Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

In vitro study of lipid metabolism in the mealworm larval fat body

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time-dependent release of lipid was strongly stimulated in a dose-dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6-¹⁴C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml^?1). Intestinal insulin-like peptide (ILP) also stimulated in vitro lipogenesis in a dose-dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.     

Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.Abbreviations C Craigella, isogenic line - CK cytokinin - Cls Craigella lateral suppressor - EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride - ELISA enzyme-linked immunosorbent assay - 2iP isopentenyladenine - 2iPA isopentenyladenosine - PAP peroxidase-anti-peroxidase - PFAG paraformaldehyde/glutaraldehyde mixture - Z zeatin - ZR zeatin riboside     

DNA synthesis in excised tobacco leaves after bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin spurr sections

We describe a method for localizing replicating cells in detached tobacco leaves allowed to root. The proposed protocol has shown that formalin fixation and Spurr embedding of petiole bases can be used for demonstrating DNA synthesis after bromodeoxyuridine (BrdU) incorporation. The incorporated BrdU was immunologically visualized. After resin removal, different procedures of DNA denaturation and protease digestion were tested. Combined hydrolysis with 4 N HCl for 10 min at room temperature and digestion with 0.4% pepsin for 15 min at 37 degrees C led to the best reproducible results, with either the peroxidase or the gold detection system. The method is rapid and sensitive, with precise resolution. It can be used at the light and electron microscopic levels. Its potential application is to elucidate in the same organ the role of cytokinins, a class of plant growth regulators, in dividing cells and to define the chronology of their biosynthesis in roots in relation to DNA synthesis.     

Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA,BAP and zeatin in stimulating plantlet development

In an effort to understand the causes of arrest of somatic embryo development, generally observed in grapevine (Vitis sp.), histological studies were undertaken, using two cultivars (CH76 and 41B) which differ in their ability to develop into plants. Embryos with a high conversion rate (70%; CH76) formed a well-structured and functional shoot apex between two thread-like cotyledons. In contrast, embryos with a low conversion rate (10%; 41B) formed a normal root apex but lacked a well-structured shoot apex and developed a wide range of aberrant forms in the intercotyledonary area: uncontrolled cellular proliferation, formation of adventitious buds, over-growth of cotyledonary or leaf meristems. ABA increased the conversion rate of 41B embryos from 10% to 20%, but failed to improve embryo morphology. Zeatin and BAP promoted growth of 41B somatic embryos, but generated a high level of abnormalities and failed to improve conversion rate. Applied in combination with ABA, these PGRs increased the frequency of cotyledonary embryos, but decreased the conversion rate.     

HETEROCHRONIC DEVELOPMENTAL PLASTICITY IN LARVAL SEA URCHINS AND ITS IMPLICATIONS FOR EVOLUTION OF NONFEEDING LARVAE

Preexisting developmental plasticity in feeding larvae may contribute to the evolutionary transition from development with a feeding larva to nonfeeding larval development. Differences in timing of development of larval and juvenile structures (heterochronic shifts) and differences in the size of the larval body (shifts in allocation) were produced in sea urchin larvae exposed to different amounts of food in the laboratory and in the field. The changes in larval form in response to food appear to be adaptive, with increased allocation of growth to the larval apparatus for catching food when food is scarce and earlier allocation to juvenile structures when food is abundant. This phenotypic plasticity among full siblings is similar in direction to the heterochronic evolutionary changes in species that have greater nutrient reserves within the ova and do not depend on particulate planktonic food. This similarity suggests that developmental plasticity that is adaptive for feeding larvae also contributes to correlated and adaptive evolutionary changes in the transition to nonfeeding larval development. If endogenous food supplies have the same effect on morphogenesis as exogenous food supplies, then changes in genes that act during oogenesis to affect nutrient stores may be sufficient to produce correlated adaptive changes in larval development.     

Abscisic acid, indole-3-acetic acid and cytokinin changes in buds of Pseudotsuga menziesii during bud quiescence release

Bud quiescence release, considered as the ultimate dormancy breaking phase, was achieved in Pseudotsuga menziesii (Mirb.) Franco by a 9-week cold (5°C) treatment, under short daylength (9 h) followed by a transfer to mild temperature (22°C) under long daylength (16 h). Indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin-type (Z) and isopentenyladenine-type (iPA) cytokinin (CK) levels were measured by means of an ELISA technique performed on HPLC-fractionated extracts of terminal and axillary buds. During the cold period, all hormones except IP-type CK levels decreased, whereas the opposite observation was made after transfer to mild temperature and long daylength, when buds started to grow. Some other immunoreactive compounds were also detected and quantified. The ABA-glucosyl ester (ABA-GE) level pattern was similar to that of ABA, but no accumulation occurred at mild temperatures. A putative IAA conjugate, more polar than IAA, was also detected. Its level increased transiently like IAA in terminal buds and, to a lesser extent, in axillary buds during the 10th week of the experiment. In terminal buds, isopentenyladenosine ([9R]-iP) was released by alkaline hydrolysis of a polar immunoreactive compound detected with anti-[9R]iP antibodies. This compound accumulated during the cold period and quickly dropped at 22°C. Relationships between environmental conditions and endogenous hormones are discussed.     

Some characteristics of a fast movement of auxin in intact tomato seedlings (Lycopersicon esculentum)

Using exogenous ¹⁴C-IAA, a fast movement of auxin was demonstrated in intact tomato plants ( Lycopersicon esculentum Mill, cv. Craigella) cultured in vitro. This movement is insensitive to or weakly enhanced by cold temperature (0°C) and not influenced by gravity. In short time experiments (10 min) the main part of the radioactivity which travels through the plant is IAA; in long time ones (6 h) the metabolism is much higher and leads mainly to the formation of IAAsp. The integrity of the plant is necessary for the possibility to observe such a fast movement. Isolated hypocotyl segments show only the classical polar and slow movement of auxin. Microautoradiographic techniques show that the fast movement takes place inside the plant. In the epicotyl, the vascular bundles, mainly phloem cells, are labelled; in contrast, the radioactivity detected in the hypocotyl is low, and the tracers can be visualized only inside the epidermal cells.     

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.