Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes.

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.     

Marke''s disease herpesviruses. III. Purification and characterization of Marek''s disease herpesvirus B antigen.

Sera from chickens naturally infected with Marek's disease herpesvirus (MDHV) form preciptin lines with at least two immunologically distinct soluble antigens designated MDHV-A and MDHV-B. Partial purification and characterization of the glycoprotein MDHV-A antigen was previously reported. MDHV-B was found predominantly in the sonically treated extracts of infected cells, in contrast to the predominantly extracellular MDHV-A. Analysis of these extracts from [14C]glucosamine-labeled cells by immunodiffusion with chicken anti MDHV-B serum negative for MDHV-A followed by autoradiography confirmed that MDHV-B was a common antigen between MDHV and herpesvirus of turkeys and revealed that it was also a glycoprotein. Because of their glycoprotein nature, both MDHV-A and MDHV-B bound to concanavalin A affinity chromatography columns and could then be eluted by alpha-methyl-D-mannoside and recovered for further analysis. Concanavalin A affinity chromatography was an excellent technique for initial purification of MDHV-A and MDHV-B, since approximately 5- and 15- fold purification, respectively, was achieved in a single simple step. MDHV-B was resistant to trypsin under conditions where MDHV-A was sensitive, but was similar to MDHV-A in resistance to pH 2.0 and to 1.0 or 2.0 M urea and 0.05% Brij 35. Partially purified MDHV-B was analyzed by sucrose gradient sedimentation, isoelectric focusing, and gel filtration on Sephadex G-200 in the presence of 1.0 or 2.0 M urea and 0.05% Brij 35 to purify the antigen and to determine its physical and chemical properties in comparison with those already reported for MDHV-A. MDHV-B had a much lower isoelectric point in pH 4,54, a higher sedimentation coefficient of 4.4S, and a greater molecular weight of 58,250. These data indicate that MDHV-B is physically distinct from MDHV-A antigen, although the size difference is not sufficient to allow for effective separation. In contrast, the isoelectric point difference of greater than 2 pH units makes isoelectric focusing an effective means of purifying the antigens free of one another. The four-step purification procedure achieved greater than 200-fold purification of MDHV-B. Immunization of rabbits with this highly purified antigen results in the preparation of antisera that appeared monospecific for MDHV-B in immunodiffusion.     

Electrolyte effects on bilayer tubule formation by a diacetylenic phospholipid.

A general effect by dissolved electrolytes to destabilize the curvature of bilayer tubules prepared from the diacetylenic phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine is not found. This observation discounts the role of an electrostatic interaction between polarization charges on the edges of a ferroelectric bilayer as a means by which the cylindrical curvature may be stabilized in these structures (de Gennes, P. G. 1987. C. R. Acad. Sci. Paris. 304:259-263). The solution-mediated ionic interactions of electrolytes with this phospholipid appear not to influence significantly the relative stability of the crystalline state of the tubule, but at high levels of a few salts, may affect the nucleation and growth of the crystalline bilayer. Curvature of the bilayer in these tubular structures apparently derives from an interaction that is not very sensitive to the presence of electrolytes. Cylindrical curvature may alternatively arise from a bending force within the bilayer that is intrinsic to the anisotropic packing of the lipid molecules (Helfrich, W., and J. Prost. 1988. Phys. Rev. A38:3065-3068; Chappell, J. S., and P. Yager. 1991. Chem. Phys. Lipids. In press), and may therefore be largely determined by the packing interactions within the hydrophobic region of the tubular bilayer.     

Deuterated phospholipids as nonperturbing components for Raman studies of biomembranes.

The deuterated phospholipid, 1,2-dipalmitoyl-d62-phosphatidylcholine is shown by Raman spectroscopic measurements to be useful for obtaining information concerning phospholipid conformation in complex phospholipid and lipidprotein mixtures. The Raman bands of the deuterated phospholipid are assigned, and the sensitivity of these vibrational modes to conformational changes in the bilayer is demonstrated. Deuteration of the alkyl chains reveals the CH vibrations of the head group. A change in these bands is observed at the melting temperature and is assigned to alteration of the glycerol backbone conformation upon melting.     

Detection and Growth of Enteropathogenic Escherichia coli in Soft Ripened Cheese

The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore, a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 10⁵ at room temperature and over 10³ at 4 C within 10 days. With higher initial counts, levels up to 10⁹ were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination.     

Audition in the praying mantis,Mantis religiosa L.: identification of an interneuron mediating ultrasonic hearing

1. The praying mantis possesses a single ear located in the ventral midline of the metathorax. We have studied the mantis' auditory nervous system using both extracellular and intracellular techniques and have identified anatomically and physiologically a mirror-image pair of interneurons (MR-501-T3) in the metathoracic ganglion that mediates ultrasonic hearing. 2. MR-501-T3 is tuned broadly to ultrasound with best sensitivity (55-60 dB SPL) between 25 and 45 kHz. Its tuning matches closely that of the whole tympanal nerve. 3. The physiological responses of MR-501-T3 are characterized by: (1) a phasic-tonic firing pattern with a distinctive initial burst at 500-800 spikes/s; (2) minimum latencies of 8-12 ms; (3) no spontaneous activity; (4) sigmoid intensity response curves with a small (10 dB) dynamic range; (5) accurate coding of stimulus duration and of repetition rates up to 60 pps. 4. The ascending axon of MR-501-T3 conducts action potentials at 4 m/s, a rate comparable with some giant fiber systems. 5. MR-501-T3 shows no directional capability. Sound from right and left produce identical responses in both cells of the pair. Neither cutting one tympanal nerve nor removing one hemi-ear leads to different responses in the two cells indicating that they must receive a common input, either from the auditory afferents or from interneurons. We present evidence that the two cells are not directly connected. 6. MR-501-T3 is a large, symmetrical cell with its processes primarily in the intermediate neuropil (lateral ring tract). Its integration segment crosses the midline in the supramedian commissure, and the cell body lies dorsally near the entrance of the leg nerve. The axon travels in the dorsal lateral tract and is one of the largest (17 microns) in the connective. 7. Given the strong anatomical similarities between MR-501-T3 and the G and B cells of the locust, these cells may be homologous. 8. We present arguments based on our physiological results and existing behavioral data that MR-501-T3 is part of an ultrasonic warning/escape system in the mantis. As in moths, lacewings, and crickets, this system may provide a defense against nocturnally foraging bats.     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.