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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.

Lysed bacterial colonies containing potential recombinant plasmids were mixed with molten agar and sealed into slots of an agarose gel. After electrophoresis overnight, the gel was stained with ethidium bromide, which clearly reveals recombinant plasmids. Xenopus laevis ribosomal DNA and histone DNA of Psammechinus miliaris were ligated into pCRI plasmids and screened by this method.

A Xenopus laevis DNA segment containing the structural gene for tRNA1Met and 22 base pairs at the 5' side of the gene is active in tRNA production. The DNA segment was ligated to sea urchin histone DNA and was also inserted into plasmid pCR1. Both recombinant DNAs were shown to produce mature tRNA1Met at a high rate when injected into centrifuged Xenopus oocytes.

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.

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.

We have made deletions in a human interleukin 1 beta cDNA cloned in a yeast vector capable of directing the expression and secretion of foreign protein. Deletions at either the 5' or 3' end of the cDNA result in a dramatic drop in yield of protein. An investigation of some possible causes points to instability of the truncated peptides as a major factor contributing to low yield. The results suggest that correct folding of mature interleukin 1 into a protease resistant form requires almost the entire polypeptide.

Metabolism of (7 alpha-3H)testosterone has been measured in 111 human breast cancers and compared retrospectively with the degree of apocrine differentiation in the tumors. Cancers in which apocrine characteristics were a marked feature metabolized significantly more testosterone precursor than those in which apocrine features did not predominate. Higher metabolism was accounted for by increased conversion to 5 alpha-reduced products such as 5 alpha-dihydrotestosterone and 5 alpha-androstanediols.

An improved contact hybridization technique for the analysis of labelled RNA species is presented. The method combines high sensitivity of detection with the high resolution of polyacrylamide gel electrophoresis and should be especially useful for the characterization of transient RNA precursor molecules. Its application to gene mapping is illustrated.

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.

2-Dimethylallyl- 5,9-dimethyl-2'-hydroxybenzomorphan (Win 20,228) was found to be a weak antagonist of morphine and meperidine, whereas 2-allyl-5-ethyl-2'- hydroxy-9-methyl-6,7-benzomorphan (Win 19,362) and 2-allyl-2'-hydroxy-5,9- dimethyl-6,7-benzomorphan (Win 19,631) were about three times as potent as nalorphine. Preliminary clinical trials indicate that Win 19,362 is about twice as potent as morphine as an analgesic but, like nalorphine, is capable of producing severe psychic side effects. Win 20,228 is estimated to be about half as active as morphine as an analgesic, but no bizarre psychic effects were reported at any dose tested. Studies of addiction liability with Win 20,228 in monkeys suggest that this compound will not support morphine addiction.

Electron microscope studies have been conducted on individual fibers of human deoxyhemoglobin S (sickle cell hemoglobin). The fibers are obtained by injection of gelled samples into a large excess of glutaraldehyde, which quickly stabilizes the fibers by cross-linking. The fibers are negatively stained with phosphotungstic acid or shadowed with platinum-carbon. The fibers are approximately 200 A in diameter, and display long and short helical striations with an opposite handedness. The long striations occur at an angle of about 15 degrees from the fiber axis, and complete one turn around the helix at a distance of about 2 x 10(3) A along the fiber axis. The short striations occur at an angle of about 80 degrees from the fiber axis, with a spacing of about 65 A, and complete one turn around the helix at a distance along the fiber axis of about 130 A. The structure of the fiber appears to be a sextuple helix in terms of the long striations, and a double helix in terms of the short striations. The shadowed samples are consistent with a left-handed screw sense for the short striations, thus implying a right-handed sense for the long striations. A structural model incorporating these features is compatible with the atomic structure of hemoglobin, with individual molecules oriented with their dyad axis of symmetry perpendicular to the fiber axis and their alpha(1)-beta(1) pseudo-dyad axis roughly parallel to the fiber axis. This orientation places the two beta-6 regions of each molecule (sites of the sickle cell mutation) in contact with the beta-6 regions of the molecules above and below along the long striations. Both the long and short striations are accounted for by individual hemoglobin molecules arranged in double helical arrays with 6.4 molecules per turn in each array.

