Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer(473) and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 +/- 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased ( approximately 30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer(473) was approximately 50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer(473) and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer(473) on the improvement in insulin sensitivity requires further elucidation.     

A group of α-1,4-glucan lyase genes from the fungi Morchella costata,M. vulgaris and Peziza ostracoderma. Cloning,complete sequencing and heterologous expression

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, -1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121530 and 121971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.     

Positive supercoiling is generated in the presence of Escherichia coli SeqA protein

In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo. The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.