Biomedical subjects

Anthony Griffiths

Publications and source records attributed to Anthony Griffiths.

10 recordsLinked to original sources

PubMed2006

Mutation and divergence of the phospholipase C gene in Neurospora crassa.

In the fungus Neurospora crassa we have used RIP to obtain a presumptive null mutation of the phospholipase C-1 gene, thought to be important in intracellular calcium signaling, notably maintenance of the tip-high calcium gradient. The mutant is viable but has slow, aberrant growth and branching. Hence plc-1 is not required for polar growth at the tip, but is necessary to modulate growth to give normal form. The mutant has residual PLC activity suggesting that this enzyme function can be provided from other parts of the genome. Sequencing natural isolates has shown that the plc-1 gene is highly variable in nature. A large proportion of the variable sites are in a region that is unique to Neurospora. A phylogeny for this gene shows that New and Old World strains have diverged the most. Within the Americas, morphs are found throughout the continent suggesting extensive strain dispersal.

Calcium Signaling↗

PubMed2006

Association between urinary tract infection and postmicturition wiping habit.

Urinary tract infections are common in pregnancy. In pregnancy urinary tract infections have an associated risk of premature labour. This study looks at the incidence of urinary tract infections and the method of perineal hygiene used after urination. Wiping back to front is associated with a greater risk of developing urinary tract infection than wiping front to back.

Escherichia coli↗

PubMed2006

Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia.

Laboratory strains of herpes simplex virus lacking thymidine kinase (TK) cannot replicate acutely to detectable levels in mouse trigeminal ganglia and do not reactivate from latency. However, many pathogenic clinical isolates that are resistant to the antiviral drug acyclovir are heterogeneous populations of TK-negative (TK(-)) and TK-positive (TK(+)) viruses. To recapitulate this in vivo, mice were infected with mixtures of wild-type virus and a recombinant TK(-) mutant in various ratios. Following co-infection, the replication, number of latent viral genomes and reactivation efficiency of TK(+) virus in trigeminal ganglia were reduced in a manner related to the amount of TK(-) virus in the inoculum. TK(+) virus did not always complement the acute replication or increase the number of latent viral genomes of TK(-) mutant in mouse ganglia. Even so, TK(+) virus could still confer the pathogenic phenotype to a TK(-) mutant, somehow providing sufficient TK activity in trans to permit a TK(-) mutant to reactivate from latently infected ganglia.

Animals↗

PubMed2006

Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase.

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.

Acyclovir↗

PubMed2006

Prediction and identification of herpes simplex virus 1-encoded microRNAs.

MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. Recently, miRNAs have been identified from viruses with double-stranded DNA genomes. To attempt to identify miRNAs encoded by herpes simplex virus 1 (HSV-1), we applied a computational method to screen the complete genome of HSV-1 for sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. Using this method, we identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate, which is encoded approximately 450 bp upstream of the transcription start site of the latency-associated transcript (LAT), were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. Additionally, small RNAs, including some that are roughly the expected size of precursor miRNAs, were detected using probes for miRNA candidates derived from sequences encoding the 8.3-kilobase LAT, from sequences complementary to U(L)15 mRNA, and from the region between ICP4 and U(S)1. However, no species the size of typical mature miRNAs were detected using these probes. Three of these latter miRNA candidates were predicted to be conserved in HSV-2. Thus, HSV-1 encodes at least one miRNA. We hypothesize that HSV-1 miRNAs regulate viral and host gene expression.

Animals↗

PubMed2006

An investigation into the kerbside recycling behaviour of two Welsh case study authorities.

This paper investigates the number of observed households setting out their containers for kerbside recyclable collection in two Welsh kerbside recycling programmes; namely, Rhondda Cynon Taf County Borough Council and Caerphilly County Borough Council. Analysed data from the monitoring of approximately 114 000 households in the two separate kerbside programmes are presented, making this the largest study of kerbside recycling behaviour in the UK. The average household set-out rates of individual households per electoral division and per street in each programme were compared. Average household set-out rate for a particular electoral division and street was then related to certain socio-economic indicators. The study was performed using the 'ArcView' geographical information systems (GIS) package. The model identified a moderate link between household set-out rate (%) and socio-economic status at electoral division level. However, there was no correlation between socio-economic status and average household set-out rate in a street. Household set-out rates for both programmes fluctuated significantly in streets with similar socio-economic status. Another significant factor affecting set-out rate, herein named the 'Nant-y-Gwyddon effect', is also described.

Conservation of Natural Resources↗

PubMed2005

The role and applications of ultrasound in ambulatory gynaecology.

Ultrasound has changed gynaecological practice and continues to do so. One of the earliest applications of abdominal scanning in gynaecology was for monitoring follicular development during fertility treatment with clomiphene citrate or gonadotrophins in the 1960s and 1970s. Subsequently, it was natural that with the introduction of in vitro fertilization, abdominal and transvaginal ultrasound played a key role in the development of oocyte retrieval techniques. These were truly the first interventional ultrasound-guided ambulatory procedures in gynaecology. In this chapter, the reader will be introduced to the roles that the various ultrasound modalities play in our current daily practice, and how they have changed the management of numerous gynaecological conditions in both diagnostic and therapeutic contexts. We will also outline the recent developments and the 'hot' research topics in this field.

Ambulatory Care↗

PubMed2005

An unusual internal ribosome entry site in the herpes simplex virus thymidine kinase gene.

We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-eIF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single-base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs.

Animals↗

PubMed2003

High-frequency phenotypic reversion and pathogenicity of an acyclovir-resistant herpes simplex virus mutant.

A double-guanine-insertion mutation within a run of guanines in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. This mutation was engineered into strain KOS, and stocks were generated from single plaques. Plaque autoradiography revealed that most plaques in such stocks exhibited low levels of TK activity, while approximately 3% of plaques exhibited high levels of TK activity, indicating a remarkably high frequency of phenotypic reversion. This virus was able to reactivate from latency in mouse ganglia; a fraction of the reactivating virus expressed a high level of TK activity due to an additional G insertion, suggesting that the observed genetic instability contributed to pathogenicity.

Acyclovir↗

PubMed2003

Translational compensation of a frameshift mutation affecting herpes simplex virus thymidine kinase is sufficient to permit reactivation from latency.

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.

Acyclovir↗