Identification of neoschaftoside as 6-C-β-d-glucopyranosyl-8-C-β-l-arabinopyranosylapigenin

The structure of neoschaftoside is shown for the first time to be 6-C-β-d-glucopyranosyl-8-C-β-l-arabinopyranosylapigenin. A variety of chemical and spectroscopic techniques are involved.     

Current Practice in the Referral of Individuals with Suspected Dementia for Neuroimaging by General Practitioners in Ireland and Wales

Objectives

While early diagnosis of dementia is important, the question arises whether general practitioners (GPs) should engage in direct referrals. The current study investigated current referral practices for neuroimaging in dementia, access to imaging modalities and investigated related GP training in Ireland and North Wales.

Methods

A questionnaire was distributed to GPs in the programme regions which included approximately two thirds of all GPs in the Republic of Ireland and all general practitioners in North Wales. A total of 2,093 questionnaires were issued.

Results

48.6% of Irish respondents and 24.3% of Welsh respondents directly referred patients with suspected dementia for neuroimaging. Irish GPs reported greater direct access to neuroimaging than their Welsh counterparts. A very small percentage of Irish and Welsh GPs (4.7% and 10% respectively) had received training in neuroimaging and the majority who referred patients for neuroimaging were not aware of any dementia-specific protocols for referrals (93.1% and 95% respectively).

Conclusions

The benefits of direct GP access to neuroimaging investigations for dementia have yet to be established. Our findings suggest that current GP speciality training in Ireland and Wales is deficient in dementia-specific and neuroimaging training with the concern being that inadequate training will lead to inadequate referrals. Further training would complement guidelines and provide a greater understanding of the role and appropriateness of neuroimaging techniques in the diagnosis of dementia.     

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora,Prostomatea)

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.     

Differential stability of a human mini-chromosome in mouse cell lines

A 4 Mb human mini-chromosome, ΔΔ2, was transferred from Chinese hamster ovary (CHO) cells into a mouse L cell line. The mini-chromosome could be transferred intact into the L cells, with 112/119 clones maintaining a mini-chromosome of the same size as the original. Ten clones were grown for 30 days in continuous culture. The mini-chromosomes were maintained stably with or without selection at a copy number of 1–2 per cell and none experienced any size alterations, as determined by pulsed-field gel electrophoresis. Thus ΔΔ2 is structurally and mitotically stable in L cells. This contrasts with results in embryonic stem cells, in which ΔΔ2 is highly unstable. These findings indicate that established somatic cell lines, such as L cells and CHO cells, have less stringent controls over centromeric function than do normal embryonic cells. Received: 4 December 1997; in revised form: 25 February 1998 / Accepted: 14 April 1998     

Effect of land use on pollution status and risk of fish endocrine disruption in small farmland ponds

To study whether the intensity of agricultural activities affects pesticides loads in pond environment, a large number of Belgian farmland ponds were surveyed in spring 2004. Temporal distribution of pollutants was also investigated over restricted survey ponds sampled three times round year 2007. Sedentary pond Prussian carp juveniles were also captured to determine their brain aromatase activity (AA) and plasma vitellogenin (VTG) levels. Heavy metal distribution was also examined in various pond matrices. Amongst the pesticides analysed, only herbicides were detected. Contamination of pond water by atrazine was frequently observed during spring 2004, while isoproturon and glyphosate were detected round year 2007. Levels of herbicides were inversely related to the distance of ponds to crop field, and values peaked in April or October. Absence of endocrine disruptors in pond water was confirmed by lack of modulation in VTG and AA in male fish. Heavy metals were present in all the pond matrices, but overall contamination levels were low. The results demonstrated that Belgian ponds were mainly contaminated by herbicides and that pond sedentary fish were not affected by endocrine disruptors. They also demonstrated a marked effect of land-use intensity on herbicide pollution which can be mitigated by an adjustment of the buffer zones.     

A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil

Microbial cell concentration is a particularly important bioindicator of soil health and a yardstick for determining biological quotients which are likely to gain in ecological significance if they are calculated in relation to the viable, rather than total, microbial density. A dual-staining technique with fluorescent dyes was used for the spectrofluorimetric quantitative determination of the concentration of viable microbial cells present in three different soil types. This is a novel and substantially modified application of the dual-staining procedure implemented in the LIVE/DEAD BacLight viability kit which has never been successfully applied to the quantification of naturally occurring soil microbial communities. Indigenous microbial cell concentrations were quantified using an internal standard, i.e. spiking environmental samples with suspensions containing different concentrations of live E. coli cells, and external calibration, by comparing fluorescence emission by indigenous bacteria and known concentrations of E. coli in nutrient saline. Two types of environmental samples were tested: bacterial preparations obtained by density gradient centrifugation and soil suspensions. In both cases, prior dilution of the sample was necessary to minimise fluorescence quenching by soil particulate matter. Spectrofluorimetric measurements of indigenous cell concentration in bacterial preparations were in close agreement with those found using epifluorescence microscopy. Limits of detection of 5x10(6) for the soil bacterial preparations and 8x10(7) for the soil suspensions were estimated. Deviations observed when soil suspensions are dealt with are likely due to the selection of a unique bacterial strain for standardisation and calibration. Thorough testing of a variety of reference bacteria and fungi is suggested to determine a more accurate average fluorescence enhancement per microbial cell or mass unit.     

Trafficking through the Rev/RRE pathway is essential for efficient inhibition of human immunodeficiency virus type 1 by an antisense RNA derived from the envelope gene

A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminal-repeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.     

Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8-11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.     

Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase

Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.