On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

Primary alveolar macrophages exposed to diesel particulate matter increase RAGE expression and activate RAGE signaling

Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.     

Quantitative and Multiplexed Study of Endothelial Cell Inflammation

Endothelial inflammation plays major roles in all phases of the atherosclerotic process, the leading cause of death by cardiovascular disease. Both innate immunity and endothelial adhesion molecules contribute to endothelial inflammation. In this work, we applied multiple antibodies (Abs) to measure changes in expression levels of six proteins in response to inflammatory stimulation. These six proteins include toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) representing innate immunity and four endothelial adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin. We observed two different types of dynamic behaviors among these proteins upon inflammatory stimulation. Increased expression of toll-like receptor 2 (TLR2), P-selectin, E-selectin, and TLR4 peaked relatively early (after 4 h of stimulation) while VCAM-1, and ICAM-1 showed a more gradual and consistent increase in expression with stimulatory time. The magnitude of this increase was significantly greater for VCAM-1 and ICAM-1. The multiplexed detection developed in this study using fluorophore-conjugated primary Abs provides an approach for live cell and in vivo imaging of endothelium inflammation for quantitative characterization of multiple proteins within a network.     

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Transcriptional activity of heterocysts isolated from Anabaena variabilis

Nitrogenase activity, RNA synthesis, and protein synthesis were measured in heterocysts of Anabaena variabilis. Heterocysts labelled in situ for 4 h with [¹⁴C]uracil accumulated label in rRNA and tRNA to the same specific activity as RNA from vegetative cells. With isolated heterocysts, however, assimilation of [³H]uracil into RNa occurred at about 10% the rate in vegetative cells, and ceased 90 min after isolation. Pulse-chase experiments indicated that heterogeneous, high-molecular-weight RNA synthesized during the first 30 min of incubation was turned over during a 2 h chase, howver there was no accumulation of label in rRNA and tRNA as was seen with heterocysts labelled in situ and with vegetative cells. Assimilation of [³H]glycine into protein by isolated heterocysts was linear up to about 60 min, then proceeded at a slower rate for an additional 180 min. Maintenance of protein synsthesis and nitrogen fixation were both blocked by chloramphenicol and rifampicin. The data suggest that differentiated heterocysts continue to synthesize RNA and proteins and that these processes may contribute to the functional lifetime of heterocysts.     

I - insulin transfer to mitochondria

The aim of this study was to determine if insulin is transferred to mitoplasts by insulin-degrading enzyme (IDE). Hepatic mitochondria were isolated and controlled by electron microscopy. IDE was obtained from rats muscle by successive chromatography steps. Insulin accumulation in mitoplasts and outer membrane + intermembrane space (OM + IMS) was studied with ¹²⁵I-insulin. Mitochondrial insulin accumulation and degradation was assayed with Sephadex G50 chromatography, insulin antibody and 5 % TCA. Mitoplasts and OM + IMS were isolated with digitonin. Insulin accumulation was studied at 25 °C at different times, without or with IDE, Bacitracin, 2,4-dinitrophenol, apyrase or sodium succinate + adenosine diphosphate. Insulin accumulation in mitoplasts and OM + IMS after mitochondrial cross-linking was studied with electrophoresis in SDS-PAGE, immunoblots of IDE, insulin or TIM23 (inner mitochondrial transporter) and autoradiography. The studies showed that addition of IDE increased insulin transfer from OM + IMS to mitoplasts, and the insulin accumulation in mitoplast was IDE dependent. Bacitracin and 2,4-dinitrophenol decreased this transfer. The [Insulin-IDE] complex and [Mitoplasts] was studied as a bimolecular reaction following a second order reaction. The constant “k” (liter.mol^?1 s^?1) showed that IDE increased and Bacitracin or 2,4-dinitrophenol decreased the velocity of insulin transfer. SDS-PAGE and immunoblots studies showed bands and radioactivity coincident with IDE, insulin and TIM23. Non degraded insulin was demonstrated in immunoblot after IDE immunoprecipitation from mitoplasts. Confocal studies showed mitochondrial colocalization of IDE and insulin. The results showed that insulin at 25 °C were transferred from OM + IMS to mitoplasts by IDE or that the enzyme facilitates this transfer, and they reach the matrix together.     

