Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.

A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ.

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.

Response of Japanese quail fed seed meal from sunflowers grown on a municipal sludge-amended soil: elevation of cadmium in tissues.

Sunflowers were grown on soil amended with 224 metric tons/ha of municipal sewage sludge from Syracuse, N.Y. The yield of sunflower seeds was reduced by 47.2% by the sludge addition. The harvested seeds contained 1.71 ppm dry weight of cadmium. Deoiled seed meal was incorporated as 25 and 50% of semipurified diet and fed to male and female Japanese quail. The concentrations of cadmium were higher in kidney, liver, muscle, and eggs of birds fed the sludge-grown seed meal as compared to control quail. Tissue concentrations of cadmium increased with increasing dietary levels of sludge-grown seed meal. No significant differences (p greater than 0.05) were observed between dietary treatments in the activity of hepatic microsomal p-nitroanisole O-demethylase or aminopyrine N-demethylase in the male birds. Additionally, no mutagenic activity, either direct or with metabolic activation, was found in quail eggs. No observable changes in tissue ultrastructure were observed under electron microscopy in any of the treatment groups. There were no significant (p greater than 0.05) differences among the dietary treatment groups in feed intake, growth rate, egg production, or egg hatchability.

Toxicologic studies with lambs fed sugar beets grown on municipal sludge-amended soil: lowered relative hemoglobin in red blood cells and mutagens in blood and excreta.

Sugar beets grown on municipal sludge-amended soil were fed to growing lambs for 66 days. The relative hemoglobin content was significantly lower (P less than 0.05) in the lambs fed the sludge-grown sugar beets. The concentration of direct-acting mutagens was significantly higher (P less than 0.05) than controls in blood and urine of the lambs fed the sludge-grown beets. Cadmium concentration was higher, but not significantly (P greater than 0.05) in the livers and kidneys of the lambs fed the sludge-grown beets as compared with controls. Significant differences between treatment groups were not observed in active or passive K+ influxes in RBC; in the activity of hepatic microsomal aniline hydroxylase in p-nitroanisole-O-demethylase, aminopyrene-N-demethylase, or arylhydrocarbon hydroxylase; in tissue ultrastructure of kidney, liver, or muscle as examined by electron microscopy; or in carcass weight, dressing percentage, quality, or yield grade.

Three proton pumps, morphology and movements.

The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the delta pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 micrograms per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.

Toxicologic studies with male sheep grazing on municipal sludge-amended soil.

Growing sheep were grazed for 152 d on grass-legume forage growing on soil that had been amended with municipal sewage sludge from Syracuse, N.Y., at 224 metric tons per hectare. Cadmium was higher, but not significantly (p greater than 0.05), in tissues of sheep fed the sludge-grown forage as compared to controls. No significant differences between the sludge or control treatments were found in weight of the complete or cauda epididymis or in percent progressive motility of cauda epididymal sperm. The sludge-treatment group had significantly larger testes (p less than 0.025) when expressed as a percentage of body weight, and higher blood uric acid values (p less than 0.05). There were no observable changes in tissue ultrastructure of liver, kidney, muscle, or testes as examined by electron microscopy in either of the treatment groups. There were no significant differences for rate of animal weight gain, carcass weight, dressing percentage, or quality or yield grade of the carcases between the treatment groups.

The characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical.

We have compared the characteristics of glucose-6-phosphatase (EC 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. The latency of mannose-6-P hydrolysis, permeability to EDTA, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). Latency of mannose-6-P hydrolysis was demonstrated to provide a quantitative measure of the degree of nuclear membrane disruption. Electron micrographs confirmed the existence of substantial regions of the envelope in vitro where the permeability barrier to EDTA was intact (i.e. an "intact component"). The kinetics of glucose-6-phosphatase catalyzed by the intact component was obtained by subtracting the contribution of enzyme in disrupted regions from the total enzymic activity of untreated nuclei. The characteristics of glucose-6-phosphatase in intact and fully disrupted membranes of nuclei were indistinguishable from microsomes with respect to (a) the kinetics of glucose-6-P hydrolysis, (b) the effects of incubations with mannose-6-P, N-ethylmaleimide, and protease from Bacillus amyloliquefaciens, (c) the extremely high latency of carbamyl phosphate:glucose phosphotransferase activity, and (d) both the patterns of response of activity and the change in latency of glucose-6-phosphatase induced by fasting, experimental diabetes, and cortisol injection. Our results show clearly that apparent differences in the glucose-6-phosphatase activity of untreated preparations of nuclei and microsomes are simply expressions of significant differences in the degree of intactness of their respective permeability barriers. Since flattened cisternae, characteristic of the rough endoplasmic reticulum in situ, are preserved in intact regions of the envelope of isolated nuclei, the present findings constitute the most direct and definitive evidence to date that the properties of glucose-6-phosphatase in the endoplasmic reticulum in situ are faithfully reproduced with intact microsomes.

Toxicologic studies with swine fed corn grown on municipal sewage sludge-amended soil.

