Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

An experimental arrangement was described that enables nuclear magnetic resonance spectra of compressed plant cells to be recorded while circulating a medium through the sample. The system provided a convenient arrangement for monitoring by 31P NMR the behavior of plant cells over a long period of time under different conditions such as sucrose starvation. Perfusion of compressed sycamore cells with sucrose-free culture medium triggered a progressive decrease in the glucose 6-P and uridine-5'-diphosphate-alpha-D-glucose resonances over 30 h. When almost all the intracellular carbohydrate pool had disappeared the nucleotide triphosphate resonances decline progressively. These changes were accompanied by a Pi accumulation in the vacuole and a phosphorylcholine (P-choline) accumulation in the cytoplasm. The very long lag phase observed for ATP and P-choline evolution was comparable with that observed for the progressive intracellular digestion of cytoplasmic constituents (Journet, E., Bligny, R. and Douce, R. (1986) J. Biol. Chem. 261, 3193-3199). Addition of sucrose in the circulating system after a long period of sucrose starvation led to a disappearance of the cytoplasmic Pi resonance and a marked increase in that of glucose 6-P. Under these conditions the vacuolar Pi pool did not fluctuate to buffer the Pi in the cytoplasm. The results suggest that Pi which has been sequestered in the vacuole during the course of sucrose starvation is not restored to the cytoplasm for rapid metabolic processes. Furthermore, the presence of P-choline in plant cells in large excess should be considered as a good marker of membrane utilization after a long period of sucrose starvation and is very likely related to stress.     

Interleukin-1alpha-induced chemokines in mouse granulosa cells: impact on keratinocyte chemoattractant chemokine, a CXC subfamily

IL-1 is well known to be involved in the immune system and have a role in ovarian inflammation as well as exhibiting inhibitory effects on steroidogenesis and folliculogenesis. Because multiple aspects of ovarian function have also been shown to involve cytokine/chemokine networks, IL-1alpha-induced chemokine gene expression in mouse granulosa cells was investigated. Granulosa cells from immature mice at 28 d of age were cultured with IL-1alpha (10 ng/ml). IL-1alpha induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine, a CXC subfamily. KC chemokine mRNA and protein were increased 1-2 h after IL-1alpha and then gradually decreased. The KC promoter (-701/+30) containing three nuclear factor (NF)-kappaB sites was fully responsive to IL-1alpha, whereas deletions and mutants of the NF-kappaB sites lowered the responsiveness to IL-1alpha. The proximal NF-kappaB site (-69/-59) played a critical role in regulating IL-1alpha-induced KC chemokine promoter activity. Overexpression of the inhibitor of NF-kappaB (IkappaB) blocked KC promoter activity induced by IL-1alpha, whereas overexpression of p65, a component of NF-kappaB, increased promoter activity and mRNA of KC chemokine. In addition, FSH did not affect NF-kappaB signaling or IL-1alpha-induced KC chemokine promoter activity. Within 1-3 h after ip injection of lipopolysaccharide (100 mug/mouse), a product known to stimulate release of IL-1, KC chemokine was localized in the ovary to granulosa cells as well as the thecal-interstitial layer. The results of this study indicate that KC gene is a chemokine induced acutely by IL-1alpha via NF-kappaB signaling in mouse granulosa cells.     

Expression of Activated PIK3CA in Ovarian Surface Epithelium Results in Hyperplasia but Not Tumor Formation

Background

The Phosphatidylinositol 3′-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.

Methodology/Principal Findings

Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.

Conclusions/Significance

While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.     

In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man.     

Transformation of chondroblasts by Rous sarcoma virus and synthesis of the sulfated proteoglycan matrix.

The presence of the extracellular matrix synthesized by chondroblasts provides a barrier to virus penetration. Chondroblasts can be infected and transformed following treatment with proteolytic enzymes. Using a temperature-sensitive transformation mutant of Rous sarcoma virus and rearing the cells at permissive temperature, we demonstrate that transformed chondroblasts stop synthesizing their cell-unique sulfated proteoglycan. If such transformed chondroblasts are shifted to nonpermissive temperature, the cells reinitiate the synthesis of their cell-unique sulfated proteoglycan.     

Cell Surfaces in Plant-Microorganism Interactions : III. In Vivo Effect of Ethylene on Hydroxyproline-Rich Glycoprotein Accumulation in the Cell Wall of Diseased Plants

Ethylene production and cell wall hydroxyproline-rich glycoprotein (HRGP) biosynthesis are greatly enhanced in melon (Cucumin melo cv. Cantaloup charentais) seedlings infected with Colletotrichum lagenarium. Short-term experiments performed in the presence of specific inhibitors of the ethylene pathway from methionine, namely l-canaline and amino-ethoxyvinylglycine, indicate that under non-toxic conditions, both ethylene and [¹⁴C]hydroxyproline deposition in the cell wall of infected tissues are significantly lowered. On the contrary, treatment of healthy tissues with 1-aminocyclopropane 1-carboxylic acid, a natural precursor of ethylene, stimulates both the production of the hormone and the incorporation of [¹⁴C]hydroxyproline into cell wall proteins.     

Insulin resistance and decreased spexin in Indian Patients with Type 2 Diabetes Mellitus

Spexin is novel biomarker, which plays a potential role in glucose and lipid metabolisms. However, there was paucity of serum spexin levels in obesity and diabetes mellitus subjects. Hence the current study was aimed to find the relationship between the serum spexin levels in type 2 Diabetes mellitus (type 2 DM) with extrapolation of cardiovascular disease (CVD) risk. A cross-sectional study included 330 participants, subdivided as control (n=110), type 2 DM (n=110) and type 2 DM with CVD groups (n=110). HbA1c, insulin, lipid profile, spexin & leptin including blood pressure and body mass index were analyzed from all the participants. The serum spexin levels (ng/ml) were significantly decreased in type 2 DM (mean ± sd: 0.65 ± 0.03) and type 2 DM with CVD (0.48 ± 0.02) groups compared to the control (0.79 ± 0.03) group (p<0.001). The decreased spexin levels were observed in type 2 DM, and further more decreased in type 2 DM with CVD patients compared to controls indicating that spexin levels could be served as an early prediction of obesity-induced T2DM with CVD risk.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.