In vitro release of endogenous monoamines and amino acids from several CNS regions of the rat

The in vitro release of endogenous norepinephrine (NE), dopamine (DA), serotonin (5-HT), GABA, glutamate (GLU), aspartate (ASP), glycine (GLY), taurine (TAU) and alanine (ALA) from superfused slices of cerebral cortex (CTX), striatum (STR), hippocampus (HIP), hypothalamus (HYPO), midbrain (MB), thalamus (THAL), nucleus accumbens (ACC), pons-medulla (PM) and spinal cord (SC) was studied. Under resting conditions or with 60 mM K⁺ in the absence of Ca²⁺, there was little or no release of NE, DA, 5-HT, GABA, GLU or ASP from any region. In most regions, there was a measurable resting release of ALA, GLY and TAU; of these three amino acids, only GLY in the PM and SC showed an increased release in the 60 mM K⁺ plus 2.5 mM Ca²⁺ medium. In 8 of the regions studied, the release of both GABA and GLU were stimulated by 60 mM K⁺ in the presence of 2.5 mM Ca²⁺. For the amino acids, no reliable data were obtained for release from the ACC because of its small size. The highest amount of K⁺-stimulated, Ca²⁺-dependent release of GABA was found with slices from the HYPO, THAL and MB while the highest amount of GLU was released from slices of STR, HIP and CTX. In those regions where reliable levels of K⁺-stimulated, Ca²⁺-dependent release of ASP were observed (STR, CTX, THAL), the amount of ASP was at least 5-fold lower than the values for GLU. A K⁺-stimulated, Ca²⁺-dependent release of NE, DA and 5-HT was observed for all 9 CNS regions studied. The highest release of (a) DA occurred from slices of CTX, STR and ACC; (b) NE was found in the HYPO and ACC; and (c) 5-HT occurred in the HYPO. The data (a) do not support a transmitter role for ALA and TAU in the CNS; (b) support a major transmitter function for GLY only in the PM and SC; and (c) support a transmitter role for GABA, GLU, NE, DA and 5-HT in the CNS regions examined (with the exception of GABA and GLU in the ACC where no data were obtained).     

Control of RNA level and of RNA ratios in the latex ofHevea brasiliensis Müll. Arg. effect of latex tapping and of growth regulators

The association between latex RNA and latex production was examined using MAK column chromatography techniques. In young untapped trees the introduction of tapping or the treatment of bark with growth regulators resulted in an increase of RNA level and of rRNA/tRNA ratio in the latex. In regularly tapped trees an increase in rRNA but not in tRNA was brought about by increasing the tapping frequency. Treatment with growth regulators had the same effect but essentially only through the related enhancement of latex export from latex vessels. During latex flow, the highest RNA level was registered in latex fractions originating from the most heavily drained areas of bark. Using³²P labeling, evidence was obtained that the export of latex results in an enhancement of rRNA migration into the inner latex containing space of the vessels. This is considered as the reason of the generally observed association of high RNA level and of high rRNA/tRNA ratio with high latex yield. It is proposed that in controlling the RNA level and RNA proportions in the latex an important role is played by changes in turgor pressure associated with the loss of latex which may influence the export of RNA from the nucleus through related induction of pressure disequilibrium between the nucleoplasm and the latex cytoplasm.     

Imaging Functional and Structural Brain Connectomics in Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopment disorders in childhood. Clinically, the core symptoms of this disorder include inattention, hyperactivity, and impulsivity. Previous studies have documented that these behavior deficits in ADHD children are associated with not only regional brain abnormalities but also changes in functional and structural connectivity among regions. In the past several years, our understanding of how ADHD affects the brain’s connectivity has been greatly advanced by mapping topological alterations of large-scale brain networks (i.e., connectomes) using noninvasive neurophysiological and neuroimaging techniques (e.g., electroencephalograph, functional MRI, and diffusion MRI) in combination with graph theoretical approaches. In this review, we summarize the recent progresses of functional and structural brain connectomics in ADHD, focusing on graphic analysis of large-scale brain systems. Convergent evidence suggests that children with ADHD had abnormal small-world properties in both functional and structural brain networks characterized by higher local clustering and lower global integrity, suggesting a disorder-related shift of network topology toward regular configurations. Moreover, ADHD children showed the redistribution of regional nodes and connectivity involving the default-mode, attention, and sensorimotor systems. Importantly, these ADHD-associated alterations significantly correlated with behavior disturbances (e.g., inattention and hyperactivity/impulsivity symptoms) and exhibited differential patterns between clinical subtypes. Together, these connectome-based studies highlight brain network dysfunction in ADHD, thus opening up a new window into our understanding of the pathophysiological mechanisms of this disorder. These works might also have important implications on the development of imaging-based biomarkers for clinical diagnosis and treatment evaluation in ADHD.     

