Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development.

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.


Interaction of electrically charged lipid monolayers with malate dehydrogenase.

Malate dehydrogenase was adsorbed onto monomolecular lipid films, using a multicompartment trough. The quantity of adsorbed protein and its enzymatic activity were studied with monolayers of various electrical charge densities and subphases of various electrolyte compositions. A closely packed layer of enzyme molecules was adsorbed onto negatively charged films, whereas considerably less protein was adsorbed onto neutral and positively charged monolayers. Electrolytes reduce the quantity of adsorbed protein. The adsorption was found to be irreversible even at high ionic strength. When adsorbed to uncharged lipid films the enzyme is nearly inactive, whereas negatively charged lipid headgroups enhance the specific activity of the enzyme.


Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.


Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.


[Optical properties and molecular structure of nucleic acids and their components. X. Infrared spectra and molecular structure of denatured DNA with different water content and degree of protonation].

IR-spectra (4000-900 cm-minus-1) of heat denatured DNA (d-DNA) of calf thymus have been obtained in the range of relative humidity of 0-93% and of the guota of protoned bases alpha of 0-0,5. fron these spectra the diagram of d-DNA states is plotted as the relationships of intensities and the location of some bands with and relative humidity. At alpha approximately 0,2 a compact structure S2-1 with firm hydrogen bonds between protoned and unprotoned bases which is similar to those observed earlier while protoning homopolynucleotides poly-A and poly-C. At G1'-large values of alpha S2-1-conformation destroyed and a loose structure (alpha approximately 0,35) appears. the latter is characterized by a minimum H-binding of the base and increased hydration. Further increase of alpha brings about the formation of sufficiently compact G2-1-STRUCTURE WHICH IS CHARACTERIZED BY SOLID H-bonds between PO2-groups and protoned bases. A specific effect of the structures found on the state of absorbed water and the structure of hydrate envelope is revealed.


Effect of uncertainty on emotionality and ethanol self-selection in monkeys with cortical ablations.

Alcohol selection and emotionality were examined in rhesus monkeys with frontal cortical ablations which had been subjected to stress via an "uncertainty" paradigm. Uncertainty produced a higher selection of alcohol as compared to nonstress situations. Controls and dorsolateral subjects when exposed to uncertainty selected more of an alcohol solution than either orbital stressed or control-nonstressed animals. Emotionality changes during alcohol-loading and control conditions were measured by recording the number of aggressive or aversive behaviors. Dorsolateral ablations increased the number of aggressive responses while decreasing the number of retreats in the alcohol-loading condition. In the other groups, alcohol loading did not affect the incidence of aggressive or aversive behaviors although they exhibited more retreats than advances across both conditions. Control-nonstressed subjects yielded the lowest number of total aggressive behaviors and the highest number of total retreats.


[Interaction of gangliosides with serotonin].

It has been shown that ganglioside micellae were able to reversible interaction with serotonin; the interaction is determined by their composition. Ganglioside and ganglioside-serotonin micellae were equal in sizes if pH, the ionic strength and the type of the buffer, the temperature and serotonin concentration were given. When the ganglioside micellae were saturated with serotonin the micallae became able to jumping reconstruction forming the structure able to bind more serotonin than the first one. As the serotonin concentration was increased CCM of mixed serotonin-ganglioside micellae was reached. It has been suggested that the reconstruction of the ganglioside micelle due to its interaction with serotonin can be considered as a model of a cooperative transfer of the postsynaptical membrane when a nervous impulse passes through a synapse.


Dominant inheritance of hemophilia A in three generations of women.

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients' plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.


A comparison of the effects of cytosine arabinoside and adenine arabinoside on some aspects of brain growth and development in the rat.

1. Treatment of pregnant rats with cytosine arabinoside (ara-C, 50 mg/kg, i.p.)at 14 days of gestation severely impaired both prenatal and postnatal whole brain growth in their offspring, although the cerebellum was relatively less affected than whole brain. 2. Rats treated at 5 days of age with ara-C (250 mg/kg, i.p.) showed an impairment in growth of the cerebellum relative to the rest of the brain. 3. Adenine arabinoside (ara-A) treatment, either prenatally or postnatally, had negligible effect on brain growth, even at doses considerably higher than those of ara-C. 4. Adult rats, previously treated with ara-C (50 mg/kg, i.p.) at 14 days of gestation, showed an impairment in discrimination learning when tested in a water T-maze. 5. These results are discussed in relation to the proposed use of ara-C or ara-A as antiviral agents, particularly against intrauterine infection with cytomegalovirus.


[Spectrophotometric study of the system DNA-lipid].

