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ObjectiveFlathead catfish are rapidly expanding into nonnative waterways throughout the United States. Once established, flathead catfish may cause disruptions to the local ecosystem through consumption and competition with native fishes, including species of conservation concern. Flathead catfish often become a popular sport fish in their introduced range, and so management strategies must frequently balance the need to protect native and naturalized fauna while meeting the desire to maintain or enhance fisheries. However, there are currently few tools available to inform management of invasive flathead catfish (Pylodictis olivaris). We describe a suite of microsatellite loci that can be used to characterize population structure, predict invasion history, and assess potential mitigation strategies for flathead catfish.ResultsOur panel of 13 microsatellite loci were polymorphic and appear to be informative for population genetic studies of flathead catfish. We found moderate levels of diversity in four nonnative collections of flathead catfish in the Pennsylvania and Maryland sections of the Susquehanna River and the Schuylkill River, Pennsylvania. Analyses suggested patterns of genetic differentiation within- and among-rivers, highlighting the utility of this marker panel for understanding the structure and assessing the degree of connectivity among flathead catfish populations.

Low-grade endometrial stromal sarcoma (LGESS) is a rare malignant uterine tumor with the potential to metastasize years after the primary resection. There is a broad differential diagnosis for endometrial stromal sarcomas (ESS), including both benign and malignant entities. Herein, we present the case of a 64-year-old female with metastatic LGESS to the lung, diagnosed by cytology, 24 years after her initial presentation. This report discusses the cytomorphologic and histopathologic characteristics, and ancillary studies including immunohistochemical stains and recent advances in molecular diagnostics of ESS. Accurate diagnosis of spindle cell lesions in the lung can be challenging. As such, this case highlights the instrumental role of ancillary testing and molecular diagnostics to achieve a more definitive diagnosis.

Summary Glucocorticoids are important mediators of energy utilization for key physiological processes, including immune function. Much work has focused on the effects of energy limitation and stress for key physiological processes such as reproduction and immunity. However, it is unclear how stress alters energy use across different energy states, and the physiological ramifications of such effects are even less clear. In this study, we altered energy and stress states of an infrequent feeder, the terrestrial gartersnake (Thamnophis elegans), using fasting and repeated restraint stress (chronic stressors) to test how these challenges interacted to affect immune function, energy metabolites and glucocorticoid reactivity (a traditional indicator of stress state) to restraint stress, a standardized, acute stressor. After this acute stressor, the snakes which had received chronic stress had increased glucocorticoid reactivity, and both treatments altered energy metabolite use and storage. Evidence of interaction of food restriction and chronic stress treatments on innate immune function and energy metabolites (triglycerides and glycerol) suggests that stress alters energy use in a manner dependent on the energy state of the animal. Snakes have a remarkable ability to maintain functionality of key physiological processes under stressful conditions but are still susceptible to multiple simultaneous stressors, a situation increasingly prevalent in our ever-changing environment.

The energetic cost of immunity depends on many factors, including the type of challenge, the timing of the response, and the state of the animal. We measured changes in the standard metabolic rates of side-blotched lizards (Uta stansburiana Baird and Girard, 1852) in response to different immune challenges and nutritional states. In the first experiment, lizards were randomly assigned to one of four treatments: lipopolysaccharide (LPS) injection (to stimulate the response to a pathogen), cutaneous biopsy (as a proxy to a superficial wound), both injection and biopsy, or neither (control). Four and five days later, we measured the standard metabolic rates of the lizards. In response to healing a cutaneous wound, lizards reduced metabolic rate and lost body mass. Healing rate was also inversely related to weight loss, but LPS had no effect on body mass or metabolic rate. In the second experiment, a new set of lizards were randomly assigned to a high-food or low-food diet and administered a cutaneous biopsy. As in the first experiment, we observed a reduction in metabolic rate after wounding; moreover, this decrease was positively correlated with the rate of healing. We observed higher rates of metabolism in lizards that ate more food, but food consumption was unrelated to the decrease in metabolic rate following the biopsy. These experiments demonstrate the dynamic nature of the immune response in response to immune challenge and the state of the organism.

