Comparing the survival rate of juvenile Chinook salmon migrating through hydropower systems using injectable and surgical acoustic transmitters.

Acoustic telemetry is one of the primary technologies for studying the behavior and survival of fishes throughout the world. The size and performance of the transmitter are key limiting factors. The newly developed injectable transmitter is the first acoustic transmitter that can be implanted via injection instead of surgery. A two-part field study was conducted to evaluate the performance of the injectable transmitter and its effect on the survival of implanted fish. The injectable transmitter performed well and similarly to the proceeding generation of commercially-available JSATS transmitters tested concurrently. Snake River subyearling Chinook salmon smolts implanted with the injectable transmitter had a higher survival probability from release to each of eleven downstream detection arrays, because reach-specific survival estimates were significantly higher for the injectable group in three of the eleven reaches examined. Overall, the injectable group had a 0.263 (SE = 0.017) survival probability over the entire 500 km study area compared to 0.199 (0.012) for the surgically implanted group. The reduction in size and ability to implant the new transmitter via injection has reduced the tag or tagging effect bias associated with studying small fishes. The information gathered with this new technology is helping to evaluate the impacts of dams on fishes.

An injectable acoustic transmitter for juvenile salmon.

Salmon recovery and the potential detrimental effects of dams on fish have been attracting national attention due to the environmental and economic implications. In recent years acoustic telemetry has been the primary method for studying salmon passage. However, the size of the existing transmitters limits the minimum size of fish that can be studied, introducing a bias to the study results. We developed the first acoustic fish transmitter that can be implanted by injection instead of surgery. The new injectable transmitter lasts four times longer and weighs 30% less than other transmitters. Because the new transmitter costs significantly less to use and may substantially reduce adverse effects of implantation and tag burden, it will allow for study of migration behavior and survival of species and sizes of fish that have never been studied before. The new technology will lead to critical information needed for salmon recovery and the development of fish-friendly hydroelectric systems.

A molecular biomarker system for assessing the health of gastropods (Ilyanassa obsoleta) exposed to natural and anthropogenic stressors.

We developed a Molecular Biomarker System (MBS) to assess the physiological status of mud snails (Ilyanassa obsoleta) challenged by exposure to high temperature, cadmium, atrazine, endosulfan and the water-accommodating fraction of bunker fuel #2. The MBS is used to assay specific cellular parameters of the gastropod cell that are indicative of a non-stressed or stressed condition. The MBS distinguished among responses to each stressor and to non-stressed control conditions. For example, the biomarkers metallothionein and cytochrome P450 2E1 homologue distinguished between metal and non-metal stresses. MBS data from this study corroborate toxicological studies of organismal responses to endosulfan, atrazine, fuel and cadmium stresses. The MBS technology aids in the accurate diagnosis of the snail's health condition because the physiological significance of the changes of each biomarker is well known. This technology is particularly relevant for environmental monitoring because gastropods are used as key indicator species in many estuarine, marine, freshwater and terrestrial ecosystems. Finally, the Molecular Biomarker System technology is relatively inexpensive, easy to implement, precise and can be quickly adapted to an automated, high-throughput system for large sample analysis.

Assessing the health of grass shrimp (Palaeomonetes pugio) exposed to natural and anthropogenic stressors: a molecular biomarker system.

We developed a molecular biomarker system (MBS) to assess the physiological status of Palaomenetes pugio (grass shrimp) challenged with exposure to heat stress, cadmium, atrazine, and the water-accommodating fraction of either diesel fuel or bunker fuel No. 2. The MBS assayed 9 specific cellular parameters of shrimp that are indicative of a nonstressed or stressed condition: heat-shock protein 60, heat-shock protein 70, alpha B-crystallin homologue, lipid peroxide, total glutathione level, ubiquitin, mitochondrial manganese superoxide dismutase, metallothionein, and cytochrome P-450 2E homologue. Using these 9 parameters, the MBS can distinguish between the responses to each stressor, and to the nonstressed control conditions. The MBS was able to determine the structural integrity of the cell as defined by protein turnover, protein chaperoning, and lipid composition via lipid peroxide levels, and the status of key metabolic processes such as cytoskeletal integrity and glutathione redox potential. This technology aids in the accurate diagnosis of the health of shrimp because the physiological significance of changes of each parameter is well known. This technology is particularly relevant for environmental monitoring because grass shrimp are used as key indicator species in many estuarine ecosystems. Finally, this system is easy to implement, precise, and can be quickly adapted to an automated high-throughput system for mass sample analysis.

A molecular biomarker system for assessing the health of coral (Montastraea faveolata) during heat stress.

Using a novel molecular biomaker system (MBS), we assessed the physiological status of coral (Montastraea faveolata) challenged by heat stress by assaying specific cellular and molecular parameters. This technology is particularly relevant for corals because heat stress is thought to be an essential component of coral bleaching. This phenomenon is widely believed to be responsible for coral mortality worldwide, particularly during 1997-1998. Specific parameters of coral cellular physiology were assayed using the MBS that are indicative of a nonstressed or stressed condition. The MBS distinguished the separate and combined effects of heat and light on the 2 coral symbionts, a scleractinian coral and a dinoflagellate algae (zooxanthellae). This technology aids in the accurate diagnosis of coral condition because each parameter is physiologically well understood. Finally, the MBS technology is relatively inexpensive, easy to implement, and precise, and it can be quickly adapted to a high-throughout robotic system for mass sample analysis.

Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR.

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.

In situ detection of hepatitis A virus in cell cultures and shellfish tissues.

An in situ transcription method was developed to detect hepatitis A virus RNA in both cell cultures and shellfish tissues. Radiolabeled cDNA copies were synthesized in situ by reverse transcriptase-directed transcription after annealing with a specific primer to the viral RNA. Both tritium (3H) and 35S were useful in the in situ transcription reaction, but the use of 3H resulted in a lower background and finer detail in the localization of viral particles. Application of the method to different organs of oysters which had bioaccumulated hepatitis A virus allowed the first in situ localization of the virus, specifically in stomach and hepatopancreatic tissues.

Active kallikrein, preprokallikrein, and kallikrein-inhibitor complex.

Active kallikreins isolated from various exocrine and endocrine tissues were identified by a monoclonal antibody in Western blot analyses to be approximately 38,000 dalton proteins. Kallikreins isolated from rat pancreas, kidney, submandibular gland, brain, spleen and urine were indistinguishable with respect to molecular weight and immunological characteristics. Preprokallikreins were synthesized in a cell-free translation system directed by mRNAs and immunoprecipitated by affinity-purified kallikrein antibody. Analysis of the precipitates by SDS-polyacrylamide gel electrophoresis revealed a approximately 37,000 dalton polypeptide in kidney, brain and submandibular gland translation products. This 37,000 dalton kallikrein precursor was hybrid-arrested by a kallikrein cDNA encoding tissue kallikrein which was isolated from a rat submandibular gland cDNA library. The immunoprecipitates of products directed by pancreatic mRNA showed a major protein with Mr of approximately 30,000. An endogenous approximately 92,000 dalton component in rat urine and kidney was also identified by a monoclonal antibody to tissue kallikrein and represents a kallikrein-inhibitor complex. These results indicate that tissue kallikreins can be initially synthesized as 37,000 or 30,000 dalton prepropeptides and then converted into a 38,000 dalton active form by proteolytic processing and glycosylation. The active kallikrein is capable of binding to an inhibitor to form a 92,000 dalton complex.

A monoclonal antibody to rat tissue kallikrein: use in biochemical and immunohistochemical studies.

A monoclonal antibody (V4G6) to rat tissue kallikrein (EC 3.4.21.35) has been developed and characterized. This clone showed no cross-reactivity with rat tonin, rat esterase A or human urinary kallikrein in either radioimmunoassay or enzyme-linked immunosorbent assay. The monoclonal antibody used in the direct radioimmunoassay detects purified rat urinary kallikrein in a range of 0.32 to 40 ng per tube. The displacement curves for rat submandibular gland, pancreatic and kidney extracts and urine were parallel with the standard curve of purified rat urinary kallikrein. Analysis of immunoprecipitates from [14C] DFP labeled submandibular gland extract with SDS-polyacrylamide gel electrophoresis, demonstrates that this antibody recognizes only one 38,000 dalton serine protease while polyclonal antiserum identifies multiple species. Using this specific monoclonal reagent, tissue kallikrein was localized immunohistochemically in the granular convoluted tubules and striated ducts of the rat submandibular gland and in the acinar cells of the rat pancreas. The results showed that the monoclonal antibody (V4G6) can specifically recognize a single kallikrein in the tissue extracts without cross-reacting with other kallikrein-related serine proteases. This monoclonal antibody can be used as a specific reagent for quantitation, identification and immunohistochemical studies of tissue kallikrein.

Characterization of monoclonal and polyclonal antibodies to human tissue kallikrein.

Monoclonal antibodies to purified human urinary kallikrein have been developed. Selection of antibody producing clones was based on 125I-kallikrein binding activity of hybridoma media in both radioimmunoassay and enzyme-linked immunosorbent assay. Three clones (2 IgG1, 1 IgG2b) were subcloned, characterized, and compared with the polyclonal antiserum generated in rabbits immunized with the purified kallikrein. With radioimmunoassay, mouse ascitic fluids or rabbit antisera dilutions showing 50% binding to 125I-kallikrein were 1:1.2 X 10(6) (E7A9), 1:1.2 X 10(5) (H6A6), 1:8.0 X 10(4) (E12H1), and 1:1.4 X 10(6) (the rabbit antisera). With enzyme-linked immunosorbent assay, mouse ascitic fluids from clones E7A9 and H6A6 showed half-maximal absorbance at dilutions of 1:2.1 X 10(5) and 1:1.0 X 10(5) respectively, and the polyclonal antiserum showed half-maximal absorbance at a dilution of 1:2.0 X 10(4). These monoclonal antibodies showed no cross-reactivity with rat tissue kallikrein, rat urinary plasminogen activator, or dog pancreatic kallikrein, while the polyclonal antiserum showed some cross-reactivity. The binding of monoclonal or polyclonal antibodies to 125I-human urinary kallikrein was not affected by human plasma kallikrein, thrombin, or urokinase in a competitive radioimmunoassay. By using purified human urinary kallikrein immobilized to agarose, antibodies produced by clones E7A9 and H6A6 and in the rabbit antisera were purified to homogeneity. Each of these affinity-purified antibodies inhibited the esterase activity, and two of the three inhibited the kininogenase activity, of human urinary kallikrein. A sandwich immunosorbent assay was developed to measure this kallikrein using monoclonal antibody from the clone E7A9 in conjunction with the polyclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.

Immunological analysis of rat pancreatic prokallikrein activation.

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.