Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

Anti-idiotypic antibody response to monoclonal anti-CD4 preparations in nonhuman primate species.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Fluoro‐Sorafenib (Regorafenib) effects on hepatoma cells: Growth inhibition,quiescence, and recovery

To evaluate the growth‐inhibitory properties of the potent multi‐kinase antagonist Regorafenib (Fluoro‐Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, apoptosis, and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration‐ and time‐dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho‐ERK and phospho‐JNK and its target phospho‐c‐Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin‐1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications. J. Cell. Physiol. 228: 292–297, 2013. © 2012 Wiley Periodicals, Inc.     

Chemical Profile and Antioxidant Properties of Extracts and Essential Oils from Citrus × limon (L.) Burm. cv. Femminello Comune

Citrus × limon cv. Femminello Comune (Rutaceae) from Rocca Imperiale (Italy), one of the six Protected Geographical Indication (PGI) Italian lemon crops, has been recently received renewed interest. In this work, fresh and dried peels and leaves were extracted by hydrodistillation, supercritical fluid extraction (SFE), and Soxhlet apparatus. Chemical profile was assessed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Except for leaves extracts obtained by Soxhlet apparatus, the monoterpene hydrocarbons fraction dominated. Limonene, γ‐terpinene, and β‐pinene were the main identified compounds. The antioxidant activity was investigated using different in vitro assays namely 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS, ferric reducing ability power (FRAP), and β‐carotene bleaching test. In DPPH test, the essential oil obtained by hydrodistillation of fresh peel exhibited the highest activity (IC₅₀ of 1.17 mg/ml). Leaves extracted by SFE showed a good activity in both DPPH and β‐carotene bleaching test with IC₅₀ values of 2.20 and 6.66 mg/ml, respectively. Monoterpene hydrocarbons fraction exhibited a positive Pearson's correlation coefficient with all antioxidant assays. Leaves, often considered waste material, should be considered from a different point because they represent a matrix of indisputable interest.     

Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (G_αgust) and α-transducin (G_αtran) G protein subunits. This study tested whether G_αtran and G_αgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, G_αgust and G_αtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, G_αgust and G_αtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of G_αtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of G_αgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included G_αtran / 5HT-IR and G_αtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as G_αgust /5-HT-IR or G_αgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in G_αtran-IR cells in the duodenal crypts and a significant increase of G_αgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of G_αtran-G_αgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of G_αtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of G_αgust / G_αtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.