[Participation of the hypothalamus in conditioning and emotional behavior. (1) Behavior model in rabbits and methods of its analysis]. An attempt was made to induce conflict behavior in rabbits by conditioning under hypothalamic electrical stimulation. Behavioral analysis was performed using our newly devised technique which employs the use of a vibration apparatus fixed to head and waist, and a recording of various behavior. Conditioning was carried out by sensory stimuli flight behavior. Reinforcement was as follows: the CS used was flickering light photic (10 Hz, 10 musec.,) and acoustic (250 Hz, 15 dB) and UCS electrical stimulation (100 Hz, 1 msec., not exceeding 2 v) in the medial hypothalamic area and peri-fornix. The restrained animals were placed on a table in an acoustically isolated room with a discrete monotonous background noise. CRs were obtained within a short time as were responses: (1) During the autonomicsomatic responses, (2) continuous run and (3) non-continuous run. Data obtained during the non-continuous run represent a conflict-induced type of behavior which may be applied in studies related to the psychopharmacological actions of drugs.


[Pharmacological studies on degeneration and regeneration of peripheral nerves. (1) Effects of methylcobalamin and cobamide on EMG patterns and loss of muscle weight in rats with crushed sciatic nerve]. Experiments were performed to investigate the effects of Vitamin B12, i.e., methylcobalamin and cobamide, on the neural degeneration and regeneration. Male Wistar rats (140 to 150 g) under conditions of experimental unilateral sciatic nerve crushing were treated consecutively with methylcobalamin (50 and 500 mug/kg/day i.p.), cobamide (50 and 500 mug/kg/day i.p.) or saline. EMG recordings were periodically carried out and rats of each group were sacrificed to determine the weight-loss of denervated muscles 1, 2, 3 and 4 weeks after crush. Neither methylcobalamin nor cobamide exerted any significant effect on body-weight gain of the nerve-crushed rats with a daily injection of 50 and 500 mug/kg i.p.. The EMG pattern of the denervated biceps femoris muscle showed a total lack of fibrillation for 2 days after the nerve-crush. Thereafter, the fibrillation appeared and continued for 10 to 14 days until the nerve had regenerated, as evidenced by the appearance of a complex NMU voltage. The occurrence of fibrillation voltage was slightly delayed in methylcobalamin group (500 mug/kg/day) as compared with the saline control group. The re-appearance of normal NMU voltage was more rapid in the methylcobalamin 500 mug/kg group than in controls and other experimental groups. Neither methylcobalamin nor cobamide had any significant effect on the weight-loss of the gastrocnemius and tibialis anterior muscles following crush of the sciatic nerve. However, a daily injection of 500 mug/kg of methylcobalamin produced a significant increase in the weight of the soleus muscle which recovered to the extent of being the same weight of the contralateral 4 weeks after the nerve-crush. These results suggest that methylcobalamin may have an inhibitory effect on Wallerian degeneration and also a facilitatory effect on the neural regeneration of the crushed sciatic nerve of rats.


[Pharmacological studies on degeneration and regeneration of the peripheral nerves. (2) Effects of methylcobalamin on mitosis of Schwann cells and incorporation of labeled amino acid into protein fractions of crushed sciatic nerve in rats]. Male Wistar rats (140 to 150 g) in which the unilateral sciatic nerve had been crushed were treated consecutively with methylcobalamin (5, 50 and 500 mug/kg/day i.p.) or saline immediately after the nerve-crush. Thereafter, they were periodically sacrificed for biochemical and histological examinations. At different intervals after the nerve-crush, L-leucine-4,5-T (20 mu Ci/100g, specific activity 15 mCi/m mole) or L-leucine -14C(U) (15 muCi/100g, specific activity 270 mCi/m mole) was given i.p. to some rats of each group and 3 hr later they were sacrificed to determine the rate of leucine incorporation into protein fractions of the crushed nerve and the denervated muscles. The nerve and muscles of the contralateral side served as control. Longitudinal sections of proximal and distal stumps of the sciatic nerve were prepared and stained with hematoxylin and eosin. As compared with saline group, repeated injections of 5, 50 and 500 mug/kg/day of methyl-cobalamin caused a significant increase of the in vivo incorporation of radioactive leucine into the protein fraction of the crushed sciatic nerve 5 to 7 days after the crush. In contrast, a recovery of the increased incorporation of leucine into the crushed nerve was more rapid in methylcobalamin groups than in the saline group. On the other hand, methylcobalamin (5 approximately 500 mug/kg/day i.p.) had no significant effect on the leucine incorporation into the denervated muscles (m. gastrocnemius, m. tibialis anterior and m. soleus). In addition, consecutive injections of methylcobalamin (5 approximately 500 mug/kg/day) did not affect the mitosis of Schwann cells during the period of Wallerian degeneration of the crushed sciatic nerve. These results suggest that methylcobalamin possesses a stimulating effect on proteosynthesis in Schwann cells at the initial stage of axon regeneration and it may facilitate neural regeneration.


[Pharmacological studies on basic ethers (R111 and R97) with antispasmodic activity]. Pharmacological properties and acute toxicity of 2-tolyl 1-phenyl-3-(2-methylpiperidino) propyl ether methyl bromide (R111) and 2-chlorophenyl 1-phenyl-3-(2-methylpiperidino) propyl ether methyl iodide (R97) were examined. The results obtained were as follows: (1) In the analgesic effects, RIII and R97 inhibited markedly the acetic acid-induced writhing in mice, but in reducing pain induced by heat, R111 and R97 showed negative results. The local anesthetic effect of R111 was approximately equal to that of procaine. R111 and R97 showed no effects on spontaneous locomotion, the convulsion induced by strychnine or pentetrazol, and normal body temperature. (2) R111 and R97 antagonized acetylcholine, barium chloride, nicotine and serotonine-induced spasm, but not that of histamine and bradykinin. In particular they possessed marked anti-barium chloride activity, where their effects were 20 to 30 times more active than that of papaverine. (3) R111 and R97 indicated weak mydriatic activity. (4) R111 and R97 showed inhibitory effects on the pilocarpine-induced sialic secretion and the propulsive movements of the small intestine, but their inhibitory effects on the gastric secretion were relatively weak. (5) R111 and R97 displayed protective effects in Shay's ulcer, but had no curative effects on acetic acid ulcer. (6) R111 and R97 induced temporary reduction of arterial blood pressure and blood flow immediately after the administration of the test compounds in anesthetized rabbits. However, these agents induced no change in ECG, heart rate and respiration. (7) Intraperitoneally administered R111 and R97 were effective in inhibiting the carrageenin-induced edema in the hind paw of rats. From the above results, it may be considered that R111 and R97 have together strong cholinergic blocking and muscotropic antispasmodic effects, moreover, no significant effects on the central nervous system.


[Pharmacological studies of 2-(5H-[1]benzopyrano[2,3-b]pyridin-7-yl) propionic acid (Y-8004) (II). General pharmacological action]. This anti-inflammatory agent proved to have a therapeutic margin wider than that of indomethacin. Y-8004 exhibited little effect on the central nervous system, somatic and autonomic nervous system. On the cardiovascular system in dogs, Y-8004 induced a slight transient fall in blood pressure followed by a pressor response, increase in heart rate and coronary vasodilation at a dose of 30 mg/kg given intravenously, but no remarkable change was seen on the electrocardiogram. Y-8004 showed no effect on isolated smooth muscles such as vas deferens, ileum and atria, but inhibited gastrointestinal propulsion at a dose of 100 mg/kg given orally in mice and spontaneous contractions of the pregnant uterus of rats at a concentration of more than 10(-6)M. This agent also decreased urine volume and electrolytes in rats. This property was considered to be common to other acidic nonsteroidal anti-inflammatory agents. On the other hand, in guinea pigs, Y-8004 inhibited the bradykinin-induced bronchoconstriction and the collagen-induced platelet aggregation to a greater extent than did indomethacin. These findings suggest that Y-8004 is an active compound with properties similar to acidic non-steroidal anti-inflammatory agents such as indomethacin.


[Pharmacological properties of lopramine]. Pharmacological properties of LOP were compared with those of imipramine(IMP). LOP had little or no effect on electroconvulsive shock and chemoconvulsions in mice, conditioned avoidance response in rats, pain threshold in mice and rats and body temperature in rabbits. LOP, unlike IMP,  showed relatively weak effects on general behavior in mice, spontaneous EEG in cats and spontaneous motor activity in mice. LOP prevented oxotremorine-induced hypothermia but not tremor, while IMP antagonized both the responses in mice. In anesthetized dogs, LOP caused a respiratory stimulation and a fall in blood pressure, left ventribular pressure and left ventricular dp/dt without a noticeable effect on heart rate. LOP was less potent than IMP in the depressor and cardiodepressing effects, antispasmogenic activity and in antagonizing the depressor response to acetylcholine. LOP, like IMP, potentiated pressor response to norepinephrine and reduced that to tyramine in anesthetized dogs, but neither antidepressant produced norepinephrine potentiation in isolated guinea-pig vas deferens. Both drugs inhibited spontaneous motility of the jejunum without reducing the gastric motility in anesthetized dogs. These results indicate that, compared with IMP, LOP is characterized by weak general pharmacological activities.


