Ribulose 1, 5-bisphosphate Carboxylase/ Oxygenase Activities in Leaves of Greenhouse Roses

An enzyme assay was developed to measure the initial and Mg²⁺–CO₂activated forms of Ribulose 1,5-bisphosphate Carboxylase/Oxygenase(Rubisco) in rose leaves. The assay was verified by co-extractionof the leaflets with partially purified spinach Rubisco andthrough correlation with net photosynthetic rates of individualleaflets (r²=0.7324). Changes in activities were measured asa function of depth of leaves in the canopy for two cultivarsof greenhouse hybrid tea roses. Initial Rubisco activity declinedwith increasing canopy depth for both cultivars. The activatedform of the enzyme, however, remained constant with canopy depthfor cv. Red Success; but increased with canopy depth, then declinedafter mid-canopy in the cv. Royalty. Rubisco activities werealso measured in the cv. Red Success grown in CO₂ enriched environments(100 mm³ dm^–3) at three humidity levels. The activitieswere not significantly affected by humidity treatment. However,there was a trend for plants grown at lower humidity to havehigher activated activities. Key words: Humidity, Rubisco, Rosa ? hybrida, Royalty, Red Success     

Osmoregulation in Larvae of the Land-Crab, Cardisoma guanhumi Latreille

Daily studies were made of the osmoregulatory abilities of Cardisomaguanhumi from hatching to the end of larval development. Theseresults were compared to those of similar research on osmoregulationby larvae and megalopa of four species of estuarine, littoral,and sub-littoral crabs. The comparison shows that larvae ofC. guanhumi possess the same kinds of adaptations for waterintake at the time of molt that were found in Rhithropanopeusharrisii. Land-crab larvae hyperregulated in 10 p.p.t. sea waterand hyporegulated in water of 40 p.p.t. for experimental periodsfor 2 hr during the first one-third of their development. Duringthe remainder of larval life, they hyporegulated against 15p.p.t. in intermolt periods and became isosmotic with, or hyporegulatedagainst, 10 p.p.t. at the time of molting. From the time ofhatching, the osmoregulatory pattern of developing C. guanhumifits them for deep penetration of estuaries and for crossingsteep saline gradients. This pattern is evidence for a strongerand more enduring control of water balance, especially at thetime of molting, than we have found in non-terrestrial species.     

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

New Silurian cooksonias from dolostones of north-eastern North America

New specimens of Cooksonia and Hostinella are described from the Bertie Group of Ontario and New York State, which is dated by faunas as latest Silurian (Přídolí). The rare plant fossils are unusual in that they are preserved in fine-grained, slightly argillaceous dolostones ('waterlime') rather than clastic rocks. At least two species of Cooksonia are present, one with ± globular sporangial morphology close to C. hemisphaerica Lang. Those with ellipsoidal/discoidal sporangia are compared with C. pertoni Lang, C .  paranensis Gerrienne et al . and C. bohemica Schweitzer, the latter represented by a single specimen from the Přídolí of the Czech Republic. However, the paucity of specimens, which prevents assessment of taphonomic influences on shape, combined with the absence of any anatomical features and the gross morphological simplicity of the fossils, precludes specific assignment. Specimens of Hostinella include one in which apices and a lateral basal structure resembling a root are preserved. It is concluded that the Laurentian assemblage of Ontario and New York State is less diverse and disparate than coeval assemblages, which are also preserved in marine rocks. Its preservation in limestones may have been facilitated by the hypersalinity inferred from various sedimentary features, which would restrict the activity of many decomposers.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 399–413.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Visfatin is regulated by rosiglitazone in type 2 diabetes mellitus and influenced by NFκB and JNK in human abdominal subcutaneous adipocytes

Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin''s regulation by insulin and RSG, potentially acting through NF-κB and JNK mechanisms, with only rh IL-6 modestly affecting visfatin regulation. Taken together, these findings suggest that visfatin may represent a pro-inflammatory cytokine that is influenced by insulin/insulin sensitivity via the NF-κB and JNK pathways.