The effect of some physical and chemical factors on the system DNA-lipid and its components was studied by spectrophotometry. A change of optical density (D) of watersalt suspensions of lipid total fraction in the range of wavelengths from 200 to 350 nm with the change of ionic strength from 0 to 1 M NaC1 and temperature from 20 to 100 degrees C was investigated. Measurements were carried out under various temperature regemes. In all cases a bell-like irreversible dependence of D on temperature was observed. Its parameters were essentially dependent on the temperature heating of the samples and medium composition. In the presence of DNA the parameters of the curve were changed significantly and depended on the ratio DNA:lipid. An inhibiting effect of DNA on the oxidation of lipids and stabilization of liposomes as well as thermic destruction of DNA stabilized by lipids were observed under certain conditions. The data obtained allowed a conclusion concerning the formation of lipodesoxinucleic complex. It was shown that when a large number of lipid oxidation products was accumulated in the system a chemical modification of DNA with the change of its secondary structure went on.


[Nuclear spin relaxation of water adsorbate protons in biological matrices].

The structure and motility of water molecules adsorbed on collagen and ovalbumen have been investigated by spin-echo method. The differentiation of adsorbate to two subsystems, one of which is the hydrate shell of the macromolecules has been found. The molecular exchange between subsystems takes place with temperature-dependent intensities.


Probabilistic application of plasma carcinoembryonic antigen assay in cancer patients.

Plasma carcinoembryonic antigen (C.E.A.) levels in inpatients proved at necropsy to be cancer free were used to assess the ability of the C.E.A. assay to distinguish benign and malignant disease. The patients had a mean C.E.A. level significantly greater than that for young healthy people. In view of the considerable overlap of the ranges of plasma C.E.A. concentration in cancer patients and patients with non-malignant disease a probabilistic interpretation is advocated rather than the use of a simple cut-off between positive and negative. On the basis of the cancer-free control group, 19 out of 64 untreated patients with various solid tumours had plasma C.E.A. levels considered to correspond to a greater than 95% probability of cancer.


High intestinal lactase concentrations in adult Arbs in Saudi Arabia.

The maximum rise in blood glucose after 50 g lactose by mouth was determined in 40 adult Arabs. Out of 30 Bedouin, urban Saudi, and Yemeni and 9 of mixed ancestry (usually partly African), 25 (83%) and 2 (22%) respectively showed an increase of over 1-1 mmol/1 (20 mg/100 ml). In common with most northern Europeans and Hamitic people of northern Africa, Arabs in Saudi Arabia usually have high intestinal lactase concentrations in adult life. This persistence of high levels probably originated in the Arabian peninsula. Its selective advantage may have been associated with the fluid and calorie content of camels' milk, which is important for survival in desert nomads.


[Hypersomnia by isthmic lesion in cat. Study of the metabolism of cerebral monamines].

(1) A significant increase of 5-HT synthesis is observed in several areas of the cat brain 24 h after the bilateral destruction of the dorsal noradrenergic bundle in the isthmus. This stimulation of the synthesis was simultaneously observed at the level fo serotoninergic cell bodies (anterior part of the raphe system) and of 5-HT terminals (cortex, thalamus, mesencephalon, medulla oblongata). Conversely, a significant decrease of 5-HT synthesis was found in the caudal part of the raphe and in the hypothalamus. The possibility of a catecholaminergic control of 5-HT synthesis by neurons passing through the isthmus is discussed. (2) in the same experimental conditions, an important decrease of endogenous dopamine content without any subsequent change of noradrenaline concentration was observed in the thalamus, the geniculate body and the pons. This important decrease could be due to a greater utilization of dopamine into hypothetical dopaminergic terminals localized in these structures.


The replacement of some of the soybean meal by the first limiting amino acids in practical broiler diets. I. The value of special supplementation of chick diets with methionine and lysine.

1. Three trials were carried out in order to determine the amount by which dietary protein concentration can be reduced while maintaining the dietary concentrations of the first  two limiting amino acids, methionine and lysine. 2. In all trials growth rate and food utilisation declined as the protein concentration of a well-balanced standard chick diet was lowered by replacing soybean meal with sorghum grain (milo), but this trend could be partly or completely prevented when methionine or methionine and lysine levels were restored. 3. Addition of 0-06 to 0-07% methionine, above that considered to be normal, is sufficient to replace approximately 0.9% soybean protein. 4. In diets containing approximately 3 percentage units less soybean protein than required, a supplementation of about 0.15% each of methionine and lysine can replace nearly 2-0% soybean protein, but cannot bring about chick performance equal to that obtained on the control ration, indicating an insufficiency of a third, or third and fourth, limiting amino acid in such low-protein diets.


The replacement of some of the soybean meal by the first limiting amino acids in practical broiler diets. 2. Special additions of methionine and lysine as partial substitutes for protein in finisher diets.

1. Four trials were conducted to determine to what extent dietary protein concentration could be lowered while maintaining the concentrations of methionine and lysine, the first two limiting amino acids. 2. When the protein concentrations of well-balanced standard finisher diets were lowered by replacing soybean meal with sorghum grain (milo), there were progressive decreases in growth rate and increases in food consumption in spite of the substitution raising the energy content of the diets. 3. Maintaining methionine and lysine concentrations partly or completely reversed these trends. 4. Adding 0-12% methionine (above that generally considered to be the normal level in finisher diets) plus 0-20% lysine replace about 3 to 4 percentage units of soybean protein. 5. The recommended level of lysine supplementation may be exaggerated, since preliminary data indicate satisfactory results with a special supplementation of 0-10% each of methionine and lysine.