[Pharmacokinetics and general pharmacological actions of lithium salts administered singly or repeatedly]. Absorption, distribution and excretion after oral administration of lithium carbonate (Li2CO3) and/or lithium chloride (LiCl) were studied in Wistar rats and beagle dogs. The maximum level of concentration in the blood was seen within 4 hr after administration of Li2CO3, and a greater part of the orally dosed Li2CO3 was excreted into the urine. The blood and urine Li levels after the administration of LiCl were similar to those seen with Li2CO3. In dogs, Li2CO3 was more slowly excreted into the urine than it was in rats. Li was selectively incorporated into the thyroid and pituitary a short time after administration, and was not detected in any organ 7 days after cessation of repeated dosing for 19 days. The movement of Li into the brain was slow and relatively low levels were achieved after a single administration, but high and constant levels were shown after repeated administration. Effects of Li salts on behavior of ddy mice with repeated administration were investigated. The spontaneous motor activity was suppressed with Li2CO3 more strongly than with LiCl. The high dose of Li2CO3 suppressed not only the stimulating actions of methamphetamine and cocaine, but the ptotic and hypothermic action of reserpine. From these results, it is concluded that the repeated administration of Li salts reveals higher levels of Li ion in the brain than does a single administration, and also more responsive action on the central nervous system.


[Studies of gamma-oryzanol (1). Effects on stress-induced ulcer]. In clinical trials, it is well known that gamma-oryzanol is effective against the syndromes of autonomic nervous unbalance and climacteric disorders. The authors studied the action of gamma-oryzanol on restraint, water-immersion stress ulcer under various conditions in rats. The drug, given 1 to 100 mg/kg s.c. daily for five days, reduced the ulcer index dose-dependently, and slightly prevented the rate of increase in serum level of 11-OHCS. These effects were observed in adrenalectomized as well as sham operated rats. It is likely that the antiulcerogenic action of gamma-oryzanol is due to participation of the autonomic nervous system, but not the hypophyseoadrenal axis.


Rhizosphere mycoflora of wheat after foliar application of chlorocholine chloride, urea and 4-chloro-2-methylphenoxyacetic acid. A single-step spraying of wheat during shooting under field conditions with solutions of CCC (chlorocholine chloride), CCC and urea, CCC and Aminex (ammonium salt of 4-chloro-2-methylphenoxyacetic acid), or CCC, urea and Aminex caused changes both in numbers and composition of the rhizosphere mycoflora. The numbers both in the rhizosphere of differently treated plants and in the free soil decreased during vegetation. A more pronounced effect in the number of fungi was demonstrated in plants treated only with CCC. The difference was more considerable during first 10 days after the spray. As far as the relative occurrence of individual genera in the rhizosphere soil is concerned, fungi of the genera Penicillium Link ex Fr., Fusarium Link ex Fr., Verticillium Nees and Trichoderma Pers were most influenced after the treatment with the used agents.


Mutants of Emmonsia crescens--their pathogenicity and size of adiaspores in vivo. The pathogenicity of seven morphological mutants of Emmonsia crescens was tested by means of intraperitoneal inoculation in mice. All mutants caused adiaspiromycosis. Adiaspores were isolated from granulomas after 2 months and their diameters were determined. Adiaspores from granulomas caused by five mutants (M-5, M-6, M-8, M-9 and M-16) were significantly smaller than adiaspores from granulomas caused by the wild strain, from which the mutants were derived. Two mutants (M-6 and M-9) produced adiaspores of the smallest diameter (130.5 and 119.9 mum) with the lowest variance of values, differing thus most from the original wild strain with adiaspores of 230.4 mum in diameter. A positive correlation was found between the size of the adiaspore in vivo and growth rate of the mycelial stage of Emmonsia crescens in vitro. The mutation characterized by the decreased growth rate of the mycelial stage is phenotypically manifested in the adiasporic stage of the life cycle of Emmonsia crescens, i.e. by the smaller average size of adiaspores in granulomas.


Antibiotic-induced changes of mycelial growth of Botrytis cinerea. The effect of antibiotics and metabolic inhibitors on mycelial growth of Botrytis cinera was followed. Inhibitors of protein synthesis, chloramphenicol, erythromycin and tetracycline inhibit growth or sporulation of Botrytis cinera. Ethidium bromide, 5-fluorouracil, phenylethylalcohol and K 20 cause granulation, vacuolization and undulation of hyphase. 2,4-Dinitrophenol, boromycin, macrotetrolides, monensin, scopathricin and TX2 at subfungistatic concentrations induce intensive branching of hyphal tips i.e. at the site of synthesis of the cell wall. In older hyphase grown in the absence of the antibiotics the branching begins after their addition, particularly in the septum region. When comparing the results referred to here with those obtained previously and on the basis of literature data it may be assumed that the changes in polarity of growth of Botrytis cinerea might be caused primarily or secondarily by impairing membrane functions and formation of cell walls.


Assimilation spectrum of the yeast Candida utilis 49 used for producing fodder yeast from synthetic ethanol. Oxidizing and assimilating ability of the yeast Candida utilis 49 was tested with 21 different low-boiling organic compounds which come as components of raw synthetic ethanol. The highest yields of yeast dry weight were obtained with ethanol (72.0%), propanol (48.2%), ethyl acetate (43.4%) and acetic acid (34.2%). To a minor extent, the yeast was capable of utilizing also 2-propanol, butanol and 2-butanol; it oxidized most of the compounds tested.


[Clinical picture and pathology of the so-called vinyl chloride disease]. About 26 years ago symptoms of liver and spleen were described for the first time by Tribukh et al. in laborers exposed to vinyl chloride. Besides these symptoms sclerodermia, acroosteolysis and Raynaud's phaenomenon are known to be typical for the VC disease. Rather high concentrations of VC seem to be necessary to cause this disease. After cessation of exposure the symptoms may improve partly. Haemangiosarcoma of the liver may occur as the most serious effect of prolonged exposure to vinyl chloride. Presently 48 cases induced by VC exposure are observed all over the world. Diagnosis is difficult, the prognosis of this cancer is practically always fatal. The improvement of working places by the chemical industries should further reduce the risk of acquiring VC disease as well as haemangioma of the liver due to VC exposure.


The ventricular conduction system - ultrastructure and function. The ultrastructure and function of the ventricular conducting system in animals are briefly reviewed. As the ventricular conducting cells are resistant to anoxia, an adequate ultrastructural preservation has been obtained in human autopsy material. In a case of cardiomyopathy, abnormalities in the conducting cells are related to current theories of abberant cardiac rhythm. We propose that further knowledge of the human conduction system can be obtained by extended studies of this kind.


Susceptibility to leprosy and serum atypical pseudocholinesterase. The  pseudocholinesterase levels and the nature of the enzyme as shown by the dibucaine number (D.N.) were estimated in 720 controls and 420 lepromatous leprosy patients, and 301 tuberculoid leprosy patients. There was no statistical difference in the esterase levels between leprosy patients and normal controls. But the distribution of D.N. was significantly different in the leprosy patients compared to the normal population studied. The D.N. below 40 indicates the samples with the atypical pseudocholinesterase--the presence of which is genetically determined. The distribution of samples with D.N. below 40 was significantly higher in the lepromatous leprosy patients compared to the normal population or tuberculoid leprosy patients. It is proposed that since there is a greater incidence of the atypical enzyme in lepromatous leprosy cases, the presence of this enzyme or the deficiency of the typical enzyme may make a person more susceptible to leprosy.


Peripheral vascular deficit in leprosy.

Peripheral vascular deficit in leprosy. There is considerable controversy regarding the frequency and significance of vascular lesions in leprosy. Thirty-five patients of leprosy under 40 years of age, without any local and systemic arterial disease, with normal looking hands and feet, were subjected to brachial arteriography. One patient also had posterior tibial arteriography. Diagnosis of leprosy was confirmed by bacteriologic and histopathologic technics. Biopsy material was studied from the area of radiographic abnormality, similar material was obtained from nine matched control subjects at autopsy. Skin thermometry and reflex vasodilatation were studied in eight cases. Various fractions of serum lipids and cholesterol were estimated in all patients and found to be within normal range. Arteriographic abnormalities such as occlusion, narrowing, tortuosity, dilatation, poststenotic dilatation, irregularity and incomplete filling of the lumen by radio-opaque material, were seen in more than two vessels in 50% of the arteriograms in wrist and palm; digital vessels showed abnormality in 75% to 94% of cases. The ulnar artery was more frequently involved (74%) than the radial (50%). Superficial and deep palmar arches were equally affected. Increased vascularity in hypothenar eminence area was seen in three arteriograms. Marked increase in arcuate vessels was noted in 60% of patients. Some arcuate vessels were serving as collaterals. Histologically, all grades of vessels including capilaries showed changes in nearly half of the patients. Reflex vasodilatation was lost in three patients of dimorphous leprosy, and impaired or absent in three of five patients of lepromatous leprosy. There was no predilection for any form of leprosy to show arterial changes more than others. This study clearly demonstrates that the vascular involvement in leprosy is very frequent and must be playing an important role in causing mutilations and deformities of hands and feet.


Vitamins C and E in spontaneous abortion. A group of 50 spontaneously aborting women was compared with the same number of pregnant women whose pregnancies terminated uneventfully. Biochemical parameters were studied and particular attention was paid to the vitamin C and E levels in both groups. A significantly higher percentage of aborting women had individual values of serum alpha-tocopherol above the 0.50 mg/100 ml normal limit. There was no significant difference in overall frequency distribution of vitamin C values in both groups. However, at the end of the first trimester the mean values of vitamin C were significantly higher in the aborting group. This could not be interpreted as an evidence of causal association.


Occupational therapy in leprosy.

Occupational therapy in leprosy. This paper presents a broad discursive assessment of the philosophy and practices of occupational therapy as related to leprosy. It stresses the role of society, self-care by the patient, integration, vocational training, rehabilitation and the amotivational syndrome, and presents some illustrative original inovations. In conclusion some new approaches are suggested.


[Congenital craniopharyngioma].

