the third leaf using a ¹⁴CO₂ steady-state feeding method. In the plants that were raised under 90 W m⁻², the photosynthetic rate increased when the light intensity was increased to 180 W m⁻², whereas no increase occurred in the plants that were raised under 36 W m⁻². Although the total amount of carbon fixed during the 8-h light period showed a large difference between plants grown at the two initial light intensities, the proportion of carbon exported during the light period did not differ apparently, irrespective of the change in light intensity. However, the amount of carbon exported during the time course was higher in plants that were raised under 90 W m⁻² than those raised under 36 W m⁻², irrespective of the change in light intensity. The partitioning pattern of ¹⁴C-photoassimilates was not changed by the change in light intensity, irrespective of whether the light intensity was increased or not. However, the amount of ¹⁴C-photoassimilates accumulated in each part differed according to the two initial light intensities. The carbon transport from a source leaf was also investigated through a quantitative analysis of carbon balance.     

Involvement of autocrine CXCL12/CXCR4 system in the regulation of ovarian carcinoma cell invasion

Ovarian carcinomas are often highly invasive, especially in the peritoneal cavity; however, the mechanism involved in invasion is not yet fully understood. In the present research, we studied the role of NF-κB in the invasiveness of ovarian carcinoma cells by using (−)-DHMEQ, a specific inhibitor of NF-κB. (−)-DHMEQ inhibited invasion in vitro and the expression of CXCL12 and CXCR4. We found that neutralizing antibody against CXCR4 or knockdown of CXCR4 suppressed the invasion. Proteomic analysis revealed that CXCR4-siRNA treatment lowered the secretion of several invasion-related proteins, such as MMP-9 and uPA. These data imply that (−)-DHMEQ suppressed ovarian cell invasion via inhibition of the NF-κB-regulated autocrine system of CXCL12-CXCR4.