An experimental investigation of preorgasmic reconditioning and postorgasmic deconditioning. The effects of pre- and postorgasmic presentation of moderately erotic cues were assessed in an analogue study. Eight heterosexual male volunteers (18 to 23 years) participated in three assessment (baseline, termination-of-treatment, and two- to three-month followup) and eight masturbatory conditioning sessions. Three slides of nude females of initially equal erotic value were paired respectively with the plateau, refractory, and resolution phases of the subjects' sexual cycles. Over treatment, stimuli paired with the plateau phase increased significantly in penile tumescence indices of eroticism; conversely, stimuli paired with the refractory phase decreased significantly. The conditioned effects on tumescence were largely extinguished at followup. While treatment did not alter short-term subjective indices of eroticism, stimuli presented during the refractory phase were rated significantly less erotic than the other stimuli at followup. The findings suggest that the "pairing" model of orgasmic conditioning is insufficient to account for previously reported clinical findings. A broader conceptualization of the mechanisms of orgasmic conditioning, and implications for treatment are discussed.


Severe classroom behavior problems: teachers or counsellors. This experiment was designed to determine the relative effectiveness of teacher and counselling approaches in the reduction of disruptive or inappropriate classroom behavior. Inappropriate classroom behavior frequencies of 12 academically low achieving, seventh-grade, black male students, with a reported high rate of inappropriate classroom behavior, were recorded. Three groups, with nearly equal mean inappropriate behaviors, were randomly assigned to one of three treatment conditions: behavioral counselling, client-centered counselling, or no counselling. Each counselling group received fifteen 30-minute counselling sessions, at a rate of two to three times a week. In addition to counselling, all students subsequently received teacher approval within the classroom. Results indicated that the teacher was able to reduce inappropriate behavior more than any counselling group. There were also indications that behavioral counselling, but not client-centered counselling, was moderately helpful in reducing inappropriate classroom behavior.


Quantitative determination of polysorbate to in non-standard salad dressings. Six samples of non-standard salad dressing, containing 0.10 to 0.60% polysorbate 60, were submitted to 6 collaborators. The salad dressing is extracted and the extract is saponified and acidified, and the acids are removed. The aqueous, polyol solution is desalted and the polyoxyethylated polyols are precipitated as a highly insoluble heteropoly acid complex. The polysorbate 60 content is calculated from the weight of the precipitate, using a gravimetric factor. Average recoveries from the collaboration samples ranged from 105 to 130%, with standard deviations from 0.016 to 0.047. The method has been adopted as official first action.


Gas-liquid chromatographic determination of nitrate and nitrite in cheese, ham, fish sausage, cod roes, and salmon roes. The 2,4-xylenol method was modified and a gas-liquid chromatographic (GLC) method was developed for nitrate and nitrite determinations in several foods. Either a flame ionization (FID) or an electron capture detector (ECD) can be used. Proteins and fats are removed from warm alkaline water with zinc sulfate and filtered. The filtrate is evaporated to dryness, redissolved in water, and reacted with 2,4-xylenol in the presence of sulfuric acid to form 6-nitro-2,4-xylenol. Interfering chlorides are precipitated with silver sulfate and the nitrosylenol is extracted with hexane, concentrated, and injected. Nitrite in the filtrate is distilled at pH 5, collected in alkaline solution, and dried. The residue is oxidized to nitrate with permanganate in the presence of sulfuric acid, and then chromatographed in the same manner as nitrate. Recoveries from several foods were 83--100% for nitrate and 80--100% for nitrite. The limit of sensitivity was 0.1 ppm for both residues.


A rapid gas-liquid chromatographic method for the multi-determination of antioxidants in fats, oils and dried food products. A rapid quantitative method for determining 8 antioxidants in various food products is described. Two procedures are employed. The first involves the use of a glass wood precolumn to separate 3(2)-tert-butyl-4-hydroxyanisole, 3,5-di-tert-butyl-4-hydroxytoluene, 4-hydroxymethyl-2,6-di-tert-butylphenol, and mono-tert-butylhydroquinone (TBHQ) from nonvolatile residues resulting from direct injection of diluted sample or sample extracts into the gas-liquid chromatographic (GLC) column. In the second procedure, the antioxidants TBHQ, 3,3'-thiodipropionic acid, n-propyl gallate, 2,4,5-trihydroxybutyrophenone, and nordihydroguaiaretic acid are isolated from food products by extraction with 70% ethanol. The antioxidant residues are then converted to trimethylsilyl derivatives, and determined by GLC, using a flame ionization detector. Recoveries of all 8 antioxidants from 28 food samples fortified at either 10 or 100 ppm ranged from 70 to 105%.


Potential uses of lead isotope ratios in gunshot cases. The determination of lead isotope ratios in 14 bullets, and in material taken from 9 hand swabs and 5 primers shows that there are potentially valuable forensic uses for such a method. While a more complete study is required, this method could possibly be used to prove (or disprove) relatiohships between bullets and manufacturers, weapons, or persons firing the weapons. Sample size requirements (1 microgram or less) are such that damaged or fragmented bullets, or minute particles therefrom, may be used for the required analyses. An experiment showed that gunshot residue from a test-fired weapon was detectable even after washing the hands.


Thin layer chromatographic screening method for sulfadiazine residues in calf tissues, plasma, and urine. Because of the lack of specificity of the Bratton-Marshall procedure for assaying sulfonamides, a sensitive, specific tissue residue assay for sulfadiazine (SDZ) was developed. The methodology has been extended to provide a highly sensitive screen for sulfonamide residues, which employs 2-dimensional thin layer chromatography in conjunction with fluorescamine derivatization. The procedure described, which has been developed for SDZ in calf tissues, involves direct ethyl acetate extraction of tissue homogenates. Following evaporation of the organic phase, a portion of the residue is spotted on a 20 X 20 cm silica gel 60 plate, which is then developed in 2 dimensions with solvent systems devised to separate SDZ from endogenous substances as well as from 12 other sulfonamides that might be present in calf tissues. The presence of SDZ at a concentration of 0.1 ppm or its absence is easily demonstrated in calf kidney, liver, muscle, plasma, and urine. The basic method can be modified for a particular sulfonamide in a target tissue and can be used as a quantitative assay for sulfonamide residues.


Gas-liquid chromatographic determination of ethopabate in feed premixes. A gas-liquid chromatographic method is presented for determining ethopabate in 0.8 and 8.0% premixes. A sample is extracted with tetrahydrofuran containing an internal standard, by sonication or overnight soaking. The extract is clarified by centrifugation, diluted if necessary, and injected into a gas chromatograph equipped with a flame ionization detector. Average per cent recoveries for spiked blank samples were 100.6 at the 0.8% level and 100.4 at the 8.0% level. Precision, as indicated by replicate analyses of several premixes, ranged from 0.5 to 1.7% relative standard deviation.


Improved microbiological assay for penicillin residues in tissues and stability of residues under cooking procedures. The microbiological assay for penicillin residues was improved by using centrifugation to remove physical barriers to diffusion, a small buffer/meat extraction ratio, and a more sensitive 2-layer assay system. Recoveries from muscle, kidney, and liver tissues ranged between 70.1 and 86.7% with measurable levels of 0.03--0.05 unit/g. By comparison, the Food and Drug-suggested methodology yielded recoveries of 45.9--54.0% and levels of detectability of 0.08--0.10 unit/g. Cooking of hamburger, steaks, and port chops indicated that procaine penicillin withstood cooking conditions, and significant levels of the original activity remained.


Isolation and purification of moniliformin. A bulk purification procedure is described for moniliformin, a mycotoxin produced by Fusarium moniliforme. The method involves methanol extraction of suitably molded maize, aqueous extraction of the methanol-free residue, ion exchange chromatography with a NaCl concentration gradient, desalination, and crystallization. A pKa value of 1.70 as well as molar absorptivities of 19100 (lambda (H2O) max 229 nm), 5600 (lambda (H2O) max 260 nm), and 4700 (lambda (MeOH) max 260 nm) L/mole/cm are reported for moniliformin.


Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn. A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.


High pressure liquid chromatographic determination of xanthomegnin in corn. A method is described for the detection and quantitative analysis of xanthomegnin in corn samples. Initial extraction with CHCl3 in the presence of 0.5M H3PO4 is followed by additional purification using silica gel column chromatography. A high pressure liquid chromatograph equipped with a microparticle silica gel column and a 405 nm absorbance detector is used for detection and quantitation of the xanthomegnin. The identity of xanthomegnin is confirmed by thin layer chromatography on silica gel plates developed with benzene-methanol-acetic acid (90 + 5 + 5). The recovery of xanthomegnin added to corn samples at levels of 0.75--9.6 mg/kg averaged 41% with a coefficient of variation of 25%.