Carboxylesterases (EC 3.1.1). Amino acid composition of liver carboxylesterases.

The amino acid compositions of the carboxylesterases from chicken, ,orse, ox, sheep, and pig livers are reported and compared. As would be expected for this homologous series, the compositions show a general similarity. However, there are some significant differences, but the degree to which particular pairs of enzymes differ is consistent with the evolutionary history of the species from which they were isolated.


DNA from eukaryotic cells contains unusually long pyrimidine sequences.

Analysis of DNA samples from several different organisms revealed the presence of unexpectedly long pyrimidine tracts (polypyrimidine), ranging in size from about 25 to 200 nucleotides in length. This fraction of the DNA accounted for 0.07-0.8 percent of the total thymidine residues. Pyrimidine clusters of this length and amount would not be expected on a random basis, suggesting they may have been conserved during evolution because they are required for a specialized function(s).


Cardiac lesions in rats fed rapeseed oils.

Fully refined rapeseed oils containing different amounts of erucic acid (1.6%, 4.3% and 22.3%) were fed, at 20% by weight of diet, to weanling male and female Sprague-Dawley rats for periods up to 112 days. Transient myocardial lipidosis characterized by accumulation of fat droplets in myocardial fibers was marked in male and female rats fed oxidized and unoxidized rapeseed oil containing 22.3% erucic acid, moderate with rapeseed oil containing 4.3% erucic acid and very slight in rats fed rapeseed oil containing 1.6% erucic acid. Peak intensity of myocardial lipidosis occurred at three to seven days and regressed thereafter. Focal myocardial necrosis and fibrosis occurred in male rats fed rapeseed oils containing different levels of erucic acid for 112 days. The incidence of myocardial necrosis and fibrosis was markedly lower in female rats, and the incidence of these lesions in either sex was not affected by the state of oxidation of these oils. In a second experiment, male rats were fed diets containing crude, partially refined or fully refined rapeseed oils. There was no correlation between the number of foci of myocardial necrosis and fibrosis and the state of refinement of the oils, but there were generally fewer lesions in rats fed those oils having the lowest levels of erucic acid.


Pathology of spontaneous hemorrhagic enteritis of turkeys.

Thirteen turkeys naturally affected with hemorrhagic enteritis were studied pathologically. The main gross lesions were splenomegaly and hemorrhagic contents in the gut. The main histological lesions were intranuclear inclusion bodies in largemononuclear cells in many visceral organs and in reticular cells around the sheathed arteries of the spleens and varying degrees of lymphocytic hyperplasia in most tissues. The inclusions were frequently present in areas of the lymphocytic hyperplasia. The large mononuclear cells with the inclusions frequently showed a degenerative change.


Epinephrine induced hyperglycemia in bulls and its relationship to polioencephalomalacia.

Data on blood glucose concentration in bulls affected with molasses associated polioencephalomalacia are controversial. It has been suggested that the brain lesions are related to a "hypoglycemic state" during the development of polioencephalomalacia. This paper reports the mobilization of glucose by means of the epinephrine test in three bulls fed two diets, one forage based and the other molasses based. The results showed significantly greater  hyperglycemic responses in the animals during the molasses diet than during the forage one. This probably means that glucose stores (as glycogen) are higher in cattle consuming molasses than those consuming forage. Such hepatic glucose output is in disagreement with the hypoglycemia theory as the cause of the early stages of brain lesions and focuses the probable cause as being related to glucose utilization.


Trichinae in tail musculature of swine.

Pepsin digestion of the tail musculature of ten trichinous swine revealed from zero to 2,384 trichinae. The role of tail chewing in the transmission of trichinosis in swine is briefly discussed.


The limit dextrinases from ungerminated oats (Avena sativa L.) and ungerminated rice (Oryza sativa L.).

The limit dextrinases from ungerminated oats and rice have been purified, and their substrate specificity compared with a bacterial isoamylase preparation. Both cereal enzymes could hydrolyse (1 yields6)-alpha-D-glucosidic linkages in oligosaccharide alpha-dextrins, pullulan, amylopectin, and the beta-limit dextrins of amylopectin and glycogen. However, under comparable conditions, they were unable to attack glycogens.


Carbohydrate-binding specificity of the so-called galactose-specific phytohemagglutinins.

The carbohydrate-binding specificities of various so-called galactose-specific phytohemagglutinins were investigated by means of hemagglutination-inhibition assays. As hapten inhibitors, glycopeptides prepared by pronase-digestion of various glycoproteins (porcine submaxillary mucin, bovine submaxillary mucin, and porcine thyroglobulin), and several glycosides of D-galactose and 2-acetamido-2-deoxy-D-galactose were employed. The results indicate that these galactose-specific phytohemagglutinins may recognize the sugar residue penultimate to D-galactose or 2-acetamido-2-deoxy-D-galactose residues of the sugar chain with which they interact, and that they can be classified into three groups based on the type of sugar sequence which they primarily recognize.


