The complete mitochondrial DNA sequence of the pig (Sus scrofa)

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disorder caused by a germline inactivating mutation of the adenomatous polyposis coli (APC) gene. Patients with FAP sometimes develop various extracolonic manifestations including adrenocortical neoplasms. We present a 14-year-old boy with FAP who had an adrenocortical tumor with atypical histopathologic features, ie, sex-cord-like differentiation. Immunohistochemical studies of adrenal 4 binding protein (Ad4BP) and steroidogenic enzymes showed the capacity of these tumor cells to produce steroids. Genetic analysis of the tumor disclosed a two-hit mutation in APC: a germline 5-base pair deletion accompanied by a loss of the normal allele. Because there were no reports of genetic alterations in adrenocortical tumors developed in FAP patients, we examined 10 sporadic adrenal tumors (four carcinomas and six adenomas) for mutations in APC. However, no mutations were found in these 10 sporadic adrenal tumors. These results suggest that mutation of APC is also responsible for some fraction of the adrenocortical tumors: the tumor in this case is included.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS-/-) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS+/+ animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+ cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 microM). Similarly to PARS inhibition with INH2BP, the PARS-/- cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS-/- phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS-/- phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS-/- cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26-35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inflammatory effects in vivo.     

Protection by inhibition of poly (ADP-ribose) synthetase against oxidant injury in cardiac myoblasts In vitro

Peroxynitrite and hydroxyl radical are reactive oxidants produced during myocardial reperfusion injury. In various cell types, including macrophages and smooth muscle cells, peroxynitrite and hydrogen peroxide cause DNA single strand breakage, which triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), resulting in cytotoxicity. Using 3-aminobenzamide and nicotinamide, inhibitors of PARS, we investigated the role of PARS in the pathogenesis of myocardial oxidant injury in H9c2 cardiac myoblasts in vitro. Peroxynitrite (100-1000 microM), hydrogen peroxide (0.3-10 microM) and the NO donor compounds S-nitroso-N-accetyl-DL-penicillamine (SNAP) and diethyltriamine NONOate all caused a dose-dependent reduction of the mitochondrial respiration of the cells, as measured by the mitochondrial-dependent conversion of MTT to formazan. Peroxynitrite and hydrogen peroxide, but not the NO donors caused activation of cellular PARS activity. The suppression of mitochondrial respiration by peroxynitrite and hydrogen peroxide, but not by the NO donors, was ameliorated by pharmacological inhibition of PARS. The protection by the PARS inhibitors diminished at extremely high concentrations of the oxidants. Hypoxia (1 h) followed by reoxygenation (1-24 h) also resulted in a significant activation of PARS, and caused a suppression of mitochondrial respiration, which was prevented by inhibition of PARS. Similar to the results obtained with the pharmacological inhibitors of PARS, a fibroblast cell line which derives from the PARS knockout mouse was protected against the suppression of mitochondrial respiration in response to peroxynitrite and reoxygenation, but not to NO donors, when compared to the result of cells derived from wild-type animals. Based on our data, we suggest that activation of PARS plays a role in the myocardial oxidant injury.     

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

The complete nucleotide sequence of the domestic dog (Canis familiaris) mitochondrial genome

The synthesis, antibacterial activity, and stability to human dehydropeptidase-1 (DHP-1) of a novel series of (5R,6S)-6-[(1R)-1-hydroxyethyl]-2-heterocyclylcarbapen-2-em-3-carb oxylates are described. Of the compounds investigated 1,5-disubstituted pyrazol-3-yl and 3-substituted isoxazol-5-yl derivatives have the best combination of antibacterial activity and stability to DHP-1. They are particularly active against community-acquired respiratory tract pathogens and have stabilities to DHP-1 superior to that of meropenem.     

The wheat poly(A)-binding protein functionally complements pab1 in yeast

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.     

The aminoacceptor stem of the yeast tRNA(Lys) contains determinants of mitochondrial import selectivity

The effect of quinine, a cinchona alkaloid, was studied on gastrointestinal transit in mice. Intraperitoneal (i.p.) administration of quinine inhibited the intestinal propulsion of a charcoal suspension at a dose of 100 mg/kg, comparing favorably with 5 mg/kg morphine. In an attempt to probe into the mechanism underlying this inhibition, a possible modulation by minoxidil (1 mg/kg, p.o.) and glibenclamide (1 mg/kg, p.o.), the drugs that, respectively, open and close ATP-sensitive K+ channels was tested on gastrointestinal transit in animals treated or not with quinine or morphine. While minoxidil produced no significant change of normal transit, glibenclamide significantly increased it. However, both drugs blocked the quinine-induced reduction in gastrointestinal transit. In contrast, the inhibitory effect of morphine on gastrointestinal transit was not modified by either drug. The effects of quinine as well as of morphine on gastrointestinal transit were significantly antagonized by naloxone (2 mg/kg, s.c.), a mu-opioid receptor antagonist but not by yohimbine (1 mg/kg, i.p.), an alpha2-adrenoceptor antagonist. Furthermore, quinine at a lower dose (25 mg/kg) that showed no per se effect on gastrointestinal transit, significantly potentiated the response to 2.5 mg/kg morphine. Although the role of ATP-sensitive K+ channels in the action of quinine and morphine was not clarified by the present results, a possible involvement of endogenous opioid(s) in the quinine-induced inhibition of gastrointestinal transit can be suggested.     

Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease

3'-Exonucleolytic removal of the poly(A) tail is the first and often rate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplasmic extract from HeLa cells, the poly(A) tail of mRNA was degraded from the 3'-end. In agreement with earlier in vivo observations, prominent decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for this poly(A) shortening activity was purified from calf thymus. A polypeptide of 74 kDa copurified with the activity. The deadenylating nuclease (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly dependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly(A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. At either salt concentration DAN and PAB I reconstituted poly(A) shortening with the same pattern of intermediates seen in cytoplasmic extract. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.     

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.     

Mitochondrial encephalomyopathy associated with a novel mutation in the mitochondrial tRNA(leu)(UUR) gene (A3243T)

We report a new mutation, an A-->T transition at nt 3243 in the mitochondrial tRNA(leu)(UUR) gene, in a 9-year-old girl who presented with muscle weakness of 3 years duration complicated by rapidly progressive encephalopathy. In muscle, the activity of the mitochondrial respiratory chain complexes I, III, and IV was markedly reduced. The mutation, involving a highly conserved base pair in the dihydrouridine loop, was heteroplasmic in muscle (81.4%), skin (69.3%), and blood (13.8%) and was not present in blood of 50 healthy individuals. The mitochondrial 3243 base is a "hot spot" for mutations; an A-->G transition at this position is found in a high proportion in most MELAS patients. Since the A-->T transition creates a new recognition site for the restriction enzyme TspRI, both ApaI and TspRI should be used to exclude a mutation at nt 3243.     

Inhibition of entry of HIV into cells by poly(A).poly(U)

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A).poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A).poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I).poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A).poly(U) or other polyanions. These results confirm the potential of poly(A).poly(U) as an antiviral drug against HIV infection.