Efficacy and Safety of Human Bone Marrow-Derived Mesenchymal Stem Cells according to Injection Route and Dose in a Chronic Kidney Disease Rat Model.

Background and Objectives: We compared the efficacy and safety of human bone marrow-derived mesenchymal stem cells (hBMSC), delivered at different doses and via different injection routes in an animal model of chronic kidney disease. Methods and Results: A total of ninety 12-week-old rats underwent 5/6 nephrectomy and randomized among nine groups: sham, renal artery control (RA-C), tail vein control (TV-C), renal artery low dose (RA-LD) (0.5×106 cells), renal artery moderate dose (RA-MD) (1.0×106 cells), renal artery high dose (RA-HD) (2.0×106 cells), tail vein low dose (TV-LD) (0.5×106 cells), tail vein moderate dose (TV-MD) (1.0×106 cells), and tail vein high dose (TV-HD) (2.0×106 cells). Renal function and mortality of rats were evaluated after hBMSC injection. Serum blood urea nitrogen was significantly lower in the TV-HD group at 2 weeks (p<0.01), 16 weeks (p<0.05), and 24 weeks (p<0.01) than in the TV-C group, as determined by one-way ANOVA. Serum creatinine was significantly lower in the TV-HD group at 24 weeks (p<0.05). At 8 weeks, creatinine clearance was significantly higher in the TV-MD and TV-HD groups (p<0.01, p<0.05) than in the TV-C group. In the safety evaluation, we observed no significant difference among the groups. Conclusions: Our findings confirm the efficacy and safety of high dose (2×106 cells) injection of hBMSC via the tail vein.

Identification of Cardiovascular Disease-Related Genes Based on the Co-Expression Network Analysis of Genome-Wide Blood Transcriptome.

Inference of co-expression network and identification of disease-related modules and gene sets can help us understand disease-related molecular pathophysiology. We aimed to identify a cardiovascular disease (CVD)-related transcriptomic signature, specifically, in peripheral blood tissue, based on differential expression (DE) and differential co-expression (DcoE) analyses. Publicly available blood sample datasets for coronary artery disease (CAD) and acute coronary syndrome (ACS) statuses were integrated to establish a co-expression network. A weighted gene co-expression network analysis was used to construct modules that include genes with highly correlated expression values. The DE criterion is a linear regression with module eigengenes for module-specific genes calculated from principal component analysis and disease status as the dependent and independent variables, respectively. The DcoE criterion is a paired t-test for intramodular connectivity between disease and matched control statuses. A total of 21 and 23 modules were established from CAD status- and ACS-related datasets, respectively, of which six modules per disease status (i.e., obstructive CAD and ACS) were selected based on the DE and DcoE criteria. For each module, gene-gene interactions with extremely high correlation coefficients were individually selected under the two conditions. Genes displaying a significant change in the number of edges (gene-gene interaction) were selected. A total of 6, 10, and 7 genes in each of the three modules were identified as potential CAD status-related genes, and 14 and 8 genes in each of the two modules were selected as ACS-related genes. Our study identified gene sets and genes that were dysregulated in CVD blood samples. These findings may contribute to the understanding of CVD pathophysiology.

Safety of autologous bone marrow-derived mesenchymal stem cells in erectile dysfunction: an open-label phase 1 clinical trial.

BACKGROUND AIMS: The efficacy of phosphodiesterase type 5 inhibitors (PDE5Is), which are commonly used to treat erectile dysfunction (ED), is not satisfactory in patients with denervation of the cavernous nerve due to pelvic surgeries and diabetes mellitus (DM). Pre-clinical studies using bone marrow-derived mesenchymal stem cells (BMSCs) to treat ED have shown promising results. The authors conducted a phase 1 clinical trial with autologous BMSCs in patients with ED due to radical prostatectomy or DM. METHODS: Ten patients (five with post-prostatectomy ED and five with DM-associated ED) who could not perform sexual activity despite taking the maximum dose of a PDE5I were enrolled. The brief clinical trial protocol was registered with the US National Institutes of Health on ClinicalTrials.gov (NCT02344849). The primary outcome was the safety of stem cell therapy, and the secondary outcome was the improvement of erectile function. RESULTS: Of the 13 patients screened, 10 were registered in the clinical trial and received autologous BMSCs and nine completed the clinical trial. One patient with post-prostatectomy ED experienced two treatment-emergent adverse events (TEAEs) (pyrexia and back pain), and two patients with DM-associated ED experienced a total of five TEAEs (one case each of viral upper respiratory tract infection, prostatitis and pruritus and two cases of hyperglycemia). Of these patients, one with DM-associated ED experienced two serious TEAEs (two instances of hyperglycemia). All TEAEs were considered not to be related to autologous BMSC therapy. In addition, no clinical significance was identified related to other safety measures, such as laboratory tests and vital signs. The mean International Index of Erectile Function score increased significantly at 1 month versus baseline (24.9 versus 18.1, P = 0.0222). CONCLUSIONS: This phase 1 clinical trial confirmed the safety and potential efficacy of autologous BMSC therapy in patients with ED. The authors' results need to be confirmed by a phase 2 clinical trial.

