Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.     

How do we educate young people to balance carbohydrate intake with adjustments of insulin?

The dietary management of childhood diabetes is complex. Is it possible to educate young people to balance carbohydrate with their insulin? Can dietary knowledge be translated into lasting behaviour change? Do present teaching methods provide the skills necessary for children and parents to adjust their insulin therapy adequately? Evidence shows great variation in glycaemic control between centres and countries but the impact of dietary education methods is poorly evaluated and its links with clinical and psychosocial outcomes is virtually unknown. There is also little evidence to suggest cohesive teamworking with clear dietary targets for glycaemic control, lipids, incidence of hypoglycaemia, compliance, effect on peer and sibling relationships, and evaluation of individual dietary components, e.g. fibre, fat, antioxidants. There is wide variation in methods of dietary education, which are often based on historic practice. They include rigid counting of grams of carbohydrate, carbohydrate portion assessments, qualitative diets, low glycaemic index diets and the more recent 'intensified' carbohydrate measures with daily adjustments of insulin (the basis also of pump management). This last method has many benefits although it requires extensive nutrition education, it allows greater flexibility and variety of food intake, is sensitive to the varying daily energy expenditure of childhood and it addresses postprandial glycaemic excursions, all of which are inadequately managed by conventional therapy. However, one of the problems of overemphasizing carbohydrate measurement is that total carbohydrate intake may be suppressed, with a resulting increase in fat, this may contribute to an increase in cardiovascular risk. The ISPAD Consensus Guidelines 2000 contain dietary recommendations but scientific evidence is often lacking. Limited dietary studies show that some countries can meet guidelines more successfully than others. There are many reasons for this, such as food availability, types of food eaten, food preferences and family/cultural/religious influences. Educational methods must be adapted to local customs. Is there enough evidence to recommend a particular dietary education method? What outcomes do we hope to achieve? The workshop explored these issues in order to develop a deeper understanding of the complexity of dietary modification in childhood diabetes.     

Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy

The environmental scanning electron microscope (ESEM) provides a highly relevant and controllable environment in which to study hydrated systems without the artefacts of other highly prepared specimens. The instrument facilitates control of turgor through hydration using different chamber vapour pressures. Deformation of a simple plant tissue-upper epidermal layers in Allium cepa (onion)-was observed at the scale of the two principal failure mechanisms: cell breakage; and cell separation induced by treatment with a chelating agent. Cell rupture and release of contents occurred at cellular junctions ahead of an imposed growing notch, indicating that disruption of cells occurred remotely from the creation of a new surface. Cells that separated usually maintained their turgor and the separation process took place through progressive failure of middle lamellar material seen as strands between separating cells. These mechanisms were compared with the rupture of excised Chara corallina walls that occurred by formation and breakage of strands between separating wall layers. This study provides in situ visual characterization of wall rupture and cell separation at the microscopic level in hydrated plant material.     

FT-IR study of the Chara corallina cell wall under deformation

Fourier-transform infrared (FT-IR) microspectroscopy was used to investigate both the chemical composition of, and the effects of an applied strain on, the structure of the Chara corallina cell wall. The inner layers of the cell wall are known to have a transverse cellulose orientation with a gradient through the thickness to longitudinal orientation in the older layers. In both the native state and following the removal of various biopolymers by a sequential extraction infrared dichroism was used to examine the orientation of different biopolymers in cell-wall samples subjected to longitudinal strain. In the Chara system, cellulose microfibrils were found to be aligned predominantly transverse to the long axis of the cell and became orientated increasingly transversely as longitudinal strain increased. Simultaneously, the pectic polysaccharide matrix underwent molecular orientation parallel to the direction of strain. Following extraction in CDTA, microfibrils were orientated transversely to the strain direction, and again the degree of transverse orientation increased with increasing strain. However, the pectic polysaccharides of the matrix were not detected in the dichroic difference spectra. After a full sequential extraction, the cellulose microfibrils, now with greatly reduced crystallinity, were detected in a longitudinal direction and they became orientated increasingly parallel to the direction of strain as it increased.     

The Utility of Carbon and Nitrogen Isotope Analyses to Trace Contributions from Fish Farms to the Receiving Communities of Freshwater Lakes: a Pilot Study in Esthwaite Water,UK

A pilot study was conducted to assess the potential for stable isotope analyses to reveal the fate of waste pelleted food material from fish farms in freshwater food webs. Esthwaite Water (Cumbria, UK) was selected as the study site, as it hosts an established salmonid farm, and a wealth of complementary limnological data exists. Salmonid pellet feed consists of primarily marine-derived material and thus exhibits carbon and nitrogen stable isotopic compositions distinct to most freshwater organic material. Comparison of the isotopic ratios of organisms at the cage site with an unaffected control site, supports incorporation of pellet-derived material to the diet of planktonic and benthic communities. Moreover, after allowing for a number of trophic transfers, stable isotope analyses revealed the predatory cladoceran Leptodora kindti also utilised pellet material, while roach were probably short-circuiting the food chain by directly consuming particulate pellet material, as well as via ingestion of their zooplankton prey. Isotope data substituted into a simple two-source mixing model suggested that approximately 65% of Daphnia, and >80% of roach body carbon may be derived from pellet material in the plankton, and that chironomid larvae may incorporate >50% in the sediment environs. However, contributions calculated from both ¹³C and ¹⁵N values were inconsistent, which may simply be due to the constraints of the model and parameters used, but may also reflect different routing of isotopes from the original pellet source, via soluble or particulate routes.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser⁷⁴⁸ autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4⁻). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser⁷⁴⁴, a prominent and rapid PKC transphosphorylation site, and Ser⁷⁴⁸, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4⁻ potently induced PKD activation and Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser⁷⁴⁸ phosphorylation induced by AlF4⁻, and largely abolished Ser⁷⁴⁴ phosphorylation. In contrast, Ser⁷⁴⁸ phosphorylation was almost completely intact, and Ser⁷⁴⁴ phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser⁷⁴⁸ phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser⁷⁴⁸.     

Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth

Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.     

Insulin sensitivity, fat distribution, and adipocytokine response to different diets in lean and obese cats before and after weight loss

Obesity is a major health problem in cats and a risk factor for diabetes. It has been postulated that cats are always gluconeogenic and that the rise in obesity might be related to high dietary carbohydrates. We examined the effect of a high-carbohydrate/low-protein (HC) and a high-protein/low-carbohydrate (HP) diet on glucose and fat metabolism during euglycemic hyperinsulinemic clamp, adipocytokines, and fat distribution in 12 lean and 16 obese cats before and after weight loss. Feeding diet HP led to greater heat production in lean but not in obese cats. Regardless of diet, obese cats had markedly decreased glucose effectiveness and insulin resistance, but greater suppression of nonesterified fatty acids during the euglycemic hyperinsulinemic clamp was seen in obese cats on diet HC compared with lean cats on either diet or obese cats on diet HP. In contrast to humans, obese cats had abdominal fat equally distributed subcutaneously and intra-abdominally. Weight loss normalized insulin sensitivity; however, increased nonesterified fatty acid suppression was maintained and fat loss was less in cats on diet HC. Adiponectin was negatively and leptin positively correlated with fat mass. Lean cats and cats during weight loss, but not obese cats, adapted to the varying dietary carbohydrate/protein content with changes in substrate oxidation. We conclude that diet HP is beneficial through maintenance of normal insulin sensitivity of fat metabolism in obese cats, facilitating the loss of fat during weight loss, and increasing heat production in lean cats. These data also show that insulin sensitivity of glucose and fat metabolism can be differentially regulated in cats.