The recently developed technique of aerosol chemical ionization mass spectrometry is used to study the reaction of ozone with particles consisting of unsaturated organic molecules, including oleic acid, linoleic acid, oleyl alcohol, and 1-octadecene. The reactive uptake coefficients, γ, for polydisperse aerosols with mean diameters of 800 nm are determined from the rates of loss of the particle species to be (7.5 ± 1.2) × 10-4 for oleic acid, (1.1 ± 0.2) × 10-3 for linoleic acid, (7.5 ± 1.3) × 10-4 for oleyl alcohol, and (2.4 ± 0.4) × 10-4 for 1-octadecene. The ozonolysis products of oleic acid particles are studied in detail with simultaneous detection of the four primary products: nonanal, nonanoic acid, 9-oxononanoic acid, and azelaic acid. All four products are found to exist in the reacted particles, though nonanal is also detected in the gas phase indicating that it partially evaporates. The yield of azelaic acid is determined to be 0.12 ± 0.04, and the yields of nonanal and 9-oxononanoic acid are ...

A novel photoacoustic spectrophotometer (PAS) for the measurement of gas-phase and aerosol absorption over the UV-visible region of the spectrum is described. Light from a broadband Hg arc lamp is filtered in eight separate bands from 300 to 700 nm using bandpass interference filters (centered at 301 nm, 314 nm, 364 nm, 405 nm, 436 nm, 546 nm, 578 and 687 nm) and modulated with an optical chopper before entering the photoacoustic cell. All wavelength bands feature a 20-s detection limit of better than 3.0 Mm(-1) with the exception of the lower-intensity 687 nm band for which it is 10.2 Mm(-1). Validation measurements of gas-phase acetone and nigrosin aerosol absorption cross sections at several wavelengths demonstrate agreement to within 10% with those measured previously (for acetone) and those predicted by Mie theory (for nigrosin). The PAS instrument is used to measure the UV-visible absorption spectrum of ambient aerosol demonstrating a dramatic increase in the UV region with absorption increasing by 300% from 405 to 301 nm. This type of measurement throughout the UV-visible region and free from artifacts associated with filter-based methods has not been possible previously, and we demonstrate its promise for classifying and quantifying different types of light-absorbing ambient particles.

The alkaloid 12-epi-hapalindole E isonitrile, from a cyanobacterial Fischerella species, and the indolophenanthridine calothrixin A, from Calothrix, inhibited Escherichia coli RNA polymerase competitively with respect to ATP, and non-competitively with respect to UTP. The inhibition was dependent on the order of addition of the inhibitors. The K(I) values, with ATP as the variable substrate, were 1.3+/-0.2 mM and 0.23+/-0.11 mM, respectively. Based on comparisons with the sensitivity of whole cells to these inhibitors, it is concluded that other targets in addition to RNA polymerase may also be implicated in their action.

Mechanisms that terminate signals from activated receptors have potential to influence the magnitude and nature of cellular responses to insulin. The aims of this study were to determine in rat liver endosomes (the subcellular site of insulin signal termination) whether dissociation of insulin from its receptor was a pre-requisite for ligand degradation and whether the state of receptor phosphorylation influenced the dissociation and hence endosomal degradation of insulin and/or receptor recycling. Following in vivo administration of 125I-[A14]-insulin or analogues (native, X10 or H2, relative binding affinities 1:7:67) livers were removed and endosomes prepared. In the endosomal preparations a significantly greater percentage of both analogues were receptor-bound than native insulin with concomitantly less ligand degradation. When rats were injected with protein-tyrosine phosphatase inhibitors (peroxovanadium compounds bpV(phen) or bpV(pic)) before insulin, endosomal insulin receptor phosphotyrosine content, assessed by Western blotting, was increased as was receptor-bound 125I-[A14]-insulin, whilst insulin degradation was decreased. Peroxovanadiums also completely inhibited recycling of insulin receptors from endosomes. However, treatment of freshly isolated endosomes with acid phosphatase which completely dephosphorylated the insulin receptor, did not return the rate of insulin dissociation and degradation to control values, suggesting that peroxovanadium compounds elicit their effect on binding and degradation via a mechanism other than as protein-tyrosine phosphatase inhibitors. We conclude that promotion of sustained receptor binding decreases endosomal insulin degradation and extends the half-life of the activated endosomal receptor, which in turn would be expected to potentiate insulin signalling from this intracellular compartment.

