A comparison of stability of chemical analytes in plasma from the BD Vacutainer® Barricor™ tube with mechanical separator versus tubes containing gel separator.

BACKGROUND: There is a need of prolonged stability of certain chemical analytes in lithium heparin tubes with separators. A new tube with a mechanical separator has recently been launched (Barricor™), which according to the manufacturer may have these benefits. The aim of this study was to evaluate stability performance of this tube in comparison with plasma gel tubes under clinically realistic circumstances. METHODS: Blood was collected in tubes containing lithium heparin with different separators; gel separator (Vacutainer® PST™, Becton Dickinson and Vacuette® , Greiner bio-one) and mechanical separator (Vacutainer® Barricor™, Becton Dickinson). All tubes had an aspiration volume of 3 mL and were centrifuged at similar time and force. Tubes were transported manually or by car. Seven analytes from 122 patients were analyzed after 3 to 80 hours by Cobas c701 (Roche). RESULTS: The Barricor™ tube showed increased stability of phosphate and potassium and similar stability of aspartate aminotransferase, glucose, homocysteine, lactate dehydrogenase, and magnesium compared with gel tubes. Maximal allowable bias for phosphate was exceeded after 68 hours for Barricor™ tubes compared with 29 or 35 hours for gel tubes and for potassium after 40 hours for Barricor™ tubes vs 9 or 12 hours for gel tubes. Transportation did not affect stability. Hemolysis index was slightly lower in Barricor tubes than in gel tubes (P = .01). CONCLUSION: Implementing the new Barricor™ tube will improve stability of potassium and phosphate in plasma. Blood sampling facilities far from the laboratory may benefit from using these tubes, thus diminishing preanalytical errors.

Effects of prolactin on platelet activation and blood clotting.

Increased levels of prolactin often coincide with an increased risk for thromboembolic events, but it is unclear whether a direct causal relation exists. Our aim was to examine the effect of prolactin on platelet function. In addition to using recombinant prolactin for experiments in vitro, we analyzed platelet function by flow cytometry in a group of 13 females with hyperprolactinaemia and 18 healthy female controls. Platelet activation was measured by P-selectin expression and by the amount of platelet-bound fibrinogen after stimulation with adenosine di phosphate (ADP), collagen-related peptide and the protease activated receptor (thrombin receptor) (PAR)-activating peptides PAR4-AP and PAR1-AP. Free oscillation rheometry was used to measure clotting time in whole blood. No significant effect on platelet activation or clotting time could be seen in in vitro experiments by adding recombinant prolactin. However, significantly lower P-selectin expression was found in the hyperprolactinemic group when platelets were activated by ADP (5 and 10 µM) or PAR4-AP. The expression of fibrinogen did not differ between the two groups for any of the activators used. For all samples, inverse significant correlations between P-selectin expression and prolactin concentration were found for both 5 µM ADP (r = - 0.61, p < 0.01), 10 µM ADP (r = - 0.62, p < 0.001) and PAR4-AP (r = - 0.69, p < 0.001). Thrombin cleavage of recombinant prolactin resulting in a 16 kDa C-terminal fragment did not alter the P-selectin expression upon activation. We found an indirect inhibitory effect of prolactin on platelets in hyperprolactinemic patients, suggesting that prolactin might have a protective role in thromboembolic disease.

In vitro cultured rat islets express genes that both prevent and promote angiogenesis.

CONTEXT: Endogenous pancreatic islets are supported by a dense sinusoidal capillary system which is disrupted following isolation and culture in vitro. A rapid and accurate revascularization is therefore crucial for the survival and functioning of the transplanted islet. Although a blood flow is established in islet grafts within 1-2 weeks, these islets show poor development of intra-islet capillaries. To improve the revascularization process and the arrangement of the new blood vessels, islet production of the factors governing these processes needs to be further characterized. OBJECTIVE: To study the expression of factors which regulate angiogenesis in cultured rat islets. DESIGN AND MAIN OUTCOME MEASURES: Rat islets were isolated and cultured for one week. After 6 hours of exposure to normoxic (21% O2) or hypoxic (1% O2) conditions, mRNA expression was evaluated by the GEArray Angiogenesis 1 and 2 systems. The expression of the vascular endothelial growth factor (VEGF), the tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1 (Tie1) and acidic fibroblast growth factor (aFGF), was further evaluated by semi-quantitative RT-PCR. RESULTS: We found the expression of 19 genes that code for factors either promoting or preventing angiogenesis. Only VEGF and Tie1 were upregulated in response to hypoxia. CONCLUSION: Hypoxia-induced islet vascularization may involve VEGF and Tie-induced signaling events. The results also show that cultured islets express genes which prevent angiogenesis concurrently with genes coding for factors stimulating angiogenesis. The balance between these factors is probably of vital importance for the revascularization process in transplanted islets. Thus, pharmacologic or genetic attenuation of islet-derived angiostatic factors may prove beneficial in promoting islet revascularization in future transplantation trials.

The role of PTB in insulin mRNA stability control.