Establishment of NOAEL for intracavernous injections of human bone marrow-derived mesenchymal stem cells in rats.

Purpose: To assess the possible negative health effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on fertility and early embryonic development following intracavernous injections in rats. Materials and Methods: A total of 88 Crl:CD(SD) male and female rats were equally divided into 4 groups in a random manner: control group (normal saline), low-dose group (2×105 hBMSCs), moderate-dose group (1×106 hBMSCs), and high-dose group (2×106 hBMSCs). hBMSCs or normal saline was injected into the penis of the rats 3 times at 2-week-intervals prior to mating. We compared each group with respect to parameters of reproduction and histopathology. Results: For male rats, various degrees of flushing and swelling were observed at the penile injection site in all the groups, although the severity increased in a dose-dependent manner in the hBMSC injection groups. There were no statistically significant differences in mean body weights and food consumption among all the groups of both sexes. There were no statistically significant differences in reproductive parameters among all the groups of both sexes. The absolute and relative organ weights did not significantly differ among the groups. At the time of necropsy, no remarkable findings were observed in gross examinations in all groups. On histopathological analysis, minimal mononuclear cell infiltration was observed in the right epididymis of each rat in the moderate- and high-dose groups. Conclusions: The non-toxic amount of hBMSCs for male fertility and early embryogenesis in rats under the test conditions was determined to be 2×106 cells/head.

Mesenchymal Stem Cell-Mediated Delivery of an Oncolytic Adenovirus Enhances Antitumor Efficacy in Hepatocellular Carcinoma.

Oncolytic virotherapy is a promising alternative to conventional treatment, yet systemic delivery of these viruses to tumors remains a major challenge. In this regard, mesenchymal stem cells (MSC) with well-established tumor-homing property could serve as a promising systemic delivery tool. We showed that MSCs could be effectively infected by hepatocellular carcinoma (HCC)-targeted oncolytic adenovirus (HCC-oAd) through modification of the virus' fiber domain and that the virus replicated efficiently in the cell carrier. HCC-targeting oAd loaded in MSCs (HCC-oAd/MSC) effectively lysed HCC cells in vitro under both normoxic and hypoxic conditions as a result of the hypoxia responsiveness of HCC-oAd. Importantly, systemically administered HCC-oAd/MSC, which were initially infected with a low viral dose, homed to HCC tumors and resulted in a high level of virion accumulation in the tumors, ultimately leading to potent tumor growth inhibition. Furthermore, viral dose reduction and tumor localization of HCC-oAd/MSC prevented the induction of hepatotoxicity by attenuating HCC-oAd hepatic accumulation. Taken together, these results demonstrate that MSC-mediated systemic delivery of oAd is a promising strategy for achieving synergistic antitumor efficacy with improved safety profiles. SIGNIFICANCE: Mesenchymal stem cells enable delivery of an oncolytic adenovirus specifically to the tumor without posing any risk associated with systemic administration of naked virions to the host.

Millimeter-Scale Growth of Single-Oriented Graphene on a Palladium Silicide Amorphous Film.

It is widely accepted in condensed matter physics and material science communities that a single-oriented overlayer cannot be grown on an amorphous substrate because the disordered substrate randomizes the orientation of the seeds, leading to polycrystalline grains. In the case of two-dimensional materials such as graphene, the large-scale growth of single-oriented materials on an amorphous substrate has remained unsolved. Here, we demonstrate experimentally that the presence of uniformly oriented graphene seeds facilitates the growth of millimeter-scale single-oriented graphene with 3 × 4 mm2 on palladium silicide, which is an amorphous thin film, where the uniformly oriented graphene seeds were epitaxially grown. The amorphous palladium silicide film promotes the growth of the single-oriented growth of graphene by causing carbon atoms to be diffusive and mobile within and on the substrate. In contrast to these results, without the uniformly oriented seeds, the amorphous substrate leads to the growth of polycrystalline graphene grains. This millimeter-scale single-oriented growth from uniformly oriented seeds can be applied to other amorphous substrates.