Insulin degradation within isolated rat liver endosomes was studied in vitro with the aid of three 125I-insulin isomers specifically labelled at tyrosine (A14, B16 and B26). Chloroquine and 1,10-phenanthroline were used to minimize insulin proteolysis during endosome preparation, whereas the manipulation of endosomal processing of insulin in vitro by Co2+ ions (to activate) and 1,10-phenanthroline (to inhibit) permitted the study of degradation intermediates and their time-dependent production. Structural and kinetic analysis of intermediates isolated from both intra- and extra-endosomal compartments allowed the determination of major cleavage sites and the probable sequence of proteolytic events. It was found that 125I-tyrosine is the ultimate labelled degradation product of all iodo-insulin isomers, suggesting that endosomal proteases are able to degrade insulin to the level of its constituent amino acids. 125I-tyrosine was also the only radiolabelled product able to cross the endosomal membrane. Intra-endosomal insulin degradation proceeds via two inter-related cleavage routes after metalloendoprotease cleavage of the B-chain. One pathway results from an initial cleavage in the centre region of the B-chain (B7–19), probably at B14-15, whereas the major route results from a cleavage at B24-25. B24-25 cleavage removes the B-chain C-terminal hexapeptide (B25–30), which is subsequently cleaved by an aminopeptidase activity to produce first the pentapeptide B26–30 and then 125I-tyrosine. The isolation of intact radiolabelled A-chain from the degradation of 125I-[A14]-insulin suggests that further degradation of proteolytic intermediates containing cleaved B-chain proceeds via interchain disulphide reduction. The A-chain is then processed by several cleavages, one of which occurs at A13-14.

Indicator dilution curves of 51Cr-EDTA and 125I-insulin injected into perfusate entering the rat liver in vivo are used as a basis for developing a mathematical model of insulin distribution and uptake within the organ. EDTA is not taken up by liver cells and therefore serves as a "volume marker" whose dilution curve reflects the characteristics of perfusate flow through the organ and through the cannulae. These two components are modelled separately but the same approach is used in each case, that is, the minimum number of delayed exponential terms sufficient to reproduce the dilution curves is determined and used as the basis for modelling. This allows the cannula, vascular and sinusoidal volumes to be identified and described as corresponding compartmental configurations. The shape of the insulin dilution curve is additionally influenced by binding to, and uptake by, liver cells. Binding of insulin to receptors on hepatocyte plasma membranes and subsequent internalization of the insulin-receptor complex is modelled by the introduction of additional compartments but this is found to be insufficient unless non-specific binding is also taken into account. The accuracy of determination of the rate constants for insulin-receptor dissociation and for endocytosis is improved by a sudden reduction in the pH of the perfusate about 100 sec after injection of the insulin bolus. This releases any residual receptor bound insulin and is modelled by a sudden shift in the insulin-receptor dissociation rate constant. Matching of the complete model to individual pairs of 51Cr-EDTA and 125I-insulin dilution curves allows vascular and sinusoidal volumes to be determined together with the binding and endocytic rate constants. Use of the model to investigate the effect of substances that modify these rate constants is briefly illustrated in the case where the liver is preperfused with 5 mM indomethacin. The model can also be used to simulate the internal distribution and uptake of insulin with any nominated input function and any set of parameters; this is illustrated by comparing the impulse response in the normal case and that in which indomethacin has been preperfused. Although the present study is confined to insulin, the model and methodology that is developed should be applicable to other ligands for which the hepatocyte carries specific receptors.

Quinones such as Calothrixins A and B display a range of biological properties. As part of our ongoing studies to elucidate the mechanism of action of the Calothrixins, several related quinones were synthesized and tested for biological activity. The results of the structure–activity relationship (SAR) studies are reported here.

Most laser-based aerosol mass spectrometers rely on a single ultraviolet laser to both ablate and ionize the aerosol particle. This technique produces complex and fragmented mass spectra, especially for organic compounds. The approach presented here achieves a more robust and quantitative analysis using a CO2 laser to evaporate the aerosol particle and a vacuum ultraviolet laser to ionize the vapor plume. Vacuum ultraviolet laser ionization produces little fragmentation in the mass spectra, making the identification of an aerosol particle's constituents more straightforward. An analysis of simple, three-component mixtures of aniline, benzyl alcohol, and m-nitrotoluene shows that the technique also provides a quantitative analysis for all the components of the mixture. Furthermore, the detection of predominantly parent ion signal from anthracene particles demonstrates the utility of the technique in the analysis of lower vapor pressure, solid-phase aerosols. Finally, we discuss the potential and limitations of this technique in analyzing organic atmospheric aerosols.

