Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N.

Complete sequence of the closed circular extrachromosomal element of De Jonckheere (strain Singh).

The DNA encoding the ribosomal RNA in is encoded on closed circular extrachromosomal ribosomal DNA-containing elements (CERE) in the nucleolus. In this report, we describe the sequence of the CERE of De Jonckheere (strain Singh).

Complete sequence of the closed circular extrachromosomal element (CERE) of De Jonckheere (strain PP 397).

Ribosomal RNA is not encoded in chromosomal DNA in amoebae of the Naegleria genus but the rRNA genes are located on closed circular extrachromosomal ribosomal DNA (rDNA)-containing elements (CERE). In this report, we describe the sequence of the CERE of De Jonckheere (strain PP397).

Complete Sequence of the Closed Circular Extrachromosomal Element of Naegleria jadini Willaert and Ray (Strain ITMAP400).

Amoebae of the genus carry all ribosome-encoding DNA on closed circular extrachromosomal elements (CERE). We report the sequence of the CERE of Naegleria jadini (strain Willaert and Ray).

Persistent Enterovirus Infection: Little Deletions, Long Infections.

Enteroviruses have now been shown to persist in cell cultures and in vivo by a novel mechanism involving the deletion of varying amounts of the 5' terminal genomic region termed domain I (also known as the cloverleaf). Molecular clones of coxsackievirus B3 (CVB3) genomes with 5' terminal deletions (TD) of varying length allow the study of these mutant populations, which are able to replicate in the complete absence of wildtype virus genomes. The study of TD enteroviruses has revealed numerous significant differences from canonical enteroviral biology.

Complete Genome Sequence of the Naegleria fowleri (Strain LEE) Closed Circular Extrachromosomal Ribosomal DNA Element.

The circular extrachromosomal ribosomal DNA (rDNA) element of strain LEE was molecularly cloned and fully sequenced. The element comprises 15,786 bp and contains a single copy of the organism's rDNA cistron. The nonribosomal sequence contains five potential open reading frames, two large direct repeat sequences, and numerous smaller repeated-sequence regions.

Mucin-1 is required for Coxsackie Virus B3-induced inflammation in pancreatitis.

The Muc-1 oncoprotein is a tumor-associated mucin often overexpressed in pancreatic cancer. We report that knockout of Muc-1 reduced the degree of pancreatic inflammation that resulted from infection with Coxsackievirus B3 (CVB3) in a mouse model. CVB3-infected Muc-1-deficient (Muc-1) mice had significantly reduced infiltration of macrophages into the murine pancreas.

Mapping the Single Origin of Replication in the Naegleria gruberi Extrachromosomal DNA Element.

The genes encoding the ribosomal RNA (rRNA) subunits of the amoeba Naegleria gruberi are encoded in a relatively uncommon arrangement: on a circular extrachromosomal DNA element with each organism carrying about 4,000 copies of the element. As complete sequence analysis of the N. gruberi chromosomal DNA revealed no copy of the rRNA genes, these extrachromosomal elements must therefore replicate autonomously.

Complete Genome Sequence of the Circular Extrachromosomal Element of Strain EGB Ribosomal DNA.

The circular extrachromosomal element of strain EGB was linearized, molecularly cloned, and fully sequenced. The sequence comprises 14,007 bp and encodes the organism's rRNA genes, two potential open reading frames, and numerous repeated sequence regions.

Reversion to wildtype of a mutated and nonfunctional coxsackievirus B3CRE(2C).

The cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] of enteroviruses (EV) facilitates the addition of two uridine residues (uridylylation) onto the virus-encoded protein VPg in order for it to serve as the RNA replication primer. We demonstrated that coxsackievirus B3 (CVB3) is replication competent in the absence of a native (uridylylating) CRE(2C) and also demonstrated that lack of a functional CRE(2C) led to generation of 5' terminal genomic deletions in the CVB3 CRE-knock-out (CVB3-CKO) population. We asked whether reversion of the mutated CRE(2C) occurred, thus permitting sustained replication, and when were 5' terminal deletions generated during replication.

