GFP-visualized immobilized enzymes: degradation of paraoxon via organophosphorus hydrolase in a packed column.

A versatile gene-fusion technique for immobilizing and visualizing biologically active enzymes which includes from the N to C-termini, an affinity histidine tag, the green fluorescent protein (GFP), a proteolytic enzyme (enterokinase, EK) cleavage site and the enzyme of interest, were developed. Specifically, the organophosphorus hydrolase was bound to the affinity (His(6))-reporter(GFP)-EK fusion elements. Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. In the case of immobilized OPH, paraoxon was rapidly degraded when pumped through a packed column. In reaction mixtures containing CHES buffer at pH 6.9, a continual decay in OPH activity was observed and importantly, this was monitored by GFP fluorescence. This decay in activity was fully restored, along with fluorescence, upon washing with PBS buffer. Many subsequent experiments were performed at varied pH and in different background buffer solutions. In all cases when there was OPH activity there was also marked fluorescence from the GFP fusion partner. Likewise, when OPH activity was lost, so was GFP fluorescence and, importantly, both were regenerated when washed in the presence of the kosmotropic salt, phosphate. Recently, Waldo et al. (1999) showed that GFP fluorescence from whole cells indicated the extent of proper folding of normally aggregated proteins designed via directed evolution. The present work demonstrates an application wherein GFP fluorescence indicates stability and activity of its fusion partner.     

Serum fatty acid analysis and digestibility study in the Vancouver Island marmot (Marmota vancouverensis) fed a captive diet supplemented with α‐linolenic acid

Mammals that hibernate must rely on endogenous lipid reserves to survive over winter. This study was conducted to compare the difference in serum fatty acid composition, dietary intake, and apparent digestibility in the Vancouver Island marmot (N = 6) fed the Metro Zoo lagomorph diet supplemented with α‐linolenic acid [C(18:3) n‐3 (α‐LA)]. The experiment was designed as a 3 × 3 Latin square with three 17‐day collection periods. The test diets contained 12.16, 14.85, and 17.05% α‐LA as a percentage of fatty acids in the diet supplied through the addition of flaxseed oil (～53% α‐LA). Across treatments, dry matter intake (g/d), dry matter digestibility, apparent fat digestibility, and apparent neutral detergent fiber digestibility did not differ significantly (P > 0.05). There were no significant differences in serum α‐LA concentration between the three levels of α‐LA supplementation. However, this supplementation did elevate serum α‐LA, eicosapentaenoic acid [C20:5 (n‐3)], and docosahexaenoic acid [C22:6 (n‐3)] levels compared with feeding the basal zoo lagomorph diet (P < 0.05). Thus, supplementation of the basal zoo lagomorph diet with α‐LA elevated the serum levels of essential fatty acids in the Vancouver Island marmot. Zoo Biol 20:251–259, 2001. © 2001 Wiley‐Liss, Inc.     

Draft genome sequence of the extremely acidophilic biomining bacterium Acidithiobacillus thiooxidans ATCC 19377 provides insights into the evolution of the Acidithiobacillus genus

Acidithiobacillus thiooxidans is a mesophilic, extremely acidophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and inorganic sulfur compounds. Here we present the draft genome sequence of A. thiooxidans ATCC 19377, which has allowed the identification of genes for survival and colonization of extremely acidic environments.     

Developmental mechanisms and experimental models to understand forebrain malformative diseases

The development of the central nervous system can be divided into a number of phases, each of which can be subject of genetic or epigenetic alterations that may originate particular developmental disorders. In recent years, much progress has been made in elucidating the molecular and cellular mechanisms by which the vertebrate forebrain develops. Therefore, our understanding of major developmental brain disorders such as cortical malformations and neuronal migration disorders has significantly increased. In this review, we will describe the major stages in forebrain morphogenesis and regionalization, with special emphasis on developmental molecular mechanisms derailing telencephalic development with subsequent damage to cortical function. Because animal models, mainly mouse, have been fundamental for this progress, we will also describe some characteristic mouse models that have been capital to explore these molecular mechanisms of malformative diseases of the human brain. Although most of the genes involved in the regulation of basic developmental processes are conserved among vertebrates, the extrapolation of mouse data to corresponding gene expression and function in humans needs careful individual analysis in each functional system.     