The concentration-dependence of the inhibition of whole-cell hydrogen formation by oxygen has been measured in the trichomonads Trichomonas vaginalis and Tritrichomonas foetus, and compared with the oxygen inhibition of the in situ hydrogenase activity as measured by a tritium exchange assay. The inhibition profiles closely paralleled each other, suggesting that hydrogenase is the primary site of inhibition of anaerobic fermentative metabolism. In addition the inhibition profile for isolated hydrogenosomes was measured and shown to be similar to that for whole organisms. Ascorbate peroxidase was shown to be present in both organisms whereas catalase was confirmed to be present only in Tritr. foetus. The kinetic parameters of both enzymes were measured and their respective roles in oxygen protection discussed.

Indomethacin inhibits autophosphorylation of the insulin receptor. The lack of a consensus as to whether phosphorylation of the insulin receptor is necessary for its endocytosis prompted an investigation into the effects of indomethacin on the uptake and subcellular processing of insulin in the perfused rat liver. Indomethacin did not affect total uptake of insulin by the liver, but there was a concentration dependent inhibition of transfer from the plasma membrane to the endosomes (1mM, 32% inhibition; 5mM, 90% inhibition). Compartmental analysis showed the endocytic rate constant to be inhibited by 82% at 5mM indomethacin (0.0084 to 0.0015 sec-1). The similarity between the level of inhibition of autophosphorylation and the inhibition of endocytosis suggest that phosphorylation of the receptor is necessary for endocytosis. Indomethacin at 5mM completely abolished efflux of insulin degradation products from the perfused liver, suggesting that internalisation is an absolute requirement for degradation.

Les derives de l'hematoporphyrine agiraient en augmentant la proportion de membrane rugueuse dans le reticulum endoplasmique de foie de rat, ce qui pourrait etre le reflet d'une augmentation de l'activite de synthese proteique

Counter-current partition, sucrose density gradient centrifugation and enzymic analysis were used to explore the changes in rat liver organelles induced by phenobarbital. There was a small increase in partition coefficient and marker enzyme activity of the endoplasmic reticulum. The modal density of the marker enzyme showed a significant decrease reflecting the proliferation of the smooth endoplasmic reticulum. The mitochondria showed a bimodal distribution with a small reduction of marker enzyme activity. In contrast, lysosomes and peroxisomes were relatively unaffected by phenobarbital treatment. Phenobarbital caused a small but statistically insignificant increase in γ-glutamyl transferase activity: density gradient centrifugation studies indicated that the increased activity was predominantly in the biliary canalicular elements. In contrast, cytosolic γ-glutamyl hydrolase activity was strikingly reduced by phenobarbital treatment.

SUMMARY: The cyanobacterium Anabaena cylindrica was found to have nickel-dependent urease activity. Addition of nickel to a nickel-free culture led to recovery of urease activity. This was not prevented by chloramphenicol. Cell-free extracts of nickel-depleted cells did not regain activity upon addition of Ni2+. Enzyme synthesis was constitutive; similar urease activities occurred in urea-, NO− 3- or NH3-grown cultures or those grown under N2-fixing conditions. The urease activity had hyperbolic kinetics, with an apparent K m with respect to urea of 1.3 ± 0.6 (SD) mM. Enzyme activity was enhanced up to two fold by MgSO4 and MnSO4 at pH 7.5. At 10 mM-urea, cells hydrolysed urea and excreted NH3 in excess of biosynthetic requirements.

A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments. Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. Gamma-Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5'-nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.

gamma-Glutamyl transferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, ED 2.3.2.2) has been partially purified from both whole rat liver (600-fold) and from isolated biliary tract (1200-fold). The most highly purified fraction gave two protein bands on polyacrylamide gel electrophoresis, the major band alone having enzyme activity. The enzyme purified from biliary tract appears identical to that from whole liver preparation according to molecular weight, kinetic parameters and the effects of various inhibitors. Three liver cell-types; parenchymal, Kupffer and biliary tract were isolated by perfusion of the rat liver in situ with collagenase, followed by selective cell isolation. Approx. 80-90% of the total recovered enzyme activity was found in the biliary tract. Nearly 50% of the apparent enzyme activity in the parenchymal cell was attributable to a nonspecific hydrolase.