[Congenital craniopharyngioma]. Case report of a congenital craniopharyngioma in a full-term male stillborn infant. The tumour was remarkable for its size (10.5 X 9.5 X 6.5 cm) which exceeds those already published in the literature, and for the fact that hydrocephaly was minimal. Histologically, there were long, thin spaces lined by a cubic-like epithelium, and a large number of keratinized epidermic pearls containing peripheral keratohyalin granules. The clinical and neuropathological aspects of this tumour are compared with those of four other cases published in the literature.


Observations on the involvement of wildlife in an epornitic of chlamydiosis in domestic turkeys. In an investigation of potential wildlife reservoirs of Chlamydia psittaci at the site of an acute, highly fatal epornitic of chlamydiosis in domestic turkeys in Texas, various species of wild birds and rodents were captured and tested for chlamydial serum antibodies and chlamydiae in their tissues. Thirteen (65%) of 20 blackbirds (Agelaius sp), 4 (44%) of 9 killdeer (Oxyechus vociferus), 3 (27%) of 11 sparrows (Passer sp) and 1 of 4 mourning doves (Zenaidura macroura) were seropositive, but chlamydiae were not isolated from their tissues. Two of 3 rats (2 cotton rats, Sigmodon hispidus, and 1 roof cat, Rattus rattus) were seropositive, but 7 gophers (Citellus sp), 1 ground squirrel (Tamias sp), an opossum (Didelphis virginiana), and a domestic cat (Felis domesticus) were seronegative; however, chlamydiae were recovered from the livers and spleens of the opossum and domestic cat, both of which had been observed scavenging carcasses of turkeys dead of chlamydiosis. Cultures of these isolants were inoculated experimentally into turkeys and produced lesions of chlamydiosis that were indistinguishable from those caused by the strain originally recovered from diseases turkeys on the premises. Nine of 50 domestic goats quartered near the diseased turkeys were tested, and all were seropositive.


Vaccine-induced canine distemper in a lesser panda. A fatal disease occurred in a lesser panda (Ailurus fulgens) 2 weeks after vaccination with modified live distemper vaccine. The disease clinically resembled canine distemper. Pathologically there was giant cell pneumonia, with canine distemper viral inclusion bodies in pulmonary and digestive tract epithelium. Viral isolates were indicative of an attenuated strain rather than virulent types.


Fatal vaccine-induced canine distemper virus infection in black-footed ferrets. Four black-footed ferrets that were live-trapped in South Dakota and transported to the Patuxent Wildlife Research Center died within 21 days after vaccination with modified live canine distemper virus. Immunofluorescence, European ferret inoculation, virus isolation attempts, and serum-neutralization tests indicated insufficient attenuation of the vaccine for this species.


Sperm-egg interactions in the pig: monospermy, extensive polyspermy, and the formation of chromatin aggregates. The process of incorporation and metamorphosis of the sperm head within the vitellus has been examined by phase-contrast microscopy in a large series of pig eggs exhibiting either normal monospermic fertilization or extensive polyspermy. This latter condition was induced in vivo after increasing the numbers of capacitated spermatozoa in the Fallopian tubes by pre-ovulatory surgical insemination. Attention was drawn in monospermic eggs to the initial fusion between the spermatozoon and vitelline surface which produced a characteristic constriction in the equatorial region of the head. Immediately following cytoplasmic contact with the sperm nucleus, an increase in size was detectable in this structure, remnants of the inner acrosomal membrane having apparently been displaced during incorporation. In fixed preparations of activated eggs, there was some evidence that the morphologically transformed sperm nucleus underwent a brief period of shrinkage before commencing pronuclear formation. The most striking feature of the polyspermic condition was the number of spermatozoa that had entered the vitellus (2 to greater than 80), and the formation of aggregates of sperm head chromatin in eggs penetrated by more than 20-30 spermatozoa; the heads of at least 8 or 9 spermatozoa could participate in the formation of such an aggregate. Various unusual configurations were noted during breakdown of the midpiece in polyspermic eggs, and degeneration was also a general feature of the cytoplasm in situations of excessive polyspermy. Aspects of the block to polyspermy are discussed, and it is inferred that highly polyspermic pig eggs can still exhibit a zona reaction.


Effect of cerulenin on the growth and differentiation of Dictyostelium discoideum. The growth of Dictyostelium discoideum Ax-2 was inhibited completely by cerulenin at a concentration of 5 mug/ml. This inhibition of growth was found to be due to the inhibition of fatty acid synthesis. Acetate incorporation into a long-chain fatty acid was inhibited completely by cerulenin, and the growth inhibition could be reversed by inclusion of certain saturated fatty acids in the medium. Unsaturated fatty acids and sterols failed to reverse the inhibitory effect. The fatty acid and sterol compositions of cerulenin-treated cells were determined to establish whether the drug could be used to manipulate the organism's lipid composition. Only relatively small manipulations were obtained under the conditions employed in this study. Cerulenin inhibited differentiation but only at high concentrations (150 mug/ml). This inhibition could be reversed by palmitic acid, suggesting that the prime cause of the inhibition was an inhibition of fatty acid synthesis. Thus, it appears that continued fatty acid synthesis is required for the cellular process of differentiation in D. discoideum.


Effects of light and temperature on macrocyst formation in paired mating types of Dictyostelium discoideum. Light-dark and temperature shifts performed on myxamoebal populations of mixed mating types indicate that there is early differentiation of the population leading to the macrocyst formation in Dictyostelium discoideum.


Thermal adaptation in yeast: growth temperatures, membrane lipid, and cytochrome composition of psychrophilic, mesophilic, and thermophilic yeasts. The temperature limits of growth of a number of yeast species were examined, and on this basis the organisms were classified into different thermal categories. The following species were examined: Leucosporidium frigidum and Leucosporidium nivalis, psychrophilic, temperature limits of growth, -2 to 20 degrees C; Canadian lipolytica mesophilic, temperature limits of growth, 5 to 35 degrees Candida parapsilosis and Saccharomyces telluris, thermotolerant, temperature limits of growth, 8 to 42 degrees C; Torulopsis bovina and Candida slooffi, thermophilic, temperature limits of growth, 25 to 45 degrees C and 28 to 45 degrees C, respectively. The membrane lipid and cytochrome composition of mitochrondrial fractions isolated from these yeasts were compared. There was a direct correlation between the growth temperature and the degree of membrane of lipid unsaturation; the lower the temperature, the greater the degree of lipid unsaturation. The membrane lipid composition of the thermophilic yeasts were distinguished by the high percentage (30 to 40%) of saturated fatty acid, as compared with the mesophilic and psychrophilic yeasts. The latter contained approximately 90% unsaturated fatty acid, 55% of which was linolenic acid, C alpha-18:3. Changes in phospholipid composition in relation to temperature were also noted. The respiratory-deficient thermophile, C. slooffi, was characterized by the absence of cardiolipin (sensitivity 0.1 mug of phosphorus) and cytochrome aa3. The absence of conventional mitochondrial structures in this thermophilic microorganism is tentatively suggested although low concentrations of cytochromes b, c, and c1 were detected by low-temperature spectroscopy. On the other hand, the respiratory-competent thermophile, T. bovina, was characterized by a high cardiolipin (25% of the total phospholipid) and cytochrome aa3 content (1 nmol/mg of mitochrondrial protein). Low-temperature spectra showed the presence of one b-type cytochrome in the thermophilic yeasts, two b-type cytochromes in the mesophilic yeasts, and three b-type cytochromes in the psychrophilic yeasts. It was concluded that a knowledge of the properties of the biological membrane is fundamental to an understanding of the ability of a microorganism to grow and reproduce in different temperature environments.


Isolation of diacylglyceryl-2-aminoethylphosphonate from bovine liver. Diacylglyceryl-2-aminoethylphosphonate was isolated from bovine liver by a combination of silicic acid column and silica gel thin-layer chromatographic techniques, and was identified from the results of elementary analysis, the infrared spectrum, chemical properties, and chromatographic behavior. This is the first isolation of a lipid-bound form of ciliatine in mammals.


The amino acid sequences of two soybean double headed proteinase inhibitors and evolutionary consideration on the legume proteinase inhibitors. Two proteinase inhibitors, C-II and D-II, were isolated from soybeans. C-II was shown to be an inhibitor of bovine trypsin [EC 3.4.21.4], bovine alpha-chymotrypsin [EC 3.4.21.1], and porcine elastase [EC 3.4.21.11], whereas D-II inhibited only trypsin. The complete amino acid sequences of the two inhibitors establishors. On the basis of the specificities of the inhibitors and their homologies with other double-headed inhibitors, the reactive sites of C-II seems to be alanine-22 for elastase and arginine-49 for trypsin (and probably also for chymotrypsin). D-II was quite unique because its both reactive sites are arginine residues and it only inhibits trypsin. It is suggested that D-II might be a primitive form of double-headed inhibitor and that the prototype single-headed inhibitor was a trypsin inhibitor with an arginine residue as the reactive site.


Kinetic analysis of biochemical differentiation in Dictyostelium discoideum. The metabolic network leading to accumulation of cellulose, trehalose, and mucopolysaccharide during development of Dictyostelium discoideum was simulated on a computer. The program consists of a metabolic map, the measured specific activity of the enzymes involved at each stage in development, and the substrate and inhibitor affinities. The Km values of four enzymes, amylase, UDP-galactose polysaccharide transferase, UDP-galactose epimerase, and cellulose synthetase, were determined for this study. At each iteration (1 min) during the period simulated (1500 min), the in vivo activity was calculated for each enzyme using Michaelis-Menten equations and new values for metabolites and end products were generated. The computed values for the concentration of both metabolites and polysaccharides were in close agreement with the measured values at all stages of development. We conclude that the in vitro measured values correlate well with the measured in vivo rates when treated in this manner. The program was modified to simulate the alterations in carbohydrate metabolism which might be expected in mutant strains with reduced activity of various enzymes. Trehalose was found to overaccumulate when either the peak value of the developmentally controlled increase in the specific activity of UDPGlc pyrophosphorylase was reduced. Trehalose accumulation was decreased in simulations of mutants lacking glycogen phosphorylase or glycogen synthetase. The interaction of these metabolic pathways is discussed.