Hypoxic Preconditioned Mesenchymal Stromal Cell Therapy in a Rat Model of Renal Ischemia-reperfusion Injury: Development of Optimal Protocol to Potentiate Therapeutic Efficacy.

Although previous and ongoing clinical studies have used stromal cells during renal ischemia-reperfusion injury (IRI), there is little consensus regarding the optimal protocol. We aimed to optimize the protocol for hypoxic preconditioned human bone marrow-derived mesenchymal stromal cell (HP-hBMSC) therapy in a rat model of renal IRI. We determined the optimal injection route (renal arterial, renal parenchymal, and tail venous injection), dose (low-dose: 1×106, moderate-dose: 2×106, and high-dose: 4×106), and injection period (pre-, concurrent-, and post-IRI). During optimal injection route study, renal arterial injections significantly reduced the decreasing glomerular filtration rate (GFR), as compared to GFRs for the IRI control group, 2 and 4 days after IRI. Therapeutic effects and histological recoveries were the greatest in the group receiving renal arterial injections. During the dose finding study, high-dose injections significantly reduced the decreasing GFR, as compared to GFRs for the IRI control group, 3 days after IRI. Therapeutic effects and histological recoveries were the greatest in the high-dose injection group. While determining the optimal injection timing study, concurrent-IRI injection reduced elevated serum creatinine levels, as compared to those of the IRI control group, 1 day after IRI. Pre-IRI injection significantly reduced the decreasing GFR, as compared with GFRs for the IRI control group, 1 day after IRI. Therapeutic effects and histological recoveries were the greatest in the concurrent-IRI group. In conclusion, the concurrent-IRI administration of a high dose of HP-hBMSC via the renal artery leads to an optimal recovery of renal function after renal IRI.

Dirac electrons in a dodecagonal graphene quasicrystal.

Quantum states of quasiparticles in solids are dictated by symmetry. We have experimentally demonstrated quantum states of Dirac electrons in a two-dimensional quasicrystal without translational symmetry. A dodecagonal quasicrystalline order was realized by epitaxial growth of twisted bilayer graphene rotated exactly 30°. We grew the graphene quasicrystal up to a millimeter scale on a silicon carbide surface while maintaining the single rotation angle over an entire sample and successfully isolated the quasicrystal from a substrate, demonstrating its structural and chemical stability under ambient conditions. Multiple Dirac cones replicated with the 12-fold rotational symmetry were observed in angle-resolved photoemission spectra, which revealed anomalous strong interlayer coupling with quasi-periodicity. Our study provides a way to explore physical properties of relativistic fermions with controllable quasicrystalline orders.

Bone marrow-derived mesenchymal stromal cell therapy in a rat model of cavernous nerve injury: Preclinical study for approval.

BACKGROUND AIMS: Although clinical studies using stem cells to treat erectile dysfunction have been performed or are ongoing, there is little consensus on the optimal protocol. We aimed to develop a protocol optimizing human bone marrow-derived mesenchymal stromal cell (hBMSC) therapy in a rat model of cavernous nerve injury. METHODS: We performed, in order, a dose-finding study, a toxicokinetic study of hBMSCs, and a study to determine the timing and number of cell injections. RESULTS: From the dose-finding study, 1 × 10(6) cells were selected as the dose per hBMSC injection. From the toxicokinetic study, 14 days was selected as the interval between repeat treatments. In the final study, the ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the control group than in the sham group (23.4% vs. 55.1%, P <0.001). An immediate single injection of hBMSCs significantly improved erectile function compared with the control group (39.8%, P = 0.035), whereas a delayed single injection showed improvement with a marginal trend (38.1%, P = 0.079). All histomorphometric changes were significantly more improved in the immediate or delayed single injection groups than in the control group. Repeat treatments did not provide any benefit for the recovery of erectile function and histomorphometric changes. CONCLUSIONS: Intracavernous injection of 1 × 10(6) hBMSCs results in a recovery of penile erection and histomorphometric changes in a rat model of cavernous nerve injury, even when treatment was delayed until 4 weeks after cavernous nerve injury.

Epitaxial growth of a single-crystal hybridized boron nitride and graphene layer on a wide-band gap semiconductor.