Determination of zearalenone in cornflakes and other corn-based foods by thin layer chromatography, high pressure liquid chromatography, and gas-liquid chromatography/high resolution mass spectrometry. A simple cleanup procedure based on pH adjustments was used to obtain extracts of corn foods. The method gave good recoveries of zearalenone determined by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). As little as 5 ng zearalenone was detected by TLC, using Fast Violet B Salt as the spray reagent; the lower limit of detection in cornflakes was about 20 microgram/kg. With HPLC on Spherisorb silica (5 micrometer) and detection by fluorescence at an excitation maximum of 310 nm as little as 5 microgram zearalenone/kg cornflakes could be determined. While the TLC method was also applicable to corn chips, cornmeal, popcorn, and frozen corn, an interference was observed in HPLC of the latter 3 products. This interference was separated from zearalenone by adding a second HPLC analytical column (Spherisorb ODS). Gas-liquid chromatography coupled with mass spectrometric single ion monitoring at high resolution, although of limited availability, was shown to be the most sensitive and selective method for determining zearalenone in corn foods. The natural occurrence of zearalenone in a sample of cornflakes (13-20 microgram/kg) was demonstrated by all 3 detection procedures.


Thymidine kinase heterozygous L5178Y mouse lymphoma forward mutational assay. The biological basis and protocol for the thymidine kinase heterozygous L5178Y mouse lymphoma forward mutational assay (TK+/-) are described. The TK+/- assay is used for the detection of mutations produced by chemicals or radiation.


High pressure liquid chromatographic determination of strychnine, using a reverse phase solvent system. A simple, rapid extraction and subsequent determination for strychnine, using high pressure liquid chromatography (HPLC) with a reverse phase solvent system, is described. Stomach contents or grain bait containing strychnine were made alkaline with sodium hydroxide and extracted with chloroform. Extract filtrates were injected directly into a liquid chromatograph without further preparation except for dilution, if necessary. Peaks were resolved within 3.5 min and peak heights were used for quantitation. HPLC of strychnine was carried out on a 30 cm X 4 mm id stainless steel column packed with micronBondapak C18. The solvent program was 0.005M phosphate buffer-methanol (60+40) at a flow of 1.5 ml/min. Recovery from spiked stomach contents was 93.9 +/- 3.5%. The detection capability for strynchnine, using the 254 nm ultraviolet detector, was 5 ng. The strychnine peak was collected and subjected to thin layer chromatography with strychnine standards for confirmation.


Atomic absorption spectrometric determination of cyhexatin in apples. A rapid method using atomic absorption spectrometry has been developed for determining residues of cyhexatin in apples. Each analysis takes less than 30 min and the method is sensitive to less than 0.1 mg cyhexatin/kg. Recoveries varied from approximately 100% at 0.25 and 0.05 mg/kg to 70% at 5 mg/kg. No acid digestion or cleanup is required for the analysis.


Analytical behavior data for chemicals determined using AOAC multiresidue methodology for pesticide residues in foods. Analytical methods capable of detecting more than one pesticide residue simultaneously (multiresidue methods) become more effective with an increase in the number of chemicals whose behavior through the various steps of the method has been documented. Since 1970, the method behavior data related to the AOAC official method for residues of 25 chlorinated and phosphated pesticides and polychlorinated biphenyls have been extended to include information on twice as many chemicals as was previously available. The value of having a large bank of method behavior data is outlined and the experimental protocol by which the data were collected is described. A complete listing is included of the available data on the analytical behavior of over 300 pesticidal and/or industrial chemicals.


Direct analysis of lead in fruit juice by anodic stripping voltammetry with no prior sample preparation. A method is presented for determining lead in fruit juice in less than 3 min after instrument calibration. The range examined is 0.01 to 1.3 ppm lead. The method is compared with 3 other methods in general use. Standard error of estimates between the methods compared range from 0.023 to 0.051 ppm for a set of 50 samples and from 0.037 to 0.091 ppm for a set of 9 samples. Regression correlation coefficients between methods range from 0.968 to 0.995. Judged by the comparisons, the direct method is precise and accurate over greater than a 100-fold range of lead concentrations.


Flameless atomic absorption spectroscopic determination of heavy metals in whole-fish samples. Flameless atomic absorption spectroscopy with a carbon rod atomizer was used to determine lead, cadmium, and chromium in whole-fish samples. Samples were dry-ashed, and the metals were separated by solvent extraction with ammonium pyrrolidine dithiocarbamate in methyl isobutyl ketone, and then back-partitioned into an aqueous acid solution for analysis. The back-partitioning step allows a direct comparison of sample solutions with aqueous solutions of the standard. Recoveries of the metals from fortified samples averaged 91% (+/-9.6) for lead and 100% (+/-5.6) for chromium at the 0.1-1 ppm level, and 100% (+/-13.3) for cadmium at the 0.01-0.1 ppm level.


High pressure liquid chromatographic separation and quantitation of chlordiazepoxide.HCl and two of its related compounds. A method is described for the separation and quantitative determination of chlordiazepoxide. HCl and 2 related compounds, 7-chloro-1-3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one-4-oxide (CBO) and 2-amino-5-chlorobenzophenone (ACB), by high pressure liquid chromatography. The 3 compounds are separated on a reverse phase microparticulate column packing, using a mobile phase of acetonitrile and aqueous ammonium acetate. Bulk powders or capsule formulations are quantitated by direct comparison of their peak heights to those of appropriate reference standards. Data are presented showing the limit of detectability for the related compounds to be less than 80 ng CBO/ml and less than 40 ng ACB/ml. Based on peak heights, the relative standard deviation for the precision for chlordiazepoxide.HCl is 0.42%. Recoveries from laboratory-prepared samples for chlordiazepoxide.HCl were 99.4%; CBO, 102.4%; and ACB, 99.7%.


Gas-liquid chromatographic quantitation of cocaine.HCl in pouders and tablets: collaborative study. Eight laboratories collaboratively studied a method for the quantitative gas-liquid chromatographic (GLC) determination of cocaine.HCl in solid forms. The cocaine.HCl and other water solubles are dissolved in dilute HCl. The aqueous solution is made weakly basic with K2HPO4, and the organic solubles are extracted into CHCl3 for the GLC determination of cocaine. Tetracosane (n-C24) is incorporated in the extracting CHCl3 as an internal standard. Five samples of known cocaine.HCl concentration and one sample of commercial ground tablets were collaboratively studied. The cocaine.HCl content ranged from 6 to 100%. Recoveries ranged from 98.7 to 103%; coefficients of variation ranged from 0.89 to 3.16. The method was adopted as official first action.


High pressure liquid chromatographic method for probenecid in flavored oral suspensions containing ampicillin: collaborative studies. A high pressure liquid chromatographic method for determining probenecid in oral suspensions of amoxicillin was applied to the determination of probenecid in oral suspensions of ampicillin. Three preparations containing various known amounts of probenecid in synthetic mixtures of ampicillin oral suspensions were analyzed by 5 chemists in an intralaboratory study, with satisfactory results. Blind duplicates of 3 prepared oral suspensions were sent to 12 collaborators, who were instructed to analyze the samples in a fixed random order. The standards showed a satisfactory linear response. Average recoveries of probenecid in the interlaboratory study for the 6 mixtures ranged from 95.2 to 99.1%, and the coefficients of variation ranged from 1.63 to 4.9%.


Measurement of the bioavailability of iron, using the rat hemoglobin repletion test. Variations in the AOAC official first action rat hemoglobin repletion test for iron were studied. These changes included (1) use of a simplified basal diet to eliminate ingredients which sometimes contribute too much iron; (2) increased fortification of the basal diet with vitamin E, pantothenic acid, and pyridoxine; (3) increased dietary copper; (4) variations in the carbohydrate source in the basal diet; (5) changes in the length of the depletion and repletion periods; and (6) comparison of prophylactic and curative procedures. The changes yielded results comparable to those obtained with the present official method. Further study may reveal that the depletion period can be shortened or eliminated. To fully meet the rat's vitamin requirements, increased levels of vitamin E, pantothenic acid, and pyridoxine are recommended. It is further recommended that the present method remain in official first action status, and that study be continued.