Studies on the disulfide bonds of glycoprotein hormones. Course of reduction of bovine luteinizing hormone, bovine thyroid-stimulating hormone, and their subunits. The five disulfide bonds of isolated alpha subunits of luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) are completely reduced at pH 8.5 in 15 min with no denaturant required and with only a slight excess of reducing agent. At pH 7.0, reduction is complete after 6 to 10 h. These results together with an earlier study concerning the positions of the two most readily reduced bonds (Cornell J.S., and Pierce, J.G. (1974) J. Biol. Chem. 249, 4166-4174) show that, in the isolated alpha subunit, all disulfides are readily accessible, although it is possible that a change in conformation, after rapid initial reduction of two disulfides, makes the remaining three more susceptible to reduction. No partially reduced and S-carboxymethylated intermediates were found at pH 7.0 other than those seen at pH 8.5, nor were additional intermediates found at pH 8.5 when reduction was initiated in the presence of alkylating agent. In contrast, reduction of the beta, hormone-specific, subunits of LH and TSH, while complete at pH 8.5 after 2 to 6 h, does not proceed to completion at pH 7.0 even after 24 h or upon addition of 6 M urea or large concentrations of reducing agent, and partially reduced intermediates useful in location of disulfide bridges can be trapped (e.g. Reeve, J.R., Cheng, K.-W., and Pierce, J.G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155). Little or no reduction of the intact hormones is found at pH 7.0 in the absence of denaturing agents. This protection by the intact structure shows that the two most readily reduced disulfides of the alpha subunit and the single most readily reduced sidulfide of the beta subunits are either in regions of subunit-subunit contact or that these bonds become more reactive in the isolated subunits because of different influences by neighboring groups. At pH 8.5, intact LH is completely reduced after 6 h, but intact TSH is more resistant to reduction, which may reflect a higher affinity between subunits than exists in LH.


Studies on the disulfide bonds of glycoprotein hormones. Complete reduction and reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone. Reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone (LH) after their complete reduction both in the presence and absence of denaturing agent yields a product which is indistinguishable from the native subunit in its electrophoretic pattern on polyacrylamide gels and in its ability to recombine with the beta subunits of both luteinizing hormone and thyrotropin. The circular dichroism spectrum of the reoxidized alpha subunit is essentially identical to that of native alpha subunit except that its maximum at 233 nm is smaller than observed with native LHalpha. The intact hormone preparations obtained by recombination of reoxidized alpha subunit with native LH-beta exhibit electrophoretic patterns in polyacrylamide gels, elution profiles on gel filtration, binding activities to a membrane fraction from rat testes, and circular dichroism spectra identical to those of native LH and recombinants of native LH-alpha with the beta subunit. Recombinants of native or reoxidized LH-alpha with the beta subunit of thyrotropin are also indistinguishable in their electrophoretic patterns on polyacrylamide gels and in their in vivo activities of stimulating 32P uptake in thyroids of day-old chicks. While this study does not preclude that the alpha subunit may be biosynthesized as part of a larger precursor protein, the data demonstrate that sufficient information is present in the linear sequence of the alpha subunit to allow folding and formation of disulfide bonds to yield a functional alpha subunit.


Mitogen receptors in chick embryo fibroblasts. Kinetics, specificity, unmasking, and synthesis of 125I-insulin binding sites. Insulin, a mitogen for cultured chick embryo fibroblasts (Temin, H.M. (1968) Cancer 3, 771-787), has been employed to characterize the effects of mitogen/cell membrane interactions as it relates to growth. The specific binding of 125I-insulin to substratum-attached cells is time- and temperature dependent and is optimum at a pH of 7.0. Fetal calf and chicken sera, somatomedin "A/C mixed," and desalanine or native porcine insulin compete with 125I-insulin for membrane-binding sites. Proinsulin, although competing less effectively than native insulin for binding, is more effective than desoctapeptide insulin. Unrelated polypeptide hormones do not compete for 125I-insulin binding. The lowest concentration of insulin at which specific binding is detected is 0.1 nM. Scatchard plot analysis of the binding data indicates that there are two types of binding sites in confluent cultures of fibroblasts: one of high affinity (K1 = 2 to 6 X 10(8) M-1) and low capacity, the other of low affinity (K2 = 0.8 to 3.0 X 10(7) M-1) and high capacity. Approximately 1.9 and 7.1 X 10(3) molecules of insulin are bound at each site, respectively. A 10-min incubation at 24 degrees of the fibroblasts with 10 mug/ml of trypsin causes a 2-fold stimulation of specific 125I-insulin binding and a similar 2-fold increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation. Neuraminidase treatment also produces a 37% increase in specific 125I-insulin binding but treatment with alpha-chymotrypsin or phospholipase C are without significant effect. The results of this and additional experiments support the hypothesis that trypsin treatment of chick embryo fibroblasts leads to an unmasking of 125I-insulin binding sites. Serum starvation of fibroblasts for 12 or 24 h produces a 2.5- to 5-fold increase in specific 125I-insulin binding. This increase is the result of an increase in the number of hormone-binding sites from 9 X 10(3) to 6 X 10(4) per cell which are predominantly of the low affinity type. There is no change in the affinity constants. The presence of camptothecin, or cordycepin, or cycloheximide in the incubation medium completely blocks the increase in number of 125I-insulin-binding sites resulting from serum starvation. The addition of native insulin to the medium of serum-starved cultures also blocks this increase. The magnitude of insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation correlates with the levels of occupancy of the low affinity 125I-insulin-binding sites in untreated fibroblasts. In fibroblasts cultured in the absence of serum, the marked increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation parallels the increase in number of mitogen receptors. The concentration of insulin that produces a half-maximum stimulation of thymidine incorporation is calculated to be 5 X 10(-8) M. At this concentration of insulin, 42% of the receptor sites are occupied.


Biosynthesis of glycosaminolgycans during corneal development. Glycosaminoglycan biosynthesis was studied in developing chick corneas, with particular attention paid to keratan sulfate I, the major glycosaminoglycan of this tissue. This polysaccharide is unique to the cornea and may be required for the development and maintenance of corneal transparency. Corneas from 5-to 20-day chick embryos were labeled in vitro with D-[6- 3H] glyhucosamine and H(2)35SO(4)35SO(4) and the amount of label in each glycosaminoglycan was determined. The data indicate that, contrary to previous suggestions, keratan sulfate biosynthesis in the cornea begins at the time of fibroblast invasion of the primary stroma, at least 8 days prior to the onset of corneal transparency, which occurs on Day 14 of the development in the chick. The rate of incorporation of radioactivity into keratan sulfates, on a dry weight basis, increases rapidly after Day 6 and levels off on Day l4. The proportion of 3H and 35S in keratan sulfate reaches nearly maximal levels as early as Day 9. In contrast, the proportion of radioactivity in corneal heparan sulfates declines rapidly after Day 5. However, the rate of incorporation of radioactivity into heparan sulfates, on a dry weight basis, increases or remains the same during early development. On and after Day 14, keratan sulfates appear to become more highly sulfated. Moreover, the ratios of 4-sulfated to 6-sulfated chondroitin sulfates increase during development, reaching a maximum on Day 14. These changing patterns of glycosaminoglycan biosynthesis during corneal development may play an important role in corneal morphogenesis and the achievement of corneal transparency


Studies of chemical modifications of proteins by carbon 13 neuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodine. The reaction of iodine with aromatic residues of hen egg white lysozyme is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. In the unfractionated product of the reaction at PH 5.5 (with I2/lysozyme molar ratios of 0.5, 1.0, and 2.5), the only detectably modified aromatic residues are Trp-108 and either Tyr-20 or Tyr-23 (probably the latter). The rates of reaction at the two sites are similar. The extents of modification (at each site) are approximately 25%, 50%, and approximately greater than 80% for I2/lysozyme molar ratios of 0.5, 1.0, and 2.5, respectively. At pH 4.5, the rates of reaction of both residues are about one-third or less of the rates at pH 5.5. When the reaction is carried out at pH 8.5 (with an I2/lysozyme molar ratio of 1.0), only the tyrosine residue is modified. Resonances observed in the spectra of the modified protein mixtures (but not in the spectrum of intact lysozyme) indicate that the modified Trp-108 residue is not oxindolealanine, but either delta1-hydroxytryptophan or an ester thereof. This result is consistent with previous evidence which indicates that the modified tryptophan is the Glu-35 ester of delta1-hydroxytryptophan-108 (Imoto, T., and Rupley, J.A. (1973) J. Mol. Biol. 80, 657-667; Beddell, C. R., Blake, C. C. F., and Oatley, S. J. (1975) J. Mol. Biol. 97, 643-654). The spectra also indicate that the modified tyrosine residue is predominantly monoiodinated. The spectra of modified protein samples subjected to denaturation with 6M guanidinium chloride for 24 h at 37 degrees (and the renatured) indicate that residue 108 is converted to about equal amounts of the two diastereoisomers of oxindolealanine. However, incubation in 6M guanidinium chloride for 2 h at 25 degrees does not cause measurable hydrolysis of the Glu-35 ester of delta1-hydroxytryptophan-108.


