Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.     

Tocopherol biosynthesis is enhanced in photomixotrophic sunflower cell cultures

α-Tocopherol is the most biologically active component of vitamin E and is synthesized only by photosynthetic organisms. Two heterotrophic cell lines of sunflower (Helianthus annuus L.) of differing α-tocopherol biosynthetic capability, three-fold higher in the high synthesizing cell line, HT, than in the low synthesizing one, LT, were previously identified. To investigate the relationship between α-tocopherol biosynthesis and photomixotrophic culture conditions, a new photomixotrophic sunflower cell line HS3 was established by selecting HT cells able to grow in the presence of a ten-fold reduced sucrose concentration in the culture medium. The photosynthetic properties of HS3 cells were characterized in comparison with HT and LT cells, revealing an increase in chlorophyll content, chloroplast number, and level of the photosynthesis related enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Furthermore, an enhanced expression of the gene encoding for the tocopherol biosynthetic enzyme geranyl-geranylpyrophosphate synthase (GGPPS) was observed in HS3 cells. HS3 cells also revealed a 25% and a more than three-fold higher tocopherol level than HT and LT, respectively, indicating a positive correlation between α-tocopherol biosynthesis of sunflower cell cultures and their photosynthetic properties. These findings can be useful for improving the tocopherol yields of the sunflower in vitro production system.     

Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.     

Seasonal fluctuations in the response of Italian Mediterranean buffaloes to synchronization of ovulation and timed artificial insemination

A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI.     

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.     

Antioxidant Activity of Inulin and Its Role in the Prevention of Human Colonic Muscle Cell Impairment Induced by Lipopolysaccharide Mucosal Exposure

Background

Fructans, such as inulin, are dietary fibers which stimulate gastro-intestinal (GI) function acting as prebiotics. Lipopolysaccharide (LPS) impairs GI motility, through production of reactive oxygen species. The antioxidant activity of various fructans was tested and the protective effect of inulin on colonic smooth muscle cell (SMC) impairment, induced by exposure of human mucosa to LPS, was assessed in an ex vivo experimental model.

Methods

The antioxidant capacity of fructans was measured in an in vitro system that simulates cooking and digestion processes. Human colonic mucosa and submucosa, obtained from disease-free margins of resected segments for cancer, were sealed between two chambers, with the mucosal side facing upwards with Krebs solution with or without purified LPS from a pathogenic strain of Escherichia coli (O111:B4) and inulin (Frutafit IQ), and the submucosal side facing downwards into Krebs solution. The solutions on the submucosal side were collected following mucosal exposure to Krebs in the absence (N-undernatant) or presence of LPS (LPS-undernatant) or LPS+inulin (LPS+INU-undernatant). Undernatants were tested for their antioxidant activity and the effects on SMCs contractility. Inulin protective effects on mucosa and submucosa layers were assessed measuring the protein oxidation level in the experimental conditions analyzed.

Results

Antioxidant activity of inulin, which was significantly higher compared to simple sugars, remained unaltered despite cooking and digestion processes. Inulin protected the mucosal and submucosal layers against protein oxidation. Following exposure to LPS-undernatant, a significant decrease in maximal acetylcholine (Ach)-induced contraction was observed when compared to the contraction induced in cells incubated with the N-undernatant (4±1% vs 25±5% respectively, P<0.005) and this effect was completely prevented by pre-incubation of LPS with Inulin (35±5%).

Conclusions

Inulin protects the human colon mucosa from LPS-induced damage and this effect appears to be related to the protective effect of inulin against LPS-induced oxidative stress.     

LMNA Knock-Down Affects Differentiation and Progression of Human Neuroblastoma Cells

Background

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.

Methodology/Principal Findings

Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.

Conclusions/Significance

We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.     

Genetic and Environmental Factors Influencing the Placental Growth Factor (PGF) Variation in Two Populations

Placental Growth Factor (PGF) is a key molecule in angiogenesis. Several studies have revealed an important role of PGF primarily in pathological conditions (e.g.: ischaemia, tumour formation, cardiovascular diseases and inflammatory processes) suggesting its use as a potential therapeutic agent. However, to date, no information is available regarding the genetics of PGF variability. Furthermore, even though the effect of environmental factors (e.g.: cigarette smoking) on angiogenesis has been explored, no data on the influence of these factors on PGF levels have been reported so far. Here we have first investigated PGF variability in two cohorts focusing on non-genetic risk factors: a study sample from two isolated villages in the Cilento region, South Italy (N = 871) and a replication sample from the general Danish population (N = 1,812). A significant difference in PGF mean levels was found between the two cohorts. However, in both samples, we observed a strong correlation of PGF levels with ageing and sex, men displaying PGF levels significantly higher than women. Interestingly, smoking was also found to influence the trait in the two populations, although differently. We have then focused on genetic risk factors. The association between five single nucleotide polymorphisms (SNPs) located in the PGF gene and the plasma levels of the protein was investigated. Two polymorphisms (rs11850328 and rs2268614) were associated with the PGF plasma levels in the Cilento sample and these associations were strongly replicated in the Danish sample. These results, for the first time, support the hypothesis of the presence of genetic and environmental factors influencing PGF plasma variability.