Phosphorus-31 nuclear magnetic resonance spectroscopy of extracellular, yeast O-phosphonohexoglycans.

P nuclear magnetic resonance spectra of a number of purified yeast O-phosphonohexoglycans were recorded. The data therefrom were correlated with established chemicals aspects of individual and collective polymer structures, permitting (a) conclusions to be drawn regarding the chemical environment of the phosphate groups of these polymers, and (b) assignment of anormeric configurations to the hexosyl phosphate residues.


Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.


A study of the kinetics of microsporogenesis in Campelia zanonia (L.) H.B.K.

During maturation, microspores pass through a series of morphologically distinguishable stages or compartments. A study has been made of the systematic fluctuations in the frequency of microspores in these compartments, when plants are grown under rigidly controlled conditions. A new approach to the construction of cumulative flux rate curves is described; these give the number of cells passing the compartment boundaries per unit time. The curves obtained indicate that simple models, which assume constant flux rates and compartment transit times, will not explain the observations. It is evident that not only do microspores mature at different rates, but that the maturation rate of individual microspores varies during the developmental sequence. The overall process may be controlled by the intimate relationship which exists between the microspores and the tapetal periplasmodium in the Tradescantiae.


Mutant chinese hamster cells with a thermosensitive hypoxanthine-guanine phosphoribosyltransferase.

By selecting variants of Chinese hamster cells that were resistant to 6-thioguanine at 39 degrees C, but which would continue to grow in HAT medium at 33 degrees C, we have isolated cell lines with thermosensitive phenotypes. These clones form colonies in HAT medium and incorporate 14-C-hypoxanthine much more efficiently at 33 degrees C than at 39 degrees C. The specific activity of hypoxanthine-guanine phosphoribo-syltransferase is at least 10 times higher in variant cells grown at 33 degrees C than in those grown in 39 degrees C, and the enzymes from the variant clones are inactivated in vitro at 39 degrees C 7-9 times more rapidly than is the enzyme from wild-type cells. The results are consistent with the conclusion that the selected clones have missense mutations in the structural gene for the enzyme.


Temperature-sensitive cell cycle mutants: a chinese hamster cell line with a reversible block in cytokinesis.

A thermosensitive line (TS 111) was isolated from a suspension culture of Chinese hamster fibroblasts, using a BUdR suicide selection technique. In this line, cytokinesis is blocked at 39 degrees C. DNA and protein synthesis are not arrested but keep on at a steady rate. Giant cells are produced which accumulate either numerous nuclei or one big nucleus with several nucleoli and more than a hundred chromosomes. At each nuclear cycle, all the chromosomes in the cell appear to condense in a synchronous manner, although it is possible that not all the sets of chromosomes duplicate. When the culture is returned to the permissive temperature (34 degrees C) after a prolonged arrest at the restrictive temperature, cytokinesis resumes with early extrusion of karyoplasts from multinucleated cells. The division block is independent of cell density in suspension culture and is not prevented by cell contact when cells grow attached to Petri dishes. At 34 degrees C, a residual expression of the mutation is indicated by the presence of binucleate and up to 30% anucleate cells. A remarkable similarity and some synergism exists between the mutation and cytochalasin B effects.


Isolation of heat- and cold-sensitive mutants of chinese hamster lung cells affected in their ability to express the transformed state.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.


The temporal structure of S phase.

DNA synthesis in the S phase of V79 and CHO Chinese hamster cells was examined in detail using an automated system for selection and subculturing of mitotic cells and four different assays for DNA synthesis. Flow microfluorometric (FMF) analysis showed that the selected populations were highly synchronous with few noncycling cells. In CHO cells changes in mean and modal fluorescence in the FMF suggested that DNA content increased in a saltatory fashion with 10-20% of the DNA replicated in early S, 40% in mid S, and 40-50% in late S. Pulse labeling and acid precipitation revealed a repeatable pattern of fluctuations in the rate of isotope incorporation with the maximum rate occurring late in S both V79 and CHO. Autoradiography proved to be the best means of accurately determining the beginning of S phase. Cumulative labeling from mitosis to points in S exaggerated the differences in rate between early and late S, so that significant DNA synthesis in early S might easily be overlooked using this method. In CHO cells DNA-specific fluorescence by the Kissane and Robins assay supported the isotopic incorporation data and the FMF analyses by exhibiting a stepwise increase. In V79 cells, S phase lasts only 5 or 5.5 hr, and consequently the mid S and late S steps in fluorescence are compressed. In V79, greater than 80% of the increase in DNA-specific fluorescence occurred between 4.5 and 7 hr after mitotic selection. In both cell lines, fluorescence in early S phase frequently increased transiently to maximum and then decreased.


