Acute hibernation decreases myocardial pyruvate carboxylation and citrate release

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.     

The old and new biochemistry of polyamines

Polyamines are ubiquitous positively charged amines found in all organisms. These molecules play a crucial role in many biological functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are observed to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the critical roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compounds have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochemically integrated link between iron and polyamine metabolism.     

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.     

Regulation of PDGF production and ERK activation by estrogen is associated with TSC2 gene expression

Mechanisms that regulate the growth response to estrogen (17beta-estradiol, E2) are poorly understood. Recently, loss of function of the tuberous sclerosis complex 2 (TSC2) gene has been associated with E2-related conditions that are characterized by benign cellular proliferation. We examined the growth response to E2 in vascular smooth muscle cells (VSMCs) that possess wild-type TSC2 and compared them with ELT-3 smooth muscle cells that do not express TSC2. In TSC2-expressing VSMCs, growth inhibition in response to E2 was associated with downregulation of platelet-derived growth factor (PDGF), PDGF receptor (PDGFR), and limited activation of extracellular signal-regulated kinase (ERK). In contrast, the growth-promoting effect of E2 in TSC2-null ELT-3 cells was associated with induction of PDGF, robust phosphorylation of PDGFR, and sustained activation of ERK. Furthermore, in ELT-3 cells, cellular growth and ERK activation by E2 were inhibited by the PDGFR inhibitor tyrphostin AG 17 and by PDGF-neutralizing antibody. These results demonstrate that autocrine production of PDGF and augmentation of the ERK pathway leads to estrogen-induced cellular proliferation in TSC2-null cells, a pathway that was downregulated in cells that express TSC2. Understanding the mechanisms that regulate the diverse responses to the steroid hormone estrogen could lead to novel approaches to the treatment of estrogen-related diseases that are characterized by aberrant cell proliferation.     

Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat

Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4-8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na(v)1.3 (TTX-S) and Na(v) 1.7 (TTX-S) and decreases in the expression of Na(v) 1.6 (TTX-S) and Na(v)1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na(v) 1.6 and In Na(v)1.8 increased in response to diabetes. addition, increased tyrosine phosphorylation of Na(v)1.6 and Na(v)1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.     

Maternal mortality and women's condition: “The distress in being a woman”

For millenaria, maternal mortality has been considered as a fatality inherent to women's condition. Thanks to the progress of the obstetric technology, the world M.M.R. fell from 2.000 to 400 per 100.000 births during the past 150 years. At the same time women's condition improved chiefly because of a better level of education. Is there any relationship between them: the decrease of Maternal Mortality and the amelioration of women's general status? Or, in other words, can the decrease of Maternal Mortality Rate be considered as an “indicator” of the situation of women? After a historical review of the importance of the maternal mortality drama as it occured in older times before the “obstetric era”, which began with the improvement of the obstetric technology, this paper will present the actual situation of maternal mortality in relation with the obstetric coverage, the female literacy level and some other socio-economic variables, and in relation with the Gross National Product (G.N.P.) as an indicator of governmental interest in the specific female problems of giving-life.     

Kinetic and mechanistic considerations in the gelation of genipin-crosslinked gelatin

The gelling properties (gel time (t_gel) and gel strength) of a 10% (w/w) gelatin sol were investigated as a function of genipin (GP) concentration (0–15 mM) and temperature (25–55 °C) to discern mechanisms and optimal conditions for fixation. Gel time increased with increasing temperature, reached a maximum, and then declined as temperature was raised further. By contrast, network strength data followed the opposite trend. From the thermal behavior of t_gel and network strength, it was inferred that gelation in the low-temperature regime was dominated by hydrogen bonding, while in the high-temperature regime it was dominated by covalent crosslinking. At higher temperatures, crosslinking was described by an Arrhenius rate law expression, with activation energies between 63.2 and 67.8 kJ/mol, depending on GP concentration. In the low temperature regime, an Arrhenius plot resulted in negative activation energies of −75.8 and −64.4 kJ/mol in the presence of 10 and 15 mM GP, respectively. With an increase in both GP concentration and temperature, the gelatin network gradually shifted from being dominated by hydrogen bonds (physical crosslinks) to covalent crosslinking (chemical crosslinks).