Time-dependent changes in Dictyostelium discoideum adenylate cyclase activity upon incubation with ATP. In this study we report that preincubation of Dictyostelium discoideum membrane-bound adenylate cyclase with ATP over the concentration range 0.5 to 100 mM results in a loss of catalytic activity and that this effect persists even after removal of ATP. An analysis of the time course of this effect shows that, at 25 mM ATP, a 5- to 10-min preincubation results in 50% loss of activity. Additional studies on this effect showed that anhydride bond cleavage of ATP occurs during the preincubation. However, loss of catalytic activity is not porduced by ADP, AMP, cAMP, adenosine, pyrophosphate, or phosphate either separately or in pairs. Further, using the structural analogs adenosine 5'-(alpha, beta-methylene)triphosphate and adenyl-5'-yl imidodiphosphonate, we show that there is a direct correlation between alpha-beta-phosphoanhydride bond cleavage and the loss of catalytic activity. These results can be interpreted in terms of two classes of reaction mechanisms: either those involving covalent modifications or those involving a ligand-induced slow conversion of the adenylate cyclase from an active to an inactive form. Additional studies show that the addition of AMP to the reaction mixture, as well as removal of the membrane-bound 5'-nucleotidase activity, can prevent the loss of cyclase activity. These results suggest not only that adenylate cyclase activity is related to the AMP:ATP ratio but that the cyclase activity can be modified by the level of 5'-nucleotidase activity. Studies on the duration of the loss of activity produced by ATP show that following removal of ATP and additional incubation, a gradual recovery of cyclase activity is observed. This result suggests that under appropriate conditions the cyclase inactivation by ATP is reversible.


Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+. As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.


Proteins and glycoproteins of membranes from developing chick red cells. Membrane vesicles produced when chick erythroid cells are disrupted by nitrogen cavitation were isolated by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Sodium dodecyl sulfate-polyacrylamide gel analysis of isolated membranes shows eight to ten proteins and four to five glycoproteins. Membranes must be prepared with protease inhibitors, otherwise an endogenous activity degrades high molecular weight polypeptide components. Red cells from several stages of development (5- and 17-day embryos and adult chickens) all appear to have the same major embrane proteins. However, primitive erythroid cells from 5-day embryos lack a Mr = 40,000 glycoprotein that is present in definitive erythrocytes from 17-day embryos and from adult chickens; erythrocytes from young chicks show a decrease in the amount of a glycoprotein of Mr = 50,000. Lactoperoxidase-catalyzed iodination of intact 5-day embryonic red cells detects three surface components which comigrate with the membrane glycoproteins on sodium dodecyl sulfate polyacrylamide gels.


Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro. Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.


Microtubules and control of insect egg shape. This study evidence for tension transmission by microtubules and desmosomes in the follicular epithelium during anisometric growth of certain insect eggs. Most insect oocytes, and the follicles which surround them, grow anisometrically as they assume shapes which approximate to those of long prolate spheroids. Surface growth is most rapid in directions which parallel the polar axis of an oocyte and slowest in circumferential directions at right angles to this axis. The longitudinal axes of microtubule bundles in follicle cells of the gall midge Heteropeza and the cockroach Periplaneta are oriented circumferentially with respect to the surfaces of developing eggs and at right angles to the polar axes of eggs. At cell boundaries, the tubules appear to be attached to spot desmosomes. It is suggested that microtubules and desmosomes form a mechanical continuum throughout a follicular epithelium which transmits tensile forces around the circumference of a growing egg. Follicular resistance to circumferential expansion may be largely responsible for defining the elongate form of insect eggs.


Concanavalin A and wheat germ agglutinin receptors on Dictyostelium discoideum. Their visualization by scanning electron microscopy with microspheres. The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.


Initiation of brain tubulin assembly by a high molecular weight flagellar protein factor. A protein factor found within the flagella of Chlamydomonas and sea urchin sperm is capable of stimulating the initiation of calf and chick brain tubulin dimer assembly in vitro.


Changes in the topography of the sea urchin egg after fertilization. Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but  microvillar elongation was blocked by cytochalasin B treatment.


Clonal lines of teratocarcinoma cells in vitro: differentiation and cytogenetic characteristics. Clonal lines of embryonal carcinoma cells have been established in culture from four independently-derived transplantable teratocarcinomas of mice: three from strain C3H and one from strain 129/Sv. Cells from all lines retain the capacity to differentiate into a variety of tissue types both in tumors formed following the injection of cells into syngeneic animals and in vitro under appropriate culture conditions. Analysis of their G-banded chromosomes indicated that the four lines have near-diploid but not absolutely normal karyotypes. The same chromosomal abnormalities were often present in more than one line. Tetraploid embryonal carcinoma cells made by Colcemid or cytochalasin B treatment were also pluripotential in spite of chromosomal instability. Hybrid cells were readily obtained between diploid or tetraploid embryonal carcinoma cells and mouse 3T3 fibroblasts. Hybrid cells failed to differentiate and were contact inhibited like the 3T3 parent.


Aggregation in Polysphondylium.

Aggregation in Polysphondylium. Dense aggregating fields of P. violaceum and P. pallidum exhibit propagated waves which may be analogous to those seen in Dictyostelium discoideum. As in D. discoideum the wave velocity is determined by an intracellular delay between stimulation and response, rather than by the diffusion coefficient of the acrasin. The frequency of the propagated wave overlaps that of aggregating D. discoideum, so that absence of coaggregation reported by Raper & Thom is unlikely to be based on specificity of the propagated wave parameters, but rather on a difference in the chemical acrasin itself, as argued on independent evidence by Bonner. Despite this difference in the chemical nature of the acrasin, the presence of propagated waves in Polysphondylium with frequencies similar to those in D. discoideum, taken together with evidence for a diffusible acrasin, suggests that the mechanism of aggregation is substantially the same in both genera.


Cyclic adenosine monophosphate and the development of Polysphondylium. Centre formation in Polysphondylium violaceum is delayed for 2 h on buffered agar containing 10(-3) M c-AMP, and for up to 22 h on unbuffered agar with the same c-AMP concentration. With ambient c-AMP concentrations as low as 10(-6) M, P. pallidum forms numerous, small, atypical aggregates which do not fruit. This effect is independent of whether the agar is buffered. P. violaceum amoebae are weakly attracted to the tip of a microelectrole containing 10(-3) or 10(-4) M c-AMP, but the electrode cannot compete when natural centres form nearby. P. pallidum amoebae are not attracted. Aggregates of P. violaceum and of P. pallidum are strongly attracted to a microelectrode releasing c-AMP. The observation of Shaffer that Polysphondylium grex switch over from secreting an acrasin that attracts homologous amoebae to one that attracts the larger Dictyostelium species suggests that the second acrasin might by c-AMP. The above results strengthen this conjecture. As c-AMP inhibits centre formation, the secretion of c-AMP by older aggregates may explain an inhibition of centre formation in the 'overlay' experiments of Shaffer.


Differentiation and positioning of nuclei in eggs of the cecidomyid Heteropeza pygmaea. During the first three cleavage divisions of the egg nuclei a precise sequence of spindle orientation and elongation parallel to the longitudinal axis of the egg is apparently involved in positioning one nucleus among the polar granules at the posterior pole of the egg. The size of this nucleus, and the position at which the egg cleaves when pole cell formation occurs, appear to constitute part of the mechanism which ensures that only one nucleus is included in the first pole cell. Blastoderm formation occurs without a well-defined migration of nuclei to the egg surface. Nuclei are so large in relation to the size of the egg that uniform spacing and distribution of nuclei ensures that a large proportion are situated near the egg surface. Those nuclei which are near the egg surface divide synchronously to form a layer of blastoderm nuclei, while membranous cleavage furrows invaginate from the egg surface between them. Nuclei in the central region of the egg chamber condense to form yolk nuclei before blastoderm nuclei have been separated from the rest of the egg by the completion of the cleavage membranes. Polar granules provide the only evidence of fine-structural differences in different regions of the egg chamber cytoplasm. They are found near the posterior pole of the egg from an early stage of oogenesis. They undergo a specific sequence of structural changes and increase in size as the egg grows. No microtubular or microfibrillar arrays have been found in the egg chamber which might form a cytoskeletal basis for spindle orientation or for the spatial differences which develop during differentiation of the uncleaved egg cytoplasm.


The effects of dietary sodium on the diurnal activity of the renin-angiotensin-aldosterone system and the excretion of urinary electrolytes. Diurnal variations of five normal men were tested over three 24 h consecutive periods. The first experiment began at 0900 h after the subjects had fasted for 12 h and a normal sodium diet of about 70-80 mEq was given at 0900 h, 1200h, and 1630 h (total of about 220 mEq of Na). Significant variations in the plasma renin activity (PRA), in the plasma aldosterone (PA), and in the urinary Na and K outputs were found. The second experiment began at 1200 h with the first feeding time at 2100 h after fasting about 24 h and the subjects were given a normal sodium diet as in the first experiment, but with the meals given at 2100 h, 2400 h, and 0430 h. The diurnal variations in PRA, plasma aldosterone, and urinary electrolytes disappeared. From this study, it appears that the diurnal variation in urinary electrolyte excretion is a factor of the diurnal variation in PRA and plasma aldosterone. The diurnal variation in PRA  and plasma aldosterone are related to the timing of sodium ingestion.


An androgen binding protein in the cyst fluid of patients with polycystic ovary syndrome. Androgen binding proteins (ABP) have not been described in the ovary. We have studied cyst fluids from three patients with the Polycystic Ovary Syndrome and one patient with choriocarcinoma for the presence of ABP. All contained a high affinity, low capacity binding protein for dihydrotestosterone and 17beta-estradiol. Comparison of this protein with the plasma androgen binding protein (TeBG) in polyacrylamide gel electrophoresis and equilibrium dialysis did not reveal any significant differences in molecular size (Kg) net charge (Yo), radius R, molecular weight (mol wt), heat stability, steroid specificity, or binding affinity (Ka). Cyst fluid and plasma could not be distinguished from each other on the basis of protein composition as measured by cellulose acetate electrophoresis. In the one patient studied, the binding capacity for DHT in cyst fluid and plasma was 58.1 and 16.1 ng/Gm protein, respectively.


