Effect of estracyt on the rat prostate.

Effect of estracyt on the rat prostate. Two types of experiments were performed to elucidate the estrogenic effect of estracyt on the ventral and immature rats that had received injections of testosterone; then changes in weight of accessory rats and changes in weight of the total body, the ventral and the dorsolateral prostates, and adrenal gland, and changes in activities of testosterone 5alpha-reductase, alkaline phosphatase, and arginase of both lobes of the prostates were examined. In the second experiment, estracyt was injected into castrates rats and immature rats that had received injections of testosterone; then changes in weight of accessory sex organs were determined. Because similar changes were induced by treatment of animals with estradiol-17beta, it was concluded that the effect of estracyt on the prostates was most likely attributable to the estrogenic effect of the drug.


[Modern aspects in the pathogenesis of urticaria with special reference to intolerance phenomena]. The chronic urticaria remains till now a "vexing problem" (Sheldon, 1954), because its etiopathology can be clarified only in about 20-30%. Five different immunologic mechanisms, two nonimmunologic mechanisms and two important manifestation factors are found. The physical triggering (12-17%) and the so-called aspirine/additiva provocation (26%) seem to be the most frequent causes of urticaria. Exogenic antigens are identified only in 3-8% of cases. The unspecific reaction phase of urticaria can be different too, although the degranulation of mast cells with histamine-deliberation is found very often. In hereditary urticaria and in pressure urticaria histamine-deliberation seems not to be important.


[Clinical picture of recurring aphthae in the mouth mucosa]. Various classifications of recurrent aphthous ulcers, their differential diagnosis, especially the herpetic ulcers of the oral mucous membranes, are discussed. The clinical and histological pictures show three variants, the minor form, the major form or periadenitis mucosa necrotica recurrens (Sutton), and the herpetiforme aphthous ulcers. Recent investigations discussing the etiology of these ulcers assume that an auto-antigen might be responsible for the immuno-pathological mechanism. This auto-antigen is supposed to be formed by the contamination of the oral mucosa with microorganisms, particularly streptococci.


[Transient acantholytic dermatosis (Grover)]. The clinical, histopathological, and electron microscopical characteristics of "transient acantholytic dermatosis" (Grover, 1971) are described. Clinically, there are papules with smooth or keratotic surfaces and sometimes papulovesicles. The lesions are mainly distributed on the trunk predominantly of male patients past the age of 40. The disease usually disappears within weeks, but rarely may be protracted. Histologically, focal changes with the epidermis of Dariertype, Hailey-Hailey-type, pemphigus (vulgaris or foliaceus)-type, or spongiotic-acantholytic type, often in combination, are found. This disease seems to be common. Its distinctiveness should be confirmed by further observations.


[Necroses following the use of Pyoktanin]. In a patient suffering from chronic balanitis, necrotic lesions of the glans penis and the internal part of the prepuce occurred when treated with 1% gentian violet. Necrosis after treatment with gentian violet is a rarely seen complication which should be borne in mind.


[Skin necroses following the use of a 2-per-cent Pyoctanin solution]. The case of a child is reported in whom a circumscribed necrotic skin reaction developed in the gluteal fold after application of 2% crystal violet (Pyoktanin) in aqueous solution. Similar cases are described after topical treatment with 1% solutions of dyes. For direct application to intertriginous areas, triphenylmethane dyes should be used in lower concentrations.


[Echo-6-virus as the pathogen of hand, foot and mouth exanthema]. Seven cases of typical hand-foot-mouth-disease are reported. As causative agent Echovirus Type 6 was isolated.


Electron microscopical demonstration of adenylate cyclase activity in nervous tissue. Adenylate cyclase (EC4.6.1.1) activity stimulated by norepinephrine and dopamine was demonstrated histochemically by electron microscopy in the cerebral cortex and caudate nucleus of the rat. The precipitating agent in the histochemical reaction was cobalt, which was shown biochemically to increase the adenylate cyclase activity. The reaction product was located in the synapses, being contiguous attached to the postsynaptic membrane. It was also located in the plasma membrane of some nerve fibers. Alloxan, the specific inhibitor of adenylate cyclase, inhibited the reaction in the cerebral cortex and caudate nucleus, and haloperidol had a somewhat similar effect in the caudate region.


Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting, refeeding, and gallstone formation. A light microscopic autoradiographic and quantitative electron microscopic study. The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H3-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H3-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbaldder mucosa. Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly dimished the volume density of the Golgi apparatus. Refeeding the amimals restored the volume density values to normal levels, In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.


The occurrence and localisation of heavy metals and glycogen in the earthworms Lumbricus rubellus and Dendrobaena rubida from a heavy metal site. The lead content of whole earthworms, highest in contaminated site specimens, was significantly higher in Dendrobaena rubida than Lubricus rubellus and a species difference in zinc was also recorded. Selective feeding and differential absorption are discounted and a species difference in maximum tolerance to body lead is suggested. Copper was low in all specimens. Chloragocytes and intestinal tissue showed significantly higher lead levels in contaminated earthworms than in control material. Ultrastructurally, chloragocytes from contaminated earthworms showed electron dense flecks associated with the chroragosome peripheries and within the debis vesicles. Very fine flecks occurred in the nuclei, but mitochondria and Golgi were indistingushable from control material. Preliminary X-ray microanalysis of contaminated chloragocytes revealed lead and phosphorus. The deposits within the chloragocytes might represent unbound lead precipitated by phosphate buffer; flecks being absent from contaminated, citrate buffered material and from control material. The chloragosomes are proposed as possible sites for sequestered lead. Chloragocyte and intestinal glycogen levels were significantly higher in control material where the chloragocyte cytoplasm was rich in alpha-glycogen rosettes, these being absent from lead contaminated cells. The glycogen-lead correlation suggests that the metabolism of contaminated chloragocytes is directed towards lead sequestration, though differing nutritional states cannot be ignored.


Cholinesterase patterns in the cerebellum of reptiles. In the cerebellum of four species belonging to the three main reptilian orders the histochemical localization of cholinesterases has been studied. The use of different substrate-inhibitor combinations permits to record the distribution patterns of acetylcholinesterase and pseudocholinesterase, mainly revealed as butyrylcholinesterase activity. From the neurological point of view it is interesting to note that acetylcholinesterase activity shows three different distribution patterns in reptilian cerebellum, thus confirming the characteristic variability previously noticed in the cerebellar cortex of other vertebrates.


The distribution of acetylcholinesterase in the central nervous system of web-building spiders (Arachnida, Araneae). The distribution and activity patterns of acetylcholinesterase in the central nervous system of araneid and agelenid spiders have been studied. Enzyme activity was limited to the neuropile mass. The araneid and agelenid species investigated exhibited largely similar acetylcholinesterase activity in different parts of the nervous system. Enzyme reactions were relatively less intense in the optic ganglia; the central body showed only weak to moderate reaction stainings.


[Azoindoxyl methods for the investigation of hydrolases. II. Biochemical and histochemical studies of acid beta-galactosidase (author's transl)]. The determination of various reaction constants yields the following assay for the photometric evaluation of acid beta-galactosidase (measurement of the azoindoxyl dye at 540 nm after extraction with dimethylformamide or -acetamide): 1.5 mM 5-Br-4-Cl-3-indolyl-beta-D-galactoside (1 mg dissolved in 0.05 ml dimethylformamide) and 0.01-0.015 ml hexazotized p-rosaniline/ml in 0.1 M citric acid-phosphate buffer, pH 4. By means of this procedure it becomes evident that the activity of the enzyme differs considerably in various rat organs; NaCl does not influence acid beta-galactosidase. -- Similar results were obtained with the indigogenic method; indigo can be dissolved and measured photometrically as the azoindoxyl dye. The enzyme is suppressed by high concentrations of hexazotized p-roaniline to 50%; low concentrations do not inhibit; the same is true for ferricyanide-ferrocyanide employed in the indigogenic media. -- The effect of glutar- and formaldehyde on acid beta-galactosidase cannot be investigated with the azoindoxyl reaction since the azoindoxyl dye partially withstands extraction from fixed blocks of tissue. On the basis of the biochemical findings the azoindoxyl technique can be recommended for the histochemical demonstration of acid beta-galactosidase: 7.5 mg (1.5 mM) 5-Br-4-Cl-3-indolyl-beta-D-galactoside (dissolved in 0.25 ml dimethylformamide) and 0.05-0.15 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid-phosphate buffer, pH 4. After incubation the sections can be treated with osmium tetroxide followed by dehydration and mounting in resins or can be mounted without prior osmification of the azoindoxyl dye in glycerin jelly. The osmium chelate resists treatment with organic solvents; the stability of the chelate depends on the concentration of hexazotized p-rosaniline. After fixation in glutaraldehyde or in a mixture of form- and glutaraldehyde acid beta-galactosidase can be exactly localized in the lysosomes of many rat organs. In comparison with the indigogenic, the metal precipitation and the simultaneous azocoupling reactions for the in situ detection of acid beta-galactosidase the azoindoxyl procedure is superior if fixed material is used; it is equivalent or inferior in connection with membrane technique. The biochemical azoindoxyl assay represents a useful method for combined qualitative and quantitative studies of acid beta-galactosidase.


Enhanced susceptibility of DNA-synthesizing nuclei of epithelial cells of the intestine to acid hydrolysis. Albino mice were injected intraperitoneally with tritiated thymidine and killed 1/2 and 30 h later. Pieces of ileum were excised and fixed. Tissue sections were hydrolyzed with 5 N HCl at 21 degrees C for 1/2, 1, 2, 3, 4 and 6 h, some sections remained unhydrolyzed. Both the hydrolyzed and nonhydrolyzed sections were autoradiographed. Grain counts per labelled nucleus of either cryptal (DNA-synthesizing) or villous (DNA-nonsynthesizing) cells nonhydrolyzed and hydrolyzed for various time intervals were recorded. The results indicate, that the grain count of nonhydrolyzed, labelled nuclei from cryptal cells was by 1.49 higher than that of villous cells demonstrating the rate of grain counts diminution caused by cell divisions. Hydrolysis caused a diminution of grain count of cryptal cells by approximately 15% higher than that of the grain count of villous cells.