Atomic absorption spectrometric and colorimetric determination of iron in seafoods. Iron levels in 14 seafoods were determined by atomic absorption spectrometry (AAS) on freeze-dried composites. Samples were prepared for analysis after dry-ashing at 550 degrees C and wet digestion in HNO3-HClO4. Paired analysis of wet digests were accomplished by AAS and use of the colorimetric reagent, ferrozine. There was no significant difference in iron levels of seafoods due to sample preparation. While individual species levels were not significantly different between the AAS and colorimetric procedures, evaluation of all determinations indicates that ferrozine gives lower values (P less than 0.005) by 8%. Iron levels in seafoods in microgram/g dry weight (mg/100 g wet weight) determined by AAS on wet-digested samples were: 8 species of white finfish, 16.3 +/- 4.2 (0.31 +/- 0.08); Pacific shrimp, 12.3 +/- 1.4 (0.29 +/- 0.03); canned tuna, water pack, 16.6 +/- 2.9 (0.49 +/- 0.09); sockeye salmon, 29.0 +/- 5.5 (0.89 +/- 0.25); American shad, 29.1 +/- 1.5 (0.97 +/- 0.05); Pacific oysters, 391 +/- 45 (6.54 +/- 1.39); and Dungeness crab, 17.1 +/- 2.5 (0.35 +/- 0.05).


Automated fluorometric analysis of calcium pantothenate in multivitamin preparations. An automated fluorometric procedures is described for assaying calcium pantothenate in multivitamin preparations. Sample extracts containing calcium pantothenate are treated on-line with a slurry of magnesium trisilicate which removes any interfering riboflavin that may be present. The filtrate is resampled, mixed on-line with a slurry of Dowex 50W-X4 (H+) which removes any interfering beta-alanine that may be present, and dialyzed. Dialysates are hydrolyzed in an alkaline medium and reacted with a mixture of o-phthalaldehyde and 2-mercaptoethanol in boric acid solution. The fluorescence intensity due to the formation of a fluorogenic compound is measured at 455 nm after excitation at 350 nm. The procedure developed is capable of analyzing 20 samples/hr. Analytical data indicate that calcium pantothenate is assayed reliably both from real and synthetic multivitamin preparations. The mean recovery of calcium pantothenate added to sample solutions of tablet composites was 95.4%.


Application of the official AOAC chloresterol Method to a wide variety of food products. This paper presents analytical data on the cholesterol content of selected consumer food products including some fats and oils. The cholesterol data are grouped by food products. To determine the reproducibility of cholesterol values, recovery data were obtained from samples of pure vegetable shortening spiked at 40 and 20 mg cholesterol (as cholesteryl palmitate)/100 g fat. Average recoveries were 97.1% and 95.5% with coefficients of variation of 1.27 and 3.24%, respectively.


Spectrophotometric determination of vitamin A in oils and fats. A spectrophotometric method for determining vitamin A based on interaction with 50% trichloroacetic acid solution in dichloromethane was developed. The blue reaction product had a maximum absorption at 620 nm and obeyed Beer's law over the concentration range of 0.5-5.0 microgram retinol/ml solution. The molar absorptivity of the reaction product was 1.58 X 10(5). As much as 100 microgram vitamin D2, and beta-carotene up to 12 times the vitamin A concentration, did not interfere with the determination. The results obtained from the determination of vitamin A in cod liver oil and butter showed excellent agreement with the Carr-Price method, 43.013(d).


Bacterial leaching patterns on pyrite crystal surfaces. Selected pyrite crystals were placed as a bacterial energy source into stationary cultures of Thiobacillus ferroxidans. Scanning electron microscope studies performed after a period of 2 years on these crystals revealed bacterial etching pits in characteristic patterns; they include pit arrangements in loose statistical disorder, in pairs, in clusters, and most remarkably in pearl-string-like chains. It has previously been confirmed that the chemical processes of bacterial leaching occur mainly in the region of contact between bacteria and the sulfide surface. The evidence presented in this experiment strongly suggests that the observed bacterial distributions are critically dependent on crystal structure and on deviations in the crystal order (fracture lines, dislocations) of the leachable substrate.


Biochemical and genetic properties of site-specific restriction endonucleases in Bacillus globigii. Bacillus globigii contains two site-specific endonucleases, BPGLI AND BglI. A rapid technique for selection of mutants deficient in each of these enzymes was developed using sensitivity to infection by bacteriophage SP50 as an indication of the levels of enzyme. Mutants defective in BglI, BglII, and both BglI and BglII retained the wild-type modification phenotype. Genetic and biochemical studies have established that these enzymes are involved in restriction in vivo. Simplified purification procedures for BglI and BglII using these mutants are described.


Cleavage maps of a tetracycline plasmid from Staphylococcus aureus. The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.


Spheroplast fusion and heterokaryon formation in Mucor racemosus. Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to spheroplasts by using commercial chitinase and Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed in a buffered Ca(NO3)2 solution and fusion occurred. After cell wall regeneration, prototrophs were isolated. The frequency of heterokaryon formation was 1.45 X 10(-4). Prototrophic isolates segregated parental nuclei at a high frequency, indicating that heterokaryons had formed.


Lipid and fatty acid composition of Gluconobacter oxydans before and after intracytoplasmic membrane formation. Gluconobacter oxydans differentiates by forming quantities of intracytoplasmic membranes at the end of exponential growth, and this formation occurs concurrently with a 60% increase in cellular lipid. The present study was initiated to determine whether this newly synthesized lipid differed from that extracted before intracytoplasmic membrane synthesis. Undifferentiated exponential-phase cells were found to contain 30% phosphatidylcholine, 27.1% caridolipin, 25% phosphatidylethanolamine, 12.5% phosphatidylglycerol, 0.4% phosphatidic acid, 0.2% phosphatidylserine, and four additional unidentified lipids totaling less than 5%. The only change detected after formation of intracytoplasmic membranes was a slight decrease in phosphatidylethanolamine and a corresponding increase in phosphatidylcholine. An examination of lipid hydrolysates revealed 11 different fatty acids in the lipids from each cell type. Hexadecanoic acid and monounsaturated octadecenoic accounted for more than 75% of the total fatty acids for both cell types. Proportional changes were noted in all fatty acids except octadecenoate. Anteiso-pentadecanoate comprised less than 1% of the fatty acids from undifferentiated cells but more than 13% of the total fatty acids from cells containing intracytoplasmic membranes. These results suggest that anteiso-pentadecanoate formation closely parallels the formation of intracytoplasmic membranes. Increased concentrations of this fatty acid may contribute to the fluidity necessary for plasma membrane convolution during intracytoplasmic membrane development.


Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.


Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores. Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.


Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis. The relationship between ATP and energy charge was studied in individuals of Cirolana borealis under heavy metabolic stress caused by anoxia or exposure to toluene. Prolonged anoxia led to a lowering of the ATP content to about 10% after 4 days, with a simultaneous decrease in energy charge to about 0.25. A lowering of the total adenylate pool reduced the fall in energy charge somewhat, but this effect was marked only in late anoxia when the individuals had become inactive. Exposure to 0.14 mM toluene for 8 days led to a similar decrease in ATP and energy charge. Exposure to 1.4 mM toluene for 24 h led to only slight changes in the adenine nucleotide pool, although the individuals became narcotized within a few hours. The energy charge associated with moribund individuals thus varied much. The mechanism of energy charge stabilization through reduction of the adenine nucleotide pool as ATP declined seemed to be of little significance for the survival of the individuals.


Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles. Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.


Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h, the level of histone acetylation is greatly increased as compared to control cells (Riggs, M.G., Whittaker, R.G., Neumann, J.R., and Ingram, V.R. (1977) Nature 268, 462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity.


UDP-glucose pyrophosphorylase. Stereochemical course of the reaction of glucose 1-phosphate with uridine-5'[1-thiotriphosphate]. Of the two diastereoisomers of uridine-5'[1-thiotriphosphate] differing in configuration at Palpha, UDP-glucose pyrophosphorylase catalyzes the reaction of the one designated isomer B (Eckstein, F. (1975) Angew. Chem, Int. Ed. Engl. 14, 160-166) with glucose-1-P to produce uridine-5'[1-thiodiphosphate]glucose. Mild acid hydrolysis of the latter, under conditions known to be minimal for the hydrolysis of aldose phosphates, produces diastereoisomer A of uridine-5'[1-thiodiphosphate]. Therefore, the reaction of uridine-5'[1-thiotriphosphate](B) with glucose-1-P catalyzed by UDP-glucose pyrophosphorylase occurs with stereochemical inversion at Palpha to produce uridine-5'[1-thiodiphosphate]glucose(A). The 31P NMR spectra of these nucleotides are presented and used to elucidate the stereochemical course of the reaction. The apparent Km exhibited by this enzyme for uridine-5'[1-thiodiphosphate]glucose isomer A at 2 mM pyrophosphate and pH 7.6 is 75 micron, which is only 2.2 times that for UDP-glucose, 35 micron. The maximal velocity for UDP-glucose is 11 times that for uridine-5'[1-thiodiphosphate]glucose isomer A under these conditions.


