Streptozotocin induces neurite outgrowth via PI3K-Akt and glycogen synthase kinase 3ß in Neuro2a cells.

Streptozotocin (STZ), a naturally occurring chemical, is toxic to the various kinds of cells such as insulin-producing beta cells. However, the beneficial effect of STZ on neuronal cells such as neurite outgrowth-inducing activity has been unknown. In this study, we examined the effect of STZ on neurite outgrowth in mouse neuronal Neuro2a cells. STZ (0.01 mM~5 mM) exerted remarkable neurite outgrowth-inducing activity in Neuro2a cells in a concentration dependent manner. STZ also had the same neurite outgrowth-inducing activity as that of retinoic acid (RA), which is well known neurite outgrowth inducer. As with the result of RA treatment, STZ administration increased MAP2-positive cells. The MAP2-positive cells reflect neurite outgrowth-induced cells. STZ (0.01 mM~5 mM) did not induce cell death, but significantly decreased cell proliferation. The serine/threonine kinase Akt, a downstream target of phosphatidylinositol-3 kinase (PI3K), was transiently phosphorylated at Ser473 and at Thr303 by STZ (5 mM) administration. Glycogen synthase kinase 3ß (GSK3ß), which has been reported to be inactivated by Akt, was also transiently phosphorylated at Ser9 by STZ (5 mM) administration. In addition, a blocker of PI3K, LY294002 (10 µM), significantly attenuated STZ-induced neurite outgrowth. These results suggest that STZ induces neurite outgrowth via activation of PI3K-Akt signaling pathway and GSK3ß inhibition.

Genome-wide association study of degenerative bony changes of the temporomandibular joint.

OBJECTIVES: To identify susceptibility genes underlying degenerative bony changes of the temporomandibular joint (TMJ). MATERIALS AND METHODS: Bony changes of the TMJ condylar head were diagnosed by examination of panoramic radiographs and/or magnetic resonance images and/or computed tomography images. We conducted a genome-wide association study (GWAS) of 146 cases with TMJ degeneration and 374 controls from East Asian populations using an Illumina HumanOmniExpress BeadChip. After rigorous quality-control filtering, approximately 550,000 single nucleotide polymorphisms (SNPs) were used for tests of associations with disease status. RESULTS: Forty-one SNPs at 22 independent loci showed association signals at P < 1 × 10(-4). The SNP rs878962, which maps on an intron of TSPAN9 on chromosome 12, showed the strongest association (combined OR = 1.89, 95% confidence interval = 1.43-2.50, P = 8.1 × 10(-6)). According to in silico predictions of the 41 SNPs, two intronic SNPs of APOL3 (rs80575) and MRC2 (rs2460300) may fall within regulatory elements and affect DNA-protein interactions. We could not replicate SNPs located on genes that have been reported to be associated with temporomandibular disorder or temporomandibular osteoarthritis in previous studies at P < 1 × 10(-4). CONCLUSIONS: Our GWAS identified 22 independent loci showing suggestive association signals with degenerative bony changes of the TMJ. These loci provide good candidates for future follow-up studies.

A functional SNP upstream of the beta-2 adrenergic receptor gene (ADRB2) is associated with obesity in Oceanic populations.

OBJECTIVE: Obesity is a growing health concern in the Oceanic populations. To investigate the genetic factors associated with adult obesity in the Oceanic populations, the association of single nucleotide polymorphisms (SNPs) of the beta-2 adrenergic receptor (ADRB2) gene with obesity was examined in 694 adults living in Tonga and Solomon Islands. RESULTS: A screening for variation in 16 Oceanic subjects detected 17 SNPs in the entire region of ADRB2, of which nine SNPs including two non-synonymous ones, rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu), were further genotyped for all subjects. The rs34623097-A allele, at a SNP located upstream of ADRB2, showed the strongest association with risk for obesity in a logistic regression analysis adjusted for age, sex, and population (P=5.6 × 10(-4), odds ratio [OR]=2.5, 95% confidence interval [CI]=1.5-4.2). The 27Glu was also significantly associated with obesity in the single-point association analysis (P=0.013, OR=2.0, 95%CI=1.2-3.4); however, this association was no longer significant after adjustment for rs34623097 since these SNPs were in linkage disequilibrium with each other. A copy of the obesity-risk allele, rs34623097-A, led to a 1.6 kg/m(2) increase in body mass index (BMI; defined as weight in kilograms divided by height in meters squared) (P=0.0019). A luciferase reporter assay indicated that rs34623097-A reduced the transcriptional activity of the luciferase reporter gene by approximately 10% compared with rs34623097-G. An electrophoretic mobility shift assay demonstrated that rs34623097 modulated the binding affinity with nuclear factors. An evolutionary analysis implies that a G>A mutation at rs34623097 occurred in the Neandertal genome and then the rs34623097-A allele flowed into the ancestors of present-day humans. CONCLUSION: The present results suggest that rs34623097-A, which would lead to lower expression of ADRB2, contributes to the onset of obesity in the Oceanic populations.