Vertical and lateral heterogeneous structures of two-dimensional (2D) materials have paved the way for pioneering studies on the physics and applications of 2D materials. A hybridized hexagonal boron nitride (h-BN) and graphene lateral structure, a heterogeneous 2D structure, has been fabricated on single-crystal metals or metal foils by chemical vapor deposition (CVD). However, once fabricated on metals, the h-BN/graphene lateral structures require an additional transfer process for device applications, as reported for CVD graphene grown on metal foils. Here, we demonstrate that a single-crystal h-BN/graphene lateral structure can be epitaxially grown on a wide-gap semiconductor, SiC(0001). First, a single-crystal h-BN layer with the same orientation as bulk SiC was grown on a Si-terminated SiC substrate at 850 °C using borazine molecules. Second, when heated above 1150 °C in vacuum, the h-BN layer was partially removed and, subsequently, replaced with graphene domains. Interestingly, these graphene domains possess the same orientation as the h-BN layer, resulting in a single-crystal h-BN/graphene lateral structure on a whole sample area. For temperatures above 1600 °C, the single-crystal h-BN layer was completely replaced by the single-crystal graphene layer. The crystalline structure, electronic band structure, and atomic structure of the h-BN/graphene lateral structure were studied by using low energy electron diffraction, angle-resolved photoemission spectroscopy, and scanning tunneling microscopy, respectively. The h-BN/graphene lateral structure fabricated on a wide-gap semiconductor substrate can be directly applied to devices without a further transfer process, as reported for epitaxial graphene on a SiC substrate.

Direct momentum-resolved observation of one-dimensional confinement of externally doped electrons within a single subnanometer-scale wire.

Cutting-edge research in the band engineering of nanowires at the ultimate fine scale is related to the minimum scale of nanowire-based devices. The fundamental issue at the subnanometer scale is whether angle-resolved photoemission spectroscopy (ARPES) can be used to directly measure the momentum-resolved electronic structure of a single wire because of the difficulty associated with assembling single wire into an ordered array for such measurements. Here, we demonstrated that the one-dimensional (1D) confinement of electrons, which are transferred from external dopants, within a single subnanometer-scale wire (subnanowire) could be directly measured using ARPES. Convincing evidence of 1D electron confinement was obtained using two different gold subnanowires with characteristic single metallic bands that were alternately and spontaneously ordered on a stepped silicon template, Si(553). Noble metal atoms were adsorbed at room temperature onto the gold subnanowires while the overall structure of the wires was maintained. Only one type of gold subnanowire could be controlled using external noble metal dopants without transforming the metallic band of the other type of gold subnanowires. This result was confirmed by scanning tunnelling microscopy experiments and first-principles calculations. The selective control clearly showed that externally doped electrons could be confined within a single gold subnanowire. This experimental evidence was used to further investigate the effects of the disorder induced by external dopants on a single subnanowire using ARPES.

Designed three-dimensional freestanding single-crystal carbon architectures.

Single-crystal carbon nanomaterials have led to great advances in nanotechnology. The first single-crystal carbon nanomaterial, fullerene, was fabricated in a zero-dimensional form. One-dimensional carbon nanotubes and two-dimensional graphene have since followed and continue to provide further impetus to this field. In this study, we fabricated designed three-dimensional (3D) single-crystal carbon architectures by using silicon carbide templates. For this method, a designed 3D SiC structure was transformed into a 3D freestanding single-crystal carbon structure that retained the original SiC structure by performing a simple single-step thermal process. The SiC structure inside the 3D carbon structure is self-etched, which results in a 3D freestanding carbon structure. The 3D carbon structure is a single crystal with the same hexagonal close-packed structure as graphene. The size of the carbon structures can be controlled from the nanoscale to the microscale, and arrays of these structures can be scaled up to the wafer scale. The 3D freestanding carbon structures were found to be mechanically stable even after repeated loading. The relationship between the reversible mechanical deformation of a carbon structure and its electrical conductance was also investigated. Our method of fabricating designed 3D freestanding single-crystal graphene architectures opens up prospects in the field of single-crystal carbon nanomaterials and paves the way for the development of 3D single-crystal carbon devices.

Opening and reversible control of a wide energy gap in uniform monolayer graphene.