1. The influence of adenosine cyclic 3',5'-monophosphate (3',5'-AMP) and of drugs believed to increase or decrease its concentration in the tissues has been determined on the response of vascular and uterine smooth muscles to catecholamines. Generally, drugs believed to increase tissue content of 3',5'-AMP potentiated the responses and those believed to decrease it depressed them.2. The cardiovascular responses of dogs (with major vessels occluded in the chest) to carotid occlusion were potentiated by infusions of theophylline and sodium fluoride. Infusion of theophylline also potentiated the response of the occluded abdominal vessels to noradrenaline.3. Intravenous infusions of theophylline and sodium fluoride potentiated pressor responses to catecholamines in the pithed rat. Infusions of iminazole depressed the responses in two animals and was without effect in two others.4. In spinal cats intravenous infusions of theophylline potentiated pressor responses to catecholamines, but sodium fluoride was without effect.5. Contractions of the isolated rat aortic strip to noradrenaline were always potentiated by sodium fluoride and by theophylline, and depressed by iminazole, when they were recorded isometrically. Theophylline always potentiated the contractions, when they were recorded isotonically but sodium fluoride was mostly, and iminazole always, ineffective.6. 3',5'-AMP in concentrations from 0.1 to 20 mug/ml. potentiated the responses of the isolated rat aortic strip to noradrenaline in thirty-eight experiments out of hundred. Concentrations from 10 to 500 mug/ml. sometimes depressed contractions recorded isometrically. In five experiments, exposure to low concentrations for 3 hr increased the resting tension of the preparation.7. Responses to noradrenaline of uteri from oestradiol-treated rabbits were potentiated by vasopressin and by sodium fluoride, but not by theophylline or iminazole. In progesterone-treated animals the responses were unaffected by vasopressin and sodium fluoride, but potentiated by theophylline and depressed by iminazole. 3',5'-AMP was without effect on the uterine responses.8. It is concluded that the results support the view that an increase in the tissue content of 3',5'-AMP potentiates the contraction of vascular and uterine smooth muscle in response to catecholamine. This view is supported by the observation that the nucleotide itself can potentiate the responses of the rat aortic strip to noradrenaline.

Daily intramuscular injections of cortisone, prednisolone, triamcinolone, dexamethasone, fludrocortisone and 2-methylfludrocortisone markedly reduced the histamine and 5-hydroxytryptamine contents of the skin and small intestine of rats; there was an increase however, in the amine contents of the stomach. The sex hormones were weaker in these effects while deoxycortone and corticotrophin were inactive. When the skin was depleted of half of its 5-hydroxytryptamine by prolonged treatment with polymyxin B, the rate of recovery of this amine was retarded by the glucocorticoids. The glucocorticoid activity of a steroid and its ability to produce changes in the histamine and 5-hydroxytryptamine contents of tissues are related.

1. Experiments have been performed to investigate the mechanism by which low doses of megestrol acetate have an antifertility action.2. It was found that the dose of megestrol acetate required to prevent pregnancy in nine of ten New Zealand White rabbits when given on 3 consecutive days, the last of which was the day of insemination, was 50 mug/kg daily.3. This treatment reduced the numbers of sperm recoverable from the uterus at various times after insemination, but egg transfer experiments showed that neither egg viability nor fertilization was affected on day 1 of pregnancy.4. Treatment with megestrol acetate for 3 days caused the fertilized eggs to enter the uterus approximately 48 h prematurely. Treatment with megestrol acetate for longer periods caused a similar but more marked increase in the rate of transport of eggs in the oviduct.5. Megestrol acetate did not modify oviduct opening pressure, but the volume of oviduct fluid secretion was reduced earlier and transport of fluid through the oviduct was accelerated compared with controls.6. Transfer of eggs on various days from the uteri of untreated rabbits to the uteri of treated rabbits indicated that the development of the endometrium which occurs in pregnancy may be advanced by megestrol administration. Histological examination of the endometrial proliferation induced by megestrol acetate confirmed that this was so.7. It is concluded that the antifertility action of megestrol acetate in doses that do not necessarily inhibit ovulation is due to a combination of accelerated egg transport and advanced endometrial development.