Demonstration of acetylcholinesterase by semipermeable membrane technique: estimation of soluble and fixation-labile portions in different regions of the central nervous system. A histochemical method for the demonstration of acetylcholinesterase using semipermeable membranes is described. This technique prevents any loss of enzyme activity caused by dissolution and/or fixation. The soluble and fixation-labile portions of acetylcholinesterase were estimated in several regions of the central nervous system of the rat and differences were found. The method improves the accuracy of the histochemical demonstration of the acetylcholinesterase on the light microscopical level.


The application of the "Lojda" method for intestinal lactase in intestinal biopsies from jaundiced newborn infants. A new use of the histochemical method for intestinal lactase activity is described. Peroral intestinal biopsies from newborn light-treated infants with diarrhoea were investigated for brush-border lactase. The lactase activity found in these infants by the histochemical method correlated well with the infants ability to hydrolyze lactose judged by lactose tolerance test.


Effects of ethyl alcohol on the acoustic reflex threshold. Acoustic reflex thresholds for pure tones and white nose were measured every 20 min for 4 hr after the ingestion of ethyl alcohol. The average maximum acoustic reflex threshold shift was 11 dB, occurring on the average at 100 min, and returning to base-line levesl at approximately 200 min postingestion. These data lend support to the classification of ethyl alcohol as a sedative, and should caution the clinician when interpreting acoustic reflex threshold data from persons who have ingested small amounts of alcohol.


Attemps to relieve tinnitus.

Attemps to relieve tinnitus. The major effect here has been to present two forms of treatment which have provided relief of tinnitus for some patients. Use of a properly fitted hearing aid relieves tinnitus when that tinnitus is embedded within the frequency region of the hearing loss. When the use of a hearing aid is counterindicated, then use of the tinnitus masker can bring relief for certain kinds of tinnitus. At present the only tinnitus masker available presents a band of noise of moderate frequencies, and it is capable of relieving tinnitus in that frequency region or near it. When masking of tinnitus is effective it produces a suppresion of the tinnitus which extends beyond the duration of the masker. This is termed residual inhibition, a matter about which we need to know a great deal more. For tinnitus which is intractable to medical treatment it has become a practice to instruct the patient that he must learn to live with his affliction. It is hoped that this presentation will cast doubt upon that view, for it can be seen that there are many tinnitus sufferers for whom much can be done.


The polarity of the sexes and its decline. The polarity of the sexes is rooted in the genic sex. The qualities which constitute masculinity and femininity are psychobiological secondary (sex) characteristics. They are epigenetic phenomena evolved from the interaction of man's biological givens with the civilization. The polarity of the sexes changes in our civilization faster than before. The potential for psychological change is in the process of identification primarily of the infants of both sexes with the mother. Socioeconomic and cultural changes bear influence on the structure of the family and promote the equalization of the sexes.


Nonlinear distortion in hearing aids: the need for reevaluation of measurement philosophy and technique. Conventional pure-tone tests of harmonic distortion are of dubious sufficiency in expressing the nonlinear distortion (NLD) characteristics of hearing aids. This paper reviews the strategies and problems of intermodulation distortion. Spearman rank order correlations for a group of hearing aids varied widely as a result of changes in the selected frequency and/or level of input signal. Also, the reference criteria for expressing distortion was observed to contribute to the inconsistency of rank order impression. A technique is described which incorporated synthetic vowels as test signals. Hearing aid outputs were studied on a spectrum analyzer for evidence of NLD. It is argued that efforts should be directed at devising tests for hearing aids which are directed at comprehensive NLD including both harmonic distortion and intermodulation distortion.


Periodicity pitch perception of retarded children. In order to expand the body of information concerning auditory function in the presence of the generalized cortical dysfunction associated with severe mental retardation, the discrimination of periodicity pitch signals by nonverbal severely retarded children and adults of normal intelligence was examined using an operant conditioning adaptation of the psychophysical method of constant stimulus differences. The periodicity pitch difference limens (DLs) based on changes in repetition rate were compared with frequency DLs obtained from low (150 Hz) and high frequency (2200 Hz) sinusoids. Periodicity pitch signals were generated by gating a 2200-Hz sinusoid over a range from 141 to 159 pulses per sec and passing the signals through a narrow band-pass filter to ensure no low frequency energy was present. The DLs (75% correct discriminations) were computed from the three types of discrimination performance curves. Standard errors of the means were small and retarded subjects showed good test-retest reliability. The results indicated that the simple and complex pitch discrimination abilities for both normal and retarded subjects were similar.


Reliability of the Tillman-Olsen procedure for determination of spondee threshold using recorded and live voice presentations. This study investigated the reliability of the Tillman-Olsen procedure for establishing the spondee threshold (ST). Monitored live voice (MLV) and the Auditec of St. Louis recordings of the Central Institute for the Deaf spondees were used as stimuli. Ninety-five percent of 96 normal hearing subjects had test-retest STs within 5 dB and no subject varied more than 8 dB. Although these results represent good short term reliability for the Tillman-Olsen procedure, repeatability is no better than that produced by other procedures. Thresholds obtained by MLV  were as reliable as the recorded spondees. It was concluded that recorded spondees offer no advantages over MLV testing for the experienced clinician. The recorded presentation gave STs that are very close to the normative value for speech specified by ANSl-1969. Live voice testing showed STs 4.7 dB better than when the Auditec recordings were used.


Occlusion effect in bone conduction pure tone and speech audiometry. Two experiments were conducted to determine the occlusion effect (OE) for pure tone and spondee stimuli. Results of experiment I using mastoid placement and unilateral occlusion yielded a mean OE for bone conduction speech reception thresholds of 4.67 dB, which was in agreement with the average OEs of 0.50, 1.0 and 2.0 kHz, or simply the same as the OE for 1.0 kHz. However, the magnitude of the observed OEs for test frequencies of 0.25 to 4.0 kHz was considerably less than the OEs reported in previous investigations. In an attempt to explain this discrepancy, experiment II used unilateral and bilateral occlusion with mastoid and forehead placement for pure tone stimuli. Significant differences (p less than 0.05) were found between OEs obtained with mastoid versus forehead placement and for unilateral versus bilateral occlusion.


Cold hemagglutinin disease in a dog.

Cold hemagglutinin disease in a dog. Cold hemagglutinin disease, a form of autoimmune hemolytic anemia caused by cold-acting erythrocyte autoantibodies, was diagnosed in a dog. Cold-acting antibodies differ from warm-acting antibodies in several ways, including temperature-dependent reactivity below 32 C. The autoantibody in this case (immunoglobulin M) was in the serum in higher than normal concentrations and was reactive at temperatures approaching freezing. The course of the disease was chronic and was characterized by cyanosis and necrosis of the body extremities. Immunosuppressive therapy with corticosteroids was successful in alleviating the clinical signs and reducing the autoantibody activity in the dog. The cause of this disorder was not determined.


Heatstroke in the dog: a polysystemic disorder. The early stages of heatstroke in 3 dogs were characterized by hyperthermia, hyperpnea, tachycardia, depression, vomiting, diarrhea, and dehydration. Laboratory signs of hepatocyte degeneration and necrosis were detected. Clinical and laboratory changes characteristic of acute primary renal failure developed several days after overheating. After symptomatic and supportive therapy, 2 of the dogs regained a sufficient degree of renal function to cause remission of signs of renal failure.


Effect of prostaglandin F2alpha-THAM in the bitch. The subcutaneous median lethal dosage for prostaglandin F2alpha for the Beagle bitch was determined to be 5.13 mg/kg, using a sequential experimental design. Clinical signs and gross and histologic findings were consistent with a diagnosis of shock syndrome. It was concluded that prostaglandin F2alpha could be used to develop a pharmacologically controlled shock model.


Research on the difference between generalization and maintenance in extra-therapy responding. Many authors have reported that the development of programs for producing durable extra-therapy responding lags behind the development of programs for producing initial behavior change. In Experiment I, responding was recorded continuously in both the therapy and extra-therapy settings. The results showed that one child did not generalize to the extra-therapy setting, but that other children did. However, for the children who generalized, extra-therapy responding was not maintained. Therfore, in Experiment II two variables affecting the durability of extra-therapy responding were assessed and found to be influential: (a) the use of partial reinforcement schedules in the original treatment environment; and (b) the presence of noncontingent reinforcers in the extra-therapy environment. The results suggest that there are two distinct parameters of extra-therapy responding: generalization and maintenance. A technology for producing durable extra-therapy responding is discussed in terms of different treatment procedures required for different deficits in extra-therapy responding.


Comparing treatment tactics with a hyperactive preschool child: stimulant medication and programmed teacher intervention. Two treatment tactics, food and praise contingent on appropriate play and varying doses of methylphenidate (Ritalin), were evaluated for their effects on a preschool child's activity changes. In addition, other social, verbal, and academic behaviors were monitored to examine possible side effects of the two treatment tactics. Fewer free-play activity changes occurred during contingent reinforcement phases while medication had variable effects: increasing attention to tasks but, at higher doses, decreasing intelligibility of speech and responsiveness to mands. The study outlines a replicable model for comparing medication with alternative behavioral strategies to control hyperactivity and enhance skill development.


Teaching children appropriate shopping behavior through parent training in the supermarket setting. The shopping behavior of three elementary school-age boys was analyzed and parent-child interactions assessed for one boy and his mother. Estimates of parent of consumer satisfaction with child shopping behavior were also obtained. The effects of a parent-mediated treatment package on child behavior were assessed using a multiple-baseline design. Treatment produced significant increases in appropriate shopping behavior in all three children and parents became increasingly satisfied with improved child behavior. Analysis of data for one mother also revealed that her manner of interacting with her child became more positive.