Impacts of an urban flood control infrastructure on the limnology and ichthyofauna of a basaltic Cuesta stream (southeast Brazil).

Strategies for flood control associated to extreme precipitation events in urban areas are urgent, in order to prevent not only material damages but also to avoid human losses. The construction of flood contention reservoirs ("piscinões") has become a common engineering intervention in urban and peri-urban areas. However, there is a lack of studies focused on the evaluation of environmental impacts of this type of construction. This study intended to verify the ecological effects of a retention reservoir built directly on the course of the Cascata stream, Botucatu (SP). Three sampling sites were selected, located upstream the reservoir, in the reservoir and downstream. Samplings were carried out in July (winter - dry) and November (late spring - rainy) 2020. In situ measurements were obtained through a multiparameter probe (temperature, pH, electrical conductivity, dissolved oxygen, total dissolved solids, and oxidation-reduction potential) and water samples were collected for laboratory determinations (nitrogen, total phosphorus, thermotolerant coliforms, and chlorophyll-a). For fish sampling, manual trawls, sieves and hand nets were used, with a sampling effort of 10 throws per artefact and site. Despite the small distance between the sampling points (~1,300 m) considerable changes in the limnological conditions and fish community structure were observed. The studied environment is originally a small river surrounded by riparian forest, but this characteristic was abruptly changed in the reservoir stretch, with the direct exposition of a much larger water surface to intense solar radiation and atmosphere exchanges. Consequently, as evidenced by the PCA analysis, there was a considerable (stream-reservoir increase) of temperature, dissolved oxygen and chlorophyll. However, this spatial trend was partially disturbed by an accidental sewage-pipe rupture (posteriorly fixed) adjacent to the first sampling point, due to a previous event of extreme precipitation, which resulted in increased values of nutrients, chlorophyll, conductivity and thermotolerant coliforms. Eleven fish species were collected (two non-native), belonging to seven families and five orders. The upstream reference point (despite not be pristine), was characterized by the predominance of native species, while the reservoir condition favored the development of large populations of the non-native species. Despite the urgency of effective actions to prevent floods in urban areas, construction of contention reservoirs directly on stream courses should be avoided, due to their negative ecological impacts.

Sequential-replenishment mechanism of exocytosis in pancreatic acini.

Here we report exocytosis of zymogen granules, as examined by multiphoton excitation imaging in intact pancreatic acini. Cholecystokinin induces Ca 2+ oscillations that trigger exocytosis when the cytosolic Ca 2+ concentration exceeds 1 microM. Zymogen granules fused with the plasma membrane maintain their Omega-shaped profile for an average of 220 s and serve as targets for sequential fusion of granules that are located within deeper layers of the cell. This secondary exocytosis occurs as rapidly as the primary exocytosis and accounts for most exocytotic events. Granule-granule fusion does not seem to precede primary exocytosis, indicating that secondary fusion events may require a plasma-membrane factor. This sequential-replenishment mechanism of exocytosis allows the cell to take advantage of a large supply of fusion-ready granules without needing to transport them to the plasma membrane.

A molecular chaperone inducer protects neurons from ER stress.