The interactions of benzo(a)pyrene with cell membranes: uptake into Chinese hamster ovary (CHO) cells and fluorescence studies with isolated membranes.

The interactions of benzo(a)pyrene (B(a)P) with the cell surface membrane were studied by measuring B(a)P uptake into intact mammalian cells and by determining B(a)P fluorescence in the presence of isolated cell surface membranes. It was found that 0.19 mu-g B(a)P were taken up by 10-6 Chinese hamster ovary (CHO) cells after 30 min exposure to a solution containing 0.59 mu-g/ml. Culture conditions were found to markedly alter B(a)P uptake. Low cell culture densities resulted in a four-fold increase in rate of B(a)P uptake per cell relative to confluent monolayer cultures. The uptake rate of B(a)P was reduced in the presence of bovine serum (BS) and, under some conditions, perylene. This information should be considered in the design of experiments on the biological effects of B(a)P. Another aspect of B(a)P membrane interaction was that the binding of B(a)P to cell surface membranes could be measured by fluorescence. The additional B(a)P fluorescence, found in the presence of cell surface membranes, was sufficiently large that the methods of data treatment used in the study of fluorescent probe-membrane interactions could be applied to get quantitative information on B(a)P-membrane interactions. It was found that 0.6 x 10-8 moles B(a)P were bound per mg membrane protein and that the apparent statistical dissociation constant for the complex was 3.8 x 10-7 M. The data suggest that the mechanism of uptake of B(a)P is probably passive diffusion.


[Cytological mechanism of induced tetraploidy in the newt Pleurodeles waltlii (author's transl)].

When the eggs of the newt Pleurodeles waltlii, at the beginning of the first furrow formation, are submitted for 10 minutes to a sudden increase in temperature (37.2 to 37.3 degrees C), the subsequently developing embryos are tetraploid. The percentage obtained of viable individuals is about 30% of the number of treated eggs. A temperature between 36.2 and 36.4 degrees C applied at the same period and for the same length of time may lead to viable animals diplo-tetraploid mosaics as previously reported.--Cytological examination showed that at the formation of the first furrow the egg contains two interphase nuclei. During heat treatment, cytoplasmic cleavage regresses resulting in an uncleaved egg with two interphase nuclei. About 2 1/2 hours after heat treatment the egg divides again into two cells. Tetraploidy results from doubling of chromosomes of both nuclei instead of the second mitotic division. The mechanism which leads to tetraploidy consists in inhibition of the movement of the two asters and aberration of spindle formation.--The mosaic animals develop from eggs in which only one of the nuclei became tetraploid while the other one divided normally.


Prospects for a pharmacological treatment of phenylketonuria.

The present study deals with a new tentative therapeutic approach of phenylketonuria, based on the use of Anabolic Steroids. Data have been obtained in 10 children, 3 females and 7 males, aged 4-12 years, all presenting the stigmata of true phenylketonuria. The Anabolic Steroids used were Norandrostenolone Phenylpropionate, administered intramuscularly, in increasing doses from 25 to 75 mg.a.d., for 2 years. The 3 older patients were always on a totally free diet, the 7 younger ones on a semi-free diet. The results obtained demonstrate a total normalization of the blood levels of phenylalanine and tyrosine all along the treatment. Moreover, improvements of the psychoneurological disorders were observed.


Heterogeneity of purified human pituitary luteinizing hormone: effect on hormone measurement by radioimmunoassay.

Purified preparations of human luteinizing hormone (hLH), Hartree IRC-2, and NIH LER-960, have been examined after gel filtration of unlabelled and iodinated hormone. Several peaks of radioactivity were observed corresponding to dimeric (peak I), monomeric (peak II), and sub-unit (peak III) lLH forms. The immunologic and receptor activities of each fraction have been evaluated, Receptor activity was found in peaks I and II but not in peak III. Within peak II all fractions were not equally active in the receptor assay, and maximum activity appeared to correspond to molecules with elution characteristics similar to the unlabelled molecule. All fractions of peak II were immunologically active when tested against rabbit anti-hCG in excess. Two immunoassay systems with improved specificity for hLH and hLH-alpha, respectively, have been employed to show the presence of alpha subunit in one of the hLH preparations. These data have relevance with respect to testing of immuno and receptor potencies of hLH preparations.


Continuous red light induces persistent estrus without retinal degeneration in the albino rat.

Female rats were exposed for 12 weeks to red (peak 660 nm) or Cool White continuous light (LL), or to 12 h per day (LD) of Cool White light, all at a flux density of 100 muw/cm2 (approximately 30 incident foot-candles). Either red or white LL produced significantly prolonged vaginal cornification and significantly reduced ovarian weight, as compared to LD exposure. White LL, but not red LL, produced degeneration of retinal photoreceptors.


Isotopic hydrogen exchange in reactions catalysed by cysteine lyase and serine sulphhydrase.

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of  L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.


Response of the ampullae of Lorenzini to static combined electric and thermal stimuli in Scyliorhinus canicula.

