Thresholds in development

The interpretation of gradients in positional information is considered in terms of thresholds in cell responses, giving rise to cell states which are discrete and persistent. Equilibrium models based on co-operative binding of control molecules do not show true thresholds of discontinuity, though with a very high degree of co-operativity they could mimic them; in any case they do not provide the cells with any memory of a transient signal. A simple kinetic model based upon positive feedback can account both for memory and for discontinuities in the pattern of cell states. The model is an example of a bistable control circuit, and transitions from one state to another may be brought about not only by morphogenetic signals, but also by disturbances in the parameters determining the kinetics of the system. This might explain some aspects of transdetermination in insects.An attempt is made to analyse the precision with which a spatial gradient of a diffusible morphogen could be interpreted by a kinetic threshold mechanism, in terms of the length of the field, the steepness of the concentration gradient, and the intrinsic random variability of cells. It is concluded that it would be possible to specify as many as 30 distinct cell states in a positional field 1 mm long with a concentration span of 10³. Mechanisms for reducing the positional error are considered.     

Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf.

1. When [2-3H]glycerol was supplied to developing maize-leaf laminae, label entered 3-sn-phosphatidycholine at a linear rate essentially from zero time, whereas other lipids were labelled at accelerating rates. On transfer of laminae from [3H]glycerol to unlabelled glycerol, radioactivity was rapidly lost from 3-sn-phosphatidylcholine and accumulated in other lipids, principally monogalactosyl diacyglycerol. 2. Degradation of these lipids showed that 3H was present only in the glycerol moiety of the lipids. 3. In double-labelling pulse-chase experiments with [14C]acetate, which labelled essentially only fatty acids and [3H]glycerol similar amounts of 14C and 3H radioactivity were lost from 3-sn-phosphatidylcholine and accumulated by monogalactosyl diacylglycerol. 4. The different molecular species of both lipids isolated from laminae during a double-labelled pulse-chase study were separated by argentation t.l.c., and the changes in the amount of radioactivity and the 14C/3H ratio in different species were compared. The greatest loss of radioactivity during the period in unlabelled substrates occurred from the 3-sn-phosphatidylcholine species containing oleate and from the dilinoleate species, and radioactivity accumulated by monogalactosyl diacyglycerol was mainly in the dilinolenate species. However, despite the considerable change in the radioactivity in these species during the chase, the 14C/3H ratio in each of them remained relatively unchanged. 5. It is proposed that 3-sn-phosphatidylcholine in the developing leaf may serve as a donor or linoleate-containing diacyl-glycerols which are incorporated into other lipids, principally monogalactosyl diacylglycerol.     

Production of diacylglycerol by exogenous phospholipase C stimulates CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in human neuroblastoma cells.

The involvement of endogenous diacylglycerol production in the stimulation of phosphatidylcholine synthesis by exogenous phospholipase C was examined using a neuroblastoma (LA-N-2) cell line. Phospholipase C treatment (0.1 unit/ml) of intact cells stimulated CTP:phosphocholine cytidylyltransferase activity significantly more effectively than did maximally effective concentrations of the synthetic diacylglycerol sn-1,2-dioctanoylglycerol (1 mM). When added to cells together with phospholipase C, oleic acid, but not dioctanoylglycerol, further increased cytidylyltransferase activity with respect to phospholipase C treatment alone, indicating that the enzyme was not maximally activated by the lipase. This suggests that the lack of additivity of diacylglycerol and phospholipase C reflects a common mechanism of action. The time course of activation of cytidylyltransferase by phospholipase C paralleled that of [3H]diacylglycerol production in cells prelabeled for 24 h with [3H]oleic acid. Diacylglycerol mass was similarly increased. Significant elevations of [3H]oleic acid and total fatty acids occurred later than did the increases in cytidylyltransferase activity and diacylglycerol levels. No significant reduction in total or [3H]phosphatidylcholine was elicited by this concentration of phospholipase C, but higher concentrations (0.5 unit/ml) significantly reduced phosphatidylcholine content. The stimulation of cytidylyltransferase activity by phospholipase C or dioctanoylglycerol was also associated with enhanced incorporation of [methyl-14C]choline into phosphatidylcholine. Dioctanoylglycerol was more effective than phospholipase C at stimulating the formation of [14C]phosphatidylcholine, and the effects of the two treatments were additive. However, further analysis revealed that dioctanoylglycerol served as a precursor for [14C]dioctanoylphosphatidylcholine as well as an activator of cytidylyltransferase; and when corrections were made for this effect, the apparent additivity disappeared. The results indicate that the generation of diacylglycerol by exogenous phospholipase C (and possibly the subsequent production of fatty acids via diacylglycerol metabolism) activates cytidylyltransferase activity in neuronal cells under conditions in which membrane phosphatidylcholine content is not measurably reduced.     

