Immunoregulatory activity of the lungs in relation to corpuscular and soluble antigens

The immunoregulatory activity of the lungs in normal Wistar rats has been evaluated by difference between primary and secondary immune response to the same dose of the antigen introduced into the respiratory tract or intravenously. As shown in this investigation, intratracheal immunization with corpuscular antigens is accompanied by faintly pronounced antibody formation and a high degree of delayed hypersensitivity, while the introduction of soluble antigens into the respiratory tract leads to the active production of antibodies. The immunoregulatory activity of the lungs is T-dependent. The preliminary introduction of corpuscular or soluble antigen into the respiratory tract is accompanied by the formation of the local mechanism in the lungs for suppressing immune response to the subsequent intratracheal immunization.     

Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli

To directly express native recombinant proteins in Escherichia coli, a new expression vector pSB was constructed using Ssp DnaB mini-intein. Using the vector, native proteins could be produced with the help of C-terminal self-cleavage of the intein. In this study, we cloned hIFNalpha-4 gene into pSB and used E. coli strain Origami B (DE3) as the host. Expression experiments were carried out both in Shake flasks and a 5 L bioreactor. The results indicated hIFNalpha-4 could be expressed in the form of soluble protein with correct folding in E. coli. The maximal hIFNalpha-4 content was 21.7% of total protein, and the antiviral activity of the protein was 1.2x10(8 )IU mg(-1). Overall, good effects were achieved with this system. This intein-mediated protein expression system opens up a useful method for production of native recombinant protein in E. coli.     

The detection of viral antigens and ribosomal RNA in cells by using low-temperature media

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl-embedded HIV-infected cells. A genomic probe containing ribosomal sequences and labeled with biotin was used to hybridize rRNA molecules in sections of animal cells embedded in Lowicryl. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression and viral proteins at the ultrastructural level.     

Diagnosis and treatment of viral diseases in recipients of allogeneic hematopoietic stem cell transplantation

Viral infections are important causes of morbidity and mortality after allogeneic stem cell hematopoietic transplantation (allo-HSCT). Although most viral infections present with asymptomatic or subclinical manifestations, viruses may result in fatal complications in severe immunocompromised recipients. Reactivation of latent viruses, such as herpesviruses, is frequent during the immunosuppression that occurs with allo-HSCT. Viruses acquired from community, such as the respiratory and gastrointestinal viruses, are also important pathogens of post-transplant viral diseases. Currently, molecular diagnostic methods have replaced or supplemented traditional methods, such as viral culture and antigen detection, in diagnosis of viral infections. The utilization of polymerase chain reaction facilitates the early diagnosis. In view of lacking efficacious agents for treatment of viral diseases, prevention of viral infections is extremely valuable. Application of prophylactic strategies including preemptive therapy reduces viral infections and diseases. Adoptive cellular therapy for restoring virus-specific immunity is a promising method in the treatment of viral diseases.     

Evaluation of natural food for planktivorous fish in Lakes Phewa, Begnas, and Rupa in Pokhara Valley, Nepal

Six floating 4-m³ cages stocked with 40 silver carp per cage (three cages) and 40 bighead carp per cage (three cages) were placed in each of the three subtropical Lakes Phewa, Begnas, and Rupa. The fish were cultured, without supplying artificial feed, from August 1994 to December 1995. Fish growth was positively correlated with water temperature, and a slight loss in weight occurred in January when water temperature fell below 16°C in all the lakes. Fish growth was better in Lake Phewa than in Lakes Begnas and Rupa, where food might not have been sufficient or of a quality to sustain fish growth. The fish fed on mostly indigestible phytoplankton, Microcystis aeruginosa in Lake Begnas and on Tabellaria fenestrata and Microcystis aeruginosa in Lake Rupa, which can lead to poor fish growth. The maximum growth rate of silver carp (5.8 g day⁻¹) and bighead carp (4.7 g day⁻¹) occurred in Lake Phewa when Ceratium hirundinella and Peridinium spp. were abundant from August through October. The food particles found in the gut were mostly similar in silver and bighead carp, though Cladocerans (Bosmina longirostris) were observed sparsely in the gut contents of bighead carp only. Received: May 21, 1999 / Accepted: September 28, 1999     

