Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.     

TCR signaling inhibits glucocorticoid-induced apoptosis in murine thymocytes depending on the stage of development

Signaling by either the TCR or glucocorticoid receptor (GR) induces apoptosis in thymocytes. Interestingly, it has been shown previously that hybridoma T cells escape apoptosis induced by either TCR or GR when both of these receptors signal simultaneously. Whether such mutual antagonism is present in primary thymocytes was the subject of the present study. Both glucocorticoids (GC) and anti-TCR/CD28 (or anti-CD3/CD28) mAb induced apoptosis in total thymocytes. When these signals were present at the same time, GC-induced apoptosis was partially inhibited by TCR/CD3 signaling. Costimulation by anti-CD28 enhanced the inhibitory effects of anti-CD3 on GC-induced apoptosis about 30-fold. However, subset analysis revealed that most cells rescued from GC-induced apoptosis were mature CD4+ and CD8+ thymocytes, and these cells were resistant to TCR/CD3-induced apoptosis in the absence of GC. Similar results were obtained with mature splenic CD4+ and CD8+ T cells. TCR/CD3 signaling alone, while inducing apoptosis in CD4+(CD8+)TCRlow thymocytes, rescued a small subset of CD4+(CD8+)TCRlow thymocytes from GC-induced apoptosis. Thus, TCR signaling increasingly reverses GC-induced apoptosis as thymocyte development progresses. As GC are infinitely present in vivo, these findings support a model wherein TCR signaling may be required to prevent GC-induced apoptosis both under basal and immune challenging conditions.     

Pitt-Rogers-Danks syndrome and Wolf-Hirschhorn syndrome are caused by a deletion in the same region on chromosome 4p 16.3.

Recently, a deletion of chromosome 4pter was found in three patients with Pitt-Rogers-Danks syndrome. We investigated two of these patients, by means of DNA and FISH studies, together with two additional patients with Pitt-Rogers-Danks syndrome, to determine the critical region of the deletion in these patients and to compare this with the critical region in Wolf-Hirschhorn syndrome. All four patients showed terminal deletions of chromosome 4p of different sizes. One of them appeared to have an unbalanced karyotype caused by a cryptic translocation t(4;8) in the mother, resulting in a deletion of chromosome 4pter and a duplication of chromosome 8pter. The localisation of the Wolf-Hirschhorn critical region has been confined to approximately 1 Mb between D4S43 and D4S115. Our study shows that the deletions in four patients with the Pitt-Rogers-Danks syndrome overlap the Wolf-Hirschhorn critical region and extend beyond this in both directions. This study, combined with the fact that our third patient, who was previously described as a Pitt-Rogers-Danks patient, but who now more closely resembles a Wolf-Hirschhorn patient, makes it likely that Pitt-Rogers-Danks and Wolf-Hirschhorn syndromes are different clinical phenotypes resulting from a deletion in the same microscopic region on chromosome 4p16.     

Functional role of NADPH oxidase in activation of platelets

Involvement of phagocyte NADPH oxidase in host defense response is well established. In contrast, little is known about the functional role of NADPH oxidase in platelets. In this study, we analyzed involvement of platelet NADPH oxidase in aggregation of human platelets and in amplification of production of reactive oxygen species (ROS) by activated human neutrophils. Apocynin, a known NADPH oxidase inhibitor, as well as superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-1-pyridyl)porphyrin, inhibited ROS generation by collagen-activated platelets, collagen-induced aggregation of platelets, as well as collagen-induced release of thromboxane B2. These data suggest the key role of intracellular ROS derived from NADPH oxidase in the control of thromboxane A2 (TXA2) production in platelets stimulated by collagen. Apocynin also inhibited thrombin-induced ROS production and thrombin-induced platelet aggregation. Activation of neutrophils with latex resulted in an outburst of ROS that was inhibited by apocynin. ROS production by latex-stimulated platelets was modest and also inhibited by apocynin. However, when a mixture of platelets and neutrophils was stimulated with latex, ROS production was three to six times higher in comparison with activation of neutrophils alone. Platelet-dependent augmentation of neutrophil ROS production was abrogated by TXA2 synthase inhibitor (furegrelate, 1 microM) or by aspirin (300 microM). In summary, NADPH oxidase in platelets seems to play a major role as an intracellular signaling mechanism in the activation of platelets. However, in host defense response involving neutrophils and platelets, platelets enhance ROS production by neutrophils and possibly their cytotoxic potential via the release of TXA2, which in turn in platelets is not affected by the extracellular release of free radicals.     

