Distribution and Morphological Features of Microglia in the Developing Cerebral Cortex of Gyrencephalic Mammals

Microglia have been attracting much attention because of their fundamental importance in both the mature brain and the developing brain. Though important roles of microglia in the developing cerebral cortex of mice have been uncovered, their distribution and roles in the developing cerebral cortex in gyrencephalic higher mammals have remained elusive. Here we examined the distribution and morphology of microglia in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that a number of microglia were accumulated in the germinal zones (GZs), especially in the outer subventricular zone (OSVZ), which is a GZ found in higher mammals. Furthermore, we uncovered that microglia extended their processes tangentially along inner fiber layer (IFL)-like fibers in the developing ferret cortex. The OSVZ and the IFL are the prominent features of the cerebral cortex of higher mammals. Our findings indicate that microglia may play important roles in the OSVZ and the IFL in the developing cerebral cortex of higher mammals.     

Complementing yeast rho1 mutation groups with distinct functional defects

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.     

The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G₂ phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G₂ arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G₁ arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G₂ arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G₂ arrest. G₂ arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G₂ arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.     

Purification and characterization of pig lung carbonyl reductase.

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.     

Dormancy of alpine and subalpine perennial forbs

Depth of dormancy of alpine and subalpine perennial forbs in autumn was investigated, which was judged by the number of days required for growth initiation at 24 °C. The depth of dormancy differed depending on Raunkiaer’s life-form and shoot habits. Chamaephytes with perennial shoot-axes showed shallower dormancy than hemicryptophytes with annual shoot-axes, and geophytes with annual shoot-axes showed the deepest dormancy. The results strongly suggest that the dormancy is more endogenously controlled in forbs less hardy to freezing stress. Potential growth ability of alpine herbaceous chamaephytes in autumn is an adaptive advantage, since they utilize the short vegetative period as long as possible. All of the species with annual shoot-axes had winter buds covered with scales. In plants with perennial shoot-axes, percentage of winter buds covered with scales increased with increasing depth of dormancy. The results indicate that the shoot apices are well protected by bud scales in forbs with a long endogeneous-dormant period.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Schizosaccharomyces pombe Bub3 is dispensable for mitotic arrest following perturbed spindle formation

The core proteins of the spindle assembly checkpoint (SAC), Mads, Bubs, and Mps1, first identified in the budding yeast, are thought to be functionally and structurally conserved through evolution. We found that fission yeast Bub3 is dispensable for SAC, as bub3 null mutants blocked mitotic progression when spindle formation was disrupted. Consistently, the bub3 mutation only weakly affected the stability of minichromosome Ch16 compared with other SAC mutants. Fission yeast Rae1 has sequence homology with Bub3. The bub3 rae1 double mutant and rae1 single mutant did not have defective SAC, suggesting that these genes do not have overlapping roles for SAC. Observations of living cells revealed that the duration of the mitotic prometaphase/metaphase was longer in the bub3 mutant and was Mad2 dependent. Further, the bub3 mutant was defective in sister centromere association during metaphase. Together, these findings suggest that fission yeast Bub3 is required for normal spindle dynamics, but not for SAC.