Contrasting patterns of phenotypic variation linked to chromosomal inversions in native and colonizing populations of Drosophila subobscura

In fewer than two decades after invading the Americas, the fly Drosophila subobscura evolved latitudinal clines for chromosomal inversion frequencies and wing size that are parallel to the long‐standing ones in native Palearctic populations. By sharp contrast, wing shape clines also evolved in the New World, but the relationship with latitude was opposite to that in the Old World. Previous work has suggested that wing trait differences among individuals are partially due to the association between chromosomal inversions and particular alleles which influence the trait under consideration. Furthermore, it is well documented that a few number of effective individuals founded the New World populations, which might have modified the biometrical effect of inversions on quantitative traits. Here we evaluate the relative contribution of chromosomal inversion clines in shaping the parallel clines in wing size and contrasting clines in wing shape in native and colonizing populations of the species. Our results reveal that inversion‐size and inversion‐shape associations in native and colonizing (South America) populations are generally different, probably due to the bottleneck effect. Contingent, unpredictable evolution was suggested as an explanation for the different details involved in the otherwise parallel wing size clines between Old and New World populations of D. subobscura. We challenge this assertion and conclude that contrasting wing shape clines came out as a correlated response of inversion clines that might have been predicted considering the genetic background of colonizers.     

Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all β-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations ( > 80% v/v) of TFE induced a β-sheet to -helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations ( > 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and -lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.     

Distribution of introduced and native fish in Patagonia (Argentina): patterns and changes in fish assemblages

The interaction between native fishes and salmonids introduced in Patagonia at the beginning of the 20th Century, developed at the same time as the environmental change. The phenomenon of global warming has led to the formulation of predictions in relation to changes in the distribution of species, in the latitudinal dimension, both at intralacustrine, or small streams levels. The aim of the present work includes three main objectives: a) to compose a general and updated picture of the latitudinal distribution range of native and alien fishes, b) to analyze the historical changes in the relative abundance of Percichthys trucha, Odontesthes sp., and salmonids in lakes and reservoirs, and c) to relate the diversity and relative abundance of native and salmonid fishes to the environmental variables of lakes and reservoirs. We analysed previous records and an ensemble of data about new locations along the northern border of the Patagonian Province. We compared current data about the relative abundance of native fishes and salmonids in lakes and reservoirs, with previous databases (1984–1987). All samplings considered were performed during spring-summer surveys and include relative abundance, as proportions of salmonids, P. trucha, and Odontesthes sp. For the first time, we found changes in fish assemblages from twenty years back up to the present: a significant decline in the relative abundances of salmonids and an increase of P. trucha. We studied the association between the diversity and relative abundance of native and salmonid fishes and the environmental variables of lakes and reservoirs using Canonical Correspondence Analysis. Relative abundance showed mainly geographical cues and the diversity relied largely on morphometric characteristics. Relative abundance and diversity seem to have a common point in the lake area, included into the PAR concept. Native abundance and alien diversity were negatively related with latitude. Greater native diversity was observed in lakes with high PAR compared with salmonids. Historical changes such as southward dispersion, relative abundance changes, and geographical patterns for relative abundance and diversity are basic concepts needed not only in future research but also in management design for Patagonian fish populations.     

Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10 concentration range

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng μl⁻¹ range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-μl sample in the 500 to 5-ng μl⁻¹ range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng μl⁻¹ range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng μl⁻¹ range. However, the Agilent kits require 1 μl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.     

