Molecular identification of phenylalanine ammonia-lyase as a substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts.

Phenylalanine ammonia-lyase (PAL) is a key enzyme in pathogen defence, stress response and secondary metabolism and is subject to post-translational phosphorylation. In order to address the significance of this phenomenon it is necessary to identify the protein kinase (PK) responsible and place it in its regulatory circuit. Using protoplast transient expression of Arabidopsis kinase genes coupled to immunocomplex kinase assay, it has been possible to screen for specific PAL kinase. We show here that AtCPK1 (calcium dependent PK), but not other closely related PKs could phosphorylate both a recombinant PAL protein and a peptide (SRVAKTRTLTTA) that is a site phosphorylated in vivo. Identification of the specific CDPK as a PAL kinase now opens up the possibility of exploring the calcium link in biotic stress signalling, salicylate and phytoalexin production as well as the significance of PAL phosphorylation. The protoplast transient expression system is a potentially powerful method to determine and screen for plant gene functions utilising genomic and proteomic data.

Flavor variety enhances food acceptance in formula-fed infants.

BACKGROUND: Research in humans and animal models suggests that acceptance of solid foods by infants during weaning is enhanced by early experiences with flavor variety. OBJECTIVE: We tested the hypotheses that the acceptance of novel foods by formula-fed infants could be facilitated by providing the infants with a variety of flavors at the time when beikost is first introduced and that, contrary to medical lore, infants who had previously consumed fruit would be less likely to reject vegetables when first introduced than would infants without such an experience. DESIGN: The infants' acceptance of a novel vegetable (puréed carrot) and a novel meat (puréed chicken) was evaluated after a 9-d exposure period in 3 groups of infants, some of whom had previously consumed fruit. During the home-exposure period, one group was fed only carrots, the target vegetable; a second group was fed only potatoes, a vegetable that differed in flavor from carrots; and a third group was fed a variety of vegetables that did not include carrots. RESULTS: Infants fed either carrots or a variety of vegetables, but not those fed potatoes, ate significantly more of the carrots after the exposure period. Exposure to a variety of vegetables also facilitated the acceptance of the novel food, puréed chicken, and daily experience with fruit enhanced the infants' initial acceptance of carrots. CONCLUSION: These findings are the first experimental evidence to indicate that exposure to a variety of flavors enhances acceptance of novel foods in human infants.

Short-term influence of breastfeeding on the infants' interaction with the environment.

The present study tested the hypothesis that around the age of weaning, human infants are more interactive with their environment immediately after breastfeeding. To this aim, we assessed 4- to 6-month-old infants' responsiveness to a rotating, musical mobile before breastfeeding on one test day, and after breastfeeding on another. Thirteen infants, fitted with miniature movement detectors on both their left leg and wrist, were videotaped for 4 consecutive min during which the mobile was alternately switched on and off in 1-min intervals. Although the infants showed no significant differences in limb activity as a function of the timing of a breastfeed, they looked at the mobile significantly longer when tested after breastfeeding. This finding suggests that breastfeeding has a substantial effect on 4- to 6-month-old infants' attentiveness to and interaction with their environment.

Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue.

The site of phosphorylation of phenylalanine ammonia-lyase (PAL) has been identified as a threonine residue. A Ca(2+)-stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease in Vmax in agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545 in the sequence VAKRTLTT (539-546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants.

Recent advances in understanding the origin of the apoplastic oxidative burst in plant cells.

The origin of the oxidative burst during plant-pathogen interactions remains controversial. A number of possibilities have been identified, which involve the protoplast, plasmalemma or apoplast. The apoplastic production of H2O2 requires three components, an extracellular peroxidase, ion fluxes leading to extracellular alkalinisation and release of a substrate. Fatty acids are the major compounds that appear in the apoplast following elicitation, which can activate H2O2 production by peroxidases in vitro. However, the reaction with peroxidases appears to be novel and is uncharacterised at present. The apoplastic mechanism also cannot be readily distinguished from the operation of a plasma membrane NADPH oxidase system by the use of the inhibitors diphenylene iodonium and N,N diethyl-dithiocarbamate since it is also inhibited by these. These inhibitors have often in the past been used to define the involvement of the latter in the oxidative burst. In common with the NADPH oxidase system, the peroxidase responsible has been cloned but unlike the NADPH oxidase it has been shown to function in vitro to generate H2O2. In vivo studies of the oxidative burst have shown that the alkalinisation is essential and the underlying ion fluxes may be regulated by cAMP. Calcium fluxes are also essential. Although the oxidative activity of peroxidase requires calcium the fluxes have obvious other function. These may include activation of release of substrate and through the activation of a CDPK, regulation of enzymes involved in phytoalexin and cell wall phenolic production such as PAL.

