Characterization of inositolpolyphosphate binding to myocardial membranes

Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP₃) and inositol-1,3,4,5-P₄ (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP₆). This study demonstrates that InSP₆ has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively.InsP₄ binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP₃, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP₄ to SR and SL by a mean of 30% and 20% respectively.Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP₆ bound to two separate protein peaks in both these membranes, while InsP₄ bound to only one. In SR membranes, InsP₄ bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.     

Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

The effects of insulin and β-adrenergic stimulation on glucose transport,glut 4 and PKB activation in the myocardium of lean and obese non-insulin dependent diabetes mellitus rats

Glucose uptake, glut 4 translocation and activation of protein kinase B were measured in Langendorff perfused hearts from (i) Wistar control, (ii) lean, neonatal Streptozotocin induced (Stz) and (iii) Zucker (fa/fa) obese diabetic rats of 10–12 weeks old. Hearts were subjected to stimulation with insulin, isoproterenol (-adrenergic agonist) or a combination of insulin and isoproterenol, during the perfusion protocol. Basal myocardial glucose uptake was impaired in both diabetic models, but could be stimulated significantly by insulin. In the Zucker rats, the time-course of insulin action was delayed. Insulin and -stimulation of glucose uptake were not additive. Evaluation of sarcolemmal membranes from these hearts showed that the affinity of glut 4 was significantly lower in the Zucker but not in the Stz hearts while a reduced affinity found with a combination of insulin and -stimulation in control hearts, was absent in both diabetic models. Total membrane lysates were analyzed for glut 4 expression while an intracellular component was generated to quantify translocation on stimulation as well as activity of protein kinase B (PKB). At this age, the neonatal Streptozotocin induced diabetic animals presented with more faulty regulation concerning adrenergic stimulated effects on elements of this signal transduction pathway while the Zucker fa/fa animals showed larger deviations in insulin stimulated effects. The overall response of the Zucker myocardium was poorer than that of the Stz group. No significant modulation of -adrenergic signaling on insulin stimulated glucose uptake was found. The PI-3-kinase inhibitor wortmannin, could abolish glucose uptake as well as PKB activation elicited by both insulin and isoproterenol.     

Nitric oxide: a trigger for classic preconditioning?

To determine whether nitric oxide (NO) is involved in classic preconditioning (PC), the effect of NO donors as well as inhibition of the L-arginine-NO-cGMP pathway were evaluated on 1) the functional recovery during reperfusion of ischemic rat hearts and 2) cyclic nucleotides during both the PC protocol and sustained ischemia. Tissue cyclic nucleotides were manipulated with NO donors [S-nitroso-N-penicillamine (SNAP), sodium nitroprusside (SNP), or L-arginine] and inhibitors of nitric oxide synthase (N(omega)-nitro-L-arginine methyl ester or N-nitro-L-arginine) or guanylyl cyclase (1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one). Pharmacological elevation in tissue cGMP levels by SNAP or SNP before sustained ischemia elicited functional improvement during reperfusion comparable to that by PC. Administration of inhibitors before and during the PC protocol partially attenuated functional recovery, whereas they had no effect when given after the ischemic PC protocol and before sustained ischemia only, indicating a role for NO as a trigger but not as a mediator. Ischemic PC, SNAP, or SNP caused a significant increase in cGMP and a reduction in cAMP levels after 25 min of sustained ischemia that may contribute to the protection obtained. The results obtained suggest a role for NO (and cGMP) as a trigger in classic PC.     

Mathematical modeling of hydrolysate diffusion and utilization in cellulolytic biofilms of the extreme thermophile Caldicellulosiruptor obsidiansis

In this study, a hydrolysate diffusion and utilization model was developed to examine factors influencing cellulolytic biofilm morphology. Model simulations using Caldicellulosiruptor obsidiansis revealed that the cellulolytic biofilm needs to generate more hydrolysate than it consumes to establish a higher than bulk solution intra-biofilm substrate concentration to support its growth. This produces a hydrolysate surplus that diffuses through the thin biofilm structure into the bulk solution, which gives rise to a uniform growth rate and hence the homogeneous morphology of the cellulolytic biofilm. Model predictions were tested against experimental data from a cellulose-fermenting bioreactor and the results were consistent with the model prediction and indicated that only a small fraction (10-12%) of the soluble hydrolysis products are utilized by the biofilm. The factors determining the rate-limiting step of cellulolytic biofilm growth are also analyzed and discussed.     