For graphene to be used in semiconductor applications, a 'wide energy gap' of at least 0.5 eV at the Dirac energy must be opened without the introduction of atomic defects. However, such a wide energy gap has not been realized in graphene, except in the cases of narrow, chemically terminated graphene nanostructures with inevitable edge defects. Here, we demonstrated that a wide energy gap of 0.74 eV, which is larger than that of germanium, could be opened in uniform monolayer graphene without the introduction of atomic defects into graphene. The wide energy gap was opened through the adsorption of self-assembled twisted sodium nanostrips. Furthermore, the energy gap was reversibly controllable through the alternate adsorption of sodium and oxygen. The opening of such a wide energy gap with minimal degradation of mobility could improve the applicability of graphene in semiconductor devices, which would result in a major advancement in graphene technology.

Involvement of Ca2+/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion.

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca(2+) channel opener Bay K8644. Genistein at a concentration of 50 µM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca(2+)/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca(2+)]i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.

Induction of growth phase-specific autolysis in Bacillus subtilis 168 by growth inhibitors.

Growth phase-specific autolysis of Bacillus subtilis by inhibitors of membrane permeability, inhibitors of macromolecule biosynthesis, inhibitors of cell wall biosynthesis and detergents were tested and characterized in glucose limited liquid medium. The minimum autolysin induction concentration (MAIC) of test compounds, which was at least 1/20th lower than the conventional autolysis induction concentration, induced autolysis only for cells at the glucose exhaustion point (diauxic point) of the growth phase, while it was not induced for cells at pre- and post-diauxic points. Inhibitors of macromolecule synthesis that are not known for inducing autolysis, such as chloramphenicol, rifampicin, nalidixic acid, and detergents, also induced specific autolysis. Two types of autolysis corresponding to the concentrations of compounds are distinguished: concentration-sensitive and concentration-insensitive types.

Involvement of Ca2+-mediated apoptotic signals in palmitate-induced MIN6N8a beta cell death.

The extracellular Ca(2+) chelator EGTA and L-type Ca(2+) channel blockers, such as, nifedipine and nimodipine were found to have a protective effect on palmitate-induced MIN6N8a beta cell apoptosis, whereas the Ca(2+) channel opener, Bay K8644, enhanced the apoptotic process. Moreover, the phospho-form of Bad, in conjunction with phospho-Akt, was reduced in response to palmitate and the palmitate-induced dephosphorylations of Akt and Bad were dependent on Ca(2+) influx. The transient expression of catalytically active Akt prevented MIN6N8a cells from palmitate-induced apoptosis. Deltamethrin, an inhibitor of Ca(2+)-activated phosphatase, delayed Akt and Bad dephosphorylations, and then protected MIN6N8a cells from palmitate-induced apoptosis. On the other hand, palmitate was found to induce CHOP, an apoptotic transcription factor in response to ER stress, and this induction was enhanced by Ca(2+) influx. Our studies suggested that Ca(2+) influx and subsequent Ca(2+)-mediated apoptotic signals are involved in palmitate-induced beta cell death.

Involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis.

BACKGROUND/AIMS: The death of endothelial cells may play a critical role in the development of various vascular diseases, including atherosclerosis. While free fatty acids (FFAs) may stimulate endothelial apoptosis, the molecular and cellular mechanisms of this effect have not been studied intensively. To elucidate the mechanisms involved in FFA-induced endothelial cell apoptosis, we investigated the effect of different pharmacological inhibitors on palmitate-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Interestingly, lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, showed a strong protective effect. METHODS AND RESULTS: To examine the involvement of GSK-3beta in palmitate-induced HUVEC apoptosis, its dephosphorylation at Ser9 and enzymatic activation in response to palmitate treatment were monitored by immunoblotting and in vitro kinase assays, respectively. GSK-3beta was dephosphorylated and its enzymatic activity increased in palmitate-treated HUVECs. In addition, pretreatment with other GSK-3beta inhibitors, e.g. SB216763 or TDZD-8, as well as adenoviral transduction with a catalytically inactive GSK-3beta had significant protective effects against palmitate-induced HUVEC apoptosis. CONCLUSION: These results demonstrate that the GSK-3beta signalling pathway is involved in palmitate-induced HUVEC apoptosis.

Involvement of Ca2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells.