Acute, early thermal experience alters weaning onset in rats.

We hypothesized that first ingestion of solid food (weaning onset) would be accelerated in young rats with advanced thermoregulatory development. To manipulate the pups' thermoregulatory development, we exposed rat pups, but not their dams, to a Cold (10 degrees C), Moderate (21 degrees C), or Warm (31 degrees C) ambience for 2 h/day from postnatal Day 2-14, expecting that early exposure to cooler temperatures would accelerate development of thermoregulatory capabilities and thus accelerate nest egression as well as onset of feeding. Contrary to expectation, cold exposure was associated with a profile of developmental delays in both growth and maturation. Pups in the Cold condition began feeding later than pups with Moderate or Warm thermal experiences. We then evaluated thermoregulatory status (mechanisms for heat production and temperature conservation) on Day 15-16 (just prior to weaning onset). Thermogenesis, measured by oxygen consumption, was unaltered by the thermal manipulation. In contrast, pelage development (insulation) was altered. Pups in the Warm condition had greater fur density and an increased frequency of longer hairs relative to pups in the Cold condition. Although the developmental response to early cold exposure was in the direction opposite to our predictions, the hypothesized relation of thermoregulatory development to weaning onset was supported: Thermoregulatory status correlated with weaning onset.

Effects of exposure to alcohol in mother's milk on infant sleep.

OBJECTIVE: To test the hypothesis that exposure to alcohol in breast milk affects infants' sleep and activity levels in the short term. METHODS: Thirteen lactating women and their infants were tested on 2 days, separated by an interval of 1 week. On each testing day, the mother expressed 100 mL of milk, while a small, computerized movement detector called an actigraph was placed on the infant's left leg to monitor sleep and activity patterning. After the actigraph had been in place for approximately 15 minutes, the infants ingested their mother's breast milk flavored with alcohol (32 mg) on one testing day and breast milk alone on the other. The infants' behaviors were monitored for the next 3.5 hours. RESULTS: The infants spent significantly less time sleeping during the 3.5 hours after consuming the alcohol-flavored milk (78.2 minutes compared with 56.8 minutes after feeding alcohol in breast milk). This reduction was apparently attributable to a shortening in the longest sleeping bout (34.5 compared with 56.7 minutes for sleeping after breast milk alone) and the amount of time spent in active sleep (25.8 minutes compared with 44.2 minutes after breast milk alone); the decrease in active sleep was observed in all but 2 of the 13 infants tested. CONCLUSIONS: Although the mechanisms underlying the reduction in sleep remain to be elucidated, this study shows that short-term exposure to small amounts of alcohol in breast milk produces distinctive changes in the infant's sleep-wake patterning.

Comparative biochemistry of the oxidative burst produced by rose and french bean cells reveals two distinct mechanisms

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N, N-diethyldithiocarbamate, superoxide was detected by a N, N'-dimethyl-9,9'-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.

Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants.

The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.

Postsuckling behavioral arousal in weanling rats (Rattus norvegicus).

Twenty-day-old litters and their dams were observed in seminatural habitats consisting of a nest compartment and adjacent open field that contained powdered rat chow. It was found that pups displayed marked bursts of activity after suckling. Independent feeding reliably followed nursing bout termination (Experiment 1). Nipple withdrawal, with or without milk transfer, induced behavioral arousal whereas withdrawal of thermotactile and conspecific odor cues did not (Experiments 2-3). Increased thermogenesis was observed following milk transfer (Experiment 4). Finally, preweanling pups (10- to 12-day-olds) also displayed postsuckling arousal within the confines of the nest; full locomotor expression of this arousal was not evident until weaning age (Experiment 5). It was concluded that postsuckling arousal in weanlings functions to stimulate activities performed away from the nest and suckling, propelling pups into the field where feeding begins.

Environmental temperature modulates onset of independent feeding: warmer is sooner.

Individual dams and their litters were observed from Days 14-22 in a seminatural environment consisting of a nestbox attached to a larger, open field in which powdered chow was available. Ambient temperature in the field was either warm (30 degrees C), moderate (21 degrees C), or cold (10 degrees C); nest temperature was always moderate. Behavior was monitored 12 hr/day by time-lapse video recording. The pups' egressions into the field and onset of independent feeding were temperature-related: Weaning was earliest in the warmth and increasingly late with decreasing ambient temperature. Among subjects in the cold condition, there was a positive correlation between duration in the field and duration feeding. Pup growth was unaffected by the temperature regimes. Environmental temperature has emerged as a determinant for early nest egressions and weaning onset.