Identification of a 48 S preinitiation complex in reticulocyte lysate. When the polypeptide initiation sequence is interrupted by the peptide antibiotic edeine, the sedimentation coefficient of the native small ribosomal subunit is increased from 43 S to 48 S. Formation of the 48 SN particle is absolutely dependent on the binding of both Met-tRNAf and globin mRNA. These results support the concept that Met-tRNAf binds to the native small ribosomal subunit before mRNA, and that processes involved in mRNA binding to the 43 SN preinitiation complex may be the rate-limiting step of 80 S initiation complex formation.


An endodeoxyribonuclease of human KB cells. Purification and properties of the enzyme. An endodeoxyribonuclease has been purified 750-fold from human KB cells. The purified endonuclease requires Mg2+ for maximum activity: Mn2+ was less than half as active and Ca2+ inhibited the reaction. The optimum pH is 8.8 in Tris-HCl and the optimum buffer concentration is 10 mM. KCl (and NaCl), --SH-reacting reagents, and tRNA strongly inhibit the reaction. An apparent molecular weight of 54,000 was determined by sedimentation in a glycerol gradient. The purified endonuclease cleaved native, double-stranded adenovirus 2 DNA, and the reaction proceeded stepwise during the initial stage of degradation by cleavage of the DNA substrate in half, then in half again, etc. At longer digestion times, single strand scissions were detected. RNA was not a substrate for the enzyme. Poly(dG) . poly(dC) was susceptible but poly(dA) . poly(dT) was resistant to degradation. Hydrolysis of adenovirus 2 DNA yielded double-stranded polynucleotides containing 5'-phosphoryl and 3'-hydroxyl termini with short, single-stranded regions presumably at the ends. More than 50% of the product of a limit digest had a chain length greater than 35 to 40 nucleotides. Analysis of the 5' and 3' end groups of the digestion products indicated a preference for the site of the enzymatic cleavage; thymidylic acid residues were present at the 5' end and deoxyguanosine residues at the 3' end, each with a frequency of 40 to 50%.


Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase. The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.


The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation. The refolding and reoxidation of fully reduced and denatured chymotrypsinogen A have been studied in the presence of low concentrations of guanidine HCl or urea. Renaturation yields of 60 to 70% were observed when the reoxidation was facilitated by mixtures of reduced and oxidized glutathione. Refolding occurred within a narrow range of denaturant concentration (1.0 to 1.3 M guanidine HCl and 2 M urea) in which the native protein was shown to be stable, and the reduced protein was shown to regain the correct disulfide pairing. Renatured chymotrypsinogen is indistinguishable from the native zymogen in chromatographic behavior, potential chymotryptic activity, sedimentation coefficient, and spectral properties. The kinetics of renaturation were determined. Some of the protein species obtained at various times of renaturation were characterized as incorrectly oxidized molecules which could be renatured by thiol-catalyzed interchange of disulfide bonds.


Influence of ring substituents on the binding of nitrosobenzene by ferrohemoglobin. The affinity of a substituted nitrosobenzene for ferrohemoglobin and the visible absorption spectrum of the resulting compound was influenced by the nature, position, and number of substituents on its benzene ring. Alkyl ring substituents inhibited the binding of nitrosobenzene to ferrohemoglobin, and binding was blocked by an ortho tertbutyl group or by a pair of ortho methyl groups. A single halogen atom increased binding affinity except that iodine decreased affinity, more at the ortho than at the para position. Binding occurred with a pair of ortho fluorine atoms but not with a pair of ortho chlorine or bromine atoms. These results favor a model of nitrosoarene ferrohemoglobin in which the iron of ferroheme is bonded to the nitrogen atom of the nitroso group since a bond to the oxygen atom would not be hindered by ortho substituents. The presence of a carboxylate substituent resulted in prevention of binding, which was reversed by esterification of the group. Large neutral para substituents, which cannot directly affect formation of the Fe-NO bond, inhibited binding, although not to the same degree as ortho substituents. It thus appears that the affinity of a substituted nitrosobenzene for ferrohemoglobin depends on interactions of the nonbonding part of the ligand molecule with the heme crevice as well as on the ability of its nitroso group to form a bond with the iron of heme. Nitrosoarenes also exhibited in varying degrees the property of removing an electron from ferrohemoglobin to form ferrihemoglobin.


Isolation and characterization of the sulfated neolactotetraosylceramide from hog gastric mucosa. A sulfated neolactotetraosylceramide has been isolated from the lipid extract of hog gastric mucosa. Isolation procedure involved extraction of lipids with 0.4 M sodium acetate in methanol/chloroform/water, alkaline methanolysis, column fractionation on DEAE-Sephadex, separation of the acetylated acidic glycolipids on silicic acid column, and thin layer chromatography. By partial acid hydrolysis, sequential hydrolysis with specific glycosidases, periodate oxidation, and methylation analysis of the native and desulfated compound, the structure of this glycolipid is proposed to be: Galbeta1 leads to 4GlcNAc(6 comes from SO3)beta1 leads to 3Galbeta1 leads to 4Glc1 leads to 1 ceramide.


Use of nitric oxide as a probe for assessing the formation of asymmetrical hemoglobin hybrids. An attempted comparison between alphaNObetaNOalphadeoxybetadeoxy, alpha2NObeta2deoxy, and alpha2deoxybeta2NO hybrids. It has been recently demonstrated that some nitrosyl hemoglobin derivatives have different optical spectrum according to the nature of their quaternary structure (Cassoly, R. (1974) C. R. Seances Acad. Sci., Paris 278, 1417-1420; Salhany, J. M., Ogawa, S., and Shulman, R. G. (1974) Proc. Natl, Acad. Sci. U.S.A. 71, 3359-3362; Cassoly, R. (1975) J. Mol. Biol. 98, 581-595). This property has been used in order to detect the presence of asymmetrical hybrids alphaNObetaNOalpha'O2beta'O2 in a mixture of the two hemoglobins alpha2NObeta2NO and alpha2'O2beta2'O2. When one changes, by deoxygenation, the conformation of the hybrid, there is a characteristic modification in the optical spectrum of the nitrosyl subunits. Quantitative analysis of this phenomenon shows that asymmetrical alphaNObetaNOalphadeoxybetadeoxy and symmetrical alpha2NObeta2deoxy hybrids have distinct properties. The structure-linked optical transition is different in rate and amplitude; it is faster and larger for the asymmetrical molecule. Carbon monoxide binding kinetics performed in absence of phosphate have also indicated that the allosteric equilibrium is more displaced in favor of the T state for alphaNObetaNOalphadeoxybetadeoxy by comparison with the symmetrical deoxygenated intermediates.


17beta-Hydroxy-C19-steroid dehydrogenases from male guinea pig liver. Purification and characterization. One major and six minor 17beta-hydroxy-C19-steroid dehydrogenases were isolated each in a highly pure form from male adult guinea pig liver by a combination of gel filtration and ion exchange chromatography. Molecular weight, amino acid composition, sugar content, number of sulfhydryl groups. NH2-terminal amino acid, isoelectric point, substrate specificity, pH optima, Km values, and inhibitory effect of other steroids were studied. The amino acid composition, Km values, and substrate specificity indicated two separate groups of enzymes. The first group possessed a dual coenzyme requirement and specificity for 5beta-androstanes, whereas the second group showed apparent TPN+ and 5alpha-androstane specificities. Phosphate enhanced the DPN+-related activity of the first group and evoked DPN+-linked activity in the second group of enzymes. A molecular weight of 31,000 to 32,000 with a single chain structure was estimated for four of the enzymes. The other three enzymes consisted of two components of 24,000 and 11,000 daltons.


Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions. Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random  reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quarternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P1. The two partial reactions, namely the exchange of [14C]GDP with the E.HCO3.MnGTP or E.MnGTP complex and the formation of pyruvate from the E.oxalacetate.MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.


Stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid in relation to its possible occurrence as a degradation product of penicillin by the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and the membrane-bound dd-carboxypeptidase from Bacillus stearothermophilus. The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) is hydrolyzed into N-formyl-D-penicillamine with a half-life of 3 to 4 min. On this basis, it is very unlikely that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid could be one of the end products resulting from the cleavage of benzylpenicillin by the DD-carboxypeptidase of Bacillus stearothermophilus (as reported by Hammarström and Strominger (1976) J. Biol. Chem. 251, 7947--7949). In 3 mM phosphate, pH 7.5, and at 37 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) has a half-life of 45 min. On the basis of kinetic experiments carried out under these conditions with phenoxymethylpenicillin and the DD-carboxypeptidase-transpeptidase of Streptomyces R61, it is concluded that the primary product which arises from the thiazolidine moiety of the antibiotic molecule and gives rise to N-formyl-D-penicillamine, has a half-life of 10 min, a value which is not compatible with the hypothesis that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid would be an intermediate involved in the fragmentation pathway.


A relationship between the rates of acetyl group oxidation and the oxygen consumption of cellls. A simple relationship has been derived that may be useful in determining the energy production in the tricarboxylic acid cycle in most tissues or whole organisms during fasting or exercise. The ratio of the rate of acetyl group oxidation to the rate of O2 consumption is nearly constant (0.34), irrespective of the proportion of glucose and fatty acids oxidized in a tissue or an organism. This relationship is derived by multiplying the respiratory quotient by the ratio of acetyl group oxidation to total CO2 production.


Purification of the fibroblast growth factor activity from bovine brain. The purification of the fibroblast growth factor (FGF) from bovine brain has led to the isolation of two peptides. FGF-1 with 128 amino acids and FGF-2 with 107 amino acids. The biological activity of these two peptides is acid- and heat-labile. As indicated by the amino acid composition of FGF-1 and -2, these two peptides are derived from a common precursor and bear no resemblance to pituitary FGF. The brain FGF peptides, like pituitary FGF, are mitogenic in vitro for the same wide variety of mesoderm-derived cells. Since their mitogenic activity is acid- and heat-labile, they are thereby distinguished from the platelet factor isolated from platelets and from the cationic peptide isolated from serum which has been shown to have the same molecular weight and isoelectric point as brain and pituitary FGF.


Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. The mRNA-dependent rabbit reticulocyte lysate of Pelham and Jackson (Pelham, H. R. B., and Jackson, R. J. (1976) Eur. J. Biochem. 67, 247-256) was supplemented with dog pancreas microsomal membranes and used to investigate the synthesis and processing of presecretory proteins. Highly efficient processing and segregation of the major bovine pituitary secretory proteins was observed upon mRNA translation. In contrast to the wheat germ cell-free protein synthesizing system, there was no significant inhibition of translation in the reticulocyte lysate even in the presence of high concentrations of dog pancreas microsomal membranes. Since the latter are required for processing and segregation of presecretory proteins, these reactions could be driven to virtual completion in the reticulocyte lysate without affecting the overall rate of protein synthesis.


Antibodies against intrinsic adipocyte plasma membrane proteins activate D-glucose transport independent of interaction with insulin binding sites. Plasma membrane vesicles from rat adipocytes were treated with dimethylmaleic anhydride to remove extrinsic proteins and then used to immunize rabbits. Immunodiffusion experiments performed in agarose containing Triton X-100 (0.1%) revealed a precipitin reaction between anti-membrane serum and the detergent-solubilized proteins from either intact adipocyte plasma membranes or membranes extracted with dimethylmaleic anhydride. Anti-membrane serum caused cytolysis of intact adipocytes and this effect could be eliminated by incubating the serum at 56 degrees C for 30 min to inactivate complement. Heat-inactivated anti-membrane serum caused a significant increase in 14CO2 production from D-[1-14C]glucose in intact fat cells and the partially purified immunoglobulin fraction from anti-membrane serum markedly stimulated 3-O-methylglucose transport. Maximum activation of transport occurred at a 1:5 dilution and was not additive to that achieved by a maximal dose of insulin. Under these conditions, the anti-membrane immunoglobulin fraction had no effect on the binding of 125-i-insulin to fat cells at all concentrations of the hormone tested. The data are consistent with the hypothesis that activation of hexose transport in these studies results from the binding of immunoglobulin directly to the transport system, although an indirect action is also possible.


Purification of terminal deoxynucleotidyltransferase by oligonucleotide affinity chromatography. Terminal deoxynucleotidyltransferase is an enzyme which has been found to be associated with thymus cells, bone marrow cells, as well as leukocytes from patients with acute lymphoblastic leukemia and chronic myelocytic leukemia in blast crisis. We report here the purification of terminal deoxynucleotidyltransferase by an oligonucleotide affinity (oligo(dT)12-18 cellulose) column. By using a 35 to 70% (NH4)2SO4 cut, Sephacryl S200 column and an oligo(dT) cellulose column, terminal deoxynucleotidyltransferase has been purified from calf thymus cells to a specific activity of more than 8,500 units/mg of protein. The terminal deoxynucleotidyltransferase purified by this method contains no detectable DNA-dependent DNA polymerase or endonuclease activities. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme appears to be homogeneous, with two polypeptides corresponding to the two subunits alpha (10,000) and beta (23,000) of terminal deoxynucleotidyltransferase. These data indicate that oligo(dT)12-18 cellulose can be used as a rapid and selective affinity column for the purification of terminal deoxynucleotidyltransferase.


Identical precursors for serum transferrin and egg white conalbumin. The NH2-terminal sequences of egg white conalbumin and chicken serum transferrin were examined and found to be identical. Conalbumin, when synthesized in a rabbit reticulocyte cell-free translation system, was found to contain an NH2-terminal extension of 19 amino acid residues. Sequential Edman degradation of this precursor (pre-conalbumin) labeled with radioactive amino acids revealed the following sequence: formula see text: The vertical line indicates the site at which pre-conalbumin is cleaved to yield authentic conalbumin. The sequence represents the primary translation product since the NH2-terminal methionine was shown to be derived from initiator Met-tRNAfMet. A partial NH2-terminal sequence of transferrin synthesized in vitro was also determined (underlined residues) and it is identical with that of pre-conalbumin.


Detection of two classes of Ca2+ binding sites in troponin C with circular dichroism and tyrosine fluorescence. We have observed a biphasic increase in [theta]222 and the tyrosine fluorescence of troponin C (TnC) with increasing [Ca2+]. Approximately 62% of the total helical change and 81% of the total increase in tyrosine fluorescence occurred with Ca2+ binding to a class of sites with a KCa of approximately 2.7 X 10(7) M-1. The remaining spectral changes occurred with Ca2+ binding to a class of sites with KCa of approximately 3.1 X 10(5) M-1. We interpret these biphasic responses in terms of distinct structural changes occurring in TnC with Ca2+ binding to the high affinity (KCa approximately 2.1 X 10(7) M-1) Ca2+-Mg2+ sites and to the low affinity (KCa approximately 3.2 X 10(5) M-1) Ca2+-specific regulatory sites.


Lipid composition of the gastric mucous barrier in the rat. The lipid composition of the "mucous barrier" of rat stomach was investigated. Cellular mucus of the mucous cells from gastric epithelium and surface mucus from gastric mucosa were obtained by perfusion in vivo of Ghosh-Lai rat stomachs with 2 M NaCl. Lipids extracted from dialyzed and lyophilized 2 M NaCl perfusates and 0.9% NaCl (saline) controls were quantitatively separated into single components by means of two-dimensional thin layer chromatography and compared. Significant differences in the level of cholesterol esters, di- and triglycerides, and glycolipids were found. Three to 4 times greater quantities of cholesterol esters and di- and triglycerides, and 2 to 6 times greater quantities of glycolipids were found in 2 M NaCl perfusates as compared to saline controls. The glycolipid fraction consisted of neutral and acidic glyceroglucolipids and was devoid of glycosphingolipids. The phospholipids constituted the smallest portion of the lipids present in 2 M NaCl and saline perfusates and were 2 times higher in 2 M NaCl. These data indicate that mucous barrier in addition to mucins contains considerable quantities of lipids of which glyceroglucolipids are the most prominent components.


