The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane

The nod  C genes from rhizobia encode an N -acetylglucosaminyl transferase (chitin synthase) involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate root nodule morphogenesis in legume plants. NodC proteins have two hydrophobic domains, one of about 21 residues at the N-terminus and a longer one, which could consist of two or three transmembrane spans, near the C-terminus. These two hydrophobic domains flank a large hydrophilic region that shows extensive homology with other β-glycosyl transferases. The topology of NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed using a series of gene fusions encoding proteins in which NodC was fused to alkaline phosphatase (PhoA) lacking an N-terminal transit sequence or to β-galactosidase (LacZ). Our data support a model in which the N-terminal hydrophobic domain spans the membrane in a N_out–C_in orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm. This orientation appears to depend upon the presence of the hydrophobic region near the C-terminus. We propose that this hydrophobic region contains three transmembrane spans, such that the C-terminus of NodC is located in the periplasm. A short region of about 40 amino acids, encompassing the last transmembrane span, is essential for the function of NodC. Our model for NodC topology suggests that most of NodC, including the region showing most similarity to other β-glycosyl transferases, is exposed to the cytoplasm, where it is likely that polymerization of N -acetyl glucoasamine occurs. Such a model is incompatible with previous reports suggesting that NodC spans both inner and outer membranes.     

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.     

Circadian Rhythm in Pineal Methionine S-Adenosyltransferase

Abstract: The circadian rhythm of methionine S -adenosyltransferase, which catalyzes the formation of S -adenosylmethionine, a cosubstrate for melatonin in the pineal gland, follows the pattern of hydroxyindole- O -methyltransferase. Around the middle of the dark period, methionine S -adenosyltransferase and hydroxyindole- O -methyltransferase appear to be elevated by 2.5- and 1.5-fold, respectively, and tend to fall back during the light period.     

Cyclin D2 Interacts with cdk-5 and Modulates Cellular cdk-5/p35 Activity

Abstract: Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neurone-specific activity. Experimental manipulation of cdk-5 expression in neurones has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, we used the yeast two-hybrid system to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacted with cdk-5. Immunoprecipitation experiments confirmed that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates, which was in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression led to a decrease in cdk-5/p35 activity in transfected cells. As cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurones.     

M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools

Abstract: This study shows that activation of M₁ muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)-K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca²⁺ concentration and nNOS activity. We examined the relationship between receptor-mediated increases in intracellular Ca²⁺ concentration and activation of nNOS over both short and long intervals using guanosine 3',5'-cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca²⁺ from intracellular stores in both the CHO M₁/nNOS transfected cells and in neuroblastoma (N1E-115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M₁ muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l -[³H]arginine to l -[³H]citrulline as well as cGMP formation following stimulation of M₁ muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca²⁺ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca²⁺.     

Controls on microbial CO2 production: a comparison of surface and subsurface soil horizons

Although a significant amount of the organic C stored in soil resides in subsurface horizons, the dynamics of subsurface C stores are not well understood. The objective of this study was to determine if changes in soil moisture, temperature, and nutrient levels have similar effects on the mineralization of surface (0–25 cm) and subsurface (below 25 cm) C stores. Samples were collected from a 2 m deep unsaturated mollisol profile located near Santa Barbara, CA, USA. In a series of experiments, we measured the influence of nutrient additions (N and P), soil temperature (10–35°C), and soil water potential (?0.5 to ?10 MPa) on the microbial mineralization of native soil organic C. Surface and subsurface soils were slightly different with respect to the effects of water potential on microbial CO₂ production; C mineralization rates in surface soils were more affected by conditions of moderate drought than rates in subsurface soils. With respect to the effects of soil temperature and nutrient levels on C mineralization rates, subsurface horizons were significantly more sensitive to increases in temperature or nutrient availability than surface horizons. The mean Q₁₀ value for C mineralization rates was 3.0 in surface horizons and 3.9 in subsurface horizons. The addition of either N or P had negligible effects on microbial CO₂ production in surface soil layers; in the subsurface horizons, the addition of either N or P increased CO₂ production by up to 450% relative to the control. The results of these experiments suggest that alterations of the soil environment may have different effects on CO₂ production through the profile and that the mineralization of subsurface C stores may be particularly susceptible to increases in temperature or nutrient inputs to soil.