Silver nanostructure sensing platform for maximum-contrast fluorescence cell imaging

We present herein a silver nanostructure-assisted sensing platform which consists of a combined structure of Ag nanowire (NW) and nanodot (ND) array. Highly enhanced fluorescence from fluorophore is attributed to a strongly coupled optical near-field interaction between proximately located Ag NW and NDs. We obtained enhanced fluorescence intensity with up to 140 folds, as contrasted from background intensity, reaching a theoretical maximum value. On the other hand, fluorescence lifetime was greatly reduced to 0.27 ns (from 2.17 ns for the same fluorophores without nanostructure). This novel platform can be a promising utility for optical imaging and labeling of biological systems with a great sensitivity.     

Targeting endothelial cells with heme oxygenase-1 gene using VE-cadherin promoter attenuates hyperglycemia-mediated cell injury and apoptosis

Risk factors for cardiovascular diseases include hyperglycemia, TNF, and reactive oxygen species (ROS), which collectively contribute to vascular endothelial cell dysfunction and apoptosis. We examined, in vascular endothelial cells, whether the selective expression of heme oxygenase-1 (HO-1) offers cytoprotection against glucose- and TNF-mediated cell death. An adenoviral vector expressing human HO-1 was constructed using a VE-cadherin (VECAD) promotor fragment, and cell-specific expression of the recombinant adenovirus was examined using endothelial and vascular smooth muscle cells. The effects of HO-1 transduction (Ad-VECAD-HO-1 gene) on HO-1 expression, HO activity, and the response to TNF and hyperglycemia were studied. Human HO-1 gene was selectively expressed in endothelial cells after infection with the Ad-VECAD-HO-1 vector. Selective expression of HO-1 prevented TNF- and hyperglycemia-mediated superoxide (O2-) formation, DNA degeneration, and upregulation of caspase, but increased the expression of pAkt and Bcl-xL, proteins responsible for endothelial dysfunction in diabetes. These results demonstrate that endothelial cell survival after oxidative stress injury may be enhanced by targeting HO-1 expression, thus blocking inflammation, apoptosis, and thereby attenuating cardiovascular risk factors.     

Vulvar Atypical Granular Cell Tumor in a Preadolescent Patient

BackgroundGranular cell tumor is an uncommon benign neoplasm with a predisposition for upper aerodigestive tract, skin and soft tissue involvement. Malignant and atypical granular cell tumors account for less than 2% of the lesions and in the pediatric population they are extremely rare and atypia has not been previously reported.CaseWe present a case of a rapidly growing granular cell tumor of the vulva of a 12-year-old girl exhibiting atypical histology. The lesion demonstrated prominent Ki-67 proliferation index (up to 20%), localized areas of spindling of tumor cells, scattered apoptotic bodies and p53 overexpression.ConclusionThe current histologic diagnostic criteria of atypical granular cell tumors are evaluated while physician awareness and the need for follow-up of patients for potential recurrences of this rare entity are emphasized.     

Bilirubin,a new therapeutic for kidney transplant?

In patients with end-stage renal disease, kidney transplantation has been associated with numerous benefits, including increased daily activity, and better survival rates. However, over 20% of kidney transplants result in rejection within five years. Rejection is primarily due to a hypersensitive immune system and ischemia/reperfusion injury. Bilirubin has been shown to be a potent antioxidant that is capable of potentially reversing or preventing damage from reactive oxygen species generated from ischemia and reperfusion. Additionally, bilirubin has several immunomodulatory effects that can dampen the immune system to promote organ acceptance. Increased bilirubin has also been shown to have a positive impact on renal hemodynamics, which is critical post-transplantation. Lastly, bilirubin levels have been correlated with biomarkers of successful transplantation. In this review, we discuss a multitude of potentially beneficial effects that bilirubin has on kidney acceptance of transplantation based on numerous clinical trials and animal models. Exogenous bilirubin delivery or increasing endogenous levels pre- or post-transplantation may have therapeutic benefits.