Rat intestinal 25-hydroxyvitamin D3- and 1alpha,25-dihydroxyvitamin D3-24-hydroxylase. The 24-hydroxylation of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 occurs in vitro in rat intestinal homogenates. 1alpha,24,25-Trihydroxyvitamin D3 obtained by incubating 1alpha,25-dihydroxyvitamin D3 with rat intestinal homogenates was isolated and then identified by co-chromatography of the biologically generated material with synthetic 1alpha,24R,25-trihydroxyvitamin D3 on a high pressure liquid chromatography system and by ultraviolet spectroscopy, periodate cleavage, and mass spectroscopy. 24,25-Dihydroxyvitamin D3 obtained by incubating 25-hydroxyvitamin D3 with intestinal homogenates was identified by co-chromatography of the biologically generated material with authentic 24R,25-dihydroxyvitamin D3 on a high pressure liquid chromatography system and by periodate cleavage. Tissue distribution studies indicate that the process occurs in rat intestinal homogenates but not in rat muscle, liver, or fetal calvaria homogenates. Nephrectomy does not diminish the production of 1alpha,24,25-trihydroxyvitamin D3 in vivo while it reduces markedly, but does not eliminate, production of 24,25-dihydroxyvitamin D3.


Folding of DNA by histones which lack their NH2-terminal regions. HeLa chromatin core particles were digested with trypsin to excise the NH2-terminal histone regions. The resulting nucleoprotein complexes were dissociated in 2.5 M NaCl; the DNA and polypeptides were then allowed to reassemble by lowering the NaCl concentration. Eighty per cent of the DNA reassociated with the polypeptides. The reassembled nucleoprotein complexes sediment at 9.7 S, have a molecular elipticity at 280 nm of 3000 degrees cm2/dmol of PO4, and contain DNase I-susceptible sites at 10 nucleotide intervals. The pattern of products generated by cross-linking the polypeptides with dimethylsuberimidate is very similar to the pattern generated by cross-linking native core particles. The results indicate that histones which lack their HN2-terminal regions retain both the features necessary for correct protein-protein interactions and the ability to fold DNA into a nucleoprotein complex resembling the chromatin core particle.


A cell culture assay for follicle-stimulating hormone. Cultured rat ovarian granulosa cells respond to follicle-stimulating hormone (FSH) by synthesizing and secreting plasminogen activator. The specificity of the response for FSH prompted us to explore the use of this system as an in vitro bioassay for FSH. The release of FSH by pituitary cell cultures has been examined by this method, as have been preparations of FSH of known biological activity. The results indicate that the granulosa cell system allows accurate, rapid, and convenient determinations of FSH activity. Furthermore, the method obviates metabolic clearance problems associated with whole animal assays and it is extremely sensitive: as little as 10(-15) mol (approximately 100 micronIU) of FSH can be detected.


Axial ligand effects on myoglobin stability. Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy. It is found that strong field (low spin) complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbN3), are 1.0 +/- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbH2O) and fluorometmyoglobin (MbF). This observed stability increment is essentially independent of the model chosen for data analysis. These results demonstrate the value of denaturation titration in measuring the stabilization of hemoprotein conformation by ligand binding. The denaturation of MbN3 appears complex. This complexity may be quantitatively accounted for by considering spin state equilibria. Applying this correction, MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins. This result implies metal spin state is more important than ligand stereochemistry in determining the conformational free energy of myoglobin.


An in vitro analysis of the dominance of emetine sensitivity in Chinese hamster ovary cell hybrids. The behavior of ribosomes derived from EmtR X EmtS hybrid cells in in vitro protein synthesis is similar to that observed with a 1:1 mixture of ribosomes from EmtR and EmtS cells. When mRNA (BM virus RNA) is present in limiting amounts (RNA/ribosome molar ratio = 0.1), protein synthesis in either mixture is sensitive to emetine. In contrast, when mRNA is present in excess (RNA/ribosome molar ratio = 2), the emetine resistant as well as the sensitive components are both expressed in the mixtures. These results strongly indicate that emetine resistant and sensitive ribosomes are present in the hybrid cells in about equal amounts and that the dominance of emetine sensitivity is best explained by assuming that emetine acts by blocking ribosome movement along mRNA by inhibiting the translocation step. The observed time lag in the expression of EmtRI and EmtRII mutations following mutagenesis is consistent with the above hypothesis for the mechanism of action of emetine.


Characterization of gangliosides from bovine erythrocyte membranes. Two glucosamine-containing gangliosides, sialosylhexaglycosylceramides, were isolated from bovine erythrocyte membranes. Both gangliosides were hydrolyzed by neuraminidase isolated from Clostridium perfringens to become neutral hexaglycosylceramides. Based on the results of sequential enzymatic hydrolysis and gas chromatography-mass spectrometric analyses of the methylated sugars, the structures of these two gangliosides were shown to be NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide and NeuGcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide, respectively. In addition, N-acetyl- and N-glycolylneuraminosyllacto-N-neotetraosylceramides, and N-acetyl- and N-glycolylneuraminosyllactosylceramides were also found in bovine erythrocytes. The predominant fatty acids in these two gangliosides were C 22:0 and C 24:0. C-18 sphingosine was the major base detected.


Experimental bladder substitution using a biodegradable graft of natural tissue. A controlled canine experimental study has been performed to test the application of a biodegradeable graft to vesical augmentation. The graft, after providing initial bladder enlargement, is progressively reabsorbed while serving as a foundation for the regeneration and healing of the layers of the bladder remnant. Follow-up studies at 1 year revealed no urinary infection, calculi, extravasation of urine, or rejection of the material. Normal bladder capacities were maintained throughout the study and the reconstructed bladders functioned normally. The biodegradable graft was equally successful when applied either to a histologically normal bladder remnant, or to a pathological bladder of intramural fibrosis. These results suggest that this material may be well suited to clinical bladder substitution in humans.


Development of adhesive pit and fissure sealants using a MMA resin initiated by a tri-n-butyl borane derivative. A new adhesive fissure sealant, which consists of a solution of 3% 2-hydroxy-3-beta-naphthoxypropyl methacrylate in methyl methacrylate (MMA), poly-MMA powder and an oxidized tri-n-butyl borane, a polymerization initiator, was developed. Tensile bond strength between an acid-etched bovine enamel and a poly-MMA bar joined by this sealant was 60 kg/cm2, and the value did not decrease much after 30 days immersion in water at 37 degrees C. Penetration of fuchsin into the sealant-enamel interface during a percolation test was not observed. SEM observation showed no voids between the enamel and the cured sealant.


Surface texture and strength of vitreous carbon-poly(methyl methacrylate) dental implant materials. A study has been carried out on the surface texture and the strength of several dental implant materials composed of vitreous carbon microballoons (6, 24, and 48 wt %)and poly(methyl methacrylate).  Two sizes of microballoons were used--particle size less than 100 micrometer and particle size greater than 100 micrometer. Square wafers of the materials (10 X 10 X 1 mm) were studied with the surface sandblasted in half of the specimens. SEM studies revealed a rough porous surface with scattered moderately sized deep cavities in the 6% and 24% specimens with an increase in the number of cavities in the 24% specimens. The 48% specimens revealed a rough, porous surface composed of large shallow craters. Prepared specimens of the materials, processed by a gas-fired air oven and by a microwave oven, were tested for compressive strength and tensile strength using an Instron Universal Testing Machine. Compressive strengths of the various mixtures were similar, but mixtures using VC particle size over 100 micrometer demonstrated somewhat reduced compressive strength. Tensile strengths of the mixutres decreased significantly as the amount of VC increased, with a 20% reduction at 24 wt % and a 45% reduction at 48 wt %.


Biocompatibility testing of polymers: in vivo implantation studies. An in vivo method is described for screening polymeric materials for biocompatibility. The test is based on grading acute and subacute tissue reactions at 7 and 28 days, respectively, following implantation in rats. The methods is reproducible and reliable. It is designed to provide uniform test criteria for biocompatibility assessment in the early phases of the development of surgical implant materials.


Surface finish of dental composite restorative materials. Five composite filling materials were finished using six finishing procedures currently in clinical use. The average roughness produced by each procedure was measured by means of a Surfanalyzer with four replications for each procedure on each of the five materials. Statistical analysis indicated that finishing with silicon carbide disks produced the smoothest surfaces. Acceptable surfaces were obtained with stones or with burs. Use of an alumina paste after a green stone-white Arkansas sequence did not produce a significant improvement. Two processes of filler particle abrasion were distinguished by means of electron microscopy: wear and shear. Fracture striations indicated particle shear. The roughest surfaces were produced by the use of a diamond instrument which is contraindicated. Acceptable finishing procedures for the composite materials tested include silicon carbide disks for accessible areas or 12 fluted finishing burs for more inaccessible areas.