Stimulation of RNA and protein synthesis in isolated chondrocytes by human serum. The stimulation of isolated chicken embryo chondrocytes was studied by measuring the incorporation of [3H]uridine and [3H]leucine into cold trichloroacetic acid precipitable material after exposure of the chondrocytes to serum. The doseresponse relationships for the incorporation of uridine and leucine were similar to that of thymidine previously demonstrated. Exposure of the cells to serum-containing buffer for 15 min sufficed both for the stimulation of incorporation into the cells and for the depletion of 28% of the stimulating activity from the medium. Stimulation persisted for at least 17 h after removal of the serum. Studies where actinomycin D was added to inhibit RNA synthesis suggested that prior RNA synthesis was required for most of the stimulation of protein synthesis by serum factors.


Stimulation of DNA synthesis in isolated chondrocytes by somatomedin. II. Validation of the assay for clinical use and comparison with stimulation of protein synthesis. The effects of GH on cartilage may be mediated by a variety of serum factors (somatomedins; SM). We have reported (Endocrinology 90: 1086, 1972) stimulation of thymidine incorporation in isolated chicken embryo chondrocytes by normal human serum. This was greater than that caused by serum from patients with hypopituitarism. We have now compared the stimulatory activity estimated by [3H]thymidine incorporation (SMT) with that estimated by [3H]leucine incorporation in 46 sera from children with GH deficiency; with short stature, but normal GH responsiveness; or with normal stature and normal GH responsiveness. These activities were also measured in sera from 9 normal adults and 12 acromegalics. Sera from GH deficient children had reduced SMT activity (.54 +/- .04; (mean +/- SE) P less than .01) compared to normal children (.83 +/- .08) whereas the sera from children with short stature and normal GH responsiveness had higher levels than normal (1.19 +/- .10: P less than .02). Acromegalic adults averaged higher SMT activity than normal adults (1.62 +/- .15 vs. 1.17 +/- .11; P less than .05). In sharp contrast, the leucine incorporation was essentially the same in the different groups of children. These studies have validated the use of the incorporation of thymidine into isolated chicken embryo chondrocytes as an adjunct in the evaluation of children with short stature (82.6% of the samples from children gave results that were consistent with their status as determined by provocative tests for GH). The disparity between the results with thymidine incorporation and those with leucine incorporation is as yet unexplained.


Alpha subunit contamination of human albumin preparations: interference in radioimmunoassy. Certain preparations of human serum albumin have been found to decrease the apparent titer of antisera to alpha subunit of human TSH (hTSH-alpha) and the sensitivity of the resultant radioimmunoassay for the immunologically common alpha subunit of the human glycoprotein hormones. Utilizing bovine serum albumin as the carrier protein, antisera to hTSH-alpha had 20-fold higher titers, and the resultant radioimmunoassay demonstrated 20-fold greater sensitivity for alpha subunit. Interference in the alpha immunoassay was not caused by protease since protease inhibitors did not eliminate it. Gel chromatography of human serum albumin revealed alph immunoactivity in an elution position identical to that of standard alpha subunit. Only certain human serum albumin preparations demonstrated interference in the radioimmunoassay because of the species specificity of the alpha subunit.A possible explanation for the alpha subunit contamination of human albumin preparations may be contamination of the serum source with placental blood, which contains large quantities of the alpha subunit of human chorionic gonadotropin.


An assessment of the macrophage electrophoretic mobility test (MEM) in cancer diagnosis. The results of an assessment of the macrophage electrophoretic mobility test in cancer diagnosis are described. We found that the test did not reliably distinguish between a group of 18 patients with benign disorders and a group of 25 with malignant disease.


Ontogeny of behavioral arousal in rats: effect of maternal and sibling presence. Spontaneous locomotor activity of 5- to 30-day-old rats housed either alone or in the presence of various components of the normal nest and litter stimuli was recorded by means of time-lapse videography. Developing animals observed alone showed a sharp increase in total daily locomotor activity from Day 5 to Day 15 followed by a rapid decline from Day 15 to Day 30. Individual animals observed in the context of the normal litter environment showed an entirely different pattern of development. They were relatively inactive during the first 15 days of life and then began a gradual increase of activity which continued for the next 15 days. The heightened activity characteristic of the isolated 15-day old inhibited by the presence of four siblings, an anesthetized lactating female, or an anesthetized adult male rat. Thermal conditioning, including heating of the floor to approximately nest temperature or the presence of a heated tube in one corner of the test apparatus, did not inhibit the heightened activity. These results question the generality of the ontogenetic sequence of excitation and inhibition proposed by Campbell and his associates - at least a portion of the heightened activity seem around 15 days of age was the result of isolation distress, not merely maturational changes in the brain.


Reactivation of alkaline phosphatase in ultra high-temperature, short-time processed liquid milk products. Alkaline phosphatase (enzyme) reactivation was studied in liquid milk products heated to 87.8 to 121.1 C for less than 1 s in a continuous, two-phase, slug-flow heat exchanger. The effects of magnesium concentration, pH, incubation temperature, homogenization pressure, processing temperature, fat content, and initial enzyme concentration were investigated. Jersey milk from one farm showed seasonal variations in enzyme concentration and its reactivation behavior. Increased reactivation in products with high fat content was due to high initial enzyme concentration in the product. Homogenization of products before heating decreased reactivation. Maximum reactivation occurred in products heated to 104.4 C, incubated at 34 C, and adjusted to pH 6.5. Maximum velocity of reactivation and reactivation constant varied with milk samples. Activation energy for the control and samples with magnesium was 22.646 +/- .118 and 24.100 +/- .210 kJ mole-1, respectively. The enzyme from raw and reactivated cream contained two major isozymes, and the reactivated isozyme differed from the control. The official method for differentiating residual and reactivated enzymes was modified in terms of magnesium concentration and extent of reactivation of the enzyme in the reactivated product.


Lead and cadmium in California raw milk. An atomic absorption procedure for the simultaneous measurement of lead and cadmium in raw whole milk has been developed and tested. A 100 g sample is dry ashed at a temperature not exceeding 425 C. The ash then is dissolved in hydrochloric acid and neutralized with ammonium hydroxide to pH 8.5 in the presence of ammonium citrate. Potassium cyanide is added to complex interfering ions followed by addition of ammonium pyrrolidino carbo dithioate to complex lead and cadmium. After the ammonium pyrrolidino carbo dithioate complexes are extracted in isoamylacetate, the lead and cadmium are measured in the extract by atomic absorption. Mean recoveries of lead and cadmium added to raw whole milk were 92.1 +/- 2.9% and 97.5 +/- 2.5%. A statewide survey showed that the mean concentration of lead in 350 raw milk samples was 91 mug/kg; 222 of the samples were below 100 mug/kg. The mean concentration of cadmium in 315 raw milk samples was 6.0 mug/kg; 40 samples had 1.0 mug/kg or less, and 255 of the samples were below 10.0 mug/kg.


Vitamin B12 in rumen wall and liver.

Vitamin B12 in rumen wall and liver. Samples of liver, rumen wall, and rumen mucosa (rumen wall stripped of its muscle) were assayed for vitamin B12 by specific and nonspecific methods. Mean differences between methods were negligible. Rumen mucosa and rumen wall contained approximately 10% as much vitamin B12 as liver. The B12 contents of liver and rumen mucosa were correlated whereas those of liver and rumen wall were not.


Etiology of growth failure in a clinic population. To identify the basis of growth failure, the background of preschool children whose growth fell below 97 per cent of their peers was compared with that of those who demonstrated a higher level of growth achievement. A questionnaire administered at the home of each child documented living conditions, health history, and other family characteristics. Families of the growth-retarded children had higher income, higher credit payments, and used higher dollar values of Food Stamps. A greater percentage of the growth-retarded children were low-birth-weight infants. However, neither low birth weight nor any other one factor consistently predicted poor growth achievement. The need for adequate growth standards to correctly diagnose achievement remains a primary need in the field of nutritional assessment.


Earnings, education, genetics, and environment. A major and well-recognized difficulty in estimating the effects of education on earnings is that the more educated are likely to be more able, irrespective of education. If ability also determines earnings and is not controlled, ordinary least squares will yield biased estimates of the education coefficient. In this study, we use data on identical twins to control for differences in ability that arise from genetic endowments and family environment. Not controlling for genetics and family environment may cause a large bias, up to two-thirds of the noncontrolled coefficient.