Adaptive management of an environmental watering event to enhance native fish spawning and recruitment

1. A common goal of many environmental flow regimes is to maintain and/or enhance the river's native fish community by increasing the occurrence of successful spawning and recruitment events. However, our understanding of the flow requirements of the early life history of fish is often limited, and hence predicting their response to specific managed flow events is difficult. To overcome this uncertainty requires the use of adaptive management principles in the design, implementation, monitoring and adjustment of environmental flow regimes.
2. The Barmah-Millewa Forest, a large river red gum forest on the Murray River floodplain, south-east Australia, contains a wide variety of ephemeral and permanent aquatic habitats suitable for fish. Flow regulation of the Murray River has significantly altered the natural flood regime of the Forest. In an attempt to alleviate some of the effects of river regulation, the Forest's water regime is highly managed using a variety of flow control structures and also receives targeted Environmental Water Allocations (EWA). In 2005, the largest environmental flow allocated to date in Australia was delivered at the Forest.
3. This study describes the adaptive management approach employed during the delivery of the 2005 EWA, which successfully achieved multiple ecological goals including enhanced native fish spawning and recruitment. Intensive monitoring of fish spawning and recruitment provided invaluable real-time and ongoing management input for optimising the delivery of environmental water to maximise ecological benefits at Barmah-Millewa Forest and other similar wetlands in the Murray-Darling Basin.
4. We discuss possible scenarios for the future application of environmental water and the need for environmental flow events and regimes to be conducted as rigorous, large-scale experiments within an adaptive management framework.     

Study on fish embryo responses to the treatment of cryoprotective chemicals using impedance spectroscopy

Investigations using electrical impedance spectroscopy to measure the responses of fish embryos to the cryoprotective chemicals, methanol and dimethyl sulphoxide (DMSO), were carried out. Zebrafish (Danio rerio) embryos were used as a model to study the newly proposed technique. The normalised permittivity and conductivity changes of the embryos were measured continuously over a 20-min period in a customised embryo-holding chamber. The normalised permittivity and conductivity spectra were obtained during embryo exposure to different concentrations of methanol (1.0, 2.0 and 3.0 M) and DMSO (0.5, 1.0 and 2.0 M) solutions. The results showed significant permittivity and conductivity changes after embryo exposure to methanol and DMSO at the optimum embryo loading level (six embryos). Embryos in different concentrations of methanol and DMSO also resulted in quantitative responses shown in the normalised permittivity and conductivity spectra. The results demonstrated that fish embryo membrane permeability to cryoprotective chemicals could be monitored in real-time. The measurement of permittivity at a lower frequency range (10–10³ Hz) and conductivity at a higher frequency range (10⁴–10⁶ Hz) during fish embryo exposure to cryoprotective chemicals using impedance spectroscopy can be used as a new tool for the fast screening of most effective cryoprotective chemicals. The results from the present study also demonstrated the possibility of quantifying the level of cryoprotective chemicals penetrating the fish embryos.     

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment

Aim: To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species. Methods and Results: Ultraviolet experiments at 254 nm were conducted to determine the inactivation coefficients for seven plant pathogenic species. At 200 mJ cm^?2, the individual species log reductions obtained for six Ascomycete fungi and a cereal virus were as follows: Leptosphaeria maculans (9·9‐log), Leptosphaeria biglobosa (7·1‐log), Barley stripe mosaic virus (BSMV) (4·1‐log), Mycosphaerella graminicola (2·9‐log), Fusarium culmorum (1·2‐log), Fusarium graminearum (0·6‐log) and Magnaporthe oryzae (0·3‐log). Dilution experiments showed that BSMV was rendered noninfectious when diluted to >1/512. Follow‐up large‐scale experiments using up to 400 l of microbiologically contaminated waste water revealed that the filtration of drainage water followed by UV treatment could successfully be used to inactivate several plant pathogens. Conclusions: By combining sedimentation, filtration and UV irradiation within a closed system, plant pathogens can be successfully removed from collected drainage water. Significance and Impact of the Study: Ultraviolet irradiation is a relatively low cost, energy efficient and labour nonintensive method to decontaminate water arising from a suite of higher biological containment level laboratories and plant growth rooms where genetically modified and/or quarantine fungal and viral plant pathogenic organisms are being used for research purposes.     