The effects of modelling on the contingent praise of mental retardation counsellors. A multiple-baseline design was used to evaluate the effects of a simple modelling procedure on the contingent praise of five counsellors while they conducted hygiene-training sessions in toothbrushing and hand-and-face washing with severely retarded children. After varying numbers of baseline sessions, each counsellor watched a model who conducted a series of toothbrushing sessions, in which he conspicuously praised correct toothbrushing responses and approximations to correct responses. No modelling occurred during hand-and-face washing sessions. As a result of several exposures to the model's performance, levels of response-contingent praise provided by four of the five counsellors during toothbrushing sessions increased sharply over baseline. The levels of counsellor praise showed parallel increases during hand-and-face washing sessions. A two-week followup showed that the levels of praise obtained through modelling were maintained in the model's absence.


Teaching coin equivalence to the mentally retarded. A program was designed to teach coin equivalence to mentally retarded adolescents. Coin equivalence was defined as choosing several different combinations of coins to equal specified target values. A pretest-posttest matched-groups design was employed with an experimental group receiving the monetary training, and a no-training control group. A multiple baseline across coin-counting responses was also incorporated in the experimental group. Training was divided into six stages, each teaching one specific method of combining coins to equal 10 target values from 5 cents through 50 cents. A three-component response chain was used, requiring (a) naming, (b) selecting and counting, and (c) depositing target monetary values into a coin machine. Experimental subjects improved significantly in coin equivalence performance and maintained their skill on follow up tests; control subjects did not.


Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branded beta-hydroxy acids in Desulfovibrio desulfuricans. The fatty acids obtained from extractable lipids of the anaerobic sulfate bacterium Desulfovibrio desulfuricans were identified. Saturated and monoenoic iso (C15-C19) and anteiso (C15, C17) fatty acids and saturated normal (C14-C18) and monoenoic normal (C16, C18) fatty acids were shown to be shown to be present by capillary gas chromatography-mass spectrometry. Iso and anteiso beta-hydroxy fatty acids were analyzed as trimethylsilyl ethers in the same way. The position of methyl branches in the monoenoic fatty acids was determined from characteristic fragment ions in the mass spectra of their methyl esters. Disilyloxy methyl esters, prepared by derivatization of the mono unsaturated methyl esters and analyzed by capillary gas chromatography-mass spectrometry, provided the position of double bonds. The monoenoic fatty acids identified in this way were normal (delta7-C16:1, delta9-C16:1, delta9-C18:1, delta11-C18:1), iso (delta7-C15:1, delta9-C16:1, delta9-C17:1, delta11-C18:1, delta11-C19:1), and anteiso (delta7-C15:1, delta9-C17:1). Iso delta9-C17:1 fatty acid is present as the major component. The occurrence of these monoenoic fatty acids in this bacterium is of taxonomical importance.


Chromosome replication in Caulobacter crescentus growing in a nutrient broth. The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.


Formation and function of Vibrio parahaemolyticus lateral flagella. Formation of the lateral flagella (L-flagella) of Vibrio parahaemolyticus was studied immunologically, using specific antiserum against L-flagella. On solid medium, L-flagella were formed at both high (37 degrees C) and low (25 degrees C) temperatures, although at high temperatures they became dissociated from the cells and decomposed in the medium. L-flagella were not formed in liquid or soft-agar medium. Formation of L-flagella was decreased by lowering the pH of the medium and repressed by transferring the cells from solid medium to liquid medium. Mutants possessing L-flagella but not a polar monotrichous flagellum (M-flagellum) swarmed on solid medium, whereas mutants were grown on solid medium and then transfered to liquid medium, the cells oscillated until they lost L-flagella. It is postulated that L-flagella are locomotive organelles on solid medium and in some cases also in liquid medium, whereas M-flagella are locomotive organelles only in liquid medium.


Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis. The catalase level of Bacteroides distasonis (ATCC 8503, type strain) varied with the amount of hemin supplied to the medium when the cells were grown in either a prereduced medium containing 0.5% peptone, 0.5% yeast extract, and 1% glucose or in a prereduced, defined heme-deficient medium. The effect of hemin on catalase production could not be duplicated by ferrous sulfate or ferrous ammonium citrate. Catalase activity reached peak values in late log phase, whereas superoxide dismutase specific activity remained constant throughout the culture growth cycle. The catalase was a nondialyzable, cyanide and azide-sensitive, heat-labile protein that coeluted with bovine erythrocyte catalase from Sepharose 6 B. Analysis of polyacrylamide gels stained for catalase activity and for heme showed a correspondence between the single catalytic activity band and one of three heme-protein bands. These data suggest a heme-protein of approximately 250,000 molecular weight. The superoxide dismutase was a cyanide-insensitive protein of approximately 40,000 molecular weight that migrated electrophoretically on acrylamide gels as a single band of activity.


p-cymene pathway in Pseudomonas putida: initial reactions. Initial reactions of the p-cymene pathway induced in Pseudomonas putida PL have been reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in three steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in two reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are three pieces of evidence for the physiological role of the dihydrodiol intermediate. (i) a mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate. (II) This metabolite is enzymically oxidized by a nicotinamide adenine dinucleotide-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. (iii) 3-Hydroxy-p-cumate does not support growth of P . putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulated during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the carbon 4 atom also is reported.


p-Cymene pathway in Pseudomonas putida: ring cleavage of 2,3-dihydroxy-p-cumate and subsequent reactions. It was confirmed that 2,3-dihydroxy-p-cumate is a substrate for ring cleavage in Pseudomonas putida PL-W after growth with p-cymene or p-cumate. This compound was oxidized to pyruvate, acetaldehyde, isobutyrate, and carbon dioxide by extracts of cells, and these products appear in equimolar amounts. The transient appearance of compounds and 2,3-dihydroxy-p-cumate to a yellow intermediate (lambda max, 345 nm) without decarboxylation. Extracts of the benzene nucleus; this is followed by decarboxylation to give the 393-nm species, which gives rise to isobutyrate, acetaldehyde, and pyruvate by the hydrolytic route of meta cleavage of catechols, via 4-hydroxy-2-oxovalerate. This was confirmed with a mutant of P. putida PL-RF-1 that was unable to grow with p-cymene (or p-cumate) but was able to oxidize both compounds AND 2,3-DIHYDROXY-P-CUMATE TO A YELLOW  INTERMEDIATE (LAMBDA MAX, 345 NM) WITHOUT DECARBOXYLATION. Extrats of P. putida PL-W (wild type) or a revertant of the mutant PL-RF-1 catalyzed the decarboxlation of the 345-nm intermediate with transient formation of the compound that absorbed at 393 nm. The substrate specificities of the 3,4-dioxygenative ring cleavage enzyme, and the decarboxylase were determined in crude extracts of P. putida PL-W and Pseudomonas fluorescens 007. It was conclude that 3,4-dioxygenative cleavage and decarboxylation are sequential enzyme-catalyzed reactions common to both P. putida and P. fluorescens for the oxidation of 2,3-dihydroxybenzoates. Unlike P. putida PL-W, which exclusively use the hydrolase branch, P. fluorescens 007 uses the dehydrogenase branch of the meta pathways that diverge after ring cleavage and later converge at oxoenate intermediates.


Purification and properties of L-glutaminase-L-asparaginase from Pseudomonas acidovorans. An enzyme that catalyzes the hydrolysis of both glutamine and asparagine has been purified to homogeneity from extracts of Pseudomonas acidovorans. The enzyme having a ratio of glutaminase to asparaginase of 1.45:1.0 can be purified by a relatively simple procedure and is stable upon storage. The glutaminase-asparaginase has a relatively high affinity for L-asparagine (Km=1.5 X 10(-5) M) and L-glutamine (Km=2.2 X 10(-5) M) and has a molecular weight of approximately 156,000 the subunit molecular weight being approximately 39,000. Injections of the enzyme produced only slight increases in the survival time of C3H/HE mice carrying the asparagine-requiring 6C2HED Gardner lymphoma and of white Swiss mice carrying the glutamine-requiring Ehrlich lymphoma.


Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses. The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.


Identification of a phosphorus-containing storage granule in the cyanobacterium plectonema boryanum by electron microscope x-ray microanalysis. Transmission electron microscopy of cyanobacteria couple with X-ray microanalysis has confirmed that dense deposits within these cells are composed of phosphorous.


Ultrastructure of Butyrivibrio fibrisolvens: a gram-positive bacterium. The cells of bacteria of the genus Butyrivibrio are universally described as being gram negative, and they produce an unequivocal gram-negative reaction in the standard staining procedure. However, their cell walls contain derivatives of teichoic acid, which are characteristic of gram-positive cells. In this study, the cell walls of two representative strains of Butyrivibrio were of the gram-positive morphological type, as seen by electron microscopy, but they were very thin (12 to 18 nm). The thinness of these cell walls may account for the tendency of these cells to stain gram negatively in the standard staining procedure. Ruthenium red staining revealed an extracellular structure surrounding cells of Butyrivibio sp. (strain C3). This structure was composed of individual "knobs" that sometimes mediated cell-to-cell adhesion in the culture.


Interconversion of large packets and small groups of cells of Micrococcus rubens: dependence upon magnesium and phosphate. Micrococcus rubens, a gram-positive occus, usually forms large, cubic packets of more than 500 cells that are regularly arranged in three-dimensional cell groups. In medium with extremely low concentration of Mg2+ and phosphate, in which the cells can only grow on a agar surface, it formed small groups of 2 to 20 cells. Irregularly arraged cell groups of intermediated size were obtained in culture media containing intermediated concentrations of Mg2+ and phosphate. Mutants that formed irregular cell groups of intermediate size under normal culture conditions were also obtained.


