The use of wildlife to monitor zoonoses

Wildlife are usually considered vectors, reservoirs or primary targets of infectious disease. A seldom considered epidemiological role which they can play involves their use as disease sentinels for the detection and monitoring of zoonoses. Their potential for such utilization has been demonstrated with the wild turkey (Meleagris gallopava intermedia) and St. Louis encephalitis in Texas and the white-tailed deer (Odocoileus virginianus) and California encephalitis in North America. The limitations and criteria which are important in the use of wild populations for "sentinel" duty are discussed.     

The use of microdialysis to monitor rapid changes in glucose concentration.

As a result of the Diabetes Control and Complications Trial, there is increased emphasis on the importance of blood glucose concentration self-monitoring for people with diabetes. The current methods for this are not ideal, and there are many other possible techniques currently under investigation. One of these techniques is microdialysis, which can be used to analyse subcutaneous interstitial glucose concentrations. A system with high recovery has recently been used to monitor glucose concentrations with sampling over one- or two-hour periods. We have investigated whether this system can be used to monitor rapid changes in blood glucose concentration in healthy volunteers with collection intervals of only ten minutes. The results show that microdialysis can be used to monitor rapidly changing blood glucose concentration, but in some subjects, dialysate glucose lagged behind the whole blood and plasma glucose concentrations to a degree that would be clinically significant. It would therefore be necessary to assess the system, comparing dialysate with plasma glucose concentrations in each individual, prior to use in a clinical setting.     

The use of voltage-sensitive dyes to monitor signal-induced changes in membrane potential-ABA triggered membrane depolarization in guard cells

The plant membrane potential reports on the activity of electrogenic plasma membrane transport processes. The membrane potential is widely used to report for early events associated with changes in light regime, hormone action or pathogen attacks. The membrane potentials of guard cells can be precisely measured with microelectrodes, but this technique is not well suited for rapid screens with large sample numbers. To provide the basis for large-scale membrane potential recordings, we took advantage of voltage-sensitive dyes. Using the fluorescent dyes bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC(4)(3)) and the FLIPR Membrane Potential Assay Kit (FMP) dye we followed changes in the membrane potential in guard cells and vacuoles. Based on the fluorescence of DiBAC(4)(3) a method was established for quantification of the membrane potential in guard cell protoplasts which should be considered as an excellent system for high-throughput screening of plant cells. In the absence of abscisic acid (ABA), one-third of the guard cell protoplast population spontaneously oscillated for periods of 5-6 min. Upon application of ABA the hyperpolarized fraction ( approximately 50%) of the guard cell protoplast population depolarized within a few minutes. Membrane potential oscillations were terminated by ABA. Oscillations and ABA responses were found in cell populations with active anion channels. Thus time- and voltage-dependent anion channels likely represent the ABA-sensitive conductance and part of the membrane potential oscillator. The suitability of membrane potential dyes was tested on vacuoles, too. Dye-based vacuolar membrane polarization was monitored upon ATP exposure. We conclude that voltage-sensitive dyes provide an excellent tool for the study of changes in the membrane potential in vacuole as well as guard cell populations.     

The use of gas-sensor arrays to diagnose urinary tract infections

Sensorial analysis based on the utilisation of human senses, is one of the most important and straightforward investigation methods in food and chemical analysis. An electronic nose has been used to detect in vivo Urinary Tract Infections from 45 suspected cases that were sent for analysis in a UK Health Laboratory environment. These samples were analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. An intelligent model consisting of an odour generation mechanism, rapid volatile delivery and recovery system, and a classifier system based on learning techniques has been considered. The implementation of an Extended Normalised Radial Basis Function network with advanced features for determining its size and parameters and the concept of fusion of multiple classifiers dedicated to specific feature parameters has been also adopted in this study. The proposed scheme achieved a very high classification rate of the testing dataset, demonstrating in this way the efficiency of the proposed scheme compared with other approaches. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology.     