Oxygen-dependent circulation of sickle erythrocytes. The effects of in vivo hyperoxia and hypoxia on the intravascular survival of 51Cr-labeled human sickle erythrocytes (SS RBS's) were studied after transfusion into rats and guinea pigs. The function of these animals' reticuloendothelial and complement systems had been previously inhibited by ethyl palmitate and cobra venom factor, thus allowing extension of the survival of the heterologous human RBC's. In the blood of rats breathing ambient air the 51Cr half-life survival of RBC's from 11 patients with sickle-cell anemia (mean, 7.1 hours; range, 2.0 to 16.5 hours) was significantly shorter (p less than 0.001) than that of five control subjects (mean, 17.5 hours; range, 12.0 to 26.5 hours). When rats transfused with sickle RBC's were exposed to 100 per cent O2, a mean increment of 16.5 per cent blood 51Cr activity was observed within the first 15 to 60 minutes of hyperoxia. Subsequent oxygen deprivation (7 to 8 per cent O2) resulted in an equally rapid decrease (mean, 35.6 per cent) in blood 51Cr activity. Continuation of hypoxia for up to 17 hours did not cause further acceleration of 51Cr activity. Continuation of hypoxia for up to 17 hours did not cause further acceleration of 51 Cr RBC clearance. Under these conditions the slope of the sickle RBC survival curve was similar to that in animals kept in ambient air. After hypoxic rats were allowed to breate room air again, mean 51Cr blood activity increased by 41.7 per cent. Sickle RBC's transfused to guinea pigs exhibited similar oxygen-dependent survival characteristics. The survival of 51Cr RBC's from four adult control subjects and of unlabeled fetal RBC's from three human cord blood samples was unaffected by oxygen changes. When rats that had been transfused with sickle reticulocytes labeled in vitro with 59Fe were made hypoxic, a decrease in blood 59Fe activity was observed. The extent of this decrease was comparable to that in rats transfused with 51Cr labeled RBC's from the same patients. There was increased liver and spleen 51Cr activity in animals transfused with 51Cr SS RBC's and killed during hypoxia when compared to that of hyperoxic animals. These studies suggest that a minor population of sickle cells is removed from circulation during hypoxia and circulates again upon reoxygenation of the animals. Erythrocyte aging does not appear to be responsible for this phenomenon. The oxygen-depdendent circulation of a population of SS RBC's in this animal system is probably due to reversible sickling and trapping of sickled cells in the microcirculation.


Serum corticosterone in rats with delayed anovulation. The purpose of this study was to investigate adrenal function in rats during the development of persistent oestrus to determine whether a change in blood levels of corticosterone would precede or coincide with the onset of infertility. The syndrome of delayed persistent oestrus and anovulation was induced by administration of a low dose (10 mug) of testosterone propionate (TP) at 5 days of age. Control animals were handled without injection or received the vehicle (sesame oil) only. Half of each group was ovariectomized at weaning and received Silastic implants of either oestradiol benzoate (OB) or cholesterol, 3 mm tube length/100 g body weight. Intact rats given the low TP dose showed precocious vaginal opening (27.3 +/- 2.1 days v. 37.6 +/- 2.4 (S.E.M.) days in unhandled controls) and ovulated within 2 days. Persistent vaginal cornification developed in 22 out of 26 rats by 75 days of age. The TP-treated rats had higher corticosterone values than the controls and did not show a further increase after OB implantation. Cholesterol implantation depressed corticosterone levels in the TP-treated rats. The effects of the low TP dose were not dependent upon gonadal function since they persisted in ovariectomized rats. The results suggest that early exposure to androgen can modify the sensitivity of the adrenal system to oestrogen, and can also lead to persistently high values of corticosterone which are not depressed by ovariectomy. These changes precede the onset of persistent oestrus.


Increased production of genetic mosaics in Habrobracon juglandis by cold shock of newly oviposited eggs. Eggs from an ebony stock exposed to 5-5 degrees C prior to syngamy exhibited increased production of genetic mosaics in comparison with untreated eggs from the same females. No increase in mosaic production occurred for cold-shocked cleavage-stage embryos from the ebony stock or from pre-cleavage cold-shocked eggs from a wild-type stock. Heat shock of pre-syngamy eggs also failed to increase the production of genetic mosaics. These findings are consistent with predictions based on the post-cleavage fertilization theory of mosaic origin in Habrobracon or with a hypothesis of differential mortality.


Morphogenesis of the silkworm egg.

Morphogenesis of the silkworm egg. Ovarian follicle development, which accompanies morphogenesis of the silkworm egg has three distinct phases: spheric, ellipsoidal and flattened-ellipsoid. Transitions between phases are rapid and form-stability (characterized by length/width ratio) is preserved from the beginning of the ellipsoidal phase. The geometric stability of the follicle-oocyte-ovariole system, the polarity of the egg and the determinism in form changes reveal strikingly coordinated spatial and temporal organization.


Comparison of growth in vitro and in vivo of post-implantation rat embryos. Rat embryos explanted at early head-fold stage and grown in vitro by improved culture methods were compared with littermates in vivo. Very similar rates of growth and differentiation were obtained over a period of 48 h, while the embryos developed to around the 25-somite stage.


Effects of testosterone implants in pregnant ewes on their female offspring. Pregnant ewes were implanted with 1 g testosterone between days 30-80, 50-100, 70-120 or 90-140 of gestation. Ewes treated between days 30-80 and 50-100 showed increased aggressive behaviour and clitoral enlargement, whereas this was not seen in the day 70-120 or 90-140 groups. Although the implants released similar amounts of testosterone at all stages of gestation, plasma testosterone concentrations were lower in the day 70-120 and 90-140 groups. The increased testosterone clearance in late gestation could be a result of increased placental aromatization. Masculinization of the genital tubercle was complete in female lambs in the day 30-80 group, partial in the day 50-100 and 70-120 groups, and absent in the day 90-140 group. These results therefore suggest that the 'critical period' for masculinization of the external genitalia is between days 40 and 50 of foetal life. In contrast to this anatomical masculinization, one aspect of behavioural masculinization had a later critical period, since female lambs of 30-80, 50-100 and 70-120 day groups adopted male-like urination postures, whereas the day 90-140 offspring showed the normal female pattern.


Duration discrimination by rats.

Duration discrimination by rats. A psychophysical procedure was used to determine the difference limen for the duration of a signal that ranged from .5 to 8.0 sec. The accuracy of three rats in keeping track of the duration was assumed to be limited by three factors: (a) inattention to the signal on some trials, (b) variability in starting to time the duration when the signal begins (and/or in stopping to time the duration when the signal ends), and (c) factors related to signal duration itself. A generalized Weber model provided a better approximation to the growth in the difference limen as a function of signal than a generalized Counter model.


Vaginal opening and early estrous cycles in rats raised at a low ambient temperature. Rats raised from day 2 of life at an ambient temperature of 10 degrees showed delayed vaginal opening when compared to controls raised at 22 degrees. Cold-raised animals were also lighter in weight at the time of vaginal opening and had significantly longer estrous cycles. Low temperature may cause these effects either by suppressing prolactin secretion or, more probably, by elevating thyroid hormone levels which in turn suppress serum gonadotropin concentrations.


Elevation of brain GABA concentrations with amino-oxyacetic acid; effect on the hyperactivity syndrome produced by increased 5-hydroxytryptamine synthesis in rats. Pretreatment of rats with aminooxyacetic acid (AOAA; 40 mg/kg) raised the concentration of rat brain GABA and inhibited the hyperactivity produced by increasing brain 5-hydroxytryptamine (5-HT) concentration by administration of tranylcypromine and L-tryptophan. The maximum effect was seen 90 min after AOAA injection with smaller effects 30 and 180 min after injection. AOAA did not affect the rate of 5-HT accumulation in the brain, but did inhibit the hyperactivity response which follows injection of the 5-HT agonist 5-methoxy-N,N-dimethyltryptamine, suggesting that post-synaptic 5-HT responses were being inhibited. AOAA also inhibited the locomotor activity which follows administration of tranylcypromine and L-dopa. Blockade of GABA receptors by injection of picrotoxin (2.5 mg/kg) enhanced the dopamine hyperactivity. Since a dopaminergic system has been shown to be involved in the 5-HT hyperactivity syndrome and appears to act post-synaptically to the 5-HT neurones initiating the syndrome it is suggested that inhibition of the 5-HT hyperactivity syndrome may be due to accumulation of GABA distal to the dopaminergic receptors.


Diurnal changes in dopamine-beta-hydroxylase, homovanillic acid and 3-methoxy-4-hydroxyphenylglycol in serum of man. Dopamine-beta-hydroxylase activity (DBH) and concentrations of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured in serum of 11 normal volunteers at 8 a.m., 2 p.m., 8 p.m. and 2 a.m. DBH was higher at 8 a.m. (p less than 0.05) and lower at 2 a.m. (p less than 0.0025). MHPG followed the same course, while HVA was higher at 8 p.m. (p less than 0.05). The mean values for the eleven volunteers were: DBH 65.8+/-25.8 nanomoles hydroxylated tyramine per ml serum and hour (At substrate concentration 0.05 mM), HVA 89.9+/-57.3 ng/ml serum and MHPG 10.9+/-3.4 ng/ml, with variation coefficient of 38.6, 63.8 and 30.1 respectively. The circadian variations found in serum parallel the data from estimations of MHPG and HVA in urine by other investigators. It is suggested that the diurnal variations of DBH, MHPG and HVA reflect the alternating activity of the catecholaminergic neurons in the peripheral as well as the central nervous system.


Management of intracerebral hemorrhage in idiopathic thrombocytopenic purpura. Report of four cases. There has been little comment on the specific management of intracerebral bleeding occurring in patients suffering idiopathic thrombocytopenic purpura. The authors present the cases of four children with intracerebral hemorrhage due to this coagulation disturbance. A plan of management is described based on this experience; it includes immediate control of cerebral edema, emergency splenectomy, supportive care with platelet transfusions and corticosteroids, cerebral angiogtaphy, and a definitive neurosurgical procedure. If necessary, the radiological investigation and surgical therapy can be performed with a single general anesthetic. Three of the patients have survived without major neurological sequelae.


Left heart imaging following inhalation of 15O-cabon dioxide: concise communication. Accelerator-produced C15O2 (t 1/2 = 124 sec) is a uniquely useful radiopharmaceutical because it can be introduced rapidly and selectively into the left side of the heart by the simple noninvasive process of inhalation and breath-holding. A standard scintillation camera system was used to obtain images of the left heart by this technique. The procedure involves minimal radiation dose to the patient and may be repeated within a few minutes if necessary.