The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Recent reports have shown that ER stress is involved in the pathology of some neurodegenerative diseases and cerebral ischemia. In a screen for compounds that induce the ER-mediated chaperone BiP (immunoglobulin heavy-chain binding protein)/GRP78 (78 kDa glucose-regulated protein), we identified BiP inducer X (BIX). BIX preferentially induced BiP with slight inductions of GRP94 (94 kDa glucose-regulated protein), calreticulin, and C/EBP homologous protein. The induction of BiP mRNA by BIX was mediated by activation of ER stress response elements upstream of the BiP gene, through the ATF6 (activating transcription factor 6) pathway. Pretreatment of neuroblastoma cells with BIX reduced cell death induced by ER stress. Intracerebroventricular pretreatment with BIX reduced the area of infarction due to focal cerebral ischemia in mice. In the penumbra of BIX-treated mice, ER stress-induced apoptosis was suppressed, leading to a reduction in the number of apoptotic cells. Considering these results together, it appears that BIX induces BiP to prevent neuronal death by ER stress, suggesting that it may be a potential therapeutic agent for cerebral diseases caused by ER stress.

The effect of surgical bowel manipulation and anesthesia on intestinal glucose absorption in rats.

The effects of surgical bowel manipulation and anesthesia on intestinal glucose absorption were determined in chronically catheterized rats. Total and passive rates of glucose absorption were measured using 3-O-methyl-glucose (3OMG) and L-glucose, metabolically inert analogues of D-glucose. The rates of 3OMG absorption immediately postoperative and 4 h later were 86 and 62% less than the absorption rate 6 d postoperative. The absorption rates of 3OMG 1 and 2 d postoperative were not different from 6 d postoperative. Absorption of L-glucose was not altered by bowel manipulation and anesthesia. Even after correction for the increased resistance of the unstirred water layer (UWL) after bowel manipulation, the rates of total and active intestinal glucose absorption immediately postoperative were only 11 and 15% of predicted rates of absorption. In chronically catheterized rats, > 75% of luminal 3OMG at a concentration of 400 mM was absorbed by active transport. The Km and Vmax of 3OMG active transport corrected for the resistance of the UWL were 11.3 mM and 15.6 mumoles/min, respectively. We conclude that measurements of intestinal glucose absorption performed within 24 h of surgical bowel manipulation greatly underestimate active absorption even if corrections are made to account for the increased resistance of the UWL.

Active transport of 3-O-methyl-glucose by the small intestine in chronically catheterized rats.

A method is described for determining the fraction of intestinal 3-O-methyl-glucose (3OMG) absorption that occurs by active transport in chronically catheterized rats without the influence of anesthesia or surgical bowel manipulation. That fraction was determined by simultaneously measuring portal venous-aortic blood concentration gradients (delta C) of 3-O-methyl-glucose (3OMG) and L-glucose, metabolically inert analogues of D-glucose. 3OMG is actively and passively absorbed by the same mechanisms as D-glucose, L-glucose is only passively absorbed. The fraction of 3OMG that is actively transported was calculated from the difference between 3OMG and L-glucose absorption, divided by total 3OMG absorption. We found that more than 94% of 3-O-methyl-glucose is absorbed by active transport when luminal concentrations range from 50 to 400 mM. We conclude that in unrestrained, unanesthetized chronically catheterized rats, most 3OMG is actively absorbed by the intestine even at high luminal concentrations.

Pramlintide Antagonizes Beta Amyloid (Aß)- and Human Amylin-Induced Depression of Hippocampal Long-Term Potentiation.