The use of melatonin in Alzheimer's disease

About 45% of Alzheimer's disease (AD) patients have disruptions in their sleep and sundowning agitation. Since melatonin secretion is greatly inhibited in AD patients we have used melatonin to treat sleep disorders in AD patients since 1995. In a first study [21] we reported, in 7 out of 10 dementia patients treated with melatonin (3 mg p.o. at bed time), a decreased sundowning. In a second study [22] we examined 14 AD patients who received 9 mg melatonin daily for 22 to 35 months, observing a significant improvement of sleep quality with stabilization of behavioral and cognitive parameters. In a third study [23] we reported two monozygotic twins with AD and similar cognitive impairment, one of them receiving 6 mg melatonin at bedtime daily for 3 years. Melatonin treatment improved sleep quality and suppressed sundowning. We now report the effect of melatonin (4-month-long treatment with 6 mg/day) in 45 AD patients with sleep disturbances. Melatonin improved sleep and suppressed sundowning, an effect seen regardless of the concomitant medication employed to treat cognitive or behavioral signs of AD. Melatonin treatment seems to constitute a selection therapy to ameliorate sundowning and to slow evolution of cognitive impairment in AD patients.     

The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.     

The use of glucose dehydrogenase to monitor the integrity of microsomes

Various concentrations of Tergitol NP-10 stimulate mannose-6-phosphatase and glucose dehydrogenase to the same extent in untreated rat liver microsomes. Thus, the latency of glucose dehydrogenase may be used as an alternative to mannose phosphatase as a measure of the integrity of the microsomal membrane. The advantage of using glucose dehydrogenase rather than mannose phosphatase to monitor microsomal integrity is that NADH is more easily measured than Pi.     

Application of cDNA arrays to monitor mRNA profiles in single preimplantation mouse embryos

Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report a protocolfor the rapid global amplification of embryonic mRNA that permits the generation of expression profiles from single murine blastocysts. The approach combines global PCR and 77 RNA polymerase amplification and allows the preparation of labeled, amplified RNA for array hybridization from single murine blastocysts containing approximately 1.5 pg mRNA in less than 12 h. We demonstrate that this amplification procedure is highly reproducible and does not bias original relative mRNA levels. Signal patterns from various embryonic stages of murine development revealed marked differences in mRNA expression that were in accordance with previously published data. We found genes known to be involved in embryonic apoptosis expressed at different levels in individual murine day 3.5 blastocysts. This technique can thus be used to assess embryonic viability and investigate molecular mechanisms of embryonic development.     

The use of DNA-intercalating dye to monitor cell fusion and microinjection

A conventional method for microinjection, using erythrocyte ghosts as the injection vector, has been modified to provide a protocol for the highly efficient delivery of small quantities of material into the cytoplasm of target cells. The technique is applicable for use with a variety of proteins, sugars, nucleotides and dyes. When the intercalating dye propidium iodide is included within the sealed ghosts their subsequent fusion with target cells can be continuously monitored by fluorescence spectroscopy, providing a convenient and sensitive parameter of cell-cell fusion. The protocol can be adapted for use with both adherent and non-adherent target cells, and can be used to monitor the relative effectiveness of a variety of fusogenic agents.     

The effect of apo E secretion on lipoprotein uptake in transfected cells.

To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.     

Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells.

IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross-linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

蛋白芯片生产用醛基化玻片制备的条件优化及初步检定

建立一种以玻片为基质的蛋白芯片制备的优化及检定方法。将经过清洗处理的玻片,用不同浓度的氨基硅烷试剂和戊二醛试剂,在不同的时间内,进行氨基化和醛基化处理,通过蛋白结合强度与处理时间及溶剂浓度的动力学相关性分析,确定最佳的优化条件,将不同浓度梯度的人IgG抗体结合于该玻片表面,经过洗涤、封闭,再加入Cy3荧光标记的二抗,孵育,洗涤后检测各点的荧光强度,确定其线性范围及其检测灵敏度。氨基硅烷试剂浓度为5%,作用时间为30min;戊二醛试剂浓度为2.5%,作用时间为60min时,蛋白结合强度达到饱和。蛋白芯片在2～2×4-7mg/ml浓度范围内有良好的线性,能够检测出人IgG的最低浓度为2×4-8mg/ml。     

Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.

Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins-14 and -28 [(1989) EMBO J. 8, 2911-2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins-14 and -28, respectively. Radioimmunochemical analysis of the somatostatin-related products indicated that replacement of either Arg-2-Lys-1 by Asn-2-Asn-1 or of Arg-15 by Asn-15 resulted in the exclusive production of either somatostatin-28 or -14, respectively. Moreover only prosomatostatin[1-76] was detected and no somatostatin-28[1-12] could be measured in cell extracts. Selective suppression of either somatostatin-14 or somatostatin-28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin-14 is exclusively generated by dibasic cleavage at the Arg-2-Lys-1 site of the intact precursor with concomitant production of prosomatostatin[1-76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.     