The effect of long-lasting electric currents on the Lorenzinian ampullae at constant temperatures between  and 25 degrees C was investigated in the dogfish (Scyliorhinus canicula). Steady state neural impulse patterns in single afferent units were analyzed by plotting interval length histograms and computing mean values and standard deviations for currents between -100 and +100 nA. The mean values depended on temperature and on current strength; the relative standard deviations remained almost constant (ca. 20--30%). Negative currents, inserted at the orifice of the ampullary canal led to higher, and positive currents to lower, steady impulse rates in the whole temperature range investigated here. This static component of electrosensitivity again disappeared at higher currents (of 50 nA and more; electric overstimulation). The maximum static response was two orders of magnitude less than the maximum dynamic component of electroreception. The electrosensitivity depended on temperature: the ampullae were most sensitive to electric currents between 13 and 19 degrees C. The maximal neural activity at 19 degrees C was not shifted to higher or lower temperatures by electric stimulation. A constant equivalent of electric and thermal stimulation throughout the tested temperature and current range could not be found.


The assessment of tobacco smoke toxicity in organ culture. II. Ultrastructural studies on the immediate response of foetal rabbit tracheal epithelium to short-term exposures of whole smoke.

Foetal rabbit tracheal organ cultures were exposed under defined conditions to whole cigarette smoke and fixed immediately for electron microscopy. After an exposure to 4 or 8 puffs, epithelial intercellular spaces were enlarged, the apical portion of many cells bulged out into the lumen and many cilia were swollen. An exposure to 12 puffs produced a breakdown of the epithelium.


Juvenile hormone promotes dominance behavior and ovarian development in social wasps (Polistes annularis).

Repeated topical application of juvenile hormone to workers of the primitive social wasp, Polistes annularis resulted in a disruption of colony social structure as indicated by a sharp increase in the frequency of dominance interactions. Ovarian maturation was also observed, probably as both a direct and an indirect effect of hormone treatment.


[Induced radiation resistance in cell cultures of Chinese hamster cells: induction and manner of resistance (author's transl)].

Mutations-induction followed by selection is a suitable method for developing a cell line with resistance to low doses of X-rays. In comparison with the original cell line, the derived resistant line is characterized by an enhanced variability of the chromosome number, and no higher level of cellular repair, limited to the two repair types described.


[Defect of incorporation of S in old Swan autoimmune mice thymus (author's transl)].

A diminution of the incorporation of radioactive S in epithelial cells of the thymus of auto-immune Swan mice is shown. This defect of incorporation seems to be in relation to a thymus deficiency.


Inhibition of human chlorionic gonadotrophin-induced ovarian and uterine growth in the mouse by synthetic arginine vasotocin-1.

21-day-old Swiss-Webster female mice were injected with 1 IU HCG at 09.00 h. Injection of freshly prepared arginine vasotocin (2 mug/0.1 ml/injection) every 12 h inhibited the HCG-induced hypertrophy of the ovaries at 36, 48, 60 and 72 hr after HCG-treatment while the uterine weight was depressed at 60 and 72 h.


[Electrical activity of mechanoreceptor neurons in rat mammary gland nipple skin].

Three types of the mechanosensitive units were found in the skin of the rat mammary gland nipple: receptors detecting: 1) transients, 2) velocity, 3) position and velocity. The 1st and the 2nd types were spontaneously inactive and both adapted rapidly to mechanical stimuli. The 3rd type had a spontaneous activity 1-4/sec and adapted slowly to mechanical stimuli.


Diurnal variation in the effects of insulin on blood glucose, plasma non-esterified fatty acids and growth hormone.

Insulin 0.05 mu/kg body weight was injected intravenously into 14 subjects both at 8 a.m. and 5 p.m. in random order 12 hrs after a 50 g glucose meal. Fasting glucose levels were similar in both cases but the 48percent plus or minus 10 percent fall in blood glucose in the morning was significantly greater (p smaller than 0.001) than that of 34 percent plus or minus 7 percent in the afternoon. Fasting plasma NEFA, however, varied markedly between 477 plus or minus 150 muEqlL in tth morning and 725 plus or minus 195 muEqlL in the afternoon (p smaller than 0.001) and the fall after insulin injection (64 percent plus or minus 14 percent) was greater in the afternoon than in the morning (47 percent plus or minus 15 percent) (p smaller than 0.001). There was an inverse relationship between proportional glucose disappearance and proportional NEFA disappearance (p smaller than 0.001). The calculated caloric change in plasma, the sum of the falls in glucose and NEFA, were very similar in both morning (2.2 plus or minus 0.5 Cals/1) and afternoon (2.3 plus or minus 0.5 Cals/1), i.e., in spite of the variations of glucose and NEFA metabolism produced by insulin at different times, the nett effect, in terms of energy, was the same. Plasma growth hormone response in the afternoon was found to be enhanced compared with the morning values, although the degree of hypoglycaemia was greater in the morning.


[Testicular feminization with an unusual gonadal tumour (Author's transl)].

