Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.     

Nucleotide Sequence and Genetic Analysis of the Region Essential for Functional Expression of the Gene for Ferredoxin I, fdxN, in Rhodobacter capsulatus: Sharing of One Upstream Activator Sequence in Opposite Directions by Two Operons Related to Nitrogen Fixation

Nucleotide sequencing of the region upstream of two ferredoxingenes, fdxC and fdxN, of Rhodobacter capsulatus revealed theexistence of one open reading frame (ORF), ORFU1,in the sameorientation as these genes and two other ORFs, ORFU2 and ORFU3,in the opposite orientation. Two potential –24/–12promoters were found in front of ORFU1 and ORFU2, respectively,and there was a putative upstream activator sequence (UAS) orNifA-binding site between them. The ORFs corresponded to noknown nif genes. However, analysis of their putative productsshowed that the product of ORFU1 (M_r 47,912) and that of ORFU3(M_r 19,090) flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-likedomain, respectively, and that the product of ORFU2 (M_r 20,424)was a hydrophobic protein with six potential membrane-spanningportions. Results of interposon mutagenesis and complementationexperiments indicated ORFU2 but not ORFU1 is essential for nitrogenfixation and that additional gene(s) essential nitrogen fixationmust be present in the unsequenced region adjacent to ORFU3.Translational fusion analysis involving lacZYA and fdxN or ORFU3provided evidence that the putative UAS responsible for regulationof both ORFUl-fdxC-fdxNand ORFU2-ORFU3 operons in opposite orientations,and that the control of the latter is stricter than that ofthe former. (Received August 19, 1992; Accepted November 16, 1992)     

Nucleoside derivatives of 5-methylcytosine suppress 5-azacytidine-induced reactivation of a silent transgene in suspension-cultured tobacco cells

Epigenetic modifications, including DNA methylation, are involved in the regulatory mechanisms of gene expression in animals and plants. In this study, we investigated whether the action of 5-azacytidine (5-aza-Cd), which is a well-known DNA methylation inhibitor, in suspension-cultured tobacco cells is affected by treatment with nucleoside derivatives of 5-methylcytosine (5-mCs), namely 5-methylcytidine (5-mCd) and 5-methyl-2′-deoxycytidine (5-mdCd). In a tobacco cell line, 5-aza-Cd treatment reactivated an epigenetically silenced transgene containing the cauliflower mosaic virus 35S promoter fused to the β-glucuronidase coding region and the nopaline synthase polyadenylation signal. The reactivation was evident on the fifth day of treatment and was augmented during culture with application of 5-aza-Cd at every subcultivation. This treatment, provided only once in the initial culture, resulted in transient transgene reactivation, followed by attenuation of its activity. The reactivation induced by 5-aza-Cd was suppressed by concomitant treatment with either 5-mCd or 5-mdCd. These results suggest that the 5-mCs derivatives inhibit and/or reverse 5-aza-Cd-induced reactivation of a silent transgene in tobacco cells.     

Detection of immunologically related Kunitz and Bowman-Birk proteinase inhibitors expressed during potato tuber development

Antiserum against a potato Kunitz-type proteinase inhibitor (PKPI) expressed in Escherichia coli was produced. In immunoblotting assays of proteins from potato tubers cultured in vitro, three proteins reacted to the antiserum, two of 20 kDa and one of 10 kDa. Their N-termini were sequenced. While the 20 kDa proteins showed 59 and 90% identity to PKPI, the 10 kDa one had 65% identity to soybean C-II proteinase inhibitor. Characterization of the temporal expression of these proteins showed that both could be detected from 10 days after induction of tuberization (DAI) in vitro, but the times when maximum amounts of PKPI and 10 kDa protein could be detected were different, corresponding to 22 and 32 DAI, respectively. The amounts of these proteins decreased in the following stages, and no positive reaction of the antiserum with mature tuber proteins could be found. The 20 kDa proteins were also detected in early stages of development of potato tubers grown in the field, indicating that these proteins are expressed during normal tuber development, and differ from the PKPIs reported previously.     

Corticotrigeminal motor pathway in the rat--II. Anterio- and retrograde HRP labeling

1. Horseradish peroxidase (HRP) injection into the rat cortical jaw motor area (JMA) disclosed direct projection fibers in and around the motor trigeminal nucleus (MTN), primarily contralaterally. 2. Descending axons were found in the ventromedial half of the cerebral peduncle and MTN-projecting axons were concentrated near the descending facial nerve root. 3. HRP injection into the peduncle could label 1257 cells in the JMA, 128 cells in the taste area and 1409 cells in the neck-forelimb motor area ipsilaterally. 4. Some MTN-terminated axons could be traced from the peduncle in serial sections.     

The Lotus Symbiont, Mesorhizobium loti: Molecular Genetic Techniques and Application

Lotus japonicus, orderly information on its symbiotic partner Mesorhizobium loti is still lacking. To improve this situation, we review the history of the M. loti nomenclature and compare several strains commonly used, since classification of rhizobia has been confusing during the last decade. We also refer to the large symbiotic gene cluster, the `symbiosis island', on M. loti chromosome. Then, molecular techniques for M. loti currently available and required to cope with the post-genome sequencing era will be summarized. Finally, we review current knowledge on the structure of M. loti Nod factors. Received 29 August 2000/ Accepted in revised form 25 September 2000