Strength of a dense alumina ceramic after aging in vitro. Rods of dense alumina ceramic were aged in Ringer's solution for time periods up to 12 weeks, with half of the rods being aged while mechanically stressed. Pyrex-type glass rods were similarly tested for comparison purposes. No reduction in flexural strength was observed in dense alumina rods after aging, providing no permeation of the solution into the ceramic occurred. However, a marked reduction in flexural strength was experienced by alumina rods, nominally dense and nonporous, into which some permeation of the saline solution was observed after aging; the largest reduction in strength occurred in such rods aged while mechanically stressed. The results indicate the importance and advantage of using densely sintered alumina ceramics as implants for optimum structural performance over an extended time period.


Scoliosis in arthrogryposis multiplex congenita. Scoliosis was evident in eighteen of eighty-eight patients with arthrogryposis multiplex congenita. The predominant pattern of spinal deformity was a structural thoracolumbar double curve that extended to the sacrum and was associated with pelvic obliquity and lumbar hyperlordosis. Significant contractures about the hips, dislocation of the hip, or both were present in all patients but one. Most of the curves were progressive and they became rigid and fixed at an early age. There was progression of the pelvic obliquity coincident with progression of the curve. Treatment by corrective casts or a Milwaukee brace was ineffective and if surgical treatment directed at the pelvic obliquity did not correct that deformity, spine fusion to the sacrum appeared necessary.


Subtrochanteric fractures of the femur.

Subtrochanteric fractures of the femur. The results of treatment of fifty-six patients with subtrochanteric fractures of the femur are presented and a new classification of these fractures is described. In forty-seven fractures treated with internal fixation there were nine failures of fixation. One type of lesion, the three-part spiral subtrochanteric fracture, occurred  eighteen times and accounted for eight of the failures of fixation.


Total hip arthroplasty.

Total hip arthroplasty. A review of 333 consecutive Charnley total hip arthroplasties four to seven years after operation showed that twenty-eight patients had died, and three deaths were directly related to the surgery. The survivors had an average improvement of 46.6 points in their evaluation scores to a level of 91.3 points. Four deep infections occurred for which removal of the prosthesis was required. Thirteen hips dislocated postoperatively, and five of them required additional surgery. Three were four sciatic-nerve injuries and all but one resolved. Wear of the acetabular component was evident in seventeen hips, measuring two millimeters in six and one millimeter in eleven. With the marking system used, accurate measurement of wear was not possible. Roentgenographic evidence of loosening of the femoral component  was found in 24 per cent of the hips, atributable more often to the quality of cement fixation than to the position of the components (varus-valgus). Varus loosening of two millimeters or more was associated with lower evaluation scores.


Functional fracture-bracing of long-bone fractures of the lower extremity in children. Fifty-six tibial fractures in fifty-five children, sixteen years old or younger, and seventeen femoral fractures in seventeen children in the same age group, seven of them with associated tibial fractures, were treated by fracture-bracing. The tibial fractures healed in an average of 13.2 weeks with no complications that could be attributed to failure of the method. The femoral fractures healed in an average of seventeen weeks. There was an angular femoral deformity of more than 5 degrees in seven of seventeen patients, all but one in fractures of the middle third of the femoral shaft. Of nine distal fractures, only one had angulation of more than 5 degrees. Therefore, the method should be restricted to fractures in the distal third of the femoral shaft. Fracture-bracing gave more freedom to the child and was eminently satisfactory to the parents.


Fracture-dislocation of the ankle with posterior entrapment of the fibula behind the tibia. Fracture-dislocation of the ankle with entrapment of the fibula behind the tibia is a rare condition. Prompt recognition and treatment are necessary to prevent permanent disability. In three of four patients, the injury was managed by closed reduction. If the injury is accurately diagnosed and if traction and medial rotation are applied to the foot while the fibular shaft is pushed laterally, a stable, satisfactory reduction may be achieved. If closed reduction fails, a lateral approach through a small vertical incision directly over the fractured fibular malleolus is sufficient to allow open reduction of the fibula.


Intra-articular pressure as a factor in initiating ulnar drift. To determine the possible relevance of intra-articular pressure in the production of ulnar drift, metacarpophalangeal joints of fresh cadaver specimens were injected with water to increase the intra-articular pressure. Of the thirty-two joints tested, ulnar deviation was produced in twenty-three, flexion in twenty-four, and simultaneous ulnar deviation and flexion (ulnar drift) in nineteen. In two or three instances, the displacement was in extension or radial deviation. These results suggest that elevated intra-articular pressure may be a factor in the initiation of ulnar drift in patients with rheumatoid arthritis.


The influence of design on the transmission of torque across knee prostheses. Knee prostheses of eight different designs were tested experimentally to determine the axial torque necessary to rotate the tibial component relative to the femoral component with the prosthesis positioned at or near full extension. The results represent transmitted torque as a function of axial rotation. For six designs, the transmitted torque was 11.3 newton-meters (100 inch-pounds) or greater at 6 degrees of rotation, with an axial load of 1556.8 newtons (350 pounds). These prostheses either had close conformation between femoral and tibial components, or were mechanically linked. In these prostheses, the geometry of the articulating surfaces was the primary determinant of torque. For the other two types, under similar loading conditions the transmitted torques were 2.5 newton-meters (twenty-two inch-pounds) at 6 degrees of rotation. These prostheses were designed to allow greater freedom for rotation, and hence for them the primary determinant of torque was friction between the articulating surfaces. The predictive value of this torque characteristic relative to the incidence of loosening will have to be determined by comparison with clinical experience.


Methylmethacrylate: A study of teratogenicity and fetal toxocity of the vapor in the mouse. To determine possible deleterious effects of methylmethacrylate vapor on mouse gestation, pregnant mice were exposed to average vapor concentrations of 1330 parts per million for two hours twice daily during Day 6 through Day 15 of pregnancy. A slight increase in the average weight of the fetuses of the exposed mothers were demonstrated but no other evidence of fetal toxicity or teratologic effects was found. During three total hip replacements, the concentration of methylmethacrylate in operating-room air never rose above 280 parts per million.


Cefazolin concentrations in bone and synovial fluid. A single intravenous dose of one gram of cefazolin was administered prophylactically to forty-eight patients undergoing total joint replacement. The serum half-life of the antibiotic was 1.8 hours, the same value found in normal volunteers. The mean concentration in bone during surgery was 5.7 micrograms per gram of bone. The mean concentration in synovial fluid during surgery was 24.4 micrograms per milliliter of fluid.


Musculoskeletal problems in blind children. At a regional school for the blind, 157 children were examined for musculoskeletal abnormalities. The incidence of scoliosis was 9.5 per cent; of foot deformity, 53.5 percent; and of ligament laxity, 25 per cent. Thirty-six children (23 per cent) had evidence of spastic, flaccid, or athetoid cerebral palsy or other disorders of the central nervous system. The orthopaedist should evaluate blind children carefully and repeatedly for evidence of occult cerebral palsy and progressive deformity of the spine or foot.


Traumatic closed transection of the biceps brachii in the military parachutist. Static line injury to the military paratrooper's arm occasionally produces an unusual muscle injury: closed transection of the belly of the biceps brachii. A review of twenty-eight untreated paratroopers so injured demonstrated significant residual weakness and deformity. Attempts at late repair resulted in only minimum improvement. The results of twenty fresh injuries treated by either surgical repair or aspiration followed by closed reduction and splinting are presented.


Sacral agenesis.

Sacral agenesis. Twenty-three patients with sacral agenesis evaluated at Newington Children's Hospital between 1959 and 1977 were classified according to patterns of morphologic deficiency in the bones and articulations. Motor deficit in these patients corresponded, within one level, to vertebral loss; sensory deficit did not correspond. The major orthopaedic problems often associated with sacral agenesis are spinopelvic instability, scoliosis, myelomeningocele, hip dislocation and contracture, knee contracture, and foot deformity. These can be controlled or corrected by proper orthopaedic management.


Failure of total knee arthroplasty due to loosening and deformation of the tibial component. A series of 100 consecutive UCI knee replacements showed a 7 per cent incidence of reoperation due to loosening of the prosthesis associated with permanent deformation of the tibial component. Analysis of the clinical data, roentgenograms, and removed implants showed associations between failure and radiolucency at the cement-bone interface, prosthetic obliquity, collapse of trabecular bone, change of the alignment of the extremity, and permanent deformation of the tibial component. Although no one of these factors by itself can be responsible for the mechanical failure of the arthroplasty, a predominant failure pattern exists. We think that the sequence of events is as follows: implantation of the tibial component with medial or lateral tilt; lack of firm skeletal stabilization; continual microtrabecular fractures; change in alignment of the extremity; and permanent deformation of the plastic component.


Sulfated compounds in the zymogen granules of the guinea pig pancreas. Sulfate incorporation into the guinea pig pancreas was investigated by light (LM) and electron microscope (EM) autoradiography using a system of minilobules incubated in vitro for 60 min in Krebs-Ringer bicarbonate medium (KRB) containing 35SO4(-2). In acinar cells, examined by EM autoradiography, the label was found concentrated over Golgi elements (including condensing vacuoles) and zymogen granules. 35SO4(-2) was also incorporated by the epithelial cells of the entire pancreatic duct system, the incorporation being surprisingly high in the epithelium of the major ducts. In all ductal epithelia, autoradiographic grains appeared over the Golgi complex and the plasmalemma. Since a contribution of duct epithelium to the sulfated compounds found in the discharged secretion could not be ruled out, a purified zymogen granule fraction was used as a source material for the isolation of sulfated compounds of acinar origin. The presence of 35S-radioactivity in the zymogen granules and condensing vacuoles of this fraction was ascertained by autoradiography (of sectioned pellets). From a lysate of this zymogen granule fraction, a soluble sulfated compound of low isoelectric point and high molecular weight was isolated by gel filtration under conditions that allowed its satisfactory separation from the bulk of the secretory proteins.


Electrophorus acetylcholinesterase. Biochemical and electron microscope characterization of low ionic strength aggregates. The "tailed" molecules of Electrophorus (electric eel) acetylcholinesterase aggregate under conditions of low ionic strength. These aggregates have been studied by sedimentation analysis and high-resolution electron microscopy. They consist of bundles of at least half a dozen molecules, the tails of which are packed side by side, to form the core of the structure. Although aggregation is normally fully reversible, aggregates were irreversibly stabilized by methylene blue-sensitized photo-oxidation. This process was shown to consist of a singlet oxygen oxidation reaction and probably involves methionine or histidine residues. It did not modify the structural or hydrodynamic characteristics of the aggregates.


Autoradiographic localization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fiber. Intact and slit nerve fibers of the squid Sepioteuthis sepioidea were incubated in a 50-nM solution of [125I] alpha-bungarotoxin in artificial seawater, in the absence and in the presence of D-tubocurarine (10(-4) M). The distribution of the radioactive label was then determined by electron microscope autoradiography. It was found that, in the fibers exposed solely to the radioactive toxin, the label was located mainly at the axon-Schwann cell boundary in the intact nerve fibers or at the axonal edge of the Schwann cell layer in the axon-free nerve fiber sheaths. Label was also present in those regions of the Schwann cell layer rich in intercellular channels. No signs of radioactivity were observed in the nerve fibers exposed to the labeled toxin in the presence of D-tubocurarine. These results indicate that the acetycholine receptors previously found in the Schwann cell plasma membrane are mainly located over the cell surfaces facing the neighboring axon and the adjacent Schwann cells. These findings represent a further advance in the understanding of the relationship between the axon and its satellite Schwann cell.


An improved method for the isolation of dense storage granules from human platelets. Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules  was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.


In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin. Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.


Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.


Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes. The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.


Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates. The basic stereological formulas for estimating volume (Vv) and surface (Sv) densities are strictly valid only for true infinitely thin sections; the use of "ultrathin" sections of finite thickness T introduces systematic errors, mostly in the sense of overestimation of the parameters. These errors depend on the size and shape of the structural elements and on T. Correction factors for this effect of T are derived by considering model structures that simulate the shape and arrangement of subcellular organelles: (a) spherical vesicles, (b) disks as models for rough endoplasmic reticulum (RER) cisternae, (c) cylindrical tublules as models for smooth endoplasmic reticulum (SER) tubules, microvilli, etc. For vesicles, a model of discrete convex spherical particles is assumed; the correction factors consider loss of caps due to grazing sections and size distribution of the vesicles. The disk and tubule models are used in connection with the new integral geometric formulas of R.E. Miles which consider random aggregates of "inter-penetrating" particles so that the resultant structure is non-convex and thus approximates in nature the networks characteristic of endoplasmic reticulum (ER). Some practical examples relative to liver cells show that the errors due to section thickness may be of the order of 20-40% or more. Computation formulas as well as graphs are given for the determination of the correction factors for Vv and Sv.


Mucosal surface morphology of the toad urinary bladder. Scanning electron microscope study of the natriferic and hydro-osmotic response to vasopressin. The mucosal cell surface of the toad urinary bladder was examined by scanning electron microscopy, and changes in the structure of the surface of the granular cell were correlated with specific physiological responses to vasopressin. Survey views of the mucosal surface demonstrated that there was no consistent repeating anatomical relationship between the granular cell and the mitochondria-rich cell that would support the concept of cooperativeness in the response to vasopressin. During base-line states of Na+-transport and water flux, the microvilli on the mucosal surface of the granular cell are arranged in a ridge-like network with occasional individual projections. When water flux is increased by exposing the tissue to vasopressin, in the presence of an osmotic gradient across the tissue the microvilli on the granular cell lose the ridge structure and appear, predominantly, as individual projection. Variability-of this appearance points out the necessity of examining large areas and many samples before the significance of any morphological change can be assessed. Blocking the simultaneously occurring natriferic response of the toad urinary bladder with 10(-2)M ouabain does not prevent these changes in the microvilli. When the hydro-osmotic response is blocked by eliminating the osmotic gradient, the granular cell shows no consistent change in mucosal surface morphology even when fixed at the height of the natriferic response. The mitochondria-rich and mucous cells did not show any change in morphology throughout these studies. We conclude that the changes in the mucosal surface morphology of the toad bladder seen after exposure to vasopressin are a result of the increased water flux that occurs when an osmotic gradient exists across the tissue, and are not related to the natriferic response or any specific alteration in the membrane properties.


Oriented adsorption of purple membrane to cationic surfaces. We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.


Effects of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast-like cells. Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.


Dissociation of plasminogen activator from the transformed phenotype in a 5-bromodeoxyuridine dependent mutant of Syrian hamster melanoma cells. The relationship between plasminogen activator levels and the expression of the transformed phenotype was studied in a 5-bromodeoxyuridine (BrdUrd) dependent mutant of Syrian hamster melanoma cells. In terms of cell morphology and cellular interactions, the BrdUrd dependent cells resemble transformed cells when grown in the presence of BrdUrd but resemble untransformed cells when grown in the absence of BrdUrd. It was found that the BrdUrd dependent cells release significant levels of plasminogen activator only when cultured in the absence of BrdUrd. In the presence of BrdUrd, the release of plasminogen activator by the dependent cells is suppressed, and the decreased level of plasminogen activator released in the presence of BrdUrd seems to be due to the decreased production of active enzyme. Growth tests revealed that the BrdUrd dependent cells, when attached to a substrate, required BrdUrd in order to attain high densities. Furthermore, the cells are able to grow well in soft agar only in the presence of BrdUrd. These results suggest that the production and release of high levels of plasminogen activator are not related (either as cause or effect) to the expression of the transformed phenotype in the BrdUrd dependent cells. The effect of dog serum (as a plasminogen source) on the BrdUrd dependent cells also was tested. It was found that cells cultured in medium containing dog serum exhibit a morphological alteration, but only in the absence of brdUrd. The morphological response of the cells to dog serum resembles that previously observed with virus-transformed cells. In the BrdUrd dependent cells, the morphological response to dog serum appears correlated with the release of plasminogen activator but separated from other transformed characteristics.


Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters. The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.


Enhancement of the synthesis of specific cellular polypeptides in a temperature-sensitive Chinese hamster cell line (K12) defective for entry into S phase. Temperature-sensitive Chinese hamster cells (K12) have been shown to be defective for the initiation of new rounds of DNA replication when incubated at the restrictive temperature (40.5 degrees). By temperature shift experiments with synchronous cultures, we have marked out the step at which the mutation is expressed as the four hours preceding the initiation of DNA synthesis. The block imposed by the mutation has been shown to be irreversible. In order to approach the biochemical characterization of the temperature-sensitive function in K12 cells, we have analyzed the cellular proteins synthesized under permissive (35 degrees) and restrictive temperatures. The synthesis of three polypeptides is markedly enhanced when K12 cells are incubated at 40.5 degrees. One of them (band B) has turned out to be a useful biochemical marker of the expression of K12 mutation since its synthesis is not affected in other ts-mutants or in hybrids in which K12 mutation is complemented. In addition, the alteration in band B synthesis is irreversible and occurs during the same stage of the cell cycle at which the mutated function is expressed.