Potassium, sodium, water, and Donnan potential changes in the myofibrils of intact muscle fibres after exposure to ouabain and K-free Ringer

Frog sartorius muscles were superfused for 40 min with solutions of K-free Ringer, normal Ringer containing ouabain, or K-free Ringer containing ouabain. Changes in myoplasmic K and Na were measured with ion-selective microelectrodes; changes in total fibre K and Na were measured by means of atomic absorption spectroscopy; and changes in total fibre water content were obtained from wet and dry weights. Application of a two-compartment model permitted one to calculate (i) the K, Na, and water changes in the myofibrils and in the surrounding myoplasm (extramyofibrillar space); (ii) the changes in the transmyofibrillar Donnan potential (ED); and (iii) the changes in the ratio of the apparent association constants (kNa/kK) of the myofilament charge sites to Na and K. In the resting fibres, the K, Na, and water content of the myofibrils were calculated to be 82, 87, and 80% of total fibre content, respectively; ED was calculated as -4.5 mV; kNa/kK was calculated as 1.4. After a 40-min ouabain treatment, 12 mmol (per kg fibre water) of intrafibre K exchanged with 7.5 mmol of extrafibre Na, 6.4 mmol of myofibrillar K exchanged with an equal amount of extramyofibrillar Na, ED increased to -8.3 mV, and kNa/kK remained relatively constant. After a 40-min K-free treatment, the fibres gained 5.5 mmol of Na without any change in fibre K or water, the myofibrils shifted 9.3% of their water into the extramyofibrillar space instead of exchanging K for Na, ED increased to -10.7 mV, and kNa/kK decreased to 0.47.(ABSTRACT TRUNCATED AT 250 WORDS)     

A genome‐wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.     

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.     

Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.     

The effect of fat mass on educational attainment: Examining the sensitivity to different identification strategies

The literature that examines the relationship between child or adolescent Body Mass Index (BMI) and academic attainment generally finds mixed results. This may be due to the use of different data sets, conditioning variables, or methodologies: studies either use an individual fixed effects (FE) approach and/or an instrumental variable (IV) specification. Using one common dataset, the Avon Longitudinal Study of Parents and Children, and a common set of controls, this paper compares the different approaches (including using different types of IV's), discusses their appropriateness, and contrasts their findings. We show that, although the results differ depending on the approach, most estimates cannot be statistically distinguished from OLS, nor from each other. Examining the potential violations of key assumptions of the different approaches and comparing their point estimates, we conclude that fat mass is unlikely to be causally related to academic achievement in adolescence.