125I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subceltular processing of receptor-bound internalized insulin.

Studies of the interactions between dissimilar macromolecules are of considerable importance but pose special technical problems in comparison with small-ligand binding studies, since dialysis procedures are frequently inapplicable. This communication describes a sedimentation velocity procedure for detecting the binding of proteins and polysaccharides to lipoproteins and for examining perturbations resulting from the introduction of selected ligands. It makes use of the significant difference in density between the lipoprotein and the interacting macromolecule. The procedure is illustrated by studies of the binding of serum low density lipoprotein (LDL) to concanavalin A (con A) and heparin.

Using ( A14 - 125 I)-insulin as a tracer, insulin proteolytic activity in rat liver was found to be localized both to the cytosol and the endoplasmic reticulum. The membrane-associated activity was highly latent (70–80%). Both cytosolic and particulate activities had similar K m values and M r of approx. 300 000 by gel filtration. Both were strongly inhibited by diamide (90%), but were unaffected by leupeptin or pepstatin. A comparison of the subcellular distributions with various 125 I-isomers of insulin as tracers showed that both particulate and cytosolic activities were highest with ( A14 - 125 I)-insulin.

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

The binding and uptake of insulin in perfused rat liver has been investigated with specifically labelled 125I-A14-tyrosyl insulin as a tracer and compared with a commercially available iodo-insulin preparation. The commercial preparation did not show saturation uptake kinetics and the clearance from the perfusate remained low and constant throughout a wide concentration range. A14 labelled insulin showed saturation kinetics and high clearance at low carrier concentration, falling rapidly with increasing carrier concentration and reaching a steady state value of 1 ml/min. These results emphasize the importance of using specifically labelled insulin in physiological and biochemical studies of hepatic insulin metabolism. Perfusion with A14 tyrosine-labelled insulin at 4 degrees C showed apparent saturation with binding to the plasma membrane fraction. Perfusion at 37 degrees C also showed apparent saturation with uptake predominantly to the ligandosome fraction. These results implicate the plasma membrane-ligandosome pathway in the hepatic uptake of insulin at both physiological and pharmacological concentrations of the hormone.

Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.

Fluorescence microscopy and analytical subcellular fractionation were used to investigate the hepatic localisation of haematoporphyrin derivative (HPD) after intraperitoneal administration. HPD was found to rapidly accumulate in the liver and then to slowly decline over 48 hr. Fluorescence microscopy showed that although at early times porphyrins could not be localised to a particular cell type, at 24 hr porphyrins were preferentially localised to the Kupffer cells of the liver. Subcellular fractionation studies indicated that the initial rapid uptake of HPD was to the cytosol. However, at 24 hr, porphyrins appeared to be localised to lysosomes. Lysosomal localisation was confirmed using the selective organelle perturbant, Triton WR 1339. No evidence was found either at the light microscope level or by subcellular fractionation to suggest association of HPD with other organelles. HPLC analysis showed that the porphyrins present in the plasma and in the cytosol and lysosome fractions were mainly the (RS, SR) and (RR, SS) diastereoisomers of haematoporphyrin and the two position isomers 8-(1-hydroxyethyl)-3-vinyldeuteroporphyrin and 3-(1-hydroxyethyl)-8-vinyldeuteroporphyrin. There was no evidence for the involvement of dimers such as dihaematoporphyrin ether.

Uptake of 125I-insulin by the liver of intact rats is followed by rapid translocation of label to low-density vesicles. Subcellular-fractionation studies indicate that, although the 125I associated with these vesicles is predominantly trichloroacetic acid-precipitable, there is an acid-soluble component arising from processing of the hormone in vivo. H.p.l.c. analysis indicates that the acid-precipitable 125I is not intact iodoinsulin, but may correspond to ‘clipped insulin’. Isolated low-density vesicles degrade the acid-precipitable iodopeptide intravesicularly when incubated at 37 degrees C in iso-osmotic medium at pH7. The rate constant for intravesicular degradation is consistent with the rate of insulin clearance by the liver in vivo. Pretreatment of the rats with chloroquine resulted in a decrease in proteolysis of the iodopeptide within the isolated vesicles.