The effects of gender and obesity on myocardial tolerance to ischemia

Obesity is increasing at an alarming rate globally. Several studies have shown that premenopausal women have a reduced risk of CV disease and a reduced myocardial susceptibility to ischemia/reperfusion injury. The effect of obesity on myocardial tolerance to ischemia in women has not been established. To determine how obesity affects myocardial susceptibility to ischemia/reperfusion injury in both males and females, we fed male and female Wistar rats a high caloric diet (HCD) or a control rat chow diet (CD) for 18 weeks. Rats were subsequently fasted overnight, anesthetized and blood was collected. In separate experiments, 18-week-fed (HCD and CD) rats underwent 45 min in vivo coronary artery ligation (CAL) followed by 2 hours reperfusion. Hearts were stained with TTC and infarct size determined. Both male and female HCD fed rats had increased body and visceral fat weights. Homeostasis model assessment (HOMA) index values were 13.95+/-3.04 for CD and 33.58+/-9.39 for HCD male rats (p<0.01) and 2.98+/-0.64 for CD and 2.99+/-0.72 for HCD fed female rats. Male HCD fed rats had larger infarct sizes than CD fed littermates (43.2+/-9.3 % vs. 24.4+/-7.6 %, p<0.05). Female HCD and CD diet fed rats had comparable infarct sizes (31.8+/-4.3 % vs. 23.9+/-3.3 %). We conclude that male rats on the HCD became viscerally obese, dyslipidemic and insulin-resistant, while female HCD fed rats became viscerally obese without developing dyslipidemia or insulin resistance. Obesity increased myocardial infarct size in males but not the females.     

IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4(+) and CD8(+) T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1(-/-) mice or in mice depleted of CD4(+) and/or CD8(+) cells than in normal mice. The tumors in RAG1(-/-) mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.     

Early cardiovascular changes occurring in diet-induced, obese insulin-resistant rats

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase which is composed of two catalytic α- or α'-subunits and two non-catalytic β-subunits. CK2 has been shown to be implicated in embryogenesis, spermatogenesis, and the development of certain organs but its role in basal differentiation processes is only sparsely analyzed. 3T3-L1 cells, which are murine pre-adipocytes, can be induced to differentiate into mature adipocytes within 2 weeks using a combination of insulin, dexamethasone, and isobutylmethylxanthine. We found that the activity of CK2 slightly increases until day 6 and subsequently, decreases in fully differentiated adipocytes. The decrease in activity goes along with a lower expression of all the three subunits of CK2. After inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) or 1,2,5,8-tetrahydroxyanthraquinone (quinalizarin), before day 6, 3T3-L1 cells did not differentiate into adipocytes; inhibition of CK2 after day 6 had no effect on the differentiation process. These results indicated a role of CK2 in early events of the differentiation process and that CK2 is dispensable for late stages of differentiation.     

High-affinity fluorescent ligands for the 5-HT(3) receptor

The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT(3) receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT(3)AR.     

Hindered submicron mobility and long-term storage of presynaptic dense-core granules revealed by single-particle tracking

Dense-core granules (DCGs) are organelles found in neuroendocrine cells and neurons that house, transport, and release a number of important peptides and proteins. In neurons, DCG cargo can include the secreted neuromodulatory proteins tissue plasminogen activator (tPA) and/or brain-derived neurotrophic factor (BDNF), which play a key role in modulating synaptic efficacy in the hippocampus. This function has spurred interest in DCGs that localize to synaptic contacts between hippocampal neurons, and several studies recently have established that DCGs localize to, and undergo regulated exocytosis from, postsynaptic sites. To complement this work, we have studied presynaptically localized DCGs in hippocampal neurons, which are much more poorly understood than their postsynaptic analogs. Moreover, to enhance relevance, we visualized DCGs via fluorescence labeling of exogenous and endogenous tPA and BDNF. Using single-particle tracking, we determined trajectories of more than 150 presynaptically localized DCGs. These trajectories reveal that mobility of DCGs in presynaptic boutons is highly hindered and that storage is long-lived. We also computed mean-squared displacement curves, which can be used to elucidate mechanisms of transport. Over shorter time windows, most curves are linear, demonstrating that DCG transport in boutons is driven predominantly by diffusion. The remaining curves plateau with time, consistent with motion constrained by a submicron-sized corral. These results have relevance to recent models of presynaptic organization and to recent hypotheses about DCG cargo function. The results also provide estimates for transit times to the presynaptic plasma membrane that are consistent with measured times for onset of neurotrophin release from synaptically localized DCGs.