Phylogenetic relationship of medicinally importantCnidium officinale and Japanese Apiaceae based onrbcL sequences

Cnidium officinale Makino is important medicinally and economically, but its origin is uncertain. The phylogenetic relationship ofC. officinale is provided from the analyses based on the ribulose-1,5-bisphosphate carboxylase/oxgenase gene (rbcL) sequences of 41 species which represent the 34 genera of Aplaceae, the four genera of Araliaceae, and one genus each of Pittosporaceae, Cornaceae, and Caprifoliaceae. The strict consensus tree obtained supports a close relationship ofC. officinale to the Chinese members ofLigusticum, especially toL. chuanxiong. Additionally, the tree shows (1) polyphyly of the genusLigusticum and (2) monophyly of the subfamily Apioideae. Within Apioideae, we recognized some groups in our phylogenetic tree. The grouping is discordant in several respects with the traditional tribal divisions based mainly on fruit morphology.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Autotrophic picoplankton in southern Lake Baikal: abundance, growth and grazing mortality during summer

Autotrophic picoplankton were highly abundant during the thermalstratification period in late July in the pelagic area (waterdepth 500–1300 m) of southern Lake Baikal; maximum numberswere 2 x 10⁶ cells ml^–1 in the euphotic zone ({small tilde}15m). Unicellular cyanobacteria generally dominated the picoplanktoncommunity, although unidentified picoplankton that fluorescedred under blue excitation were also abundant (maximum numbers4 x 10⁵ cells ml^–1) and contributed up to {small tilde}40%of the total autotrophic picoplankton on occasions. Carbon andnitrogen biomasses of autotrophic picoplankton estimated byconversion from biovolumes were 14–84 µg C l^–1and 3.6–21 µg N l^–1. These were comparableto or exceeded the biomass of heterotrophic bacteria. Autotropicpicoplankton and bacteria accounted for as much as 33% of paniculateorganic carbon and 81% of nitrogen in the euphotic zone. Measurementsof the photosynthetic uptake of [^l4C]bicarbonate and the growthof picoplankton in diluted or size-fractionated waters revealedthat 80% of total primary production was due to picoplankton,and that much of this production was consumed by grazers inthe <20 µ.m cell-size category. These results suggestthat picoplankton-protozoan trophic coupling is important inthe pelagic food web and biogeochemical cycling of Lake Baikalduring summer.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Collective motions in proteins investigated by X-ray diffuse scattering

We have developed theoretical models for analysis of X-ray diffuse scattering from protein crystals. A series of models are proposed to be used for experimental data with different degrees of precision. First, we propose the normal mode model, where conformational dynamics of a protein is assumed to occur mostly in a limited conformational subspace spanned by a small number of low-frequency normal modes in the protein. When high precision data are available, variances and covariances of the normal mode variables can be determined from experimental data using this model. For experimental data with lower degrees of precision, we introduce a series of simpler models. These models express the covariance matrix using relatively simple empirical correlation functions by assuming the correlation between a pair of atoms to be isotropic. As an application of these simpler models, we calculate diffuse-scattering patterns from a human lysozyme crystal to examine how each adjustable parameter in the models affects general features of the resulting patterns. The results of the calculation are summarized as follows. (1) The higher order scattering makes a significant contribution at high resolutions. (2) The resulting simulated patterns are sensitive to changes in correlation lengths of about 1 Å, as well as to changes of the functional form of the correlation function. (3) But only the “average” value of the intra- and intermolecular correlation lengths seems to determine the gross features of the pattern. (4) The effect of the atom-dependent amplitude of fluctuations is difficult to observe. © 1994 John Wiley & Sons, Inc.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.