Formation of regular packets of Staphylococcus aureus cells. Staphylococcus aureus, which usually forms grape-like clusters, has the ability to form regularly arranged cell packets. These regular cell packets are formed when the activity of its separation enzyme(s) is lost either by treatment with detergents, such as sodium dodecyl sulfate or Trition X-100, or by mutation of the cells. These cell packets consisted of 8 to 64 spherical cells that have a three-dimensional arrangement. Some irregularity in the arragement of cells in packets, however, can be observed by scanning electron microscopy. It is concluded that S. aureus fundametally divides along three definitely oriented planes that are located at right angles to each other. After cell division, the cells usually become translocated due to the action of a separation enzyme(s) to form grape-like clusters.


Biochemical and physiological properties of alkaline phosphatases in five isolates of marine bacteria. The alkaline phosphatase activities of five unique isolates of marine bacteria were found to be associated with the periplasmic space; however, the enzymes from these isolates differed with respect to their repressibility, the apparent number of isoenzymes, the necessity for Mg2 for activity, and the conditions required for their release. With three of the isolates, the enzyme was released when cells that had been washed in 0.5 M NaCl were suspended in sucrose; however, with the other two isolates, one required the additional presence of tris(hydroxymethyl)aminomethane and the other required the presence of lysozyme and ethylenediaminetetraacetic acid. In two isolates the activity was constitutive, in two it was partially repressed, and in one it was completely repressed by inorganic phosphate. The repression of activity was associated with corresponding changes of activity bands as seen by acrylamide gel electrophoresis.


Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine. Lysozyme-sensitive mutants of Mycobacterium smegmatis, isolated by nitrosoguanidine treatment, have been converted into protoplasts in a nutritionally enriched medium containing lysozyme and DL-methionine.


Characterization of a glucan from Polyporus circinatus. An alpha-D-glucan was isolated from a monokaryon of Polyporus circinatus; it has been characterized as a highly branched glycogn.


Chemical modification of spinach ferredoxin. Properties of acetylated spinach ferredoxin. The acetylation of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated. An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was indistinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferredoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity. Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1.6.7.1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the modified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.


Studies on glycosphingolipids of fresh-water bivalves. III. Isolation and characterization of a novel globoside containing mannose from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii. Three globosides were isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii by mild alkaline hydrolysis, acetone precipitation, Unisil column chromatography and preparative thin-layer chromatography. These globosides are unique in their sugar chains, since they contain mannose instead of galactose found so far in all globosides of mammals and other animals examined. The main globoside in the spermatozoa was characterized as Glc-NAcbeta(1 leads to 2)Manbeta(1 leads to 3)Manbeta(1 leads to 4)Glcbeta(1 leads to 1)-ceramide by partial acid hydrolysis, analysis of its anomeric configuration with chromium trioxide, methylation analysis and enzymatic hydrolysis. The globoside contained normal saturated fatty acids ranging in length from C16 to C21, palmitic and stearic acids being predominant. Its main long-chain was octadeca-4-sphingenine.


Soluble proteins from fowl feather keratin. I. Fractionation and properties. A method is described for the fractionation of reduced and alkylated proteins of fowl feather. Fowl feather extracts were chromatographed on a Sephadex G-75 column in 4 M urea containing 1 M NaCl and separated into four fractions, GF-1, 2, 3, and 4. The elution patterns were used to compare the components of different feather parts, barbs, rachis + medulla, and calamus. In all cases, GF-3 was the main fraction and the percentages with respect to the total peak area found for barbs, rachis + medulla, and calamus were about 65%, 74%, and 93%, respectively. Each of the fractions was examined by polyacrylamide disc gel electrophoresis and all were heterogeneous. The slowly moving bands mainly corresponded to fraction GF-1, intermediate bands to GF-2 and 3, and faster bands to GF-4. Many other polypeptide chains, which have not been found previously, were newly separated from three minor fractions of fowl body feather. The molecular weights of fractions GF-2 and 3 were estimated by calibrated gel filtration to be 33,000 and 10,500, respectively. Marked differences were found in the amino acid compositions of various fractions from fowl feather. The GF-1 fraction and insoluble residue had very similar compositions; in both cases the contents of serine, glycine, and proline were lower and those of helix-favoring amino acids, namely, lysine, tyrosine, and methionine, were higher than those found in other fractions.


Micro-scale determination of seminolipid by high performance liquid chromatography and its application for determination of the seminolipid content in boar spermatozoa. Seminolipid (1-O-alkyl-2-O-acyl-beta-3'sulfogalactosyl glycerol) was detected on a micro-scale by high performance liquid chromatography (HPLC) using a UV detector. The benzoylation method of McCluer and Evans ((1973) J. Lipid Res. 14, 611-617) was used to convert seminolipid to a derivative with UV absorption. Total lipid from germinal tissue was benzoylated and the product was separated by Florisil and Sephadex LH-20 column chromatographies. The seminolipid obtained in the effluent from the Sephadex LH-20 column was determined by HPLC. Eighty percent recovery of 35S-labelled seminolipid was obtained by this method. The method was used to determine the seminolipid content of boar spermatozoa: 980 nmoles of seminolipid was found per gram of packed cells.


The effect of aurintricarboxylic acid on RNA polymerase from rat liver. The effect of the dye aurintricarboxylic acid (ATA) on RNA polymerases [EC 2.7.7.6] solubilized from rat liver was studied. Complete inhibition of RNA synthesis in vitro was observed when 3-5 microng/ml of ATA was added to the reaction mixture at time 0, while 40-70 microng/ml of a rifampicin derivative, AF/013, was required to produce the same extent of inhibition. RNA formation, however, continued at a rate of one-half that of the control when ATA was given after the onset of RNA synthesis in a dose capable of completely blocking RNA formation if administered at time 0. ATA was found to interact with RNA polymerizing enzyme itself and competed specifically with the binding of RNA polymerase to template DNA. Preincubation of the enzyme with DNA at 37 degrees before adding dye made the DNA-enzyme complex partly resistant to the drug. RNA-synthesizing activity resistant to ATA increased when nucleoside triphosphates, especially GTP, were added to the preincubation mixture in the presence of Mn2+. However, ATA only slightly affected RNA synthesis in nuclei isolated from rat liver.


Effects of protein deficiency on the biosynthesis and degradation of ribosomal RNA in rat liver. Employing livers from rats fed on a protein-free diet for two weeks, the effects of protein deficiency on both biosynthesis and degradation of rRNA were investigated and the following results were obtained. 1. Protein deficiency led to a decrease of total liver RNA content per DNA to about 80% of that in normal rat liver. 2. From the kinetics of rRNA labelling with [14C]orotic acid in vivo, the half-lives of cytoplasmic rRNA's of normal and protein-deficient rat livers were determined to be 6.2 and 5.1 days, respectively. Furthermore, considering the pool size of rRNA in rat liver, the turnover rate of cytoplasmic rRNA was calculated to be 0.212 pmole/min/mg of nuclear DNA in normal rats and 0.240 pmole/min/mg of nuclear DNA in protein-deficient rats. 3. From the electrophoretic patterns of nucleolar RNA's of both groups of rat livers labeled with [14C]orotic acid, the time courses of the specific activities of nucleolar 45S, 32S, and 28S rRNA's were analysed and the half-life of each nucleolar RNA in both groups of rat livers was determined. Nucleolar 45S, 32S, and 28S RNA's had half-lives of 6.0, 15.9, and 26.5 min in normal rats, respectively, and 5.5, 19.4, and 22.9 min in protein-deficient rats, respectively Considering the pool size of each nucleolar RNA obtained from the leectrophoretic pattern, the turnover rates of 45S, 32S, and 28S RNA's were calculated to be the same, i.e., o.189 pmoles/min/mg of nuclear DNA, in normal rat liver and 0.372, 0.372, and 0.358 pmoles/min/mg of nuclear DNA in protein-deficient rat liver, respectively. 4. These results indicate that protein deficiency increased both the rate of degradation of cytoplasmic rRNA and that of nucleolar rRNA synthesis in rat liver. While in normal rat liver the rates of rRNA synthesis and degradation were rather similar, the rate of rRNA synthesis in protein-deficient rats was about 1.5 times higher than that of its degradation. Therefore, the decrease of total liver RNA content in protein deficiency might be accounted for by stimulated degradation of rRNA in the nucleus. 5. The activities of RNase in nuclear fractions of both groups of rat livers were compared. Both activities of nuclear acid RNase and especially that of the free form of alkaline RNase in protein-deficient rat liver were higher than those in normal rat liver.


Regulation of Protein Synthesis in Hen's oviducts. I. Removal of extracellular and intracellular proteins and their effects on protein synthesis. The minced oviducts prepared from laying hens contained considerable amounts of extracellular proteins (about 60 mg per g of the wet oviducts), which could largely be removed (about 80%) by washing four times with chilled 5rebs-Ringer-Tris buffer, pH 7.6. When the washed, minced oviducts were incubated at 37 degrees with the buffer, intracellular proteins were increasingly secreted to the extracellular fluids as the incubation time was increased and the amounts of intracellular proteins decreased concomitatnly, suggesting that the rate of secretion of intracellular proteins was faster than that of protein synthesis under the conditions used (without addition of amino acids to the medium). Repeated 5-min incubations (five to seven times) resulted in the secretion of intracellular proteins in amounts of 100 mg or more per g of the wet oviducts. The secretion rate of intracellular proteins was almost constant at 77 degrees regardless of the volume of buffer used for the incubation, and no secretion occurred at 4 degrees even after 5 h. It appeared that a 30 min period was required for the secretion of intracellular proteins from the oviducts at 37 degrees and that the rate of secretion was not affected appreciably by the amount of secreted proteins present in the extracellular fluids. The protein synthesis activity in the washed, preincubated minced oviducts was independent of the amount of intracellular proteins involved but was dependent upon that of secreted proteins present in the extracellular fluids.


