Single prenatal injections of melatonin or the D1-dopamine receptor agonist SKF 38393 to pregnant hamsters sets the offsprings' circadian rhythms to phases 180° apart

Pregnant Syrian hamsters with lesions of the suprachiasmatic nucleus (SCN) received single injections of melatonin or the D1-dopamine receptor agonist, SKF 38393 on day 15 of gestation (1 day before birth). Pups were weaned on postnatal day 20 and their freerunning activity rhythms recorded for 3–4 weeks. The pups' phases on the day of weaning were significantly clustered in both of the treatment groups, but the average phases differed by approximately 180°. The results demonstrate that a single prenatal stimulus is sufficient to set the phases of the hamsters' rhythms and that the phase established depends on the stimulus. Both c-fos mRNA and Fos protein were expressed in the fetal SCN after SKF 38393 injection but neither were expressed after melatonin injection. Simulations showed that a single stimulus could produce the observed synchrony from a population of uniformally distributed phases if the phase shifts were three to four times the magnitude of the adult hamster light phase response curve (PRC). A light pulse PRC mimicked the effect of an SKF 38393 injection and a dark-pulse PRC mimicked the effects of a melatonin injection. Together these results suggest that dopamine and melatonin either are, or mimic, maternal entraining signals that represent day and night. Accepted: 12 November 1996     

Reevaluation of the Coding Potential and Proteomic Analysis of the BAC-Derived Rhesus Cytomegalovirus Strain 68-1

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.     

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.     

Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels early oligomer states of the Alzheimer disease Aβ42 protein

The oligomerization of the amyloid-β protein (Aβ) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aβ and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aβ42 form of the protein. The mass spectrum of a mixture of PADK and Aβ42 clearly shows that PADK binds directly to Aβ42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aβ42 dodecamers, but also removes preformed Aβ42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aβ42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aβ42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.     

Integrin signalling regulates the nuclear localization and function of the lysophosphatidic acid receptor-1 (LPA1) in mammalian cells

We show that LPA1 (lysophosphatidic acid receptor-1) is constitutively localized in the nucleus of mammalian cells. LPA1 also traffics from cell membranes to the nucleus in response to LPA (lysophosphatidic acid). Several lines of evidence suggest an important role for cell-matrix interaction in regulating the constitutive nuclear localization of LPA1. First, the RGDS peptide, which blocks cell matrix-induced integrin clustering and cytoskeletal rearrangement, reduced the number of cells containing LPA1 in the nucleus. Secondly, a higher proportion of cells contained nuclear LPA1 when adhesion on fibronectin-coated glass was compared with adherence to polylysine-coated glass. Thirdly, pre-treatment of cells with the Rho kinase inhibitor (Y27632) or the myosin light chain kinase inhibitor (ML9) reduced the number of cells containing nuclear LPA1. The addition of LPA and/or Ki16425 (which binds to LPA1) to isolated nuclei containing LPA1 induced the phosphorylation of several proteins with molecular masses of 34, 32, 14 and 11 kDa. These findings demonstrate that trafficking of LPA1 to the nucleus is influenced by cell-matrix interactions and that nuclear LPA1 may be involved in regulating intranuclear protein phosphorylation and signalling.     

Methylation of E-cadherin and hMLH1 genes in Indian sporadic breast carcinomas

Hypermethylation of promoter regions leading to inactivation of tumor suppressor genes is a common event in the progression of several tumor types. We have employed a novel restriction digestion based multiplex PCR assay to analyse the methylation status of promoter regions of tumor suppressor genes (p16, hMLH1, MGMT and E-cadherin) in sporadic breast carcinomas of Indian women. The present results indicated the absence of hypermethylation in promoter region of p16 and MGMT genes. However, 6 of the 19 (31.6%) sporadic breast carcinomas showed hypermethylation in the promoters of two of the genes analysed; three in hMLH1 and another three in E-cad. Since our earlier studies have shown lack of genetic alterations such as missense mutations and deletions in the tumor associated genes-p16, ras and p14ARF in sporadic breast tumors, the epigenetic alterations of the two genes reported in the present study could be of interest and might be among the events in the genesis/progression of sporadic breast carcinomas.     

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.     

Comparison of protein active site structures for functional annotation of proteins and drug design

Rapid and accurate functional assignment of novel proteins is increasing in importance, given the completion of numerous genome sequencing projects and the vastly expanding list of unannotated proteins. Traditionally, global primary-sequence and structure comparisons have been used to determine putative function. These approaches, however, do not emphasize similarities in active site configurations that are fundamental to a protein's activity and highly conserved relative to the global and more variable structural features. The Comparison of Protein Active Site Structures (CPASS) database and software enable the comparison of experimentally identified ligand-binding sites to infer biological function and aid in drug discovery. The CPASS database comprises the ligand-defined active sites identified in the protein data bank, where the CPASS program compares these ligand-defined active sites to determine sequence and structural similarity without maintaining sequence connectivity. CPASS will compare any set of ligand-defined protein active sites, irrespective of the identity of the bound ligand.