Proteins of the sieve-tube exudate of Cucurbita maxima

Summary Sieve-tube exudate which appears on cut surfaces of stems of Cucurbita maxima as distinct droplets has been depicted in electron micrographs of longitudinal sections of the phloem. The exudate, which was produced from mature sieve tubes only, contained filaments of P-protein, but no mitochondria or vesicles of endoplasmic reticulum. The water-soluble part of the exudate contained at least 12 proteins, as shown by disc-electrophoresis. Enzymic activity was found for peroxidases, acid phosphatases, and aldolases. Color tests and assays for other enzymes, including ATPase, fructokinase, several dehydrogenases, and UDP-glucose: D-fructose-2-glucosyl transferase, gave negative results. With repeated cutting of a stem, the protein content of the exudate increased, while the amount of exudate decreased.Supported by Deutsche Forschungsgemeinschaft and Stiftung Volkswagenwerk. During part of this investigation the senior author held a U.S. National Science Foundation Senior Foreign Scientist Fellowship at the University of Wisconsin.     

The influence of externally supplied sucrose on phloem transport in the maize leaf strip

Sucrose (2,5–1000 mmol l^–1), labeled with [¹⁴C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ¹⁴C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of ¹⁴C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ¹⁴-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l^–1) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l^–1 sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.Supported by Deutsche Forschungsgemeinschaft     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua

We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. SBP activity was detected at different pH values (3-7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). In the cases of D. antartica, A. boletinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per μm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the discussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi.     

Novel object exploration in mice: not all objects are created equal

Object exploration is an increasingly popular experimental paradigm in behavioral sciences. We have begun a series of studies with mice specifically looking at the parameters that influence behaviors in this test. The aim of the present study was to examine the effect of object type on performance in the object exploration test. More specifically, adult male C57BL/6J mice were trained and tested using objects that could be climbed (CLIMB) or with those that could only be touched (TOUCH). The results show that activity is affected by the presentation of objects, with object type interacting with some of these changes. C57 mice explored objects that can be climbed over significantly longer than objects that can only be touched and a more rapid habituation was observed using objects that could only be touched. Robust recognition memory was observed in both groups of mice, however mice in the CLIMB group exhibited a significantly greater discrimination index compared to mice in the TOUCH group. Taken together, these findings demonstrate that the selection of objects is of critical importance and it is recommended that special attention be given to the functional (affordant) properties of the objects to-be-used in future studies.     

The spatial distribution of acid phosphatase activity in ectomycorrhizal tissues depends on soil fertility and morphotype, and relates to host plant phosphorus uptake

Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.