Soluble proteins from fowl feather keratin. II. Isolation of some proteins from barbs. Four fractions, GF-1, 2, 3, and 4, which had been separated from S-carboxymethylated (SCM-) proteins of fowl feathers by gel filtration, were each chromatographed on a DEAE-cellulose column in 0.05 M Tris-HCl buffer (pH 8.5) containing 8 M urea. The major fraction, GF-3, was further separated into seven peaks; the first four were shown to be single components by polyacrylamide disc gel electrophoresis. Chromatograms of GF-1 and 2 showed broad peaks which appeared at nearly the same volume as in GF-3. The components from GF-3 had very similar amino acid compositions except that the SCM-cysteine content showed a tendency to increase in the order of elution from the column. SCM-extract prepared from barbs of the wing feathers of a fowl was more heterogeneous than that taken from the body feathers. A combination of gel filtration on Sephadex G-75 and chromatography on DEAE-cellulose was found to be more effective for the isolation of soluble SCM-proteins.


I-Protein, a new regulatory protein from vertebrate skeletal muscle. I. Purification and characterization. A new minor regulatory protein designated as I-protein has been isolated from skeletal muscles of the chicken and the rabbit. The yield of purified I-protein was small: 5-10 mg per kg of muscle. I-protein was found to have a molecular weight of 50,000 and high contents of Asp and Glu, accounting for more than one third of the total amino acids.


Heme oxygenase purified to apparent homogeneity from pig spleen microsomes. Heme oxygenase was purified to apparent homogeneity from pig spleen microsomes. The purified heme oxygenase showed an apparent molecular weight of 157,000 +/- 7,000 daltons when estimated by gel filtration. On SDS-polyacrylamide gel electrophoresis, the heme oxygenase preparation gave a single protein band showing a minimum molecular weight of about 26,000 daltons. Heme oxygenase could readily bind with heme and the resulting heme complex gave an absorption maximum at 406 nm. The heme bound to the enzyme protein was found to be a good substrate for the heme oxygenase reaction.


Studies on thermolysin. I. Effects of chemical modifications on the activity of thermolysin. 1. When thermolysin was treated with a 100-fold molar excess of 2,4,6-trinitrobenzene-1-sulfonate at pH 8.0 and 37 degrees for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity. 2. When treated with a 100- to 1,000-fold molar excess of N-acetylimidazole at pH 6.5 and 23 degrees for 2 h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site. 3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide at pH 6.0 and room temperature for 1 h both in the presence and absence of 8 M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inactivated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8 M urea retarded the inactivation.


Molecular weight of hyaluronic acid from rabbit skin. Hyaluronic acid was prepared from adult rabbit skin. Defatted skin powder suspended in 0.5 M NaCl was homogenized, and total glycosaminoglycans were precipitated from this 0.5 M NaCl extract with cetylpyridinium chloride, then redissolved successively with increasing concentrations of NaCl and finally 0.5 N NaOH. Hyaluronic acid, the major acid glycosaminoglycan in the 0.5 M NaCl extract, was purified and fractionated by DEAE-Sephadex chromatography. The molecular weights ranged from 1 X 10(4) to 7.2 X 10(4). Alternatively, hyaluronic acid was obtained from adult rabbit skin without mechanical powdering and homogenizing. Defatted skin pieces were suspended in water and heated at 100 degrees, then the extract was digested with pronase followed by DNase [EC 3.1.4.5]. Glycosaminoglycans were excluded in gel filtration with Sephadex G-75. Hyaluronic acid and dermatan sulfate, the two major glycosaminoglycans of this tissue, were separated by gel chromatography on Sepharose 4B. The molecular weight of this hyaluronic acid ranged from 1.6 X 10(5) to 1.3 X 10(6). Yields of hyaluronic acid by these two methods were similar. Hyaluronic acid was probably degraded by the mechanical treatments in the first method. Other factors affecting the viscosity of the tissue extract were examined.


Static and kinetic studies by fluorometry on the interaction between gluconolactone and glucoamylase from Rh. niveus. A transition-state analog, gluconolactone, was found to partially quench the protein fluorescence of glucoamylase [EC 3.2.1.3] from Rhizopus niveus. The interaction between gluconolactone and the enzyme was studied statically and kinetically at pH 4.5 in terms of fluorescence change. The dissociation constant Kd of the enzyme-analog complex determined by fluorometric titration at 25 degrees (Kd = 1.6 mM) was in good agreement with that obtained by difference spectrophotometric titration (Ohnishi, M. et al. (1975) J. Biochem. 77, 695-703) and with the inhibitor constant determined for the hydrolysis of maltodextrin (Ohnishi, M. et al. (1976) J. Biochem. 79, 1007-1012). The kinetics of the interaction were studied by the fluorescence stopped-flow method. The dependence of the apparent first-order rate constant, kapp, on gluconolactone concentration showed a saturation curve, consistent with a two-step mechanism involving a rapid bimolecular association followed by a slow unimolecular isomerization process. The dissociation constant, KI, for the rapid bimolecular process and the forward and backward rate constants for the isomerization were obtained at 25 degrees and 5 degrees, and the activation parameters were evaluated. It was found that the isomerization process, but not the bimolecular association, is accompanied by fluorescence intensity change, indicating that the former process involves a micro-environmental change of a tryptophan residue(s) of the enzyme. Maltose was found to decrease the rate of interaction of gluconolactone with the enzyme by competing with the analog for the active site.


In the eye of the beholder. A theory of sight and memory and its application to illustration. The purpose of this article is to show how to apply principles of sight and memory to the creation of teaching illustrations. With illustrations that guide the eye and are not merely copies of photographs we can go beyond representation to the creation of pictures that will be remembered.


Plagiarism: A case history.

Plagiarism: A case history. A case of plagiarism involving duplication of the author's drawings is discussed. The chronology of events leading to an out-of-court settlement is reviewed and an attempt is made to clarify certain often misunderstood points in the Copyright Law as it applies to illustrators.


A self-instructional approach to imagery training for medical students. At the University of North Carolina School of Medicine, 110 first-year medical students were randomly assigned to experimental and control groups. Before the experimental group began the academic year, they were trained in visual imagery by means of 4 self-instructional units. Subsequently, both groups were given a criterion test to assess their ability to process and retrieve information. The investigators concluded that visual imagery can be taught and that it may help some medical students.


Role of pyruvate kinase in the regulation of gluconeogenesis from L-lactate. Glucagon and L-epinephrine stimulate gluconeogenesis from 20 mM L-lactate, the effect being about 3 times greater in liver cells from fed rats than in those from fasted rats. The rate of pyruvate kinase flux was estimated to be less than 10% of the rate of gluconeogenesis from lactate in hepatocytes from fasted rats, and neither glucagon nor epinephrine lowered the absolute rate significantly. In hepatocytes from fed rats, however, the rate of pyruvate kinase was nearly one-half that of gluconeogenesis. Glucagon caused a marked depression of pyruvate kinase flux, with 1 muM glucagon lowering the rate to nearly the level found in cells from fasted rats Epinephrine at concentrations from 10(-8) to 10(-6) M actually increased pyruvate kinase flux during gluconeogenesis from lactate in cells from fed rats. These results are in accord with the view that the effects of glucagon and epinephrine on gluconeogenesis are not identical.


Oxygenated Cytochrome o. An active intermediate observed in whole cells of Vitreoscilla. The oxygenerated form of cytochrome o can be readily observed in whole cell suspensions of Vitreoscilla because it is the predominant steady state form of the cytochrome in respiring cells. When oxygen is introduced to an anaerobic suspension of cells, the steady state is established immediately and absorption maxima are observed at 578, 545, and 425 nm in difference spectra using ammonium persulfate-oxidized cells as reference. The oxygenerated form of cytochrome o is identified as the predominant steady state species during turnover of the cytochrome in vivo from the close coincidence of these spectra with those obtained using purified cytochrome. When the dissolved oxygen is consumed, the spectrum of the oxygenated form is replaced by the spectrum of the reduced form. Glutamate increased the respiration of these cells and decreased the duration of the steady state. Spectra of the oxygenated form of cytochrome o could also be obtained by using starved cells as reference, i.e. (untreated cells) - (starved cells) difference spectra. These results implicate the oxygenated form of cytochrome o as an active intermediate in terminal electron transport of these cells. In the presence of 1 mM cyanide, which inhibits the respiration of Vitreoscilla, the duration of the steady state was increased and the steady state spectrum was altered, showing that turnover of the cytochrome was inhibitied and formation of the oxygenated form wsa decreased. All these results suggest that the cyclic changes of cytochrome o in terminal respiration involve the oxidized, reduced, and oxygenated forms.


epsilon-(gamma-Glutamyl)lysine cross-links in human stratum corneum. epsilon-(gamma-Glutamyl)lysine has been found in human stratum corneum in the fraction containing the alpha helical fibrous proteins (keratins) and other high molecular weight proteins. The S-carboxymethylated fractions were enzymatically digested with pronase, carboxypeptidase A and B, leucine aminopeptidase and prolidase, and epsilon-(gamma-glutamyl)lysine isolated from digests by gel filtration and cation ion exchange chromatography. Acid hydrolysis of the purified epsilon-(gamma-glutamyl)lysine yielded equimolar amounts of lysine and glutamic acid, and end group analysis of the peptide by dansylation (application of 5-dimethylaminonaphthalene-1-sulfonyl) confirmed the isomer assignment to be epsilon-(gamma-glutamyl)lysine. About 9 nmol of the peptide per mg of protein were found in the fraction by isotope dilution after the enzymatic digestion. These results suggest that proteins in stratum corneum may be covalently cross-linked through epsilon-(gamma-glutamyl)lysine bonds.


Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. Effects of sulfur and nitrogen sources on the formation and decay of the enzyme. The ATP sulfurylase of cultured tobacco cells is repressed during growth on readily assimiliated sulfur sources, such as sulfate, L-cysteine, or L-methionine, but it is derepressed during growth on slowly assimiliated sulfur sources, such as L-djenkolate or glutathione, or during sulfur starvation. The enzyme is not induced by sulfate. The enzyme level in the cells begins to rise 12 to 24 h after the derepression conditions are initiated and continues to rise for 3 to 4 days, up to as much as 25 times above the initial specific activity. Addition of a repressing sulfur source to derepressed cells causes the enzyme to decay. Derepression by sulfur limitation does not occur in cells starved for nitrogen, a circumstance in which turnover synthesis of protein is known to continue. Upon addition of a nitrogen source to such cells, the development of the enzyme begins within 12 h, along with the resumption of growth and net protein synthesis. Derepression occurs in cells growing on the slowly assimilated nitrogen in urea, reaching specific activities very similar to those which develop in cells grown on nitrate, in spite of the lower protein accumulation rate on urea. Thus the ATP sulfurylase of tobacco cells appears to be regulated by both a negative feedback mechanism in which an end product of the sulfate assimilation pathway is the effector, and by a positive mechanism which serves to couple the regulation of the sulfate assimilation pathway to the cells' potential for nitrogen assimilation, i.e. net protein synthesis. The sulfur compounds which affect the development of ATP sulfurylase in vivo have no effect on the enzyme activity in vitro. Furthermore, extracts with high activity contain no activator and extracts with low activity contain no inhibitor of ATP sulfurylase. Cycloheximide, at a concentration which strongly inhibits amino acid incorporation into protein, inhibits derepression. ATP sulfurylase does not decay in cells inhibited by cycloheximide. Therefore, the changes in ATP sulfurylase of tobacco cells appear to involve changes in the rate of formation or degradation of the enzyme.


Effects of chemical modification of antibodies on their clearance from the circulation. Addition of simple aliphatic compounds by reductive alkylation and carbodiimide-promoted amide formation. Anti-hapten antibodies from the ascitic fluid of inbred mice were purified by immunoadsorption and characterized immunochemically for in vivo studies of their plasma clearance rates and organ distributions after chemical modification. Following sodium borohydride-promoted reductive methylation and carbodiimide-promoted amide linkage of glycine and several other simple aliphatic compounds, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into syngeneic animals. Using double radioiodine labels, it was possible to show that stoichiometric combinations of additive and chemical reactant did not alter antibody survival time in the circulation or antigen-binding capabilities. However, modifications involving excess reagent (carbodiimide or sodium borohydride) resulted in significant decreases in both circulatory longevity and ligand binding capacities. Excess carbodiimide treatments resulted in immunoglobulin cross-linkage which could be detected by molecular sieve chromatography. Increased kidney localization found with carbodiimide-treated antibodies was apparently due to trapping of the cross-linked aggregates. Elevated clearance of highly methylated antibodies could not be attributed to a single organ. However, sodium borohydride-catalyzed reductive methylation resulted in antibody populations which were localized primarily in the liver and spleen. Results are evaluated in terms of the concept, developed in this paper, of essential groups for the circulatory longevity of glycoproteins.


Purification and characterization of two major DNA-binding proteins in human serum. The two major DNA-binding proteins (designated DNA-binding protein 1 and DNA-binding protein 2) in human serum have been purified and physically characterized. The two proteins co-purify through an ion exchange chromatographic step and DNA-cellulose affinity chromatography. Subsequently, DNA-binding protein 1 can be precipitated by 40% saturated ammonium sulfate; DNA-binding protein 2 precipitates in the 55% to saturation fraction. From these fractions, the two proteins are isolated by different protocols. Both purified proteins are homogeneous by the criteria of sodium dodecyl sulfate slab gel electrophoresis after reduction and denaturation and by sedimentation equilibrium centrifugation. DNA-binding protein 1 has a minimum molecular weight of 126,000; DNA-binding protein 2, 86,000. Amino acid analyses of the two proteins indicate that both are relatively rich in proline and cysteine and contain little methionine. Both proteins contain carbohydrate. Gel electrofocusing confirms the acidic nature of these proteins. DNA-binding protein 1 exhibits a single band upon isoelectric focusing, but DNA-binding protein 2 appears to be polymorphic, exhibiting three bands. NH2-terminal end group analysis of DNA-binding protein 2 yields two major amino acids. DNA-binding protein 1 is an alpha2-globulin as determined by immunoelectrophoresis; DNA-binding protein 2 is only weakly immunogenic. Neither of the proteins appears to be identical to any previously characterized serum protein.


Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. Two forms of proacrosin have been purified from ejacualted boar spermatozoa. The isolation method utilized benzamidine to inhibit the premeture activation of the zymogen and included pH precipitation, ammonium sulfate fractionation, and sodium chloride precipitation. Further purification was achieved by Sephadex G-200 FILTRATION OF THE PREPARATION AFTER IT WAS TREATED WITH 8 M urea. The overall proacrosin yield was 58% with a specific acitivity of 253 units/mg of protein. The molecular weights of the proacrosins determined by sodium dodecyl sulfate disc gel electrophoresis were 55,000 and 53,000. Proacrosin autoactivation followed the classical S-shaped activation curve and calcium was not required to obtain full activation. Time course activation studies in 0.1 M Tris/HCl, pH 8.4, at 0 degrees were monitored with sodium dodecyl sulfate-disc gel eletrophoresis and anlytical gel electrophoresis with staining techniques for protein and enzymatic activity. Under the conditions used, the zymogens were sequentially degraded to three different active specise of acrosin (alpha, beta, and gamma). The approximate molecualr weights of the acrosins were 49,000, 39,000, and 25,000 for the alpha, beta, and gamma forms, respectively. The autoactivation is concentration-dependent and can be proteolytically stimulated with either alpha- or beta-acrosin and trypsin, indicating the activation of proacrosin can via a bimolecular process.


Investigation of the binding site of human corticosteroid-binding globulin by affinity labeling. Demonstration of a cysteinyl residue in the binding site. This communication deals with the investigation of the binding site of purified human corticosteroid-binding globulin (CBG) by the method of affinity labeling. The design of the studies necessitated the rapid removal of unbound ligands, followed by assay of the binding capacity of CBG. We were able to accomplish this by demonstrating that CBG absorbed on DEAE-filter discs bound cortisol as if both molecules were in solution. Using this principle, 6beta-bromoprogesterone was shown to react with CBG in a time-dependent and irreversible fashion with a t1/2 = 15 min at 2 degrees. One mol of 6beta-bromo[3H]progesterone reacted with 1 mol of CBG. After completion of the reaction, one of the two sulfhydrul groups in CBG was no longer titratable with Ellman's reagent. The product of the reaction of 6beta-bromoprogesterone with cysteine is progesterone-6-S-L-cysteine. After acid hydrolysis of CBG, which had been incubated with 6beta-bromoprogesterone, a compound which migrated with the same mobility on thin layer chromatography as the model compound was observed. We conclude that 6beta-bromoprogesterone is an affinity label for CBG and that a cysteinyl residue is present in the binding site.


Hemoglobin switching in sheep and goats. Preparation and characterization of complementary DNAs specific for the alpha-, beta-, and gamma-globin messenger RNAs of sheep. Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.


Correlation between activation and dimer formation of rat renal phosphate-dependent glutaminase. Gel filtration and velocity sedimentation in sucrose gradients were used to determine the molecular weights of purified rat renal phosphate-dependent glutaminase. The purified glutaminase has a molecular weight of 160,000 in Tris or barbital buffers and forms dimers of 332,000 molecular weight in the presence of its activator, Pi. The correlation between activation and dimer formation was investigated by determining the sedimentation coefficient at various concentrations of glutaminase activators. Saturation curves for Pi and riboflavin phosphate demonstrate an excellent correlation between per cent activation and increasing S20,w with increasing concentrations of these activators. The concentrations required for half-maximal saturation were 40 to 50 mM for Pi and 10 to 15 mM for riboflavin phosphate. Correlation between activation and dimer formation was also found with other activators at subsaturating concentrations. Moreover, the activation and dimer formation were found to be reversed to a similar extent by increasing concentrations of NaCl. Finally, we studied the effects of Pi and NaCl on the stability of glutaminase activity at 37 degrees. Pi stabilized glutaminase activity by increasing the t1/2 for inactivation from 12 min in the absence of Pi to 242 at 150 mM Pi. The concentration of Pi which gave approximately half-maximal change in t1/2 was 50 mM and addition of NaCl reversed this stabilization. These results support the hypothesis that phosphate-dependent glutaminase is active only as a dimer or larger aggregate. However, we cannot exclude the possibility that binding of Pi changes the monomer conformation sufficiently to produce activation and that this new conformation leads to self-association.


Primed and unprimed synthesis of poly (dA-dT) by calf thymus DNA polymerase alpha. The primed and unprimed synthesis of poly(dA-dA-dT) by calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7) has been compared to replication of activated DNA. Synthesis of poly(dA-dT) by alpha-polymerase is both autocatalytic and exponential. The rate of synthesis of poly(dA-dT) is markedly affected by the Mg2+ concentration and has a higher temperature optimum than replication of activated DNA, implicating "slippage" as a necessary part of poly(dA-dT) replication. Calf thymus 24,000-dalton unwinding protein influences poly(dA-dT) synthesis by increasing both the exponential rate constant and the rate of linear synthesis. Single-stranded template poly(dA-dT) is provided alpha-polymerase by both "strand slippage" and melting by unwinding protein.


Polydispersity of cartilage proteoglycans. Structural variations with size and buoyant density of the molecules. Proteoglycan monomers were subfractionated according to buoyant density by dissociative CsCl density gradient centrifugation. It was shown that with decreasing buoyant density of the proteoglycan subfraction: (a) the average sizes of the molecules decreased (shown by Sepharose 2B chromatography); (b) the relative content of chondroitin sulfate decreased; (c) the relative content of protein increased; (d) the relative proportion of the amino acids glycine and serine, which occur close to the chondroitin sulfate-peptide linkage, decreased; (e) the relative proportion of the hyaluronic acid-binding region released by treatment of proteoglycans with cyanogen bromide increased; (f) the relative content of the keratan sulfate-enriched region increased. The data indicate that proteoglycans contain a nonvariable hyaluronic acid-binding region, a keratan sulfate-enriched region and a chondroitin sulfate-enriched region of variable size. It is concluded that proteoglycans vary in size mainly because of variations in the size of the chondroitin sulfate-enriched region. Additional data were obtained using subfractions of proteoglycan monomers isolated according to size differences by using Sepharose 2B chromatography. The Kav values of the subfractions on Sepharose 2B ranged from 0 to 0.54. Analyses of these subfractions showed the same variations with size of the content of chondroitin sulfate, protein, amino acids, hyaluronic acid-binding region, and keratan sulfate-enriched region, as was shown for the subfractions isolated at different buoyant densities in the dissociative gradient.


Exit transport of a cyclic nucleotide from mouse L-cells. At a concentration of 30 mum, 1,4,5,6,8-pentazaacenaphthylene, 3-amino-1,5-dihydro-5-methyl-1-beta-D-[5-14C]ribofuranosyl (NSC-154020), a tricyclic, 7-deazapurine nucleoside (TCN) is rapidly taken up by cultured mouse L-cells and converted to intracellular TCN-monophosphate, but not further metabolized. The TCN-monophosphate is also excreted by the cells into the medium. It is released by a saturable process against a concentration gradient and the release is inhibited by various inhibitors of energy production. This inhibition correlates with a depletion of the cells of ATP. Thus TCN-monophosphate excretion probably involves an active transport system. This transport system is highly temperature-dependent (the Q10 falls between 3 and 4) and is inhibited by papaverine, theophylline, Persantin, Probenecid, phenethyl alcohol and p-chloromercuribenzoate, but not by 500 muM cyclic AMP, AMP, or adenosine. Significant amounts of various natural phosphorylated intermediates (AMP, ATP, UTP, UMP, UDP-hexoses, and phosphorylcholine) are not released from the cells under similar experimental conditions either in the absence or presence of 30 muM TCN.


Purification and properties of a beta-mannosidase from Aspergillus niger. A beta-mannosidase (beta-D-mannoside mannohydrolase, EC 3.2.1.25) was purified to apparent homogeneity from the culture filtrate of the fungus, Aspergillus niger. The enzyme had an estimated molecular weight of about 120,000 and was a glycoprotein. Radioactive enzyme was prepared by growing the fungus in [14C]fructose, and this enzyme was used for the preparation of 14C-glycopeptides. The glycopeptides were purified on Sephadex G-25 and G-50 and were then hydrolyzed for sugar analysis. Two radioactive sugars were found in the glycopeptides and these were identified as mannose and glucosamine in a ratio of 2.5 or 3:1. Based on susceptibility of the enzyme to alkaline treatment and the formation of [3H]glucosaminitol in the presence of NaB3H4, the oligosaccharide is apparently attached to the protein in a GlcNAc-asparagine linkage. The beta-mannosidase had good activity on p-nitrophenyl-beta-D-mannoside but was inactive on p-nitrophenyl-alpha-D-mannoside as well as on other p-nitrophenyl glycosides. It also showed good activity on the beta(1 leads to 4)-linked trisaccharide of mannose and somewhat lower activity of the corresponding disaccharide. With each of these substrates the Km was about 1 mM, whereas with the p-nitrophenyl-beta-D-mannoside the Km was about 2 mM. The beta-mannosidase also released [14C]mannose from the Man-GlcNAc-GlcNAc trisaccharide isolated from the lipid-linked oligosaccharides of aorta and released mannose from the disaccharides, Man-(beta1 leads to 4)GlcNAc and Man-(beta1 leads to 4)ManNAc. The pH optimum for the enzyme was about 3.5 to 4.0 in glycine or acetate buffer.


Relationship of nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. Oviduct nuclei from laying hens, or from chicks given an optimal dose of estrogen, contain receptors with a high affinity for 17beta-estradiol (apparent Kd of about 3.7 nM) at a concentration of about 10,600 molecules per tubular gland cell. Chicks withdrawn from estrogen stimulation exhibit a 7-fold lower level of nuclear estrogen receptors. Radioimmunoassay of 17beta-estradiol in the serum indicates that birds withdrawn from estrogen stimulation have a serum level of 0.09 nM. A maximum concentration of oviduct nuclear receptors is achieved when the serum level reaches 0.7 nM 17beta-estradiol. Endogenous 17beta-estradiol in the serum of laying hens is also approximately 0.7 nM. The concentration of nuclear estrogen receptors achieved by administering different dosages of 17beta-estradiol, 17beta-estradiol-benzoate, or diethylstilbestrol is related to the rate of accumulation  of ovalbumin and conalbumin mRNA. The rate of conalbumin mRNA production is directly proportional to the concentration of nuclear receptors, i.e. half-maximal induction is obtained with about 5,300 nuclear receptors per tubular gland cell. In contrast, half-maximal induction of ovalbumin mRNA occurs when nuclear receptor levels are 80% of maximum; this is achieved with a dose of estrogen about 2.5 times that required for half-maximal conalbumin mRNA induction. These differential responses may be related either to different numbers of specific binding sites regulating the production of each mRNA, or to different affinities of regulatory sites for estrogen receptors.


Prostaglandin E2 biosynthesis by rabbit renomedullary interstitial cells in tissue culture. Mechanism of stimulation by angiotensin II, bradykinin, and arginine vasopressin. The mechanism of hormone-stimulated prostaglandin E2 biosynthesis was investigated in rabbit renomedullary interstitial cells in tissue culture. Angiotensin II, bradykinin, and arginine vasopressin stimulated arachidonic acid release and subsequent prostaglandin biosynthesis. Indomethacin completely inhibited prostaglandin biosynthesis without affecting arachidonic acid release. Mepacrine, a phospholipase inhibitor, inhibited arachidonic acid release without affecting subsequent synthesis of prostaglandin E2. Our studies suggest that arachidonic acid release via hormone-activated phospholipase or acylhydrolase (or both) is the mechanism of hormone-stimulated prostaglandin E2 biosynthesis.


Purification and characterization of UDP-galactose-4-epimerase from bovine tissues. Bovine liver and mammary UDP-galactose-4-epimerases have been purified to apparent electrophoretic homogeneity by a simple procedure involving the use of two affinity adsorbants, UDP-hexanolamine-Sepharose and NAD+-hexanolamine-Sepharose. The bovine thyroid epimerase has been partially purified by the same procedure. All three enzymes require NAD+ for activity, and all have a similar apparent molecular weight of approximately 40,000 as determined by gel filtration of the active enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the liver and mammary epimerases revealed similar molecular weights of 37,000 for both enzymes thus indicating that neither protein was dimeric. During development of the isolation procedure conditions were determined which would stabilize enzymatic activity in very dilute solutions. The liver and mammary enzymes, although similar in some respects, differed in amino acid composition and specific activity.


Mechanisms of lymphocyte activation. Binding kinetics of phytohemagglutinin to human lymphocytes. The interactions of phytohemagglutinin (PHA) with normal human lymphocytes were studied utilizing radioiodinated leukoagglutinin (125I-LPHA) over a concentration spectrum encompassing the entire range of lymphocyte metabolic responses. 125I-LPHA binding was temperature-, pH-, and time-dependent. Ligand association was rapid with a t1/2 of 3 to 5 min, reaching steady state in 30 min at 22 degrees. Receptor specificity was demonstrated by the high receptor affinity for 125I-LPHA and by quantitative inhibition of 125I-LPHA binding with LPHA and 127I-LPHA but not with concanavalin A or bovine serum albumin. Under our experimental conditions there was no measurable degradation of 125I-LPHA and no detectable shedding of 125I-LPHA receptors or receptor-125I-LPHA complexes. Equilibrium studies of 125I-LPHA interactions with specific lymphocyte membrane receptors generated a complex curvilinear Scatchard plot. This, added to progressive deceleration of the dissociation reaction inversely proportional to receptor occupancy by 125I-LPHA, reflects changing receptor affinity for the ligand and suggests site-site interactions of the negative cooperativity type. These interactions which appear to be common to all lymphocyte subpopulations, preclude accurate calculation of lymphocyte binding capacity for 125I-LPHA and of physically meaningful affinity constants. Although the fate and role of a small fraction of apparently nondissociable 125I-LPHA remains to be elucidated, occupancy-dependent receptor affinity for 125I-LPHA, dissociation of receptor-125I-LPHA complexes, retention of binding properties by cell-exposed 125I-LPHA, and the large numbers of spare surface receptors for 125I-LPHA might represent important mechanisms for modulating cell activation by 125I-LPHA.


Microtubular protein catalytic interactions with nucleotides. Purified tubulin prepared from pig brain in the absence of added guanine nucleotides contains 1 mol each of GDP and GTP/mol of tubulin. Incubation of a tubulin preparation with inorganic [32P]phosphate results in the incorporation of 32P into tubulin-associated GDP and GTP. Typically, in a 5-h incubation, 0.45 mM 32Pi reacts with 22 muM tubulin to form 3.3 muM [32P]GDP, and 0.3 muM [32P]GTP. The yield of labeled nucleotide is decreased as the result of a hydrolase activity associated with the preparation. The [32P]GTP is exclusively beta-labeled and is therefore formed by phosphorylation of [32P]GDP by a phosphate donor other than inorganic phosphate, most likely by nonradioactive GTP. Added GDP significantly decreases the yield of labeled GTP, but increases the yield of labeled GDP in a manner that is consistent with a partial protection against [32P]GDP hydrolysis, but not consistent with significant additional [32P]GDP formation. Added GMP is an inhibitor of both labeling activities associated with the preparation, although it has no effect on the hydrolase activity. Added ADP (0.4 MM) does not form labeled ADP or ATP and does not influence the labeling of GDP or GTP. The formation of [32P]GDP was shown to occur by an exchange mechanism rather than through net synthesis from GMP and Pi. These results provide evidence for a reversible guanidylation of the protein. The labeling activity is always specifically associated with highly purified tubulin preparations. Microtubular protein preparations are found to catalyze an exchange of oxygen from H218O into inorganic phosphate. Thus, there are two distinct catalytic properties associated with tubulin.


Partial purification and characterization of a translational inhibitor from Friend leukemia cells. A translational inhibitor has been isolated from unstimulated Friend leukemia cells. The inhibitor is a heat-labile, sulfhydryl reagent-insensitive protein with a molecular weight of approximately 214,000. It inhibits protein synthesis at a step of peptide chain initiation by preventing initiation factor-dependent binding of methionyl-tRNAf to 40 S ribosomal subunits. However, it does not interfere with the formation of the ribosome-independent ternary complex between the initiation factor IF-E2, methionyl-tRNAf, and GTP. The inhibitor preparation contains protein kinase activity which phosphorylates the smallest subunit of IF-E2. There appears to be a functional similarity between this inhibitor from Friend leukemia cells and the hemin-controlled repressor from reticulocytes.


Isolation and characterization of plasma membranes and intact nuclei from lymphoid cells. A method has been developed for the rapid large scale isolation of plasma membranes and intact nuclei from RAJI lymphoid cells utilizing hypotonic lysis of cells after intracellular loading with glycerol followed by combined flotation-sedimentation within a discontinuous sucrose gradient. Nuclei may be isolated in about 1 h and plasma membranes in about 6 h from 1 to 20 g of cells. Intact nuclei, obtained in 90 to 95% yield based on lysed cells, was isolated by differential centrifugation and contained 16% DNA and about 30% of total cell sialic acid. A crude plasma membrane fraction was isolated by centrifugation onto a cushion of 38% sucrose (d 1.1683) and subsequently resolved into two subfractions. The less dense vesicles had an average d 1.127 and showed a 7-fold increase in specific activity for thymidine phosphodiesterase while the more dense (d 1.151) had a 20-fold concentration of enzyme activity. Activity of enzymes indicative of contamination with lysosomes, microsomes, mitochondria, and cytoplasm was negligible in these plasma membrane fractions. The less dense vesicles had a cholesterol:phospholipid ratio of 0.97 which was higher than that of the more dense vesicles (0.69). Otherwise, the analytical values for the two types of membrane vesicles were similar as both fractions contained like percentages of protein (approximately 30%), lipid (approximately 30%), and carbohydrate (approximately 15%) with trace amounts of RNA and DNA. Twenty-five per cent of the total cell sialic acid was in the plasma membrane fractions.


Acetylcholine receptor metabolism in a nonfusing muscle cell line. The development and turnover of acetylcholine receptors in a nonfusing muscle cell line has been investigated using iodinated alpha-bungarotoxin as a probe for acetylcholine receptor. logarithmically growing cells do not bind toxin, while cells that have ceased cell division bind toxin at a site which has the pharmacological characteristics of an acetylcholine receptor. These binding sites are removed from the cell surface at a rate equal to 8.9 +/- 0.5% of the total surface binding sites/h and appear at a rate equal to 8.3 +/- 1.5% of the total surface binding sites/h. Appearance of new binding sites can occur for a period of 1 1/2 h in the presence of cycloheximide, during which time 15% of the surface receptors can be replaced. There is a hidden population of receptors which is not accessible to toxin without disrupting the cell. This population amounts to 35% of the Triton-extractable receptors in the cell and is composed of two classes. One class, termed a precursor receptor, appears to move from the hidden population to the cell surface, and composes about 40% of the total hidden receptor population. The second class of hidden receptors does not appear to function as a surface precursor and is neither depleted nor enriched by any of the procedures we employed. Surface receptors and hidden receptors are distinguishable on the basis of their sedimentation coefficient about 0.5 to 0.6 S lower than surface receptors. We were unable to distinguish between precursor and non-precursor hidden receptors on the basis of cursor and nonprecursor hidden receptors on the basis of their sedimentation coefficients.


Increasing activity of enzymes on pathway of triacylglycerol synthesis during adipose conversion of 3T3 cells. When preadipose 3T3 cells pass from growing to resting state, they increase their rate of synthesis of triacylglycerol from precursors and convert to adipose cells. The behavior of two enzymes involved in the synthesis of triacylglycerol, glycerophosphate acyltransferase, and malic enzyme have been examined during the adipose conversion of 3T3-F442A in surface culture and in suspension culture stabilized with methylcellulose. Glycerophosphate acyltransferase activity rises sharply during the conversion and reaches a level of 80 times higher than that of another 3T3 subline in which practically no adipose conversion takes place (3T3-C2). The activity of malic enzyme also rises during the adipose conversion of 3T3-F442A and reaches a level of 15-fold higher than that of 3T3-C2. The activity of glycerophosphate acyltransferase responds more sharply than that of malic enzyme but the rise in activity is not sustained as long, so that the relative levels of the two enzymes change during the conversion. The adipose conversion appears to be the result of increases in the activity of the synthesizing enzymes, brought about by either of the two most physiological methods available for arresting the growth of cultured cells.


Interactions of synthetic and natural surfaces with blood in the physiological environment. Efforts to explain blood compatibility with synthetic and natural surfaces based on a single parameter or a single biological test procedure have either been unsuccessful or led to misleading generalizations. The problem reflects the complex interdependence between material's properties, the composition and properties of blood, and in vivo biorheological conditions. Among the initial events that occur when materials contact blood is the very rapid adsorption of plasma proteins; this process effectively influences the subsequent interactions with the formed blood elements, especially the platelets with the proteinated surfaces. In the case of natural surfaces, when the endothelium is damaged, collagen may become exposed that may cause the activation, adhesion, and aggregation of platelets leading to thrombosis. Current evidence indicates that the platelet-aggregating ability of collagen depends on its "multimeric" or fibrillar structure, rather than on the activation of the platelet-bound enzyme system. Under normal conditions, the flowing blood is probably not in direct contact with endothelial cells that line the blood vessel walls, but with an adsorbed layer of plasma proteins. Should a formation of a multilayer of plasma proteins occur following the initial adsorption of a monolayer, this process could be influenced by changes in the solubility of the proteins, especially fibrinogen, the solubility of which is quite low in plasma. The hypothesis is proposed that such changes may be intimately related to the electrical properties of proteins present in the vascular wall and in blood. It is possible that these properties play a much greater role in thrombogenesis and in the problem of blood compatibility than is currently appreciated. Considering synthetic polymers, a number of these have been prepared that exhibit little adverse effects on blood components and, at the same time, retain their physical properties for various periods of time in the physiological environment. These combined biological and physical properties make them useful for various prosthetic and other biomedical applications in surgery and therapy.


Interaction of living cells with polyionenes and polyionene-coated surfaces. Polyionenes have been shown recently (A. Rembaum, Appl. Polym. Symp. No. 22, 299, 1973) to produce the following biological effects: 1) bactericidal action, 2) formation of insoluble complexes with DNA and heparin, 3) neuromuscular blocking action, 4) cell aggregation and lysis, and (5) cell adhesion. In present study, polyionenes of various structures (mainly I3, 3, I6, 10) were used as molecular probes to gain an understanding of the cell surface phenomena of adhesion on glass- and polyionenes-treated surfaces. Since tumor cells show different durface cell properties, including an increase in the anodic mobility, they bind preferentially to polyionene-treated surfaces. Normal human diploid WI-38 cells were found to adhere at a lower rate than SV-transformed WI-38 cells. However, cell spreading was accelerated in both cases. A study of the interaction of polyionenes in solution in vitro and in vivo and polyionenes covalently bound to polymeric microspheres with leukemic murine EL4 cells and normal thymocytes showed specific cytotoxity towards the leukemic cells.


Perfusion trials with a collagen-immobilized enzyme in an extracorporeal reactor: activity, stability, and biocompatibility. This paper is concerned with the evaluation of the in vivo performance characteristics of reconstituted bovine collagen as an insoluble carrier matrix fro therapeutic enzymes. The enzyme that was chosen as a model for this evaluation was E. coli L-asparaginase, which has been widely investigated as a soluble chemotherapeutic agent for the treatment of acute lymphocytic leukemia in humans (Oettgen et al., Cancer Res., 27, 2619, 1967 ; Beard et al., Brit. Med. J., 1, 191, 1970; Ohmuma et al., Cancer Res., 30, 2297, 1970). The results presented here were obtained from perfusion trials with a collagen-asparaginase reactor incorporated into an extracorporeal circuit attached to the vascular systems of healthy mongrel dogs. A series of 1-2 hr perfusions were conducted with a single collagen-asparaginase membrane over a period of 4 months. Serum asparagine levels were reduced by more than 98% after 15-30 min perfusion time. Red blood cell (RBC), hemoglobin, hematocrit, and fibrinogen values remained constant during each perfusion. An average decrease of 48% in white blood cell (WBC) and 24% in platelet levels was observed, but these values began to rise slowly even before cessation of the perfusion. No serious toxic or antigenic reactions or mechanical or clotting difficulties were observed. In vitro activity, when assayed between perfusions, remained constant over a period of 4 months of intermittent use and storage. The potential advantages of collagen-enzyme complexes for the administration of therapeutic enzymes is discussed.