EGb 761, a standardized form of Ginkgo biloba L. (Ginkgoaceae) leaf extract, was recently reported to increase pancreatic beta-cell function. To determine whether EGb 761 elicits insulin secretion directly, we treated INS-1 rat beta cells with EGb 761 and then measured insulin release. Treatment of EGb 761 (50 microg/ml) significantly stimulated insulin secretion in INS-1 cells, compared with untreated control (p<0.05) and the stimulatory effect of EGb 761 on insulin secretion was dose-dependent. To elucidate the mechanism of EGb 761-induced insulin secretion, we investigated the involvement of calcium. The treatment with nifedipine, an L-type calcium channel blocker, prevented EGb 761-induced insulin secretion and furthermore, EGb 761 itself elevated [Ca(2+)](i), suggesting the involvement of calcium in this process. To identity the protein kinases involved in EGb 761-induced insulin secretion, INS-1 cells were treated with different kinase inhibitors and their effects on EGb 761-induced secretion were investigated. KN62 and H89, calium/calmodulin kinase (CaMK) II and protein kinase A (PKA) inhibitor, respectively, significantly reduced EGb 761-induced insulin secretion. Immunoblotting studies showed an increase in the phosphorylated-forms of CaMK II and of PKA substrates after EGb 761 treatment. Our data suggest that EGb 761-induced insulin secretion is mediated by [Ca(2+)](i) elevation and subsequent activation of CaMK II and PKA.

Involvement of calcium-mediated apoptotic signals in H2O2-induced MIN6N8a cell death.

Reactive oxygen species are believed to be the central mediators of beta-cell destruction that leads to type 1 and 2 diabetes, and calcium has been reported to be an important mediator of beta cell death. In the present study, the authors investigated whether Ca(2+) plays a role in hydrogen peroxide (H(2)O(2))-induced MIN6N8a mouse beta cell death. Treatment with low concentration H(2)O(2) (50 microM) was found to be sufficient to reduce MIN6N8a cell viability by 55%, largely via apoptosis. However, this H(2)O(2)-induced cell death was near completely blocked by pretreatment with BAPTA/AM (5 microM), a chelator of intracellular Ca(2+). Moreover, the intracellular calcium store channel blockers, such as, xestospongin c and ryanodine, significant protected cells from 50 microM H(2)O(2)-induced cell death and under extracellular Ca(2+)-free conditions, 50 microM H(2)O(2) elicited transient [Ca(2+)](i) increases. In addition, pharmacologic inhibitors of calpain, calcineurin, and calcium/calmodulin-dependent protein kinase II were found to have a protective effect on H(2)O(2)-induced death. Moreover, H(2)O(2)-induced apoptotic signals, such as c-JUN N-terminal kinase activation, cytochrome c release, caspase 3 activation, and poly (ADP-ribose) polymerase cleavage were all down-regulated by the intracellular Ca(2+) chelation. These findings show that [Ca(2+)](i) elevation, possibly due to release from intracellular calcium stores and the subsequent activation of Ca(2+)-mediated apoptotic signals, critically mediates low concentration H(2)O(2)-induced MIN6N8a cell death. These findings suggest that a breakdown of calcium homeostasis by low level of reactive oxygen species may be involved in beta cell destruction during diabetes development.

Mammaglobin expression in lymph nodes is an important marker of metastatic breast carcinoma.

CONTEXT: Organ specificity is a desirable property of a tumor marker, especially in metastatic adenocarcinomas of unknown primary origin. Mammaglobin, a mammary-specific member of the uteroglobin family, is known to be overexpressed in human breast cancer. OBJECTIVE: We investigated mammaglobin A expression in metastatic carcinomas of lymph nodes from the breast and various other organs and its usefulness in identifying metastatic carcinoma of the breast. For comparative purposes, we also investigated BRST-1 and BRST-2 expression. DESIGN: We produced recombinant mammaglobin and polyclonal antimammaglobin antibodies. Mammaglobin expression was analyzed by immunohistochemical staining using a tissue microarray and by reverse transcription-polymerase chain reaction in 210 carcinomas, including those of the breast (n = 70), lung (n = 30), stomach (n = 30), colorectum (n = 25), hepatobiliary tract (n = 20), urinary tract (n = 10), thyroid gland (n = 10), ovary and endometrium (n = 10), and salivary gland (n = 5). RESULTS: Mammaglobin expression was observed in 59 cases (84.3%) of breast cancer and in 21 cases (15.0%) of nonbreast cancer. The BRST-1 and BRST-2 expression rates were 75.7% and 44.3% in breast cancer and 26.4% and 2.1% in nonbreast cancer, respectively. Mammaglobin is superior to BRST-1 for both specificity and sensitivity and is superior to BRST-2 for sensitivity. CONCLUSION: Our data suggest that mammaglobin is one of the first relatively mammary-specific and mammary-sensitive markers. Mammaglobin and BRST-2 appear to represent useful markers for breast cancer and should be used as a component of panels evaluating tumors of unknown primary sites.