Comparison of fluorographic methods for detecting radioactivity in polyacrylamide gels or on nitrocellulose filters

The commercial fluorographic enhancers, En3Hance or Amplify, were not as efficient as 2,5-diphenyloxazole (PPO) for detecting radioactively labeled proteins in polyacrylamide gels or on nitrocellulose filters. For most of the X-ray films tested, optimal preexposure was essential to obtain maximum sensitivity in fluorography or indirect autoradiography using intensifying screens. The best results were obtained with nitrocellulose by saturating the filters with PPO. The minimum levels of 35S/14C that could be detected on filters by autoradiography or fluorography in a 24-h exposure were 4 X 10(2) or 1 X 10(2) dpm cm-2 respectively. For 3H these levels were, respectively, 20 X 10(3) or 0.5 X 10(3) dpm cm-2.     

Bremsstrahlung radiation detection for small animal imaging using a CCD detector

The use of optical methods for the detection of radionuclides is becoming an established tool for preclinical molecular imaging experiments. In this paper we present a set of proof of principle experiments showing that planar bremsstrahlung radiation images can be detected with an intensifying screen using a small animal optical imager based on charge coupled device detector.We develop a bremsstrahlung source using a ³²P-ATP vial placed in a Plexiglas box, the source with an intensifying screen on top was placed inside a small animal optical imaging system. Bremsstrahlung radiation images were produced with the ³²P-ATP source only and also with a pair of pliers placed between the source and the screen. We found that the pair of pliers absorption image matches the shape of the object.Spatial resolution measurements were not performed however, the bremsstrahlung image of the pliers show that the resolution is relatively poor due to a large penumbra effect.We conclude that it is possible to produce planar bremsstrahlung images using optical imaging devices.     

Studies on the stability of 125I-purified porcine zona antigen (PPZA) obtained by chloramine-T glucose oxidase with lactoperoxidase and N-bromosuccinimide lodination procedures

A purified pig zona antigen (PPZA) was labeled with ¹²⁵I by chloramine-T (CL-T), glucose oxidase with lactoperoxidase (GO-LP), and N-bromosuccinimide (N-BS) iodination procedures. The ¹²⁵I-PPZA fractions thus obtained were reconstituted to a concentration of 25,000 dpm/0.1 ml and tested at weekly intervals for binding capacity to a fixed dilution (1:400,000) of PPZA antibody. The ¹²⁵I-PPZA obtained by the N-BS procedure showed greater stability compared to that obtained by CL-T and GO-LP methods. The nonspecific binding levels for the N-BS and GO-LP methods were comparable and significantly lower than for the CL-T method.     

Quantitative Studies on the Growth of Allogromia laticollaris (Foraminifera)

SYNOPSIS. The growth and reproduction of Allogromia laticollaris was studied. More schizozoites were generally produced in mixtures of food organisms than on single algal foods. In the presence of moderate numbers of bacteria, cultures with Phaeodactylum tricornutum, Chlorococcum sp., Nannochloris sp., and an unidentified chlorophyte (BL-1), added singly, were also highly productive. Schizogony was the dominant asexual form of reproduction. Binary fission and cytotomy also occurred in bacterized otherwise unfed controls. ³⁵S and ³²P are convenient labels for measuring growth of A. laticollaris when introduced into the system in the range of 1 × 10⁴ - 1 × 10⁵ dpm/ml (³²P specific activity ～ 2.03 MCi/g; ³⁵S specific activity ～ 95 μCi/g). Small allogromiids grew faster than did larger ones. By means of the Taylor series modification of the classical least-squares method, a continuous life-cycle representation was calculated for A. laticollaris for the conditions of the experiment. Four points of cell volume growth were maxima for reproduction: 1.0 × 10⁷μ per organism for curve I; 2.2 × 10⁷μ³ and 1.2 × 10⁷μ³ for curve II; and 6.7 × 10⁷μ³ for curve III.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane

Mouse leukemia L-1210 cells were iodinated with ¹²⁵I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of ¹²⁵I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ($\text{M}{}_{\mathrm{<mtext>m</mtext>}}$ 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized.     

Synthesis and in vitro evaluation of radioiodinated indolequinones targeting NAD(P)H: Quinone oxidoreductase 1 for internal radiation therapy

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase and is highly expressed in many human solid cancers. Because NQO1 can be induced immediately after exposure to ionizing radiation, we aimed to develop an NQO1-targeted radiolabeled agent to establish a novel internal radiation therapy that amplifies the therapeutic effects when combined with external radiation therapy. We designed three NQO1-targeted radioiodinated compounds including two ether linkage compounds ([¹²⁵I]1 and [¹²⁵I]2) and a sulfide linkage compound ([¹²⁵I]3) based on the selective binding of indolequinone analogs to the active site of NQO1 by the stacking effect. These compounds were successfully prepared using an oxidative iododestannylation reaction with high radiochemical yields and purity. In NQO1-expressing tumor cells, [¹²⁵I]1 and [¹²⁵I]2 were readily metabolized to p-[¹²⁵I]iodophenol or m-[¹²⁵I]iodophenol and [¹²⁵I]I⁻, whereas over 85% of the initial radioactivity of [¹²⁵I]3 was observed as an intact form at 1 h after incubation. The cellular uptake of [¹²⁵I]3 was significantly higher than those of [¹²⁵I]1 and [¹²⁵I]2. The uptake of [¹²⁵I]3 was specific and was dependent on the expression of NQO1. These data suggest that the novel NQO1-targeted radioiodinated compound [¹²⁵I]3 could be used as a novel internal radiation agent for the treatment of cancer.     

Use of diacylclycerol kinase to quantitate picomole levels of 1,2-diacylglycerol.

An extremely sensitive and specific analytic procedure is described for quantitating 1,2-diacylglycerol (DAG). Escherichia coli DAG kinase (EC 2.7.1.-) catalyzed the formation of [³²P]phosphatidic acid by the transfer of ³²PO₄ from [γ-³²P]adenosine 5′-triphosphate to DAG in linear proportion to the quantity of added DAG from 10 to 1000 pmol. This technique allowed reliable detection of as little as 2 pmol of added DAG. To assess levels of DAG in tissue lipid extracts a miniaturized method for silicic acid column chromatography was developed to separate DAG from triglycerides and phospholipids. When these procedures were applied to erythrocytes, lysis in the presence of Ca²⁺ caused a 10.6-fold rise in cellular DAG confirming not only the results obtained in an earlier investigation (1), but also the utility of this technique in the analysis of exceedingly small quantities of cellular DAG.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

An age-dependent proliferation is involved in the postnatal development of interstitial cells of Cajal in the small intestine of mice

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICCs) in the murine small intestine from 0-day to 56-day post-partum (P0–P56) by immunohistochemistry. The Kit⁺ ICCs, which were situated around myenteric nerve plexus (ICC-MY) formed a loose cellular network at P0 which changed into an intact one before P32. The density of ICC-MY increased from P0 to P12, and then decreased until P32. In contrast, the estimated total amount increased more than 15-fold at P32 than that at P0. Some Kit⁺/BrdU⁺ cells were observed at 24 h after one BrdU injection to the different-aged mice, and the number decreased from P2 to P24 and vanished at P32. Actually a few Kit⁺/BrdU⁺ cells can be observed at 1 h after one BrdU injection at P10, and the amount doubled at 24 h along with paired Kit⁺/BrdU⁺ cells. A number of BrdU⁺ ICCs were also labeled with CD34, CD44 and insulin-like growth factor I receptor. About 65% ICCs were BrdU⁺ at P32 after daily BrdU injection from P0. Our results indicate that an age-dependent proliferation is involved in the postnatal development of ICC-MY which increase greatly in cell numbers and proliferative ICCs may originate from ICCs progenitor cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Feng Mei and Jiang Zhu have contributed equally to this work.     

In vitro iodination of plant ribonucleic acids

The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 × 10⁴ to 10 × 10⁶ cpm/μg. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and ¹²⁵I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between ¹²⁵I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [³²P]chloroplast-rRNA. Thermal stability of DNA-¹²⁵I-rRNA hybrids was similar to the thermal stability of DNA-[³²P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties.     

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.     

A new model system for studying the phosphatidylinositol cycle

An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (³²P)P_i into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phosholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with K_i values ranged from 0.11 to 4.64 nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [¹²⁵I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [¹²⁵I]10 and [¹²⁵I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [¹²⁵I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [¹²⁵I]10 and [¹²⁵I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [¹²⁵I]13 exhibited high brain uptake (3.42% ID/g at 2 min) and rapid clearance from the brain (0.53% ID/g at 60 min), while [¹²⁵I]10 showed lower brain uptake (0.87% ID/g at 2 min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [¹²⁵I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Effect of short-duration constant exercise on permeability of cockerel aorta to 125I-albumin

To determine quantitatively the effect of short duration constant exercise on the rate of uptake (U) of intravenously injected 125I-labeled cockerel albumin (A) by the aorta of the adult cockerel, 24 birds divided into age-matched pairs, each pair consisting of an exercised and nonexercised control bird, were studied. The time period of heparinization, anesthesia, and time from injection of A (each member of each pair received about 50 microCi from the same batch) to the death of the animal (T) was identical for each member of each pair. The exercised animal was exercised at a constant speed of 3.2 kph at 0 degrees elevation for between 2 and 5 min on a treadmill. U was defined as accumulated wall radioactivity (dpm)/plasma radioactivity (dpm/ml) X endothelial surface area (cm2) X T (s). Free 125I in the injectate amounted to 1.29 +/- 0.31% (mean +/- SD). Free 125I in the plasma and the wall in the exercise and control animals was not significantly different: plasma 0.84 +/- 0.34% (mean +/- SD) and 0.55 +/- 0.18 (P less than 0.20); wall 3.38 +/- 5.64% and 6.42 +/- 4.72 (P less than 0.04). Injected A remaining in the blood at between 8 and 16 min after intravenous injection was 83 +/- 8.7% (n = 10) in the exercised and 82 +/- 10% (n = 7) in the control (P less than 0.2). U was greater in the exercise group in 9 out of 12 matched pairs (P less than 0.05). We conclude that U increases for short periods of constant exercise.     

Possibility for tumor detection with fatty acid analogs

Newly synthesized 17-[¹⁸F]fluoro-3-methylheptadecanoic acid ([¹⁸F]BMHDA), 16-[¹⁸F]fluoropalmitic acid ([¹⁸F]PA) and 15-(p-[¹²⁵I]iodophenyl)-3-R,S-methylpentadecanoic acid ([¹²⁵I]BMIPP), fatty acid tracers, were examined for the possibility of tumor imaging using 10 tumor models in rats and mice. The highest tumor/muscle ratios with [¹⁸F]BMHDA were 2.3 using rat tumor AH109A and 1.9 using mouse tumor B16F1 (tumor accumulation 0.41 ± 0.05, 4.29 ± 0.77% ID/g, respectively). Tumor/muscle ratios with [¹²⁵I]BMIPP and [¹⁸F]PA were lower than those with [¹⁸F]BMHDA. Labeled fatty acids seem to have a lower potential for tumor detection.     

Isolation of membranes from normal and thrombintreated gel-filtered platelets using a lectin marker

We report a technique for the isolation of plasma membranes from gel-filtered platelets exposed to thrombin, using ¹²⁵I-labeled lentil lectin as an external marker. Labeled cells not exposed to thrombin could be lysed on a gradient of glycerol. Those cells incubated with thrombin (without external Ca²⁺) were made more susceptible to breakage on a gradient of glycerol-EDTA, and homogenized with a zero-clearance homogenizer. Lysates were spun on gradients of sodium diatrizoate. The membranes obtained from such gradients have been examined by electron microscopy and by assays for enzymes and ¹²⁵I label. Membranes from platelets incubated without and with thrombin were found to be enriched as follows: lectin marker, 8- and 9-fold, respectively; phosphodiesterase, 9- and 12-fold; acid phosphatase, 2.5- and 2-fold. There is thus a particularly close correlation of lectin marker with phosphodiesterase, an enzyme characteristic of normal purified membranes.Monitoring for ¹²⁵I-labeled lentil lectin appears to be a useful procedure for following platelet membranes during isolations from relatively small quantities of blood.     

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Influence of Approach Velocity and Mesh Size on the Entrainment and Contact of a Lowland River Fish Assemblage at a Screened Irrigation Pump

Fish screens can help prevent the entrainment or injury of fish at irrigation diversions, but only when designed appropriately. Design criteria cannot simply be transferred between sites or pump systems and need to be developed using an evidence-based approach with the needs of local species in mind. Laboratory testing is typically used to quantify fish responses at intake screens, but often limits the number of species that can studied and creates artificial conditions not directly applicable to screens in the wild. In this study a field-based approach was used to assess the appropriateness of different screen design attributes for the protection of a lowland river fish assemblage at an experimental irrigation pump. Direct netting of entrained fish was used along with sonar technology to quantify the probability of screen contact for a Murray-Darling Basin (Australia) fish species. Two approach velocities (0.1 and 0.5 m.sec⁻¹) and different sizes of woven mesh (5, 10 and 20 mm) were evaluated. Smaller fish (<150 mm) in the assemblage were significantly more susceptible to entrainment and screen contact, especially at higher approach velocities. Mesh size appeared to have little impact on screen contact and entrainment, suggesting that approach velocity rather than mesh size is likely to be the primary consideration when developing screens. Until the effects of screen contacts on injury and survival of these species are better understood, it is recommended that approach velocities not exceed 0.1 m.sec⁻¹ when the desire is to protect the largest range of species and size classes for lowland river fish assemblages in the Murray-Darling Basin. The field method tested proved to be a useful approach that could compliment laboratory studies to refine fish screen design and facilitate field validation.