Disruption of Growth Hormone Receptor Prevents Calorie Restriction from Improving Insulin Action and Longevity

Most mutations that delay aging and prolong lifespan in the mouse are related to somatotropic and/or insulin signaling. Calorie restriction (CR) is the only intervention that reliably increases mouse longevity. There is considerable phenotypic overlap between long-lived mutant mice and normal mice on chronic CR. Therefore, we investigated the interactive effects of CR and targeted disruption or knock out of the growth hormone receptor (GHRKO) in mice on longevity and the insulin signaling cascade. Every other day feeding corresponds to a mild (i.e. 15%) CR which increased median lifespan in normal mice but not in GHRKO mice corroborating our previous findings on the effects of moderate (30%) CR on the longevity of these animals. To determine why insulin sensitivity improves in normal but not GHRKO mice in response to 30% CR, we conducted insulin stimulation experiments after one year of CR. In normal mice, CR increased the insulin stimulated activation of the insulin signaling cascade (IR/IRS/PI3K/AKT) in liver and muscle. Livers of GHRKO mice responded to insulin by increased activation of the early steps of insulin signaling, which was dissipated by altered PI3K subunit abundance which putatively inhibited AKT activation. In the muscle of GHRKO mice, there was elevated downstream activation of the insulin signaling cascade (IRS/PI3K/AKT) in the absence of elevated IR activation. Further, we found a major reduction of inhibitory Ser phosphorylation of IRS-1 seen exclusively in GHRKO muscle which may underpin their elevated insulin sensitivity. Chronic CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared to normal mice, likely explaining or contributing to the absence of CR effects on insulin sensitivity and longevity in these long-lived mice

Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells

Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 µg of protein with histone content of 60% –70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments

Immunoblotting analysis of somatic components of Dirofilaria immitis

SDS-PAGE analysis of Dirofilaria immitis extracts demonstrated the complexity of somatic protein component of adult male similar to that of adult female worm. Western blot analysis showed six major peptide bands of 85, 66, 42, 20, 16.2 and 14.5 kDa recognized in the sera of infected dogs. Western blotting sera from dogs with Dirofilaria immitis infection suggest that antigenic components in the low molecular weight region may be related to the anti-parasitic mechanism of the host

Neuromuscular consequences of prolonged hunger strike: an electrophysiological study

Objectives: The purpose of this study was to determine the electrophysiological consequences of neuromuscular and central nervous system involvement in a group of patients presented with the neurological complications of a long-term hunger strike (HS)

Magnetic nerve root stimulation in two types of brachial plexus injury: Segmental demyelination and axonal degeneration

Magnetic cervical nerve root stimulation was performed in 9 patients with plexopathies secondary to suspension (SP) and in 12 cases with neurogenic thoracic outlet syndrome (NTOS). The findings were compared with those of the previously reported case groups: n-hexane polyneuropathy (HPNP), inflammatory demyelinating polyneuropathy (IDP), and motor neuron disease (MND). Muscle responses elicited by magnetic stimulation had very high rates of amplitude and area loss in the neck-axilla segments of the 6 SP patients. This, along with the other electrophysiological findings, suggested the presence of segmentally demyelinating plexus lesions. In NTOS patients, magnetic stimulation findings were not significantly different from those of the controls. Neck-axilla segment amplitude and area reduction rates in SP and IDP patients were significantly higher than those found in NTOS, HPNP, and MND groups, implying that magnetic nerve root stimulation may have a role in the demonstration of segmentally demyelinating lesions involving proximal nerve segments. (C) 1997 John Wiley & Sons, Inc