Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1–29)NH₂ (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of ¹²⁵I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1–42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control.     

In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70

To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which were obtained from E. coli, the viral RNA replicon, rATP, rGTP, and a yeast cell-free extract. The in vitro assembly of the replicase took place in the membraneous fraction of the yeast extract, in which the viral replicase-RNA complex became RNase- and proteinase-resistant. The assembly of the replicase complex required the heat shock protein 70 (Hsp70 = yeast Ssa1/2p) present in the soluble fraction of the yeast cell-free extract. The assembled TBSV replicase performed a complete replication cycle, synthesizing RNA complementary to the provided RNA replicon and using the complementary RNA as template to synthesize new TBSV replicon RNA.     

Dopaminergic amacrine cells express opioid receptors in the mouse retina

The presence of opioid receptors has been confirmed by a variety of techniques in vertebrate retinas including those of mammals; however, in most reports, the location of these receptors has been limited to retinal regions rather than specific cell types. Concurrently, our knowledge of the physiological functions of opioid signaling in the retina is based on only a handful of studies. To date, the best-documented opioid effect is the modulation of retinal dopamine release, which has been shown in a variety of vertebrate species. Nonetheless, it is not known if opioids can affect dopaminergic amacrine cells (DACs) directly, via opioid receptors expressed by DACs. This study, using immunohistochemical methods, sought to determine whether (1) μ- and δ-opioid receptors (MORs and DORs, respectively) are present in the mouse retina, and if present, (2) are they expressed by DACs. We found that MOR and DOR immunolabeling were associated with multiple cell types in the inner retina, suggesting that opioids might influence visual information processing at multiple sites within the mammalian retinal circuitry. Specifically, colabeling studies with the DAC molecular marker anti-tyrosine hydroxylase antibody showed that both MOR and DOR immunolabeling localize to DACs. These findings predict that opioids can affect DACs in the mouse retina directly, via MOR and DOR signaling, and might modulate dopamine release as reported in other mammalian and nonmammalian retinas.     

An antibody-based construct carrying DNA-mimotope and targeting CR1(CD35) selectively suppresses human autoreactive B-lymphocytes

Among tumor necrosis factor (TNF) superfamily, TNF-related apoptosis inducing ligand (TRAIL) along with TNF- and FasL is known as death ligand due to its selective cytotoxicity against transformed tumor cells. TRAIL can also induce alternative angiogenic and/or proinflammatory signals other than apoptosis, however, the molecular mechanisms responsible for the alternative signals have not been detailed yet. Intercellular adhesion molecule-1 (ICAM-1) is thought to be involved in the processes of metastasis and angiogenesis in various tumors. We investigated the molecular mechanisms responsible for ICAM-1 expression by death ligands in human astroglial cells to delineate the alternative signals of these ligands. Here, we demonstrate that (1) death ligands induced expression of ICAM-1 at the mRNA and protein levels in human astroglial cells; (2) pre-treatment of z-VAD-fmk and/or SB202190 suppressed death ligand-induced ICAM-1 expression and subsequent adhesion of activated monocytic cells; and (3) inhibition of caspase suppressed death ligand-induced phosphorylation of p38 MAPK and IKK. These findings suggest biological function of death receptors other than apoptosis in human astroglial cells, and the involvement of caspase and/or p38 MAPK in alternative signaling through death receptors.     

Rats exposed to traumatic stress bury unfamiliar objects--a novel measure of hyper-vigilance in PTSD models?

Electric shocks lead to lasting behavioral deficits in rodents, and as such are often used to model post-traumatic stress disorder (PTSD) in the laboratory. Here we show that a single exposure of rats to 3 mA-strong shocks results in a marked social avoidance that lasts at least 28 days; moreover, the response intensifies over time. In an attempt to study the impact of cue reminders on the behavior of shocked rats, we administered shocks in the presence of a highly conspicuous, 10 cm-large object. This object was introduced into the home cage of rats 28 days after shock exposure. Shocked rats manipulated the object considerably less than controls. More importantly, however, the object was buried by shocked rats. This behavior was virtually absent in controls. The response strongly depended on the intensity of shocks, and was robust. Rats shocked with 3 mA currents spent 40% of time burying the object, which was often hardly visible at the end of the 5 min test. Subsequent experiments demonstrated that the response was not cue-specific as unfamiliar objects were also buried. Rats are well known to bury dangerous objects; the shock-prod burying test of anxiety is based on this response. Behavioral similarities with this test and the differences from the marble-burying behavior of mice suggest that traumatized rats bury unfamiliar objects in defense, and the response can be interpreted as a sign of hyper-vigilance. We further suggest that object burying can be used as a sign of hyper-vigilance in models of PTSD.     

Antagonistic Analogs of Growth Hormone-releasing Hormone: New Potential Antitumor Agents

Recently, new potent antagonistic analogs of growth hormone-releasing hormone (GH-RH) have been synthesized. These GH-RH antagonists bind to pituitary receptors for GH-RH and inhibit the release of GH in vitro and in vivo. This suggests that they could be clinically useful in conditions such as acromegaly. The main applications of GH-RH antagonists would be in the field of insulin-like growth factor I (IGF-I)- and IGF-II-dependent cancers. GH-RH antagonists inhibit the growth of various human cancer cell lines xenografted into nude mice, including mammary cancers, androgen-independent prostate cancers, small-cell lung carcinomas, non-small-cell lung carcinomas, renal adenocarcinomas, pancreatic cancers, colorectal carcinomas and malignant gliomas. These effects could, in part, be exerted indirectly through inhibition of the secretion of GH and the resulting reduction in levels of hepatic IGF-I. However, the principal action of GH-RH antagonists in vivo appears to be the direct suppression of the autocrine and/or paracrine production and expression of the genes encoding IGF-I (IGF1) and IGF-II (IGF2) in tumors. In vitro, antagonists of GH-RH inhibit the proliferation of mammary, prostatic, pancreatic and colorectal cancer cell lines, reducing the expression of IGF2 mRNA in the cells and the secretion of IGF-II. The presence of the GH-RH ligand has been demonstrated in human ovarian, endometrial, mammary and lung cancers, suggesting that GH-RH could be a growth factor. Further development of GH-RH antagonists should lead to potential therapeutic agents for IGF-dependent cancers.     

Mutational analysis of Bax and Bcl-2 in childhood acute lymphoblastic leukaemia

In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl-2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF (uORF) of bcl-2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in bax and bcl-2. DNA sequence analysis of the ORFs of bax and bcl-2 and of the uORF of bcl-2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl-2 that (in)activate or deregulate Bax and Bcl-2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease. Int. J. Cancer (Pred. Oncol.) 79:273–277, 1998.© 1998 Wiley-Liss, Inc.