Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

The novel nucleotide 4KNTP, in high concentrations in erythrocytes of renal failure children: a comparison with accumulation of other putative precursors in the plasma

We have measured the concentrations of metabolites related to the turnover of NAD, which accumulate in the blood of children with renal failure. One is a novel nucleotide, identified as the N1-riboside triphosphate of 4-pyridone-3-carboxamide (4PYTP), also described as 4KNTP, which accumulates in the erythrocytes in parallel with renal failure.     

Antibiotic dosing in the 'at risk' critically ill patient: Linking pathophysiology with pharmacokinetics/pharmacodynamics in sepsis and trauma patients

Background

Critical illness, mediated by trauma or sepsis, can lead to physiological changes that alter the pharmacokinetics of antibiotics and may result in sub-therapeutic concentrations at the sites of infection. The first aim of this project is to identify the clinical characteristics of critically ill patients with significant trauma that have been recently admitted to ICU that may predict the dosing requirements for the antibiotic, cefazolin. The second aim of this is to identify the clinical characteristics of critically ill patients with sepsis that may predict the dosing requirements for the combination antibiotic, piperacillin-tazobactam.

Methods/Design

This is an observational pharmacokinetic study of patients with trauma (cefazolin) or with sepsis (piperacillin-tazobactam). Participants will have samples from blood and urine, collected at different intervals. Patients will also have a microdialysis catheter inserted into subcutaneous tissue to measure interstitial fluid penetration of the antibiotic. Participants will be administered sinistrin, indocyanine green and sodium bromide as well as have cardiac output monitoring performed and tetrapolar bioimpedance to determine physiological changes resulting from pathology. Analysis of samples will be performed using validated liquid chromatography tandem mass-spectrometry. Pharmacokinetic analysis will be performed using non-linear mixed effects modeling to determine individual and population pharmacokinetic parameters of antibiotics.

Discussion

The study will describe cefazolin and piperacillin-tazobactam concentrations in plasma and the interstitial fluid of tissues in trauma and sepsis patients respectively. The results of this study will guide clinicians to effectively dose these antibiotics in order to maximize the concentration of antibiotics in the interstitial fluid of tissues.     

Effects of cage beddings on microsomal oxidative enzymes in rat liver

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.     

Validation of a 1DL earliness per se (eps) flowering QTL in bread wheat (Triticum aestivum)

Vernalization, photoperiod and the relatively poorly defined earliness per se (eps) genes regulate flowering in plants. We report here the validation of a major eps quantitative trait locus (QTL) located on wheat 1DL using near isogenic lines (NILs). We used four independent pairs of NILs derived from a cross between Spark and Rialto winter wheat varieties, grown in both the field and controlled environments. NILs carrying the Spark allele, defined by QTL flanking markers Xgdm111 and Xbarc62, consistently flowered 3–5 days earlier when fully vernalized relative to those with the Rialto. The effect was independent of photoperiod under field conditions, short days (10-h light), long days (16-h light) and very long days (20-h light). These results validate our original QTL identified using doubled haploid (DH) populations. This QTL represents variation maintained in elite north-western European winter wheat germplasm. The two DH lines used to develop the NILs, SR9 and SR23 enabled us to define the location of the 1DL QTL downstream of marker Xgdm111. SR9 has the Spark 1DL arm while SR23 has a recombinant 1DL arm with the Spark allele from Xgdm111 to the distal end. Our work suggests that marker assisted selection of eps effects is feasible and useful even before the genes are cloned. This means eps genes can be defined and positionally cloned in the same way as the photoperiod and vernalization genes have been. This validation study is a first step towards fine mapping and eventually cloning the gene directly in hexaploid wheat.     

Persistently infected cultures as a source of hepatitis A virus

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Importance of flavonoids in insect--plant interactions: feeding and oviposition

Jeffrey Harborne and colleagues have been responsible for collating the majority of data on the role of flavonoids in insect plant interactions. This article examines some of this information and assesses our knowledge about the role flavonoids play in insect feeding and oviposition behaviour. It is clear that insects can discriminate among flavonoids and that these compounds can modulate the feeding and oviposition behaviour of insects, but further work is required to understand the neural mechanisms associated with these behavioural responses. Despite the wealth of data about the diversity of flavonoids in plants, very few of these compounds have been tested against insects and their role in the evolution of host range in insect--plant interactions has yet to be determined.     

A comparative method for finding and folding RNA secondary structures within protein-coding regions

Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins, and evidence is accumulating that this phenomenon may also be found in Eukaryotes. We here present the first comparative method, called RNA-DECODER, which explicitly takes the known protein-coding context of an RNA-sequence alignment into account in order to predict evolutionarily conserved secondary-structure elements, which may span both coding and non-coding regions. RNA-DECODER employs a stochastic context-free grammar together with a set of carefully devised phylogenetic substitution-models, which can disentangle and evaluate the different kinds of overlapping evolutionary constraints which arise. We show that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD.     

Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and acquired pathologies

CSF purines were grossly elevated compared with controls only in adenylosuccinate lyase (ADSL) deficiency and TB meningitis. The former representing low permeability, the latter severe damage to the normal blood/brain barrier. By contrast, the similarity to controls, with no difference between Lesch-Nyhan disease (LND) or LND variants, would exclude hypoxia as a factor in the severe neurological deficits in LND. Similar findings in purine nucleoside phosphorylase (PNP) deficiency (although nucleosides replace the normal bases) likewise exclude hypoxia in the aetiology of the albeit milder neurological deficits.