The use of milk progesterone to monitor reproductive function in beef suckler cows

Milk progesterone analysis was used to monitor reproductive function in 134 autumn calving cross-bred suckler cows. Progesterone was measured in milk samples collected three times per week from around 4 week post-calving to around day 60 of pregnancy during 1st and 2nd lactation. The mean day of onset of luteal activity (OLA) was 40.7 +/- 1.1 with the distribution skewed towards a later return. Once cyclicity had been initiated the incidence of reproductive cycle problems (6.5%) was low, though animals with such problems (n = 14) exhibited a delayed interval to first service (P < 0.05), lower conception and calving rates (P < 0.001), increased services per pregnancy (P < 0.001) and a higher (P < 0.10) barren rate (14.3% versus 4.0%) compared to animals with normal cycles (n = 201). In conclusion, using milk progesterone analysis we found a relatively low incidence of reproductive cycle problems in beefxdairy suckler cows. However, while the incidence of cycle problems was low, those animals with problems showed significantly impaired reproductive function.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells.

A Chinese hamster ovary cell line hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene was transfected with a plasmid, pAG100, capable of correcting the endogenous aprt mutation by targeted homologous recombination. In some experiments, pAG100 was transfected in combination with one of two 'competitor' plasmids. Competitor pCOMP-A was identical to pAG100 except that the aprt sequence on pCOMP-A had the same mutation as the endogenous aprt gene. Competitor pCOMP-B was identical to pAG100 except for a 763 bp deletion in the aprt sequence encompassing the site of mutation in the endogenous gene. Neither pCOMP-A nor pCOMP-B was capable of correcting the defect in the endogenous aprt gene via gene targeting. We asked whether cotransfection of a 4-fold excess of either competitor DNA molecule with pAG100 would reduce the efficiency of targeted correction of the endogenous aprt gene. We report that while plasmid pCOMP-B did not influence the efficiency of gene targeting by pAG100, plasmid pCOMP-A reduced the number of gene targeting events about 5-fold. These observations indicate that the initial homologous interaction between transfected DNA and a genomic target sequence occurs rapidly and that targeting efficiency is limited by a step subsequent to homologous pairing.     

The use of horseradish peroxidase–lectin conjugates to monitor the purity of plasma-membrane preparations obtained from isolated cells

The plasma membranes of viable murine EL4 tumour cells were labelled with horseradish peroxidase-conjugated wheat-germ agglutinin. After disruption of the labelled intact cells, plasma-membrane purification could be monitored by ultrastructural examination of the various fractions for positive reaction product on the membrane vesicles.     

The use of trail cameras to monitor species inhabiting artificial nest boxes

1. Artificial boxes are commonly used in studies of cavity‐dwelling animals of various taxa. One advantage of nest boxes is that cameras can be used to monitor animals inside the cavity, however, most cameras used today have to be built de novo or modified or are expensive.
2. Here, we describe a method for monitoring nest boxes using off‐the‐shelf models of trail cameras that can record photographs and videos in daylight and darkness (TCM; Trail Camera Method). The cameras can record sequentially within a given time lapse or an infra‐red motion sensor can be triggered by activity in the nest box.
3. Using TCM in a Great Tit (Parus major) nest box population, we studied the hourly pattern of the first egg laying and the first egg hatching. We found that Great Tits laid eggs within 2 h of the sunrise while the timing of hatching spanned the 24‐h day. Moreover, we found that the hour of hatching affects the nestlings’ mass on the 2nd day of life, but not on the 12th day of life.
4. Comparing to traditional nest box checks, TMC requires about 75% less time to obtain data on the timing of egg laying and hatching. Moreover, the hour estimation error was several orders of magnitude greater with the traditional method.
5. Our data demonstrate that commercially available trail cameras are an affordable and convenient method of monitoring artificial cavities. Trail cameras are small, standalone, weather‐proof devices with integrated powering, memory storage, lighting, and recording systems. They could be easily swapped between boxes or removed. After small modifications of the box, they could be used to monitor a wide variety of behaviors of many animal taxa.