Three capsid amino acids notably influence coxsackie B3 virus stability.

Coxsackievirus B3 strain 28 (CVB3/28) is less stable at 37 °C than eight other CVB3 strains with which it has been compared, including four in this study. In a variant CVB3/28 population selected for increased stability at 37 °C, the capsid proteins of the stable variant differed from the parental CVB3/28 by two mutations in Vp1 and one mutation in Vp3, each of which resulted in altered protein sequences. Each of the amino acid changes was individually associated with a more stable virus.

The microbiology of human hygiene and its impact on type 1 diabetes.

The incidence of type 1 diabetes (T1D), as with several other autoimmune diseases and conditions, began to notably rise in the latter half of the last century. Most cases of T1D are not solely attributable to genetics and therefore, environmental influences are proposed to account for the difference. Humans live today in general under much more hygienic conditions than their ancestors.

Enteroviruses and type 1 diabetes.

Background: Human enteroviruses, which are transmitted via a faecal-oral route, have long been associated with type 1 diabetes onset. Increased hygiene in the 20th century may now be responsible for a decreased chance of enterovirus exposure from an early age onward. Infections with enteroviruses may also be more likely to occur at a later age; the recurrent poliomyelitis epidemics in the 20th century were linked to increased hygiene, consistent with this hypothesis.

Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment.

While group B coxsackieviruses (CVB) use the coxsackievirus and adenovirus receptor (CAR) as the receptor through which they infect susceptible cells, some CVB strains are known for their acquired capacity to bind other molecules. The CVB3/RD strain that emerged from a CVB3/Nancy population sequentially passaged in the CAR-poor RD cell line binds decay-accelerating factor (DAF) (CD55) and CAR. A new strain, CVB3/RDVa, has been isolated from RD cells chronically infected with CVB3/RD and binds multiple molecules in addition to DAF and CAR.

Functional role of the 5' terminal cloverleaf in Coxsackievirus RNA replication.

Using cell-free reactions, we investigated the role of the 5' cloverleaf (5'CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop 'b' and the C-rich sequence was inhibited.

5' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart.

Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5' non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5' viral genomic termini were detected, 5' terminal deletions ranged in size from 22 to 36 nucleotides.

Endogenous low-level expression of the coxsackievirus and adenovirus receptor enables coxsackievirus B3 infection of RD cells.

Cells in which the appropriate viral receptor cannot be detected may paradoxically act as a host to the virus. For example, RD cells are often considered to be non-permissive for infection with coxsackievirus and adenovirus receptor (CAR)-dependent group B coxsackieviruses (CVB), insofar as inoculated cell monolayers show little or no cytopathic effect (CPE) and immunohistological assays for CAR have been consistently negative. Supernatants recovered from RD cells exposed to CVB, however, contained more virus than was added in the initial inoculum, indicating that productive virus replication occurred in the monolayer.

Group B coxsackievirus diabetogenic phenotype correlates with replication efficiency.

Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types.

Myocarditis and heart failure associated with hepatitis C virus infection.

Background: The aim of study is to determine the prevalence of hepatitis C virus (HCV) infection and myocardial injury among patients enrolled in the Myocarditis Treatment Trial. HCV infection has recently been noted in patients with cardiomyopathies and myocarditis. However, prevalence of HCV infection in myocarditis and heart failure remains to be clarified.

National Institutes of Health-sponsored workshop on inflammation and immunity in dilated cardiomyopathy.

Nonischemic dilated cardiomyopathy (DCM) is an uncommon cause of heart failure but has widespread importance because it is the cause of 45% of heart transplantations. Multiple experimental and clinical lines of evidence have implicated altered immunity in the pathogenesis of DCM. However, advances in the understanding of the mechanisms of altered immunity have not affected the diagnosis or treatment of DCM.

Monoclonal antibody against mouse CAR following genetic immunization.

To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR.