Search

The role of extracellular vesicles (EVs) as vehicles for cell-to-cell communication between a tumour and its environment is a relatively new concept. The hypothesis that EVs may be critical in co-opting tissues by tumours to generate distant metastatic niches is particularly pertinent to prostate cancer (PCa), where metastatic-tropism to bone predominates over other tissue types. The potential role of EVs as a means of communication between PCa cells and cells of the bone stroma such as osteoblasts, is yet to be fully explored. In this study, we demonstrate that PCa cell EVs both enhance osteoblast viability and produce a significantly more supportive growth environment for PCa cells when grown in co-culture with EV-treated osteoblasts (p< 0.005). Characterisation of the RNA cargo of EVs produced by the bone-metastatic PCa cell line PC3, highlights the EV-RNA cargo is significantly enriched in genes relating to cell surface signalling, cell–cell interaction, and protein translation (p< 0.01). Using novel techniques to track RNA, we demonstrate the delivery of a set of PCa-RNAs to osteoblast via PCa-EVs and show the effect on osteoblast endogenous transcript abundance. Taken together, by using proof-of-concept studies we demonstrate for the first time the contribution of the RNA element of the PCa EV cargo, providing evidence to support PCa EV communication via RNA molecules as a potential novel route to mediate bone metastasis. We propose targeting PCa EVs could offer a potentially important preventative therapy for men at risk of metastatic PCa.

In common with the assessment of chemical stressors many of the methods used for the assessment of risk of non-human biota exposed to radiation use tiered approaches. The initial tier within these approaches is designed to be simple and conservative with the aim to identify sites of negligible concern which can be excluded from more detailed assessment with a high degree of confidence. In this paper we compare the outputs of the screening tiers of three tools which are freely available as software packages. Outputs were compared for terrestrial and freshwater ecosystems assuming 1 Bq per unit media. Considerable variability between the risk quotients (RQ) estimated by the three approaches used was seen for some radionuclide-organism-ecosystem combinations. Reasons for this are explored with differences in transfer parameters used by the models being a common contributor. The large variation within RQ values estimated by the approaches requires further investigation as it does not promote the level of confidence required by the user. The practicalities of applying a single generic screening dose rate in assessments are also explored.

Environmental risk assessment methodologies often require background dose-rates for wild flora and fauna to be derived in a robust and transparent way. To facilitate this task, information can be organised around reference organisms (ROs), entities that can be used as representatives of the broader ecosystem. In this study, generic datasets have been collated on levels of naturally occurring radionuclides in sediments, seawater and ROs for marine ecosystems. In some cases, data gap filling methods have been required owing to a paucity of information. Once full datasets have been acquired, dose conversion coefficients (DCCs) can be applied and dose-rate calculations can be performed probabilistically allowing the variability in the underlying datasets to be accounted for. Unweighted absorbed dose-rates for marine reference organisms lie between 0.02–1.2 µGy h-1, whereas weighted dose-rates fall in the range 0.08 µGy h-1to 8.8 µGy h-1. Except for the special case of sea anemones/corals, the most important nuclides for the total dose-rates are 210Po and 226Ra for heterotrophic ROs (i.e. all vertebrate and invertebrate animals), whereas 226Ra and 228Th contribute most for autotrophic ROs (i.e. macroalgae, phytoplankton and aquatic plants). In addition, 40K has a considerable impact on unweighted dose-rates for all ROs. Non-parametric statistical tests have been applied to show whether there are differences between activity concentrations data for ROs and ICRP-defined Reference Animals and Plants (RAPs).

The Arctic environment is unique in terms of many factors that may influence the behaviour and fate of contaminants including the presence of permafrost, low temperatures, slow breakdown of organic compounds, short food chains and long recovery times for some species. This study introduces the issues surrounding the development of a bespoke environmental impact assessment methodology specifically for the consideration of radiation impacts on Arctic ecosystems. The approach allows for the impact on plants and animals from exposure to radiation to be quantified in a robust manner through integration of our current knowledge concerning transfer in the environment, resultant dose-rates from the presence of radioactivity within biota and their media and information concerning radiobiological effects on endpoints of concern from an environmental protection perspective.

There is general international acceptance of the need to demonstrate that the environment is protected from ionising radiation. In some countries requirements and guidelines for the protection of non-human biota are already in place. As a consequence a number of models and approaches have been proposed for the estimation of the exposure of non-human biota to ionising radiation. The IAEA EMRAS programme's Biota Working Group has conducted the most comprehensive intercomparison of the predictions of these approaches to date. In this paper, we present an overview of the activities of the Biota Working Group concentrating on its conclusions and recommendations.

Determination of background radiation dose-rates is important in the process of assessing risks to the environment from exposure to human activities both in terms of deriving the incremental dose-rate and as a point of reference for evaluating the significance of the exposure level. A consideration of the available literature on naturally occurring radionuclides in wild plants and animals quickly illustrates a paucity of data coverage in numerous cases. Most notable is the lack of comprehensive information for the important dose-forming radionuclides such as 210Po and 210Pb. In order to collate data concerning these radioisotopes for components of the ecosystem, field work was conducted in a semi-natural, mountainous location in central Norway. Preliminary (since no correction was made for ingrowth from 210Pb) whole-body activity concentrations of 210Po in 2 species of small mammal were commensurate with activity concentrations reported for reindeer muscle sampled at proximate locations, falling at a level of some 10s of Bq kg-1by fresh weight. Statistical analyses of the data showed that bank vole and shrew 210Po data constitute different populations with different mean ranks. Unweighted dose-rates attributable to the presence of internally distributed 210Po were calculated to be 0.07 μGy h-1for Bank vole.

ABSTRACTGamma phage specifically lyses vegetative cells of Bacillus anthracisand serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracisstrain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4°C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 °C ± 2°C for 20 h. When tested with 51 B. anthracisstrains and 49 similar non-B. anthracis Bacillusspecies, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracisstrains as well as a rare susceptible non-B. anthracisstrain, B. cereusATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.

Methodologies have been developed to allow impacts of ionising radiation on marine ecosystems to be evaluated within selected geographical settings. The stages in the assessment require initial information about unit concentrations of radionuclides in reference media to be collated. Activity concentrations for reference groups of flora and fauna and for representatives of these groups are then derived using an equilibrium concentration factor approach. Following this, dose-rates can be calculated using relevant dose conversion factors. Impacts on the environment are evaluated by comparison with dose-rates at which selected biological effects are known to occur and the natural radiation background. This paper focuses mainly on the transfer part of the assessment drawing attention to gaps in information that have been identified through review and providing an outline of methods that may be used to fill these gaps. Biokinetic models parameterised using allometric relationships have shown their utility in this respect. Initial work looking into the application of such models has met with some success with model predictions comparing favourably with the few empirical data available. Further work will be needed to validate the models for a large range of radionuclides and to consider uncertainty in model estimates through probabilistic analyses.

A framework for protection of the environment is likely to require a methodology for assessing dose rates arising from naturally occurring radionuclides. This paper addresses this issue for European aquatic environments through a process of (a) data collation, mainly with respect to levels of radioactivity in water sediments and aquatic flora and fauna, (b) the use of suitable distribution coefficients, concentration factors and global data where data gaps are present and (c) the utilisation of a reference organism approach whereby a finite number of suitable geometries are selected to allow dose per unit concentration factors to be derived and subsequent absorbed dose calculations (weighted or unweighted) to be made. The majority of the calculated absorbed dose, for both marine and freshwater organisms, arises from internally incorporated alpha emitters, with 210Po and 226Ra being the major contributors. Calculated doses are somewhat higher for freshwater compared to marine organisms, and the range of doses is also much greater. This reflects both the much greater variability of radionuclide concentrations in freshwater as compared to seawater, and also variability or uncertainty in concentration factor values. This work has revealed a number of substantial gaps in published empirical data especially for European aquatic environments.

The objectives of this paper are the presentation of site-specific radioecological information and development of models, which can be used to calculate doses to Arctic marine biota. The methodology of calculating doses from α, β, and γ-emitters is presented. This methodology is realized in form of computer code DOSBIO, and model parameters are evaluated. The following annual doses to biota are calculated: internal dose from radionuclides incorporated in the organisms; external dose from contaminated water; external dose from contaminated bottom sediments; total dose as a sum of internal and external doses. Dose conversion factors are calculated for 15 species of marine biota, including fish, sea mammals, molluscs. The list of species includes cod, plaice, haddock, herring, redfish, Greenland halibut, char, saithe, Greenland seal, white whale, ringed seal, bearded seal, and others. The calculations are performed for adult representatives of each species. Calculated dose conversion factors are presented for 11 different radionuclides (H-3, C-14, K-40, Sr-90, Tc-99, Sb-124, Cs-137, Eu-152, Po-210, Pu-239, Am-241). The current and potential doses to marine biota from the Barents and Kara Seas are estimated.

The objectives of this paper are the presentation of site-specific radioecological information and development of models, which can be used to calculate doses to Arctic marine biota. The methodology of calculating doses from α, β, and γ-emitters is presented. This methodology is realized in form of computer code DOSBIO, and model parameters are evaluated. The following annual doses to biota are calculated: internal dose from radionuclides incorporated in the organisms; external dose from contaminated water; external dose from contaminated bottom sediments; total dose as a sum of internal and external doses. Dose conversion factors are calculated for 15 species of marine biota, including fish, sea mammals, molluscs. The list of species includes cod, plaice, haddock, herring, redfish, Greenland halibut, char, saithe, Greenland seal, white whale, ringed seal, bearded seal, and others. The calculations are performed for adult representatives of each species. Calculated dose conversion factors are presented for 11 different radionuclides (H-3, C-14, K-40, Sr-90, Tc-99, Sb-124, Cs-137, Eu-152, Po-210, Pu-239, Am-241). The current and potential doses to marine biota from the Barents and Kara Seas are estimated.

The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of β-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg^-1 day^-1 ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (ΔTm value) or toxicity in the mouse−malaria model.

It has been suggested that a high pre–conceptual waist–to–hip ratio (WHR) is a good predictor of male offspring and, thus, in cultures that value male children, an androgenous body shape may be judged as most attractive. The predictive value of WHRs is based on studies measuring women who already have children and correlating their WHRs with the proportion of existing male offspring. However, carrying a male child may alter WHRs in a different way to carrying a female child, and a high WHR may be an effect rather than a cause of male offspring. In order to test the predictive power of a pre–conceptual WHR and offspring gender, we took WHR measures from 458 women who intended to become pregnant and then correlated this with the genders of their subsequent children. We found no significant correlation. It is therefore not clear why a high WHR is preferred in some cultures. We suggest that differences in attractiveness preferences between different ethic groups are actually based on weight scaled for height (the body mass index or BMI) rather than the WHR since although there will be a preferred optimal BMI for each ethnic group, which will balance environmental and health factors, this optimal BMI may differ between groups and environments.

It is now generally accepted that activation of tamoxifen occurs as a result of metabolism to alpha-hydroxytamoxifen. In this study, alpha-hydroxytamoxifen was given to female Wistar/Han rats (0.103 or 0.0103 mmol/kg, intraperitoneally, daily for 5 days). This resulted in liver DNA damage, determined by (32)P-post-labelling, of 3333 +/- 795 or 343 +/- 68 adducts/10(8) nucleotides, respectively (mean +/- SD, n = 4). Following HPLC separation, the retention times of the major alpha-hydroxytamoxifen DNA adducts were similar to those seen following the administration of tamoxifen. However, after rats were treated with alpha-hydroxytamoxifen (0.103 mmol/kg) for 5 days and the animals kept for up to 13 months, no liver tumours developed (0/7 rats), even with phenobarbital promotion (0/5 rats). GST-P foci were detected in the liver, but only after 13 months was their number or area significantly increased over the corresponding controls. When alpha-hydroxytamoxifen was given to female lambda/lacI transgenic rats (0.103 mmol/kg orally for 10 days) and the animals killed 46 days later, there was an approximate 1.8-fold increase in mutation frequency but no significant increase in G:C to T:A transversions as described after tamoxifen treatment. It is concluded that DNA damage alone, resulting from the short-term administration of alpha-hydroxytamoxifen, is not sufficient to initiate liver tumours even with phenobarbital promotion. As with tamoxifen, long-term exposure may be required to allow promotion and progression of transformed cells.

The aim of this study was to test the performance of a published dose-rate model, investigate the contribution of individual radionuclides to the total gamma air kerma rate (GAKR) and derive external doses to man in the Ribble Estuary, NW England. GAKRs were measured and sediment cores were collected in order to determine radionuclide specific activities with depth. The latter values were used as input data for the external dose-rate model. The model has a slight tendency to over-predict the GAKR, but, on average, the model predictions fall within ±26% of the measured value. Improvements, in the present case, might be made by accounting for core shortening and variations in soil density in the input data. The model predicted that, for exposed intertidal mud sites, a range of GAKRs between 0.011 and 0.022 µGy h-1was attributable to Springfields discharges alone. The contribution due to 234mPa and 234Th ranged between 20 and 60%. An excess GAKR (GAKR arising from anthropogenic emissions alone) of 0.139-0.150 µGy h-1, used in conjunction with relevant habit-survey data (for a potential critical group) and conversion factors, yielded a dose to man of 0.029-0.031 mSv year-1.

Daunorubicin is a highly potent trypanocide in-vitro but is inactive in-vivo. When daunorubicin is conjugated to bovine serum albumin or horse spleen ferritin using Schiffs base linkages, the complex is trypanocidal in-vitro and in-vivo. We have developed novel analytical methods, using HPLC with fluorimetric detection, for the quantitation of daunorubicin and doxorubicin in biological samples, either as unconjugated drug, or when covalently linked to macromolecules or particles. Ferritin-daunorubicin conjugate (25 mg kg−1) was administered intraperitoneally to mice infected with monomorphic Trypanosoma brucei rhodesiense; peak plasma levels occurred after 1·5 h, and were 5 times higher than those resulting from administration of an equivalent amount of unconjugated daunorubicin. Plasma levels then declined rapidly (t½ for 1–6 h period was 0·58 and 0·86 h respectively for conjugated and unconjugated daunorubicin). However, higher plasma levels were seen 24 h after treatment, suggesting the distribution profile of daunorubicin when conjugated to ferritin is multiphasic with resultant high levels of daunorubicin obtained for a prolonged time period.

Daunorubicin is a highly potent trypanocide in vitro but lacks significant in vivo activity. Distribution of the drug in the blood components and in trypanosomes from mice infected with Trypanosoma rhodesiensewas therefore examined. Drug is accumulated in the trypanosomes in a similar manner to, but to a greater extent than, its accumulation in platelets; it is, however, taken up by erythrocytes to a much smaller extent. Drug concentrations in trypanosomes, platelets and erythrocytes declined with its decline in plasma whereas in white blood cells the drug was retained at a constant level. Daunorubicin concentrations in plasma were several orders of magnitude higher than the in vitro trypanocidal concentration. Fluorescence microscopy showed that although daunorubicin reaches the trypanosome nucleus, its concentration decreases rapidly so that adequate levels of drug may not be sustained in the bloodstream form of the organism for a sufficient length of time to give a trypanocidal effect in vivo.

Firefly luciferin-luciferase was microinjected into single, intact photoreceptor cells of Limulus ventral eyes. In addition, total ATP content of whole end organs was measured. An attempt was made to correlate intracellular ATP levels with either the frequency of discrete events or responsiveness to light. Luciferin-luciferase luminescence emitted from a single photoreceptor cell increased after an injection of ATP or P[NH]P and decreased after injection of apyrase. We conclude that the luciferin-luciferase luminescence is monitoring increases as well as decreases in intracellular ATP concentration (ATPi). The ATPi was between 10(-3) and 10(-4) M. Bathing ventral eyes in vanadate or fluoride increased ATP content of whole end organs, increased ATPi in single photoreceptors, and increased the frequency of discrete events. However, apyrase decreased ATPi but did not affect the frequency of discrete events or responsiveness to light until ATPi was dramatically reduced. We conclude that changes in ATP levels are not strictly correlated with changes in responsiveness to light or to changes in the frequency of discrete events.

The effect of Shiga toxin, from Shigella dysenteriae 1, on the component reactions of peptide elongation were investigated. Enzymic binding of [3H]phenylalanine-tRNA to reticulocyte ribosomes was inhibited by 50% at 7 nM toxin. Elongation factor 1 (eEF-1)-dependent GTPase activity was also inhibited. Both reactions were not restored by addition of excess eEF-1 protein. In contrast, toxin concentrations of 200 nM were required to inhibit by 50% the elongation factor 2 (eEF-2)-dependent translocation of aminoacyl-tRNA on ribosomes. Addition of excess eEF-2 restored translocation activity. The eEF-2-dependent GTPase activity was unaffected at toxin concentrations below 100 nM, and Shiga-toxin concentrations of up to 1,000 nM did not affect either GTP.eEF-2.ribosome complex-formation or peptidyltransferase activity. Thus Shiga toxin closely resembles alpha-sarcin in action, both being primary inhibitors of eEF-1-dependent reactions. In contrast, the 60 S ribosome inactivators ricin and phytolaccin are primary inhibitors of eEF-2-dependent reactions of peptide elongation.

The implementation of a Quality Assurance (QA) programme in orthodontic radiography is designed to improve the quality of the resultant radiographs and to reduce the number of repeat exposures. This is particularly desirable in orthodontic practice where the majority of patients are young and therefore more at risk from the detrimental effects of X-rays. A programme is described and QA tests are given that may be applied in the surgery. Particular emphasis is placed on QA measures for extraoral radiography, since this is frequently undertaken in the treatment of the orthodontic patient.

Five nonlinear models were used to fit weight-age data for female cattle of diverse breeding and management. Comparisons were made among these models for goodness of fit, biological interpretability of parameters and computational ease. Three models, von Bertalanffy, Gompertz and logistic, consistently overestimated weights at early ages and the logistic underestimated mature weight. A four-parameter model, Richards, more accurately fit the data but was computationally more difficult than the three-parameter models. The Richards model which has a variable point of inflection is most appropriate for fitting weight-age data when goodness of fit, especially prior to 10 months of age, is critical. The Brody model is computationally easier and fit the weight-age data for ages past 6 months almost as well as the Richards model. Parameter estimates, or functions thereof, from all models can be interpreted as measures of size and rates of gain and maturing. However, parameter estimates from different models, although having the same biological interpretation, were not perfectly correlated. Genetic variations in rates of gain and maturing and mature size were observed.

The non-auditory physiological effects of extended exposure of pilots to high intensity noise were investigated. The health records of 22 professional pilots (FAA) were examined for recorded measurements of heart rate, systolic and diastolic blood pressure, serum cholesterol, and glucose. These data were compared to records of the same measurements from 29 non-flying FAA personnel of the same age, social and economic status as the pilots. The yearly means for each parameter were analyzed for changes with time, within the same population, and for differences between the two study groups. Audiometric histories were tabulated and compared, and noise levels inside aircraft were determined. Although it was demonstrated that FAA pilots were exposed to high occupational noise levels there was no indication that their exposure produced any significant non-auditory physiological response.