Accumulation of amyloid-ß peptide (Aß) is a pathological hallmark of Alzheimer's disease (AD). We have previously demonstrated that electrophysiological and neurotoxic effects of Aß and human amylin are expressed via the amylin receptor. Recently, pramlintide, a synthetic analog of amylin, has been reported to improve cognitive function in transgenic AD mouse models. In this study, we examined the effects of pramlintide on Aß1-42 and human amylin-evoked depression of long-term potentiation (LTP) at Schaeffer collateral-CA1 hippocampal synapses. In mouse hippocampal brain slices, field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum layer of the CA1 area in response to electrical stimulation of Schaeffer collateral afferents and LTP induced by 3-theta-burst stimulation (TBS) protocol. Aß1-42 (50 nM) and human amylin (50 nM), but not Aß42-1 (50 nM), depressed LTP. Pre-application of pramlintide (250 nM) blocked Aß- and human amylin-induced reduction of LTP without affecting baseline transmission or LTP. We also examined the effects of pramlintide on LTP in transgenic mice (TgCRND8) that over-express amyloid precursor protein. In contrast to wild-type controls, where robust LTP was observed, 10- to 12-month-old TgCRND8 mice show blunted LTP. In TgCRND8 mice, basal LTP is enhanced by application of pramlintide. Our data indicate that pramlintide acts as a functional amylin receptor antagonist to reverse the effects of Aß1-42 and human amylin on LTP and also increases LTP in transgenic mice that demonstrate increased ambient brain amyloid levels. Amylin receptor antagonists may thus serve as potentially useful therapeutic agents in treatment of AD.

Plasma protein S activity correlates with protein S genotype but is not sensitive to identify K196E mutant carriers.

BACKGROUND: Protein S (PS) is an anticoagulant protein that functions as a cofactor for activated protein C (APC), and congenital PS deficiency is a well-known risk factor for the development of deep vein thrombosis (DVT). Recently, we and others identified the K196E missense mutation in the second epidermal growth factor-like domain of PS as a genetic risk factor for DVT in the Japanese population. The incidence of this mutation is high in the Japanese population. OBJECTIVES: In the present study, we investigated the relationship between plasma PS activity and the presence of the K196E mutation. PATIENTS AND METHODS: We measured PS activity as a cofactor activity for APC in 1,862 Japanese individuals and determined the PS K196E genotype in this population. RESULTS: Individuals heterozygous for the mutant E-allele had lower plasma PS activity than wildtype subjects (mean +/- SD, 71.9 +/- 17.6%, n = 34 vs. 87.9 +/- 19.8%, n = 1,828, P < 0.0001). However, the PS activity of several heterozygous individuals (n = 8) was greater than the population average. In contrast, multiple wildtype subjects (n = 26) had PS activity less than 2 SD below the population mean, indicating that other genetic or environmental factors affect PS activity. CONCLUSIONS: Plasma PS activity itself is not suitable for identifying PS 196E carriers and other methods are required for carrier detection.

Silk worm Bm1 SINE RNA increases following cellular insults.

The effect of cell stresses upon the expression of the Bm1 short interspersed element (SINE) family in cultured silk worm cells is examined. Primer extension analysis shows that Bm1 repeats are transcribed by RNA polymerase III (Pol III) into cytoplasmic RNAs. Five consecutive T residues, which would normally terminate Pol III transcription, occur within the Bm1 consensus and are included within cDNA sequences representing these transcripts. In analogy to mammalian SINEs, the level of the Bm1 transcripts increases in response to either heat shock, inhibiting protein synthesis by cycloheximide or viral infection. The lifetime of Bm1 RNA increases following cell insults so that post-transcriptional events partially account for stress induced increases in its abundance. In the case of heat shock, the increase in Bm1 RNA follows the transient increase in hsp70 mRNA indicating that this response is temporally regulated to occur later in heat shock recovery. These results support the proposal that SINE RNAs serve a role in the cell stress response that predates the divergence of insects and mammals implying that SINEs are essentially a class of cell stress genes.

Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction.

BACKGROUND AND PURPOSE: Cerebral venous ischemia often induces severe brain edema. Vascular endothelial growth factor (VEGF), which induces angiogenesis, is also known as vascular permeability (VP) factor. The present study was undertaken to investigate whether the inhibition of VEGF could reduce brain edema formation and cerebral venous infarction (CVI) in a rat 2-vein occlusion (2-VO) model. METHODS: We used 2-VO model in which 2 adjacent cortical veins were photochemically occluded. Male Wistar rats (n=25) were divided into 2 groups: one group was treated with a VEGF antagonist (antagonist group, n=10) and the second group was treated with phosphate-buffered solution (PBS) (PBS group, n=15). VEGF antagonist or PBS was injected intraperitoneally immediately after 2-VO. The developing ischemic infarct was evaluated by magnetic resonance imaging (MRI) and histology 24 hours after occlusion. RESULTS: VEGF expression was observed in the cytoplasm of neurons exclusively in the area of vasogenic edema that was shown as a high-intensity area in the apparent diffusion coefficient of water map. Ischemic volumes calculated from each MR images, which are related to infarction and/or vasogenic edema, respectively, were significantly smaller in the antagonist group as compared with the PBS group (P<0.05) CONCLUSIONS: Our study is the first to provide evidence that the inhibition of VEGF attenuates VP and reduces CVI in the acute stage. Although VEGF is a significant angiogenesis factor, we concluded that the inhibition of VEGF might be a new therapy for both brain edema formation and CVI.