Influence of level and type of dietary protein, and of level of feeding on feed utilization by rainbow trout. In the first experiment, diets with 40%, 50% and 60% protein containing herring and soybean meals, and diets with 50% and 60% protein containing herring and soybean protein concentrates were fed at 100%, 85% and 70% of the ad libitum intake. Both increasing the protein level and using the concentrated protein sources resulted in lower gains, and in increases in the amounts of digestible protein and energy consumed for the deposition of protein and energy in the fish carcasses. This difference between the herring and soybean meals, and protein concentrates was not due to differences between their digestibilities. Restriction of feed intake reduced gain, but within these lower levels of gain, the 50% and 60% protein diets have higher final body weights than the 40% protein diet. Restriction of feed intake resulted in carcasses with higher protein and lower fat contents. In the second experiment, two series of diets containing either 40%, 35%, 30% or 25% protein from both herring and soybean meals, or 35%, 30% and 25% protein from herring meal were fed. Reducing the protein level below 40% resulted in lower final body weights, higher feed: gain ratios and carcasses with more protein and less fat. Comparisons of the isonitrogenous pairs of diets showed no differences in final body weights, feed: gain ratios and carcass composition. Reducing the protein level in the diet reduced the amount of protein consumed per unit protein level in the diet reduced the amount of protein consumed per unit protein deposition, but increased the amount of gross energy per unit energy deposited in the carcass.


Hatching of Schistosoma mansoni eggs and observations on motility of miracidia. Eggs of Schistosoma mansoni were obtained from livers of mice and hatching was observed under varying conditions of light and ionic composition of the medium. Hatching occurred equally well in light and in darkness. Eggs also hatched readily in 1- to 50-m OsM solutions of urea, sucrose, sodium chloride, and glycerol, but hatching was inhibited at higher concentrations unless the eggs were left in solutions for long periods of time. Hatching readily occurred in deionized water, but the emerged miracidia did not swim longer than 5 to 10 min unless Na+ was added. Histochemistry of the egg showed DNA-positive egg granules and a polysaccharide-positive vacuole matrix. Acid mucopolysaccharides were stained in the vacuolar matrix and in the anterior sac of the miracidium. Longitudinal alignment of constituents of the egg shell is suggested by the predominance of longitudinal rents in the shell at hatching. A mechanism of hatching involving an osmotic stimulus is proposed.


Arthrostoma miyazakiense (Nagayosi 1955) Comb. n., a parasite of the raccoon-like dog, Nyctereutes procyonoides, with a key to the genus Arthrostoma (Nematoda: Ancylostomatidae). Necator miyazakiensis Nagayosi 1955, a hookworm from the raccoon-like dog, Nyctereutes procyonoides, in Miyazaki Prefecture, Kyushu, Japan, is redescribed and placed in the genus Arthrostoma Cameron 1927, and compared with the 6 other species of the genus. Arthrostoma miyazakiense comb. n. is found in raccoon-like dogs not only in Miyazaki Prefecture, but also in Kyoto and Hokkaido Prefectures. This is the first record of a species of Arthrostoma in Japan.


Transmission of microfilariae and infective larvae of Dipetalonema viteae (Filarioidea) among vector ticks, Ornithodoros tartakowskyi (Argasidae), and loss of microfilariae in coxal fluid. During studies on the acquisition and transmission of infection with the filaria Dipetalonema viteae by Ornithodoros tartakowskyi, it was found that young nymphs and starved medium-sized ticks feed on recently engorged larger ticks. In this manner young ticks acquired the infection with microfilariae, and the microfilariae thus taken developed normally and after 30 days of development were transmitted to a jird. Ticks harboring infective larvae were able to transfer them to other engorged ticks when attempting to feed on them. Although it is not known whether this occurs in nature, it could be a supplementary mechanism in the natural maintenance of this species of filaria. Ticks eliminated microfilariae in the coxal fluid in small numbers during the 1st hr after the infective meal and in increasing numbers with time, reaching a peak between 3 and 5 hr. This mechanism may prevent some ticks from becoming hyperinfected.


The influence of various aminoglycoside preparations on bilirubin/albumin binding. The effect of antibiotics of aminoglycoside structure on the albumin binding of bilirubin has been tested in homozygous (jaundiced) Gunn rats aged 3-5 days. The following drugs were investigated: different preparations of gentamycin, kanamycin, tobramycin and sisomicin. The animals received 50-75% of the LD50 of heterozygous (non-jaundiced) Gunn rats. Mortality, weight gain and changes in the plasma bilirubin concentration were recorded. It was found that the displacement of bilirubin from albumin is caused by the different stabilizers used and not by the antibiotic itself. With the exception of lyophilized preparations of gentamycin for intrathecal application all vials contain different amounts of these preservatives. Special preparations used during the newborn period contain relatively more of these stabilizers. The toxicity of the additives has already a negative influence on the LD50 for heterozygous Gunn rats when the low dosed Refobacin and Sulmicin vials are given. For Refobacin (production 1973/74) the tolerance is reduced by nearly 50%. The toxicity caused by the stabilizer alone is even more marked when given to homozygous (jaundiced) Gunn rats. It becomes evident that benzylalcohol is the substance responsible for the displacement of bilirubin from albumin. The serum concentration of bilirubin decreases for 3-24 hrs depending on the doses given to the animal. This offers the opportunity to measure the competitive displacement of bilirubin easily and exactly. The free unbound, unconjugated bilirubin tends to diffuse into the lipid of the brain with resultant kernicterus. This was shown in histochemical preparations of the cerebellum of young homozygous Gunn rats. Using enzyme reactions for lactic acid dehydrogenase and NADH2-tetrazolium reductase the cytotoxic effect of bilirubin on PURKINJE cells could be demonstrated. The effect of the stabilizers used in the other antibiotic drugs tested can be neglected under clinical conditions. Finally the steepness and duration of the decrease of plasma bilirubin after injection of the dangerous stabilizers was studied in animals of different age (3-5 days; 3-4 weeks). Different results observed can be explained by the more rapid metabolism of benzoates in older animals. However, it remains an open question at what age Gunn rats reflect most precisely the human situation in premature and newborn babies.


Chemical constituents of Gentianaceae XIX: CNS-depressant effects of swertiamarin. CNS activity of swertiamarin, a secoiridoid glucoside from Swertia chirata, was evaluated. An apparent anomaly, associated with the unanticipated finding that the alcoholic extracts (excluding mangiferin) of S. chirata significantly reversed the mangiferin-induced CNS-stimulating effects in albino mice and rats, was resolved. The results indicate that swertiamarin and mangiferin antagonize each other in vivo and thereby reverse their CNS effects.


Chemical constituents of genianaceae XX: natural occurrence of (-)-loliolide in Canscora decussata. Loliolide was isolated as a native compound from Canscora decussata Schult (Gentianaceae). Physical and spectral (UV, IR, PMR, CMR, and mass spectra) properties of the compound and its acetate derivative established its identity. The significance of the cooccurrence of loliolide with a number of carotenoids in C. decussata and the facile transformation of violaxanthin into loliolide and violoxin are discussed in the light of the biogenesis of the degraded carotenoid.


Effect of dopamine-beta-hydroxylase inhibitors on blood pressure and cardiac norepinephrine levels in rats subjected to immobilization stress. Fusaric acid and its derivative, FD-008, are selective and potent dopamine-beta-hydroxyalse inhibitors in vitro and in vivo. Effects of the drugs on blood pressure and norepinephrine levels in the heart of spontaneously hypertensive rats (SHR) and normotensive control rats (NCR) in a state of increased sympathetic nerve activity induced by immobilization stress were investigated. The blood pressure of NCR rose immediately after the onset of stress. In SHR, the blood pressure did not rise after stress in spite of a marked increase in heart rate. Fusaric acid or FD-008 (100 MG/KG P.O.) given 4 hours before stress markedly inhibited the increase in blood pressure by stress in NCR and decreased blood pressure in SHR. The increase in heart rate in SHR during stress was completely inhibited by FD-008 but the increase in NCR was not inhibited. Endogenous norepinephrine levels in the heart were decreased by immobilization stress in NCR and SHR, and further significant decreases in norepinephrine levels were caused by FD-008 in SHR and NCR of fusaric acid in SHR. The effect of FD-008  was greater than that of fusaric acid. The effects of picolinic acid derivatives on blood pressure and norepinephrine levels in the heart were more remarkable in a stressed state than in a resting state. The present data support the hypothesis that the hypotensive action of picolinic acid derivatives may be attributable to the decrease in sympathetic nerve norepinephrine release caused by dopamine-beta-hydroxylase inhibition.


Body temperature in mice: a quantitative measure of alcohol tolerance and physical dependence. Mice undergoing withdrawal after chronic ethanol consumption were found to be hypothermic if kept at room temperature. The extent of the hypothermia correlated well with the behavioral withdrawal symptoms and could be used as a quantitative measure of the severity and time course of the withdrawal syndrome. Placing mice in a cold environment (4 degrees C) exacerbated the hypothermia whereas placing animals at 34 degrees C reversed the hypothermia and produced hyperthermia. It was concluded that the temperature set point mechanism and the ability to regulate around this set point was disturbed in animals physically dependent on alcohol. During consumption of the ethanol-containing diets, mice exhibited tolerance to the hypothermic effects of an acutely administered dose od ethanol. Tolerance to the hypothermic effects of ethanol mirrored the development of behavioral tolerance as measured by performance on a tilting plane. Temperature and behavioral tolerance were both shown to extend well beyond the period of the withdrawal syndrome. Ethanol-treated mice were found to be cross-tolerant to the hypothermic effects of barbiturates but not to the hypothermia produced by the monoamine oxidase inhibitor, pargyline.