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied. 1 Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed. 2 Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products. 3 None of the weak bases changed the species of the degradation products found within the endocytic vesicles. 4 Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the ratelimiting process in degradation. 5 Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition of insulin degradation in isolated endocytic vesicles. 6 The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting by switching the receptor into a tight-binding conformation thereby slowing dissociation.

The uptake and subcellular processing of radiolabelled prolactin has been studied in male and female rats. Analytical subcellular fractionation of liver homogenates from rats injected with 125I-prolactin showed that in female rats the prolactin was primarily internalised to low density (1.12 g·cm−3) membranes. Approx. 10–15% of the total label was found in high density membranes, similar in distribution to lysosomal marker enzymes. In the normal male rat, prolactin was internalised solely to lysosomal type membranes. However, in male rats treated with estrogen, the distribution of prolactin was very similar to that seen in the female, indicating that internalisation to low density membrane is dependent on the presence of prolactin receptors. Gel exclusion chromatography showed that the prolactin internalised to the lysosomal membranes was extensively degraded whereas that associated with the low density membrane remained intact. Use of digitonin, to establish the identity of the low density membrane gave inconclusive results, but suggested that the prolactin was associated with membrane bearing NADH pyrophosphatase rather than the classical Golgi marker, galactosyltransferase.

When incubated in vitro, isolated rat liver low-density vcsicles degrade endocytosed insulin intraluminally. The rate of intravesicular degradation suggests that this pathway contributes significantly to insulin degradation in vivo. The vesicles can be selectively disrupted with digitonin at concentrations that abolish the latency of NADH pyrophosphatase, with minimal effect on the cisternal Golgi marker, Galactosyl transferase. The results suggest that latent NADH pyrophosphatase may act as a marker enzyme for the vesicles within which insulin is degraded. The possible role of insulin-glucagon protease, a candidate enzyme for insulin degradation by the liver, was investigated. The activity of latent insulin-glucagon protease associated with low-density vesicles is sufficient to account for the rate of intravesicular proteolysis. However, the rate of intravesicular proteolysis is insensitive to membrane-permeant thiol reagents under conditions which strongly inhibit insulin-glucagon protease. This shows that insulin-glucagon protease is not rate-limiting for insulin degradation by these vesicles, and is unlikely to be involved in the regulation of degradation. After disruption with Brij, internalized insulin remains associated with the membrane. Degradation is not inhibited by addition of excess unlabelled insulin to the medium, and occurs more rapidly than the degradation of an equal activity of iodo-insulin added to the disrupted membranes. This implies that degradation of endocytosed insulin occurs while it is still bound to the inner surface of the vesicles. When bacitracin is coinjected with iodo-insulin, it inhibits degradation of internalized insulin both by intact and Brij-disrupted vesicles, but not the degradation of added exogenous insulin, confirming that degradation is membrane-associated, and that it does not require the release of insulin into free solution.

Cell extracts from photoautrophic cultures of two cyanobacterial Calothrix isolates inhibited the growth in vitro of a chloroquine-resistant strain of the malaria parasite, Plasmodium falciparum, and of human HeLa cancer cells, in a dose-dependent manner. Bioassay-directed fractionation of the extracts led to the isolation and structural characterization of calothrixins A (1) and B (2), pentacyclic metabolites with an indolo[3,2-j]phenanthridine ring system unique amongst natural products, which exert their growth-inhibitory effects at nanomolar concentrations.

The specific staining observed in acinar cells raises an interesting point. These cells now appear to synthesize both secretory component and proteins such as amylase, both of which eventually appear in saliva. It is thought that the majority of secreted proteins are transported direct from the Golgi complex in secretory granules to the apical surface of the cell, where they are then secreted by exocytosis. However, in order that secretory component can bind IgA to form the secretory IgA complex found in saliva, it must travel to the basolateral membrane of the cell in order to react with the IgA found in the interstitial fluid, before being secreted at the apical surface. Thus it would seem that salivary glands must possess a protein-sorting mechanism not apparent in the previously studied gut or hepatocyte models. As such it is a useful system in which to compare possible mechanisms of IgA transport and also to extend our general understanding of any form of co-ordination that may be operative between the component sites of IgA secretion. C. S. is an M.R.C. research student. Financial support was provided by the Medical Research Council and the Wellcome Trust.

A case of functional pelviureteric junction obstruction is reported and a radiological method for detecting this entity is described. The causes of this condition are described and the reasons for the appropriate treatment are given.