1. Several nucleotide and nucleotide analogues had striking effects when pressure‐injected into Limulusventral photoreceptor cells. The poorly hydrolysable GTP analogues guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS), guanylyl imidodiphosphate (Gpp[NH]p) and guanylyl (β, γ methylene) diphosphonate (Gpp[CH2]p) produced large increases in the frequency of ‘discrete events’ that were recorded from photoreceptors in darkness. This effect was only observed after the injected cell was exposed to light. Injection of the ATP analogue ATPγS had effects similar to those of the GTP analogues.

Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM‐EGTA and no added Ca2+ (EGTA‐ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA‐ASW but did not change further when dark‐adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA‐ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA‐ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM‐Ca2+. The light‐induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark‐adapted photoreceptors were bathed in EGTA‐ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA‐ASW. The total intracellular Ca content of whole end‐organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA‐ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light‐labile store.

Two rates of broiler litter (20 and 40 mt/ha) were compared to recommended rates of inorganic nitrogen (N), phosphorus (P), and potassium (K) in a double cropping system of spring sweet corn (Zea mays L. 'Silverqueen') and fall broccoli (Brassica oleracea L, 'Southern Comet')- Sweet corn matured one week earlier both years when fertilized with 40 mt/ha of broiler litter compared to commercial fertilizer. The early maturity may be due to improved P nutrition. Similar or higher yields of fall broccoli were produced with broiler litter following sweet corn than with commercial fertilizer.

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

1. Membrane potentials were recorded from single rods in the isolated retina of Bufo marinuswhile the ionic composition of the extracellular medium was rapidly changed. Substitution of 2 m Maspartate−for Cl−produced a prompt depolarization of horizontal cells, but no modification of either resting potential or response to light in receptor cells. This implies that feed‐back from horizontal cells to receptor cells was not active.

1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulusventral photoreceptor cells to concentrations greater than 1 m M.

1. Intracellular recordings were made from the outer segments of rods in the isolated, superfused retina of Bufo marinus. Cells were impaled under observation with a compound microscope fitted with an infra‐red image converter. Changes of membrane voltage and some concomitant changes of input resistance were measured in response to light, membrane polarization and iontophoretic injections.

1. The possible role of pH changes in mediating light adaptation in Limulus ventral photoreceptor cells was studied by intracellular injection of zwitterionic pH buffers. The intracellular concentration of buffer was estimated by inclusion of a radioactive marker in the injection solution. 2. The light‐induced increase of intracellular Ca2+ concentration was monitored by intracellular aequorin. The light‐induced increase of Ca2+ concentration was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 3. The progressive decrease in responsiveness during intracellular ionophoretic injection of Ca2+ was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 4. Photoreceptors of both Limulus and Balanus were impaled with two micropipettes and voltage clamped. Membrane current induced by a prolonged steady illumination declined from an early transient to a plateau. This delayed decline of current indicates a light‐induced reduction of sensitivity (i.e. light adaptation). The wave forms were similar before and after injection of pH buffer to an intracellular concentration of about 200 mM. 5. We conclude that it is unlikely that a light‐induced change of cytosolic pH mediates light adaptation in Limulus (and Balanus) photoreceptors.

Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor‐enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1‐methyl‐3‐isobutyl xanthine or forskolin and was increased almost 100‐fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug‐induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis‐resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat‐treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc‐ mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that neither cyclic AMP nor adenylate cyclase activation is a necessary or obligatory component of the excitation mechanism in Limulus ventral photoreceptors.