Over a 3-yr period 336 MT/ha of dried sewage sludge, from a municipal waste treatment plant, was applied in liquid form to land subsequently used to grow corn. The sludge contained 115 mg Cd, 4,200 mg Zn and 538 mg Ni/kg dry matter and comprised the solids remaining after treatment of the waste waters of approximately 260 industries as well as domestic users. Corn grain harvested from the plot amended with sewage sludge (SA corn) or corn grown on a plot without sludge addition (control corn) was fed to a total of 56 pigs to determine the effect on growth performance and parameters indicative of toxicity. Each treatment consisted of seven pens of four pigs each with an average initial weight of 17.6 kg. The growth trial was terminated when pigs weighed approximately 90 kg. No differences were observed between treatment groups for average daily gain, feed: gain ratio or daily feed intake. Higher (P less than .01) concentrations of Cd, Ni and Zn were found in SA corn compared with the control corn. This resulted in higher (P less than .01) concentrations of Cd in kidney and liver and Ni in kidney of pigs fed SA corn as compared with pigs fed control corn. No significant differences were observed in Cd or Ni concentrations in muscle or in hepatic microsomal mixed-function oxidase (MFO) activity and liver:body weight ratios, which is indicative of the absence or low levels of organic toxicants in SA corn. In addition, no significant treatment effects were observed when corn, feces and urine samples were evaluated for the presence of mutagenic substances. Histopathologic analysis of various tissues for lesions demonstrated that pigs fed SA corn were not adversely affected.

Toxicologic studies in growing sheep fed silage corn cultured on municipal sludge-amended acid subsoil.

Field corn was grown on subsoil, pH 5.5, that had been amended with 100 dry tons per acre (224 metric tons per hectare) of municipal sewage sludge from Syracuse, New York. The corn plants containing 3.88 ppm dry weight of cadmium were field-chopped and ensiled, and the silage was fed to growing sheep for 225 d. The sheep fed the sludge-grown corn silage showed a significantly (10 higher feed efficiency, (2) higher hepatic microsomal p-nitroanisole O-demethylase activity, and (3) higher concentrations of cadmium in liver and kidney and nickel in kidney as compared to the control animals. No significant treatment effects were observed in mutagenic responses for animal feed or feces samples. No consistent treatment effects were noted during histopathologic examination of sheep tissues.

Location of peptide fragments in the fibrinogen molecule by immunoelectron microscopy.

Antibodies to the disulfide knot fragment of bovine fibrinogen have been used to locate the site of this fragment within the intact fibrinogen molecule. The antibodies were isolated from rabbit antifibrinogen antisera by affinity chromatography. Electron micrographs of reaction mixtures of bovine fibrinogen and antibodies against the disulfide knot fragment showed pairs of fibrinogen molecules crosslinked by antibody molecules as well as higher order antibody-fibrinogen complexes. From an electron microscopic investigation of the crosslinked material, we conclude that the disulfide knot lies within the central nodule of the trinodular fibrinogen molecule. Antibodies to fragment H were used in the same manner to locate this fragment within the outer nodules of the human fibrinogen molecule.

Toxicologic studies with sheep fed colored magazines and newsprint.

Paper containing colored inks from magazines and newspapers was fed as 23% of their ration to sheep for 175 days. A similar ration containing oat hulls in place of the paper was fed to control animals. The paper-fed animals consumed 29% more feed than the controls but the feed efficiency (kg animal weight gained/kg ration consumed) of the two rations was approximately equivalent. Lead contained in the paper accumulated in animal tissues. Hepatic microsomal mixed function oxidase activity was several fold higher in the paper-fed animals than the controls. Histopathologic examination of liver and kidney using light and electron microscopy revealed no lesions attributable to diet.

Molecular and structural characteristics of house fly brain acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the brains of house flies (Musca domestica L., tetrachlorvinphos-resistant strain) was examined for molecular and structural features, including molecular weight, Stokes radii, partial specific volumes, sedimentation coefficients and frictional ratios. Acetylcholinesterase purified by affininity chromatography was examined in the electron microscope by negative staining and three molecular forms were clearly observed (monomers, dimers and tetramers). Several tetrameric configurations were observed as well as structures of similar size showing tails. In the preparations of acetylcholinesterase so far examined, no globular structures having more than four monomeric units were observed.

Ca++-induced fusion of proteoliposomes: dependence on transmembrane osmotic gradient.

The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 A lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 mumM have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.

Identification and transmembranous localization of active cytochrome oxidase in reconstituted membranes of purified phospholipids by electron microscopy.

Cytochrome oxidase vesicles with high oxidase activity and respiratory control ratio (greater than 3.5) were characterized by the freeze-etch technique for electron microscopy. By the use of this technique, cytochrome oxidase is shown to be an inner membrane particle. By locating cross-fractured vesicles in the same preparation, cytochrome oxidase particles are shown to extend across the phospholipid bilayer membranes. When cytochrome oxidase is added to preformed liposomes respiratory control is not observed, but high oxidase activity is maintained. In this preparation the cytochrome oxidase particles are located on the outer vesicle membrane surface. These observations provide direct evidence that cytochrome oxidase is found in a transmembranous position in closed, activecytochrome oxidase vesicles having respiratory control.