Although extensively studied, there are still many unanswered questions regarding the regulation of insulin gene expression. This is important to further investigate since it will help us understand the pathophysiology of some types of diabetes. The insulin mRNA has a long half-life and changes in insulin mRNA stability, induced by glucose, are likely to be regulated through specific mechanisms. Recent findings indicate that the polypyrimidine tract binding protein (PTB), also named hnRNP I, by binding to the 3'-UTR (untranslated region) of the insulin mRNA molecule, stabilizes the messenger thereby participating in the glucose-induced increase in insulin mRNA. This review will focus on recent findings pertinent to PTB subcellular localization and function. It appears that PTB shuttles between the nucleus and the cytosol, and that protein kinase A (PKA)-mediated PTB phosphorylation promotes PTB translocation to the cytosol, an event that might enhance insulin mRNA stability. We will also review beta-cell signaling events that may control the mRNA stabilizing effect of PTB.

Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway.

Glucose-induced binding of the polypyrimidine tract-binding protein (PTB) to the 3'-untranslated region of the insulin mRNA (ins-PRS) is inhibited by rapamycin.

Despite considerable knowledge on the regulation of insulin gene transcription, little is known about the post-transcriptional control mechanisms of this gene. We have recently reported glucose- and hypoxia-regulated binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-untranslated insulin mRNA (ins-PRS), an event which may control insulin mRNA stability. The present aim was to probe for the signaling pathways that control this binding activity. Rat islets were exposed to pharmacological inhibitors against several molecules, previously shown to be involved in glucose signaling. The inhibitors used were; LY 294002 (PI3 kinase), Rp-cAMP triatylamine (the cAMP-dependent protein kinase PKA), bisindolylmaleimide I hydrochloride (PKC), PD 098059 (ERK1/ERK2), SB 203580 (p38/SAPK2a), rapamycin (mTOR) and okadaic acid (PP1/2A). PTB-binding activity to the ins-PRS was then analyzed by elecrophoretic mobility shift assay (EMSA). The glucose-induced PTB-binding was only inhibited by the mTOR inhibitor rapamycin. Rapamycin also reduced glucose-induced insulin mRNA expression. Thus, our results suggest an involvement of mTOR in glucose-induced PTB/ins-PRS binding and insulin mRNA stability.

Control of insulin mRNA stability in rat pancreatic islets. Regulatory role of a 3'-untranslated region pyrimidine-rich sequence.

Stabilization of insulin mRNA in response to glucose is a significant component of insulin production, but the mechanisms governing this process are unknown. We presently observe that insulin mRNA is a highly abundant messenger and that the content of this mRNA is mainly controlled by changes in messenger stability. We also demonstrate specific binding of the polypyrimidine tract-binding protein to a pyrimidine-rich sequence located in the 3'-untranslated region (3'-UTR) of insulin mRNA. This binding was increased in vitro by dithiothreitol and in vivo by glucose. Inhibition of polypyrimidine tract-binding protein binding to the pyrimidine-rich sequence by mutation of the core binding site resulted in a destabilization of a reporter gene mRNA. Thus, glucose-induced binding of polypyrimidine tract-binding protein to the 3'-UTR of insulin mRNA could be a necessary event in the control of insulin mRNA levels.

Nicotinamide- and caspase-mediated inhibition of poly(ADP-ribose) polymerase are associated with p53-independent cell cycle (G2) arrest and apoptosis.

Poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand breaks, is involved in DNA repair and replication but, during apoptosis, undergoes early caspase-mediated cleavage. Activation of programmed cell death in response to DNA damage may rely on functional p53 protein. Tumor cells are commonly deficient in this oncogene product resulting in resistance to many cytostatic drugs. Here we report that nicotinamide-induced inhibition of poly(ADP-ribosyl)ation and cytokine-induced nitric oxide production both result in a transient increase in p53 levels in pancreatic tumor RINm5F cells. These treatments also induce disruption of the mitochondrial membrane potential (delta psi(m)), as revealed using the mitochondrial probe JC-1, followed by PARP cleavage and apoptosis all of which are inhibited by the anti-apoptotic protein Bcl-2. Moreover, PARP-inhibition by nicotinamide or 3-aminobenzamide induces apoptosis and/or cell cycle arrest at the G2 checkpoint in all of four tested tumor cell lines of both mesenchymal and epithelial origin including mouse NIH-3T3 cells and p53 deficient human HeLa and Jurkat cells. Bcl-2 counteracts cytokine-, but not nicotinamide-induced G2 arrest. These findings indicate that both chemical and caspase-mediated inhibition of PARP activity, possibly by interfering with DNA replication and repair, may promote a p53-independent G2 arrest and apoptosis.

Promoting islet cell function after transplantation.

Engraftment (i.e., the adaptation of transplanted pancreatic islets to their new surroundings with regard to revascularization, reinnervation, and reorganization of other stromal compartments) is of crucial importance for the survival and function of the endocrine cells. Previous studies suggest that transplantation induces both vascular and stromal dysfunctions in the implanted islets when compared with endogenous islets. Thus the vascular density and the blood perfusion of islet grafts is decreased and accompanied with a capillary hypertension. This leads to hypoxic conditions, with an associated shift toward anaerobic metabolism in grafted islets. An improved engraftment will prevent or compensate for the vascular/stromal dysfunction seen in transplanted islets and thereby augment survival of the islet implant. By such means the number of islets needed to cure the recipient will be lessened. This will increase the number of patients that can be transplanted with the limited material available.