Brain proton magnetic resonance spectroscopy and brain atrophy in myotonic dystrophy.

OBJECTIVES: To evaluate by magnetic resonance spectroscopy the age-related cerebral alterations present in myotonic dystrophy (MD) and to compare these results with those obtained by magnetic resonance imaging. DESIGN: Twenty-one patients (aged 16-63 years) with MD were compared with 16 age-matched healthy control subjects. RESULTS: In magnetic resonance spectroscopy, the mean (+/- SD) ratio of N-acetylaspartate to creatine and phosphocreatine in the patients with MD (1.09 +/- 0.32) was significantly lower than that in the control subjects (1.93 +/- 0.43) (P<.001). The mean ratio of N-acetylaspartate to choline-containing compounds in the patients with MD (1.70 +/- 0.44) was also significantly lower than that in the control subjects (2.75 +/- 0.53) (P<.001). These changes could be observed already in the younger patients. In magnetic resonance imaging, the mean brain area was significantly decreased and the mean ventricular space was significantly increased in patients with MD compared with the control subjects. Although we have confirmed brain atrophy in patients with MD in previous reports, a regression analysis indicated that the brain shrinks progressively with age in patients with this disorder and in control subjects, resulting in overlapping values for younger subjects. CONCLUSION: Magnetic resonance spectroscopy indicates that the cerebral abnormalities in patients with MD may be present at an early stage, when the results of magnetic resonance imaging studies are still equivocal.

Age-related changes in homeostasis of inorganic sulfate in male F-344 rats.

Advanced age is associated with a decline in renal function including decreased glomerular filtration rate, renal blood flow and renal tubular secretion. Endogenous inorganic sulfate homeostasis is maintained by concentration-dependent active renal reabsorption in the proximal tubule. The purpose of this study was to determine the effects of advanced age on: (1) the renal mechanisms for conserving endogenous inorganic sulfate and (2) the turnover of inorganic sulfate. Awake, male Fischer 344 rats age 4-5 months and 22-23 months received i.v. acetaminophen, 300 mg/kg, followed 2 h later by i.v. sodium sulfate, 2 mmol/kg, to lower and raise, respectively, plasma inorganic sulfate in order to measure the renal clearance of this anion from plasma at sub- and supraphysiologic concentration ranges. Another group of old and young male F-344 rats received a tracer injection of [35S]sodium sulfate to determine the effect of aging on the turnover of the endogenous inorganic sulfate pool. There was no statistically significant effect of advanced age on baseline plasma sulfate concentration or on the renal clearance of inorganic sulfate from plasma. The baseline excretion rate of inorganic sulfate in the senescent animals (0.38 +/- 0.25 mumol/min/kg, mean +/- S.D., n = 7) was significantly (P < 0.05) lower than that observed in the young animals (0.64 +/- 0.19 mumol/min/kg, n = 8). There was no difference in the turnover rate constant, as measured by the change in specific activity of urinary [35S]sodium sulfate, for the endogenous sulfate pool in old and young animals. Following acetaminophen administration, plasma sulfate concentrations declined similarly in young and old animals. Under the conditions of relative inorganic sulfate depletion, the renal excretion rate of inorganic sulfate decreased to zero in 7 of 8 young rats, whereas the old animals continued to excrete sulfate anion at an average rate of 23% of the baseline value. Aged animals have a defect in active tubular renal reabsorption of sulfate under conditions of sulfate depletion. Age-related changes in the total sulfate excretion rate may reflect changes in the metabolic fate of endogenous sulfate rather than changes in the endogenous production rate of this anion.