A case of a 31-year-old patient showing the typical features of testicular feminization is presented.--Laparotomy was performed, and a large cystic tumour at the place of the right gonad was removed. Microscopically dysgenetic testicular tissue was found. The cyst was lined with cuboid epithelium. Its wall contained smooth muscle fibers.--Probably this cyst was a derivative of the Muellerian ducts.--Today, androgen resistance of the target organs is considered to be the cause of testicular feminization, while the production of hormones of the fetal gonads is normal.--In cases with rudiments of the Muellerian ducts there may be an insufficiency of the oviduct repressor.


The balance of nitrogen and composition of proteins in Pleurotus ostreatus grown on natural substrates.

The balance of nitrogen and nitrogen compounds in Pleurotus ostreatus, cultivated on waste materials, corn straw and maize residues, was investigated. The results show that this organism has a considerable ability to fix atmospheric nitrogen, fixing 312 g of total nitrogen per 100 kg dry weight. When recalculating with respect to a decrease of the substrate during growth of the organism a value of 9.7 mg per 1 g of the decrease in material is obtained. Fruiting bodies, as well as the produced substrate contain 17-19 amino acids. In the produced substrate the content of the protein nitrogen represents only 30% of the total. It can concluded that Pleurotus ostreatus yields a suitable raw material in the food industry and possibly also a fodder based on the basis of solid, cellulose-containing wastes.


Cyclic kinetics and mathematical expression of the primary immune response. Cyclic kinetics of antibody-forming cells following immunization with Salmonella typhi O-antigen.

The kinetics of antibody-forming cells (AFC) in the spleen of rats immunized with Salmonella typhi O-antigen was investigated. The number of nucleated cells of spleen and blood serum antibody titres in passive haemagglutination were determined in parallel. Cyclic changes in the number of antibody-forming cells were detected as three peaks on the 4th, 9th, and 13th days following immunization. The fluctuations of their number were not related to the total number of nucleated cells of spleen. The antibody titres reached their peak on the 10th day following immunization, decreased by the 14th day and rose again on the 16th day after immunization. Repeated increases of the number of AFC were probably due to the regular, not accidental, recruitment of committed precursors cells (memory cells).


Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei.

The immunoglobulin response of rabbits to the flagells (H), somatic (O), and protective antigens of a highly protective strain of Clostridium chauvoei was studied using antisera that had been fractionated by Sephadex G-200 chromatography. The H antigen elicited the characteristic agglutinin response to a protein antigen--early production of 19S globulin followed by persistent 7S globulin production. The O antigen stimulated a transient agglutinin response which was detected in both the 19S and 7S serum fractions. Protective antibody was assayed by passive protection tests in mice. Using these tests the protective activity of the rabbit sera was found to be confined exclusively to the 7S serum fractions. Purified immunoglobulin G, prepared by DEAE-cellulose chromatography of the above sera, was also tested and found to confer considerable passive protection on mice. It is considered that either the protective antigen fails to stimulate an immunoglobulin M response or that immunoglobulin M is relatively ineffective in conferring protection against infection in the mouse passive protection tests.


Relationship of immunotherapy, seasonal pollen exposure and clinical response to serum concentrations of total IgE and ragweed-specific IgE.

The relationship of immunotherapy, seasonal pollen exposure and clinical results to serum total IgE and ragweed-specific IgE was studied in a group of 63 highly ragweed-sensitive individuals. Both immunological parameters rose after small doses of immunotherapy. After larger doses, IgE antibodies fell. Immunotherapy inhibited the seasonal rise in ragweed IgE antibodies, which occurs in nonimmunized patients. This decrease in IgE antibodies and inhibition of seasonal rise was associated with a good clinical response. This immunological pattern and concomitant successful clinical result appeared related to high-dose immunotherapy.


Asthma in the highlands of New Guinea Total IgE levels and incidence of IgE antibodies to house dust mite and Ascaris lumbricoides.

Serum IgE levels in adults from the Highland area of Papua-New Guinea were considerably elevated above those found for Caucasian residents in Australia and most Western countries. The median IgE value for the asthmatic group in New Guinea was significantly (p less than 0.01) elevated above that of control subjects. Identification of IgE antibodies by the radio-allergosorbent test to house dust mite showed an 85-percent correlation with the results of skin reactions to extracts of the mite. 90% of the asthmatic subjects had IgE antibodies to Dermatophagoides pteronyssinus compared with 23% of patients with chronic obstructive lung disease and 6% of controls (p less than 0.0001). No significant difference was found in the incidence of IgE antibodies to Ascaris lumbricoides in asthmatic versus control subjects but the incidence was elevated (p less than 0.005) in patients with chronic obstructive lung disease. Apparently hypersensitivity to Ascaris is not a factor of importance in the aetiology of asthma in New Guinea.


Naloxone-induced jumping in morphine dependent mice: stimulus control and motivation.

