Effects of acetylcholine on the coronary artery

Acetylcholine acts on the different components of the coronary arterial wall by 1) initiating endothelium-dependent relaxation of the smooth muscle cells; 2) inhibiting the exocytotic release of norepinephrine (NE), which could result in either vasodilator or vasoconstrictor effects depending on whether the main action of NE is alpha- or beta-adrenergic, respectively; and 3) activating the contractile process of the smooth muscle cells. These different effects of the cholinergic transmitter are muscarinic in nature. Their relative importance varies among species, or when acetylcholine is given exogenously rather than released from cholinergic nerves.     

Interference of D-mannoheptulose with D-glucose phosphorylation,metabolism and functional effects: Comparison between liver,parotid cells and pancreatic islets

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.     

Opsin cDNA sequences of a UV and green rhodopsin of the satyrine butterfly Bicyclus anynana

The cDNAs of an ultraviolet (UV) and long-wavelength (LW) (green) absorbing rhodopsin of the bush brown Bicyclus anynana were partially identified. The UV sequence, encoding 377 amino acids, is 76-79% identical to the UV sequences of the papilionids Papilio glaucus and Papilio xuthus and the moth Manduca sexta. A dendrogram derived from aligning the amino acid sequences reveals an equidistant position of Bicyclus between Papilio and Manduca. The sequence of the green opsin cDNA fragment, which encodes 242 amino acids, represents six of the seven transmembrane regions. At the amino acid level, this fragment is more than 80% identical to the corresponding LW opsin sequences of Dryas, Heliconius, Papilio (rhodopsin 2) and Manduca. Whereas three LW absorbing rhodopsins were identified in the papilionid butterflies, only one green opsin was found in B. anynana.     

Dynamics of dissolved and biogenic silica in the freshwater reaches of a macrotidal estuary (The Scheldt, Belgium)

Temporal evolution of dissolved and biogenic silica concentrations along the Scheldt tidal river and in its tributaries was investigated during 1 year in 2003. In the tributaries, dissolved silica (DSi) concentrations remained high and biogenic silica (BSi) concentrations were low throughout the year. In the tidal river during summer, DSi was completely consumed and BSi concentrations increased. Overall, most of the BSi was associated with living diatoms during the productive period in the tidal river. Nevertheless, the detrital BSi was a significant fraction of the total BSi pool, of which less than 10% could be attributed to phytoliths. The tidal river was divided into two zones for budgeting purposes. The highest productivity was observed in the zone that received the highest water discharge, as higher riverine DSi input fluxes induced presumably a less restrictive DSi limitation, but the discharge pattern could not explain all by itself the variations in DSi consumption. Silica uptake and retention in the tidal river were important at the seasonal time-scale: from May to September, 48% of the riverine DSi was consumed and 65% of the produced BSi was deposited, leading to a silica (DSi + BSi) retention in the tidal river of 30%. However, when annual fluxes were considered, DSi uptake in the tidal river amounted to 14% of the DSi inputs and only 6% of the riverine silica (DSi + BSi) was retained in the tidal river.     

A Single Amino Acid Substitution in a Segment of the CA Protein within Gag That Has Similarity to Human Immunodeficiency Virus Type 1 Blocks Infectivity of a Human Endogenous Retrovirus K Provirus in the Human Genome

Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. However, no single HERV-K provirus in the human genome today appears to be infectious. Since the Gag protein is the central component for the production of retrovirus particles, we investigated the abilities of Gag from two HERV-K proviruses to support production of virus-like particles and viral infectivity. HERV-K113 has full-length open reading frames for all viral proteins, while HERV-K101 has a full-length gag open reading frame and is expressed in human male germ cell tumors. The Gag of HERV-K101 allowed production of viral particles and infectivity, although at lower levels than observed with a consensus sequence Gag. Thus, including HERV-K109, at least two HERV-K proviruses in human genome today have functional Gag proteins. In contrast, HERV-K113 Gag supported only very low levels of particle production, and no infectivity was detectable due to a single amino acid substitution (I516M) near the extreme C terminus of the CA protein within Gag. The sequence of this portion of HERV-K CA showed similarities to that of human immunodeficiency virus type 1 and other primate immunodeficiency viruses. The extreme C terminus of CA may be a general determinant of retrovirus particle production. In addition, precise mapping of the defects in HERV-K proviruses as was done here identifies the key polymorphisms that need to be analyzed to assess the possible existence of infectious HERV-K alleles within the human population.Approximately 8% of the human genome comprises endogenous retroviruses (ERVs) (33, 59). These viruses infect germ lineage cells and thereby enter the genome of the host species. Thus, endogenous proviruses (the integrated form of retroviral DNA) are transmitted from parents to offspring in genomic DNA. If ERV genomes are intact, viral particles may be generated that can reinfect the germ line and form proviruses at new positions in the host genome. However, ERVs are subject to the same mutagenic processes over evolutionary time as any cellular gene. In the absence of selective pressure on the host to maintain intact viral genomes, endogenous retroviral proviruses accrue mutations over evolutionary time that inactivate viral infectivity. Most of the ERVs in the human genome have converted to solo long terminal repeats (solo LTRs), which are the product of homologous recombination between LTRs at the ends of the complete viral genome. Other types of mutations, such as nucleotide substitutions, insertions, and deletions, can also affect ERV proviruses, and many of the retroviral proviruses in the human genome have been inactivated by such mutations, which created premature stop codons or frameshifts in viral open reading frames (ORFs). The vast majority of the ERVs present in humans today (and perhaps all of them) have incurred mutations that inactivated viral infectivity.One provirus that exists in the genome of approximately 20% of humans, human ERV K113 (HERV-K113, referred to here as K113), has full-length ORFs for all viral proteins (8, 63). However, this provirus does not appear to be infectious, as the pol and env genes of K113 do not support infectivity (9, 19, 20). K113 belongs to a subset of HERV-K called HML-2 (43). Since the human and chimpanzee lineages diverged about 6 million years ago (52), the only proviruses that entered the genome of the human lineage belong to this subgroup, although other members of this subgroup entered the germ line prior to the divergence of the human and chimpanzee lineages (8, 27, 44, 63). The human-specific proviruses of this subgroup are the most intact retroviruses in the human genome. Infectious HERV-K particles have been generated using two different approaches based on their DNA sequences. HERV-KCON (K-CON) was constructed based on the consensus sequence of human-specific HERV-K proviruses (34). Infectious HERV-K particles were also generated by combining pieces from three separate proviruses, HERV-K109 (K109) gag-pro, HERV-K115 pol, and HERV-K108 (K108) env (20). Thus, it may be that no single provirus is infectious, but recombination and/or genetic complementation among multiple genomic proviruses may be required to produce infectious HERV-K particles. This raises the questions of whether multiple functional HERV-K components exist in the human genome today and how close these components are to being able to form a functional viral genome that might be capable of reinfecting human cells.To begin addressing these issues, we examined two of the full-length HERV-K gag genes that exist in the human genome today. Like all retroviruses, HERV-K contains the four genes necessary for viral replication: gag, pro, pol, and env. The human-specific HERV-K proviruses exist in two forms, type I and type II (38, 39). The type II proviruses contain gag, pro, pol, and env plus an accessory gene, rec, that encodes a protein (Rec) that functions in nuclear export of unspliced viral RNA in a manner analogous to that of human immunodeficiency virus type 1 (HIV-1) Rev (12, 40, 41, 65, 66). In type I proviruses, the pol and env genes are fused in frame by a 292-bp deletion that includes the first coding exon of rec, and the viruses encode an additional protein called Np9 (5, 48). The gag genes are relevant to whether HERV-K components in the human genome today might form an infectious virus, as the Gag protein is sufficient to produce virus-like particles (VLPs) in the absence of other viral proteins (4). Formation of such particles is an essential step for subsequent viral replication. Therefore, we decided to investigate whether Gag proteins from K113 and a second provirus, HERV-K101 (K101), are active in functional assays.     

Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization,Biophysical Characterization,and Phylogenetic Analysis

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.Papillomaviruses (PVs) are nonenveloped, epitheliotropic, double-stranded DNA viruses that cause a variety of diseases in a multitude of hosts. Based on available whole-genome sequences and subgenomic amplicons, more than 200 human and over 55 nonhuman mammalian PV types have been described (7, 34, 35, 37, 38). To date, two avian PV types have been characterized (37, 38).The genomic organizations of the PVs are remarkably similar. The genome is ca. 8 kb in length and comprises an upstream regulatory region (URR), the early genes (E1, E2, E4, E6, and E7), and the late genes that encode the capsid proteins (L1 and L2). Although most PVs code for these seven open reading frames (ORFs), only the URR, the replicative proteins E1 and E2 (and possibly the E4 gene), and the capsid proteins L1 and L2 are strictly conserved in all PVs (11).Upon infection of the stratified squamous epithelia, PV gene expression is linked to the differentiation state of the infected epithelium cells. The expression of early PV proteins, in particular E6 and E7, primes the proliferation of the infected epithelium. This proliferation, which is absolutely required for viral replication, may become malignant depending on the PV strain considered. Several “high-risk” mucosal human PV (HPV) strains (predominantly HPV type 16 [HPV-16], HPV-18, and HPV-45) have been shown to be responsible for cervical cancer (19).The ability of PVs to induce proliferation of the infected cells has been attributed mainly to two small “oncoproteins,” E6 and E7. In genital high-risk HPVs, these proteins play a prominent role in cell immortalization and transformation (31). In most mammalian PVs, E6 is a small protein of about 150 amino acids, with two conserved N- and C-terminal zinc-binding domains, E6N and E6C, respectively (12). The solution structure of the HPV-16 E6C domain was recently determined (23). The sequence alignments pointed to a structural similarity between the E6C and E6N domains, suggesting that a single-domain protein possessing the same fold might have once existed. Earlier phylogenetic studies had suggested that gene duplication may have given rise to the current double-domain E6 proteins (5). Interestingly, although the E6 ORF has been found in most mammalian PVs (with the exception of bovine papillomavirus type 3 [BPV-3], BPV-4, BPV-6, HPV-101, and HPV-103 [3, 7]), it was not detected in the two avian PVs previously sequenced (37, 38).In this study, we present the full sequence of the genome of a novel PV from a francolin bird (Francolinus leucoscepus PV type 1 [FlPV-1]) and compare it to the two other avian PV genomes known to date (Psittacus erithacus PV [PePV] and Fringilla coelebs PV [FPV]). In light of recent structural data, we compare the unusual avian E6 and E7 ORFs to their mammalian orthologs. We describe the expression and purification of recombinant avian PV E6 and E7 proteins, their biophysical characterization, and cellular localization. Finally, we use phylogenetic techniques to investigate the evolutionary history of the E6 protein family.     

Impact of a Flood Event on the Planktonic Food Web of the Schelde Estuary (Belgium) in Spring 1998

To evaluate the effect of short-term changes in discharge on plankton dynamics, a station in the upper reaches of the Schelde estuary was monitored during 1 month in spring 1998 with a sampling frequency of 1–2 days. During this monitoring period, a flood event occurred during which discharge increased about 5-fold. This flood event had a strong effect on the composition of the planktonic community. Rotifers were strongly negatively affected by the flood event while densities of heterotrophic nanoflagellates and scuticociliates increased. Chlorophyll a concentration and abundance of bacteria, oligotrich ciliates and crustacean zooplankton did not respond clearly to the flood event. Although the flood event lasted only a few days it took more than 2 weeks for the planktonic community to return to its original composition.     

Involvement of the platelet-activating factor receptor in host defense against Streptococcus pneumoniae during postinfluenza pneumonia

Although influenza infection alone may lead to pneumonia, secondary bacterial infections are a much more common cause of pneumonia. Streptococcus pneumoniae is the most frequently isolated causative pathogen during postinfluenza pneumonia. Considering that S. pneumoniae utilizes the platelet-activating factor receptor (PAFR) to invade the respiratory epithelium and that the PAFR is upregulated during viral infection, we here used PAFR gene-deficient (PAFR-/-) mice to determine the role of this receptor during postinfluenza pneumococcal pneumonia. Viral clearance was similar in wild-type and PAFR-/- mice, and influenza virus was completely removed from the lungs at the time mice were inoculated with S. pneumoniae (day 14 after influenza infection). PAFR-/- mice displayed a significantly reduced bacterial outgrowth in their lungs, a diminished dissemination of the infection, and a prolonged survival. Pulmonary levels of IL-10 and KC were significantly lower in PAFR-/- mice, whereas IL-6 and TNF-alpha were only trendwise lower. These data indicate that the pneumococcus uses the PAFR leading to severe pneumonia in a host previously exposed to influenza A.     

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.