Functional significance of AtHMA4 C-terminal domain in planta

Background

Enhancing the upward translocation of heavy metals such as Zn from root to shoot through genetic engineering has potential for biofortification and phytoremediation. This study examined the contribution of the heavy metal-transporting ATPase, AtHMA4, to the shoot ionomic profile of soil-grown plants, and investigated the importance of the C-terminal domain in the functioning of this transporter.

Principal Findings

The Arabidopsis hma2 hma4 mutant has a stunted phenotype and a distinctive ionomic profile, with low shoot levels of Zn, Cd, Co, K and Rb, and high shoot Cu. Expression of AtHMA4 (AtHMA4-FL) under the CaMV-35S promoter partially rescued the stunted phenotype of hma2 hma4; rosette diameter returned to wild-type levels in the majority of lines and bolts were also produced, although the average bolt height was not restored completely. AtHMA4-FL expression rescued Co, K, Rb and Cu to wild-type levels, and partially returned Cd and Zn levels (83% and 28% of wild type respectively). In contrast, expression of AtHMA4-trunc (without the C-terminal region) in hma2 hma4 only partially restored the rosette diameter in two of five lines and bolt production was not rescued. There was no significant effect on the shoot ionomic profile, apart from Cd, which was increased to 41% of wild-type levels. When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect. When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL.

Conclusion

The ionome of the hma2 hma4 mutant differs markedly from wt plants. The functional relevance of domains of AtHMA4 in planta can be explored by complementing this mutant. AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.     

Incidental Findings on Brain MR Imaging in Older Community-Dwelling Subjects Are Common but Serious Medical Consequences Are Rare: A Cohort Study

Objectives

Incidental findings in neuroimaging occur in 3% of volunteers. Most data come from young subjects. Data on their occurrence in older subjects and their medical, lifestyle and financial consequences are lacking. We determined the prevalence and medical consequences of incidental findings found in community-dwelling older subjects on brain magnetic resonance imaging.

Design

Prospective cohort observational study.

Setting

Single centre study with input from secondary care.

Participants

Lothian Birth Cohort 1936, a study of cognitive ageing.

Main Outcome Measures

Incidental findings identified by two consultant neuroradiologists on structural brain magnetic resonance imaging at age 73 years; resulting medical referrals and interventions.

Primary and Secondary Outcome Measures

Prevalence of incidental findings by individual categories: neoplasms, cysts, vascular lesions, developmental, ear, nose or throat anomalies, by intra- and extracranial location; visual rating of white matter hyperintensities and brain atrophy.

Results

There were 281 incidental findings in 223 (32%) of 700 subjects, including 14 intra- or extracranial neoplasms (2%), 15 intracranial vascular anomalies (2%), and 137 infarcts or haemorrhages (20%). Additionally, 153 had moderate/severe deep white matter hyperintensities (22%) and 176 had cerebral atrophy at, or above, the upper limit of normal (25%) compared with a normative population template. The incidental findings were unrelated to white matter hyperintensities or atrophy; about a third of subjects had both incidental findings and moderate or severe WMH and a quarter had incidental findings and atrophy. The incidental findings resulted in one urgent and nine non-urgent referrals for further medical assessment, but ultimately in no new treatments.

Conclusions

In community-dwelling older subjects, incidental findings, including white matter hyperintensities and atrophy, were common. However, many findings were not of medical importance and, in this age group, most did not result in further assessment and none in change of treatment.     

Resolving spatio-temporal uncertainty in rare resource acquisition: smell the shell

Complex environments present substantial spatio-temporal uncertainty in where and when rare ecological resources become available. How animals navigate this uncertainty to turn the seemingly unpredictable into the predictable is a fundamental question in evolutionary ecology. Here we use subtidal hermit crabs (Pagurus acadianus) as a model system to experimentally test in the field how animals resolve spatio-temporal uncertainty in resource availability. Quadrat sampling within the subtidal zone revealed that hermit crabs face an extreme ecological challenge, based on the rarity of empty shells across space and time. We show how this spatio-temporal uncertainty is ultimately resolved using long-distance chemical cues, which are associated with non-destructive shell predation on living gastropods, the original source of shells. By experimentally releasing cues that simulated the chemical by-products of predation, we reveal that certain flesh cues provide fine-grained information about the precise spatial and temporal window of new shell availability. These cues were most attractive to individuals with the greatest existing resource needs, and in the absence of this information individuals were highly constrained in their ability to discover newly available resources. Broadly, these experiments reveal that exploiting simple cues from heterospecific predators can provide a solution to the general ecological challenge of finding resources that are rare in space and time.