Monoclonal antibody reveals H-2-linked quantitative and qualitative variation in the expression of a Qa-2 region determinant

We have produced a monoclonal antibody, Y-7, that reacts with a Qa-2 region-controlled determinant. Cellular and strain distribution analyses, coupled with quantitative variation in the amount of Y-7 antigen expressed among strains, provide overwhelming evidence that Y-7 reacts with the Qa-2^a determinant. The determinant detected by Y-7 is differentially expressed in T and B lymphocytes in a strain specific manner. Y-7 reacts with the majority of T lymphocytes (> 95%) and approximately one-half of B lymphocytes in certain strains (++ strains), and with the majority of T lymphocytes (> 95%) and no B lymphocytes in other strains (+ strains). T lymphocytes in + strains express approximately three fold less of the Y-7 determinant than T lymphocytes from ++ strains. In addition, we show that the Y-7 determinant is expressed in approximately one-third to one-half of Lyb-3^?, 5^? B lymphocytes. Possible mechanisms determining quantitative and qualitative variation in the expression of the Y-7 determinant in T and B lymphocytes are discussed.     

Serum IgG levels in the Storrs strain of hereditary muscular dystrophic chickens

IgG levels in sera of Storrs hereditary muscular dystrophic chickens were investigated. IgG levels in age-matched Storrs muscular dystrophic chickens varied, depending on the geographical location where the chickens were reared. IgG levels from muscular dystrophic chickens at varying ages of development were approximately 30% less than age-matched control values. Genetic analyses of F₁ hybrid, F₂ progeny, and testcross progeny showed the reduced IgG levels in the Storrs strain of muscular dystrophic chickens not to be correlated with the autosomal recessive muscular dystrophic trait, the degree of muscle destruction, nor with an autosomal recessive T cell defect. The studies reported here suggest (1) that the reduced IgG levels in the Storrs strain of muscular dystrophic chickens are due to strain differences and (2) that the mode of inheritance of serum IgG levels in the Storrs strain of muscular dystrophic chickens is polygenic.     

Regulation of androgenesis inNicotiana tabacum L. cv. White Burley andDatura innoxia Mill. Effect of bivalent and trivalent iron and chelating substances

The effect of FeSO₄.7H₂O, Fe₂(SO₄)₃.9H₂O, disodium salt of ethylene-diaminotetraacetic acid, dihydrate (EDTA) and N-(2-acetamido) iminodiacetic acid (ADA) and their combinations on the androgenesis was studiedin vitro in tobacco (cv. White Burley) and datura (Datura innoxia Mill.). Simultaneously the reversibility and irreversibility of the morphogenic process leading to the conversion of the pollen embryoid into complete plant was followed. Complete plants developed in anthers on media with trivalent iron, chelated trivalent iron, chelated bivalent iron, bivalent iron in the presence of ADA and of media with EDTA. The number of androgenic plants in anthers increased in the following order: Fe₃₊ < Fe₃₊ EDTA ≦ ≦ EDTA < Fe²⁺ EDTA. The marked brown colour of cultured anthers was due to the presence of trivalent iron in the medium. The androgenic development was most rapid on the medium containing only trivalent iron, slower on media with chelated iron and slowest on medium with EDTA. The viability of cultures with complete plants decreased in the reverse order. No complete plants grew on media without trivalent iron and without EDTA and on media containing only bivalent iron whereas globular embryoids arose and developed continuously on these media. The anthers reacted in the same way on both complete and minimal media. Isolated embryoids formed complete plants in corresponding variants on complete media only. The development of pollen embryoids into complete plants was stopped by the transfer of globular and torpedo-shaped embryoids from medium with EDTA to the medium without EDTA. Isolated greenish cotyledonar embryoids continued to grow even on the medium without EDTA.     

The effect of glucagon-like peptide-1 in the management of diabetes mellitus: cellular and molecular mechanisms

Incretins, such as glucagon-like peptide-1 (GLP)-1, have been shown to elevate plasma insulin concentration. The purpose of this study is to investigate the cellular and molecular basis of the beneficial effects of GLP-1. Normal and diabetic male Wistar rats were treated with GLP-1 (50 ng/kg body weight) for 10 weeks. At the end of the experiment, pancreatic tissues were taken for immunohistochemistry, immunoelectron microscopy and real-time polymerase chain reaction studies. Samples of blood were retrieved from the animals for the measurement of enzymes and insulin. The results show that treatment of diabetic rats with GLP-1 caused significant (P?GLP-1 (10^?12–10^?6 M) induced significant (P?GLP-1-treated rats compared to controls. GLP-1 treatment induced significant (P?GLP-1-receptor genes in diabetic animals compared to controls. GLP-1 is present in pancreatic beta cells and significantly (P?GLP-1 is co-localized with insulin and seems to exert its beneficial effects by increasing cellular concentrations of endogenous antioxidant genes and other genes involved in the maintenance of pancreatic beta cell structure and function.