The central organization of anticipatory postural adjustments was investigated by examining the influence of preparatory set on the temporal relationship between postural and arm (focal) muscle activation. Surface EMG was recorded from the right tibialis anterior, lateral gastrocnemius, anterior deltoid and posterior deltoid muscles when pushing or pulling on a stiff handle. Preparatory set was manipulated by informing the subject of the upcoming direction of responding with a 80, 50 or 20% certainty. This created high, neutral and low levels of preparatory set, respectively. All six subjects showed activation of postural muscles in advance of focal muscles for both push and pull responses. However, only three subjects showed the expected effect of preparatory set on reaction time performance, i.e., an increase of reaction time with decreasing response probability. For these three subjects, the time between the activation of postural and focal muscles was the same for the high and neutral levels of preparatory set, but increased with a low level of preparatory set. The increased postural-focal latency for the low preparatory set condition was due to a longer delay for the activation of the focal muscles but not the postural muscles. This finding suggests that anticipatory postural adjustments and the activation of focal muscles are triggered by separate motor commands.

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.

The aim of the study was to determine whether fatty acid ethyl esters, nonoxidative products of ethanol metabolism selectively present in organs damaged by ethanol abuse, are detectable in the serum after ethanol ingestion. Serum samples of hospital emergency room patients with positive (n = 32) and negative (n = 5) blood ethanol levels were assayed for fatty acid ethyl esters. In a separate study, five healthy subjects received an ethanol dose based on body weight mixed with fruit juice in a 1:2 ratio and administered by measured ingestion. Fatty acid ethyl esters were found in the serum of hospital emergency room patients with positive blood ethanol levels. The concentration of fatty acid ethyl esters in these patients correlated with the concentration of blood ethanol (r = 0.57; 95% confidence interval 0.28 to 0.77; P = 0.0002). In the controlled ethanol ingestion study with five healthy subjects, it was also determined that the serum fatty acid ethyl ester concentration began to decrease within 2 h of the time ethanol ingestion had been stopped. The fatty acid ethyl esters in the serum were bound to lipoprotein and albumin, and there was a higher percentage of saturated fatty acids in the FAEE pool than in the serum free fatty acid and triglyceride pools. These studies indicate that fatty acid ethyl esters, which have been implicated as mediators of ethanol-induced organ toxicity, are present in serum after ethanol ingestion.

1. In order to study the role of atrial pressure and atrial stretch on the release of atrial natriuretic peptide we have measured plasma atrial natriuretic peptide concentration, urine output and haemodynamic variables in eight patients during and 30 min after the relief of cardiac tamponade. This condition is characterized by high atrial pressure with little or no atrial stretch. 2. Relief of tamponade was associated with a rise in urine output (53 ± 27.9 to 101 ± 24.5 ml/h, mean ± sem; P = 0.09), systolic blood pressure (95 ± 9.6 to 126 ± 7.0 mmHg, P < 0.0001), and plasma atrial natriuretic peptide concentration (369.5 ± 70.9 to 490.3 ± 94.7 pg/ml, P < 0.05) despite a large fall in right atrial pressure (18.6 ± 1.6 to 9.5 ± 1.3 mmHg, P < 0.001). 3. These results suggest, therefore, that an increase in atrial stretch, rather than in atrial pressure, stimulates the release of atrial natriuretic peptide.

Our purpose was to determine whether the reported alteration of protein drug binding after heparin administration in man was artifactual as a result of continued in vitro activity of triglyceride lipases. The lipoprotein lipase inhibitors protamine (14 mg/ml) and ethylenediaminetetraacetic acid (10 mg/ml) were added to blood samples from 11 healthy subjects before and 15 min after 1000 USP units of intravenous heparin. Heparin elevated total nonesterified fatty acid (NEFA) concentration (P < 0.001 ) and the free fractions of lidocaine, diazepam, and propranolol (P < 0.001 for all). The presence of the lipase inhibitors diminished the heparin-induced elevation of NEFA (P < 0.001) and free fractions of lidocaine (P < 0.001) and diazepam (P < 0.001), but these values were still greater than control. The inhibitors reduced propranolol binding in control samples and did not diminish the effects of heparin. The change in NEFA concentrations correlated with the free fraction changes of all three ligands (r = 0.739 to 0.849). These data suggest that the heparin-induced protein binding changes are, to a large extent, in vitro artifacts.