Identification of restriction fragment length polymorphisms linked to genes controlling soluble solids content in tomato fruit

Summary Gene(s) conferring high soluble solids (SS) in tomato fruit had been backcrossed previously from a wild tomato species, Lycopersicon chmielewskii LA1028 ( 10% SS), into a L. esculentum cultivar, VF36 ( 5% SS), to derive a BC₅S₅ line, LA1563, similar to VF 36 but with 7–8% SS. DNAs from these lines and a tomato breeding line, H2038, were screened for restriction fragment length polymorphisms (RFLPs) using four restriction endonucleases and sixty clones chosen at random from a tomato cDNA library. Most of the cDNA clones (56) identified the same RFLP in VF 36 and LA1563 and a different RFLP in LA1028. However, two cDNA clones identified the same RFLP in LA1563 and LA1028 and a different RFLP in VF36. To determine whether RFLPs identified by these two cDNA clones were linked to SS genes, a H2038 x LA1563 F₂ population was screened for segregation of the RFLPs and for SS content. The segregation ratios of these RFLPs were consistent with ratios expected for codominant alleles at unlinked loci. Analysis of variance of SS content for different RFLP genotypic classes indicated that RFLP alleles at one of the loci were linked to genes controlling SS content. The RFLP allele from the high SS tomato line, LA1563, was associated with significantly higher SS content and, therefore, could be useful in selecting for high SS gene(s) in a tomato breeding program.     

Assessing fish metrics and biotic indices in a Mediterranean stream: effects of uncertain native status of fish

Implementation of the Water Framework Directive requires tools for measuring and monitoring the ecological status of aquatic ecosystems. Several indices are in use in the Iberian Peninsula, although there has been little comparison among them. We sampled the fish assemblage and limnological features of the Tordera stream (NE Spain) quarterly from September 2001 to May 2003 to evaluate the usefulness of several fish metrics and to compare habitat quality and biotic indices currently in use. Data for eight biotic and abiotic indices for this and three other Catalan river basins were also compiled in order to analyse the relationships among indices. In the Tordera stream, fish abundance and richness increased with stream order except in the last sampling site that had the lowest fish abundance owing to the effects of drought and water abstraction. Although most indices were positively correlated, some displayed low or null correlations particularly for the Tordera basin which is more affected by water abstraction and less by pollution; a commonly used physico-chemical index (ISQA) was the least correlated. In a regional fish index (IBICAT) under development, the brown trout (Salmo trutta) has been previously considered as introduced in the Tordera basin. Here, we report an old published record that demonstrates that trout was present before 1845 and we argue that its status should be considered as uncertain given the current information available. Whether brown trout is treated as native or introduced to this river basin has profound effects on the results of fish metrics because of its dominance in the upper reaches. We briefly discuss the role of introduced species, particularly in headwater streams, in the development of fish indices. Our study exemplifies the need for careful, basin-specific assessment of native/introduced status in the development of fish metrics. Handling editor: K. Martens     

A proteomics strategy for determining the synthesis and degradation rates of individual proteins in fish

In order to study the protein dynamics in the tissues of fish we have developed a proteomics-based strategy to determine the rates of synthesis and degradation of individual proteins. We have demonstrated the feasibility of this approach by measuring the turnover of multiple isoforms of parvalbumin (β1-7) in the skeletal muscle of common carp (Cyprinus carpio). A stable isotope-labelled amino acid ([(2)H(7)] l-leucine) was administered to the carp via the diet and its incorporation into the isoforms of parvalbumin in muscle over time was monitored by LC-MS analysis of signature peptides. The relative isotope abundance was calculated and used to deconvolute the data. The β7 parvalbumin isoform had a rate of synthesis that was greater than the rate of degradation. In contrast the rate of degradation of the β5 isoform exceeded its rate of synthesis, whilst the analysis revealed that the other parvalbumin β-isoforms (β1, β2, β3, β4 and β6) had a rate of synthesis that was equal to the rate of degradation. This work has addressed a number of technical challenges and represents the first study to use proteomic approaches to measure the turnover of individual proteins in fish.     

Breakdown of the brown trout evolutionary history due to hybridization between native and cultivated fish

Genetic changes in the population structure of brown trout Salmo trutta in the eastern Pyrenees were monitored during the 1990s. Stocking with cultivated exogenous fish has resulted in introgressed populations where those with higher introgression coefficients also have the highest values of heterozygosity. Nevertheless, this increase of local diversity reflects a decrease of genetic differentiation between populations and loss of native alleles. These changes obscure native gene pools and modify evolutionary distinctions among native brown trout populations.