Differential distribution of ferulic acid to the major plasma constituents in relation to its potential as an antioxidant.

The hydroxycinnamates, intermediates in the phenylpropanoid synthetic pathway, are effective in enhancing the resistance of low-density lipoprotein (LDL) to oxidation in the order caffeic acid > ferulic acid > p-coumaric acid. It is unclear whether the mode of action of ferulic acid as an antioxidant is based on its activities in the aqueous or the lipophilic phase. Partitioning of 14C-labelled ferulic acid into plasma and its components, LDL and the albumin-rich fractions, has been studied under conditions of maximum aqueous solubility. The majority of ferulic acid associates with the albumin-rich fraction of the plasma, although a proportion is also found to partition between the LDL and aqueous phases; however, ferulic acid does not associate with the lipid portion of the LDL particle, suggesting that it exerts its antioxidant properties from the aqueous phase. This is of particular interest since the results demonstrate that ferulic acid is a more effective antioxidant against LDL oxidation than the hydrophilic antioxidant ascorbic acid.

Differential influence of adult and juvenile conspecifics on feeding by weanling rats (Rattus norvegicus): a size-related explanation.

Weanling Sprague Dawley rat pups (Rattus norvegicus) selected between 2 safe palatable diets in concordance with the preferences of either an adult or a juvenile conspecific model (Experiment 1). Nevertheless, weanlings chose to feed more in the vicinity of an adult than in the vicinity of a juvenile, thus fulfilling the prediction of an adaptive feeding strategy (Experiment 2). The weanlings' bias for feeding in the vicinity of an adult was eliminated by increasing the magnitude of pup stimulus to 3 pups (Experiment 3). Thus, weanlings do not possess a specialization rendering them more sensitive to adults than to pups as models for diet selection. By responding to stimulus magnitude, weanlings are more likely to feed with adult conspecifics, choose foods used by them, and derive the benefits correlated with the adults' successful feeding habits.

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.).

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H2O2. The cells recover and start producing ATP immediately of H2O2 production. This indicates that the increased O2 uptake is primarily incorporated into active O2 species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M(r) 40,000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.

Sequence analysis of photoaffinity-labelled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine.

Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be Leu-Gly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137-Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137-Arg225 are Ser148-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.

Automated percutaneous discectomy: the role of the physical therapist.

A number of surgical techniques are used in the treatment of herniated intervertebral discs. The purpose of this report was to introduce the technique of automated percutaneous discectomy and suggest a postoperative physical therapy program. Automated percutaneous discectomy (APD) is a new innovative surgical technique used for the treatment of herniated lumbar discs. The procedure is performed under local anaesthesia on an outpatient basis and takes approximately one hour. The success of this procedure depends upon proper patient selection based on physical examination and differential diagnosis. The role of the physical therapist in postoperative care for the APD patient is discussed. The rationale for the suggested protocol is based on the McKenzie approach to the treatment of mechanical low back pain, emphasizing patient education and prophylaxis. The concept of spinal stabilization training is also introduced to facilitate return to daily activities. J Orthop Sports Phys Ther 1992;15(1):2-9.

An analysis of the educational role of the ward sister (implications for nurse educators).

The current scheme of nurse education in the United Kingdom has, as its foundation, the premise that student nurses learn in the clinical setting, whilst caring for patients. In order to maximise the learning potential of the clinical environment it is important for nurse educators to have an understanding of the complexities of the ward situation and in particular the role of the ward sister. Within the context of role theory, this paper presents an analysis of the educational role of the ward sister and considers the extent to which her clinical and management roles inhibit her ability to effectively fulfil her educational role. In the light of complex role expectations, the issues of role conflict and role ambiguity are addressed. The need for nurse educators to be more empathic towards the role demands imposed upon the ward sister and possible supportive measures are then considered.

Purposes, values and objectives in adult education--the post-basic perspective.

In the light of increasing calls for nurse education to adopt an education model based on the principles of adult learning this paper examines the place of objectives in adult education and how they relate to the general purposes and values in education. The development of the use of objectives is discussed from its origins in the behaviourist school of thought through to the more recent approach adopted by those purporting to embrace the principles of adult education. Implications for the use of objectives in nurse education, and in particular post-basic nurse education, are then considered.