Chemomechanical coupling of the forward and backward steps of single kinesin molecules

The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.     

Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation

In epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1-/- murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1-/- epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

Studies on the Organophosphorus Insecticides

During the course of investigation on organophosphorus insecticides, many organophosphorus compounds having methyl-, methyl-chloro-, methyl-nitro-, methyl-cyano-, methyl-thiocyano-, methoxy-nitro-, acyloxy-nitro-, and chloro-cyano-phenyl groups were prepared and their biological activities tested. As the results of these studies, two compounds were found as new low toxic organophosphorus insecticides, namely, O,O-dimethyl-O- (3-methyl-4-nitro-phenyl) and O,O-dimethyl-O- (4-cyanophenyl) phosphorothioates.     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Continuous cultures of tomato and citron roots in vitro

Summary Initial trials with tomato-root cultures disclosed the desirability of employing a gently agitated liquid medium containing iron in the chelated form. For the normal cultivars “Ace” and “Tropic”, subcultures were best achieved by utilizing sectors that possessed one or more newly emerged laterals. Continuous cultures of a nonlateral-forming tomato mutant, “Diageotropica”, and of citron were accomplished by subculturing tips of the elongating primary roots. The tomato roots were cultured in White's medium with the Fe₂(SO₄)₃ replaced by 0.03 mM NaFeEDTA. Sustained growth of citron-root tips necessitated the use of a medium containing Murashige and Skoog salts, 7.5% sucrose, 100 mg per I each of citric acid and thiamine HCl, and 5000 mg per li-inositol. The success with citron-root cultures was extendable to all cultivars ofC. medica L., but not to otherCitrus species relatives. Both citron and “Diageotropica” root cultures manifested undiminished elongation through repeated subcultures; but neither produced laterals in response to any cultural treatments. Research was supported in part by National Science Foundation Grant OIP75-10390 and Elvenia J. Slosson Fellowship in Ornamental Horticulture.     

Developmental change of an enzyme activity oxidizing γ-aminobutyraldehyde to γ-aminobutyric acid in the chick embryonic brain

An enzyme activity oxidizing -aminobutyraldehyde (ABAL) to GABA reflecting an alternative pathway for GABA synthesis was assayed in the developing chick embryonic brain and was compared with glutamate decarboxylase (GAD) activity. An enzyme activity oxidizing ABAL to GABA showed almost constant level during development in the chick embryonic brain, and was present at low levels compared with GAD activity. The results indicate that GABA synthesis via an alternative pathway is always much less than synthesis via the GAD-dependent pathway in the developing chick embryonic brain.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

A seed medium and a fermentation medium for nucleotide fermentations such as 5′ IMP, 5′GMP (plus GDP and GTP) and 5′AMP (plus ADP and ATP) with Brevibacterirm ammoniagenes ATCC 6872 were entirely chemically defined, with the use of a mixture of five amino acids.

As a result, the presence of Zn²⁺, Fe²⁺ and Ca²+ in addition to Mn²⁺ was found to be essential for the nucleotide fermentations. In particular, Zn²⁺ levels as well as Mn²⁺ affected nucleotide productions remarkably. Various fermentations proceeded favorably only when suboptimum levels of manganese (20~30 μg/liter) and zinc (100~200 μg/liter) were simultaneously present. This effect of trace metals was attributed to the fact that the excretion of R5P, a precursor of nucleotides, and those enzymes catalyzing reactions synthesizing nucleotides from R5P, ATP and purine bases were greatly stimulated by trace metals in cooperation with two vitamins, Ca-pantothenate and thiamine, and presumably high concentrations of phosphate and magnesium.

Furthermore, it was revealed that some metals were able to control the amounts of nucleotides accumulated when they were added to the broth during fermentation. For example, Hg²⁺ and Ag⁺ could increase the amounts of 5′GMP or 5′AMP, and decrease those of GTP and ATP.

Growth responses of Brevibacterium ammoniagenes ATCC 6872, capable of accumulating purine nucleotides, were investigated by the use of completely defined media.

1. Casamino acids required for its growth could be replaced by a mixture of l-histidine, l-homoserine, glycine, d, l-alanine and l-lysine. A completely defined medium for nucleotide productions was thus established by the use of this mixture.

2. High levels of phosphate inhibited growth markedly, and this inhibition was overcome by the simultaneous addition 1) of hign levels of Mg²⁺ and 2) of Mn²⁺, 3) pantothenate and 4) thiamine. Ca²⁺ had also a stimulatory effect on the growth. Therefore, a clear growth response to Mn²⁺ levels and the requirement of the two vitamins for growth emerged only under the conditions of high phosphate and magnesium salts. These 4 factors were found entirely the same as factors essential for nucleotide accumulations by Br. ammoniagenes.