Summary Dopamine D1 and D2 receptors were determined in brain tissue taken post mortem from schizophrenic patients previously known to have had tardive dyskinesia and yet who had not received neuroleptic drug treatment for over one year prior to death. In comparison with age-matched control subjects, diminished D2 receptor density was observed in striatal regions, while these receptors appeared to be increased in the pallidum, an area of the brain particularly implicated in the production of dyskinesias. D l receptors showed similar tendencies to lower numbers in the striatum in tardive dyskinesia.

Intracellular recordings were made from squid photoreceptors using micropipettes filled with 4 M potassium acetate. Resting potentials were about -55 mV, and light stimuli induced membrane voltage to become more positive (depolarized). For an intense stimulus there was a transient peak depolarization that declined to a plateau; membrane potential exceeded 0 V during the peak depolarization. The magnitude of the light response was decreased either by passing steady depolarizing current through the recording pipette or by bathing the retina in low Na+ sea water. The waveform of the response to an intense stimulus was altered by bathing the retina in low Ca++ sea water or intracellular injection of EGTA; for both of these treatments, the decline from the transient peak depolarization to the plateau was lessened and the responses became more “square”. Squid photoreceptors were found to be very sensitive to hypoxia. These results, taken together, are consistent with the interpretations that (1) the majority of the light-induced current is carried by Na+, and (2) a rise in (Ca++)in is a step in the mechanism underlying the decline from the peak transient depolarization to the plateau.

Hybridoma cell lines which produce monoclonal antibodies to Shiga toxin from Shigella dysenteriae 1 were prepared. The monoclonal antibodies were all of the immunoglobulin G1 isotype and differed in their ability to neutralize cytotoxicity and to bind to Shiga toxin in a solid-phase radioimmunoassay. When used for immunoblot analysis, these antibodies were able to identify specifically both nicked and unnicked Shiga toxin in crude lysates of S. dysenteriae.

Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.

1. Limulus ventral photoreceptor cells were voltage‐clamped with two intracellular micro‐electrodes. The light‐induced membrane current was recorded for brief stimuli. From observation of discrete waves (quantum bumps) at low stimulus energies and the early receptor potential at high energies, the stimulus energy was related to the number of rhodopsin molecules photosiomerized. 2. In the dark‐adapted cell the log (peak light‐induced current) reached almost its maximum value when about 10(3) of the 10(9) rhodopsin molecules in the cell were photoisomerized. 3. The magnitude of the maximum light‐induced current was not significantly altered after iontophoresis of EGTA into the cell. This treatment is known to counteract the Ca2+‐mediated reduction in sensitivity to light. 4. Current pulses were injected into the unclamped cell during the receptor potential. The form of the voltage deflexion (a step followed by a curve) suggested that the effective electrical equivalent of the cell was a membrane capacitance in parallel with a light‐dependent membrane resistance, Rm, and in series with another, light‐invariant, resistance, Rs. Rs ranged from 7 to 24 k omega (five cells). 5. During a receptor potential the ratio Rm/Rs was never observed to fall below 1.7 no matter how intense the light flash. Hence, it is concluded that the light‐induced current saturated essentially because Rm fell to a minimum value. 6. Charging curves gave a value for the capacitance, and hence the area, of the surface membrane. From this it was estimated that there were 10(5)‐10(6) microvilli on each cell. 7. These results show that the light‐induced increase in membrane conductance in a dark‐adapted cell comes close to its maximum value when the number of photoisomerizations is about 1/1000 the total number of microvilli. We suggest that absorption of a photon by a rhodopsin molecule in a microvillus causes an increase in membrane conductance on parts of the surface membrane beyond that microvillus. 8. In the presence of moderate background illumination the sensitivity to non‐saturating superimposed flashes was greatly decreased (e.g. by 10(3) while the saturating light‐induced current was only slightly decreased (e.g. by 15%). At higher background intensities the saturating light‐induced current was further decreased (e.g., with a background that photoisomerized 10(6.25) molecules per second the saturating light‐induced current was reduced by 47%).