Phosphorus Distribution in Red Pine Roots and the Ectomycorrhizal Fungus Hebeloma arenosa

Red pines (Pinus resinosa Ait.) were grown in a pasteurized sandy loam either unamended with phosphate or fertilized with one of two levels of phosphate (34 or 136 mg/kg) as superphosphate, and with and without addition of Hebeloma arenosa inoculum. Shoot and total dry weights of mycorrhizal seedlings grown in soil unamended with P were greater than those for nonmycorrhizal seedlings grown in the same soil, but less than the dry weights of seedlings grown in soil amended with middle to high levels of P. Mycorrhizal infection was inhibited at the highest level of P amendment. ³¹P nuclear magnetic resonance spectra of intact mycorrhizal roots showed the presence of two dominant peaks, orthophosphate (Pi) and polyphosphate (polyP). The polyP peak was absent in spectra of nonmycorrhizal roots. The ratio for areas under the two peaks, Pi/polyP, was 1.8 for mycorrhizal roots grown in both unamended soil and soil that had received middle levels of superphosphate. Apparently, the fungus strongly mediates the supply of phosphate to the tree through the production of polyP, even at growth-limiting levels of soil P, and regulates compartmentalization of P in the mycorrhizal roots.     

Single cell analysis of mesoderm formation in the Xenopus embryo

We have examined the developmental specification of individual cells in the Xenopus blastula using a new in vitro culture system. Regional differences are apparent at the mid-blastula stage when animal hemisphere cells form only ectodermal cell types, while many clones from below the pigment boundary contain mesodermal cell types. A number of clones give rise to more than one differentiated cell type indicating that the initial steps of mesoderm induction are potentially reversible. Animal hemisphere cells can be induced to form mesoderm by fibroblast growth factor (FGF). Different cell types predominate at different FGF concentrations and the neighbours in this sequence are also the pairs of cell types most usually associated in mixed clones derived from the marginal zone. We propose that the specification of individual cells depends upon both the concentration of inducing factor and on stochastic intracellular events.     

Value of outpatient follow-up after curative surgery for carcinoma of the large bowel.

The records were reviewed of 406 patients with carcinoma of the large bowel who had been treated at the Middlesex Hospital during 1958-62. Of these patients, 180 were followed up regularly in this hospital after radical surgery, and from six months to 15 years after operation they were seen 2319 times; 71 developed a recurrent carcinoma but, of these, 41 recurrences (58%) were diagnosed at times other than those of the patients'' routine outpatient appointments, although they were being regularly reviewed. Only one patient with recurrence appeared to have been cured by further surgery. For the present, adequate education of patients in the symptoms of early recurrence, with instruction to return if any of these develop, is likely to be more effective than the unsatisfactory and time-consuming routine follow-up still used in many hospitals.     

Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts.

1. Commercially available [2-14C]pyruvate and [2-14C]malonate were found to contain 3-6% (w/w) of [14C]acetate. 2. The contaminating [14C]acetate was efficiently utilized for fatty acid synthesis by isolated chloroplasts, whereas the parent materials were poorer substrates. 3. Maximum incorporation rates of the different substrates examined were (ng-atoms of C/h per mg of chlorophyll): [1-14C]acetate, 2676; [2-14C]pyruvate, 810; H14CO3-, 355; [2-14C]malonate, 19. 4. Products of CO2 fixation were probably not a significant carbon source for fatty acid synthesis in the presence of exogenous acetate.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.