Implanted bone particles induce osteoclast development in the presence of osteoblast lineage cells.

We implanted mineral-containing bone particles (BPs) in rats to investigate the involvement of osteoblast lineage cells in osteoclast development in vivo. BPs were implanted in subcutaneous regions on calvaria or artificial defects of calvaria, with or without adjacent parathyroid glands prepared from other rats. The structural characteristics of multinucleated giant cells (MGCs) induced by the BPs were investigated. The MGCs induced by subcutaneously implanted BPs showed membrane ruffling at the basolateral site, but not at the apical site, regardless of whether parathyroid glands were also implanted. In contrast, the MGCs induced by intraosseously implanted BPs showed the characteristics of osteoclasts, i.e., ruffled borders at the apical sites, clear zone, vacuoles, many mitochondria, and scattered rough endoplasmic reticulum. When BPs and parathyroid glands were implanted in bone defects, the number of MGCs was increased compared to that induced by BPs only. These MGCs showed the typical characteristics of active osteoclasts, including developed ruffled borders and stacks of Golgi succules. The number of osteoclasts was also investigated quantitatively by counting the numbers of MGCs positive and negative for tartrate-resistant acid phosphatase. In intraosseous implantation, MGCs with the characteristics of osteoclasts were observed close to osteoblastic cells characterized by developed rough endoplasmic reticulum. These results indicate that the osteoclasts were not induced solely by the subcutaneously implant BPs, but required osteoblast lineage cells for development.

Radiosensitization in vitro and in vivo by 3-nitrotriazoles.

A series of 3-nitro-1,2,4-triazole derivatives bearing various types of side chain (R) at the N1-position (AK-2000 series) were synthesized and their radiosensitizing effect and toxicity in vitro and in vivo were investigated, in comparison with those of Misonidazole (MISO), SR-2508, and RSU-1069. Of the fifteen 3-nitrotriazoles tested, all had sensitizing effects in vitro on hypoxic V79 cells. Also, all but one had definite effects on solid EMT6/KU and SCCVII tumors in vivo. For many of the triazole compounds, the degree of radiosensitization in vitro and in vivo appeared identical. However, they were generally less efficient, both in vitro and in vivo, than the corresponding 2-nitroimidazoles, whereas their aerobic cytotoxicity and toxicity to mice (LD50/7) were comparable to those of the 2-nitroimidazoles. Considering the sensitizing effect and toxicity, AK-2123 (R = CH2CONHC2H4OCH3) may be as useful as MISO, but none of the triazoles have been proved to be superior to SR-2508.

Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain.

OL-protocadherin (OL-pc) is a cell adhesion molecule that belongs to the cadherin superfamily. A previous study showed that expression of OL-pc mRNA was specific to certain brain nuclei including those of the olfactory and limbic systems, thus suggesting its involvement in neural circuit formation. Here, we examined the distribution of OL-pc protein in the postnatal mouse brain by immunohistochemistry to confirm the possibility of such a role. The results showed that the protein could be mapped to many brain compartments including brain nuclei and higher subdivisions as previously observed for the expression pattern of the mRNA. Sharp boundaries of the distribution were often seen in areas such as the interpedunclar nucleus, cerebellar cortex, and inferior olive. In addition, the protein was detected in some fibers that could not be examined by the previous study using in situ hybridization. For example, prominent staining was noted in the stria medularis, stria terminalis, fasciculus retroflexus, optic tract, and inferior thalamic radiation, structures that seem to connect OL-pc-positive brain regions. These OL-pc-positive brain nuclei and fiber tracts coincide with some local circuits of functional systems such as the olfactory system, nigrostriatal projection, olivo-cerebellar projection, and visual system. These results support the possibility that OL-pc is involved in the formation of specific neural compartments and circuits in the developing brain.

Osteosarcoma presenting as intestinal obstruction.

Osteosarcoma commonly presents with osseous and pulmonary metastases. We present an unusual case of extraosseous metastatic abdominal chondroblastic osteosarcoma presenting as intestinal obstruction.