When naloxone-precipitated abstinence occurs in morphine-dependent mice, they exhibit stereotyped jumping. The number of jumps has been used as an abstinence measure and the motivation for them described as 'escape'. Studies were designed to assess stimulus control and motivation here. Groups of ten dependent and ten saline mice were singly tested in both light and dark conditions in each of five covered cylinders (2-23 in high). Jumps were recorded automatically for 10 min after 10 mg/kg naloxone. At 2 in, which permitted tactile contact with the cover, no jumps occurred in the light or the dark; at 5-11 in, a high incidence of jumping occurred in both the light and the dark; at 23 in, too high for the animal to reach, relatively few jumps occurred in the light but many occurred in the dark. In another study, similar groups were singly tested in the light after naloxone on each of five circular platforms (18 in high, 3-48 in diameter). As the diameter increased, the number of mice jumping to the floor showed a linear decrease concomitant with an increase in latency. This suggests that jumping is controlled by visual and tactile cues; mice do not jump when escape is prevented by a cover which can be touched or a height which cannot be reached; but do jump when such cues are eliminated by intermediate heights or darkness. Latency varies with size of immediate explorable environment.


Neuroleptic action of clozapine injected into various brain areas in rats.

Bilateral microinjections of clozapine into the corpus striatum of rats antagonize amphetamine-induced stereotypy, and develops catalepsy. These two neuroleptic effects are not seen after subcutaneous or oral administration of clozapine. The effects were a little weaker than those seen after chlorpromazine. No neuroleptic effects were observed, when placebo was injected intrastriatally, nor when clozapine was injected into the hippocampus or the thalamus.


Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.


The response of iliac lymph nodes to the fetal allograft.

It is well known that large pyroninophilic cell proliferation in regional lymphoid tissue preceds and accompanies cell-mediated immunological reactions. In the present study of lymph node responses in the pregnant rat it was found that there were no significant differences between large pyronionphilic cell counts from iliac nodes (which drain the uterus) and popliteal lymph nodes, in either virgin or delivered rats. The iliac large pyronionphilic cell count, however, rose significantly in early pregnancy, fell in late pregnancy and returned to virigin levels after delivery, whereas the popliteal large pyronionphilic cell count showed no significant change during pregnancy. It is also well established that plasma cell proliferation in regional lymphoid tissue precedes and accompanies antibody synthesis. In the present studies the plasma cell count in iliac nodes rose significantly and progressively during pregnancy, while in popliteal nodes it rose significantly only in late pregnancy. There was no significant difference between the plasma cell counts of iliac and popliteal nodes in virgin animals. In delivered animals the iliac plasma cell count was significantly greater than the popliteal count, and both were significantly greater than the corresponding virgin counts. It is concluded that the mother possesses the capacity for rejecting the conceptus, but there is some mechanism which blocks rejection.


Collaborative study of a method for the extraction of light filth from crushed red peppers.

A new method was developed for the extraction of light filth from crushed red peppers. The method utilizes an isoprapanol defatting of the product followed by separation of light filth elements with mineral oil and n-heptane (85+15) in a 2 L trap flask. Collaborative studies resulted in good recoveries of light filth spike elements and clean extration papers. The method has been adopted as official first action.


Collaborative study of a method for the extraction of light filth from whole, cracked, or flaked and ground spices.

An improved method has been developed for the extraction of light filth from whole, cracked, or flaked spices (basil, bay leaves, clery leaves, chervil, chives, dill weed, mint flakes, parsley, rosemary, sage, tarragon, thyme, and vegetable flakes) and from ground spices (cloves, cumin, marjoram, mustard seed, oregano, sage, and thyme). The method involves a chloroform or isopropanol defatting, followed by a direct flotation from 40% isopropanol with Tween 80-EDTA (1+1) and mineral oil-heptane (85+15). Collaborative results show that the proposed method is more rapid to perform and yields better filth recoveries than the official first action methods for ground spices, 44.116, and whole, cracked, or flaked spices, 44.129. The method has been adopted as official first action.


Collaborative study of a method for the extraction of light filth from ground turmeric.

An improved method has been developed for the extraction of light filth from ground turmeric. The method involves an isopropanol defatting followed by a direct flotation from 40% isopropanol with Tween 80-EDTA (1+1) and hot mineral oil. Collaborative results show that the method is rapid and yields better filth recoveries than the official first action method, 44.118. The method has been adopted as official first action to replace 44.118.


Collaborative study of the determination of available lysine in proteins and feeds.

In the proposed method 1-fluoro-2,4-dinitrobenzene (DNFB) is reacted with the free epsilon-amino groups in protein of form DNFB-epsilon-amino lysine which is stable to acid hydrolysis. The sample is acid hydrolyzed and unavailable lysine is determined with an amino acid analyzer; total lysine is determined on the untreated sample. The available lysine, which was bound by DNFB, is determined by difference. The available lysine has been determined in 3 samples of 44% protein soybean meal by 5 collaborators, following the method outlined. The range for available lysine in reference standard 1 was 2.02-2.14%, in reference standard 2, 2.59-2.73% and in reference standard 3, 0.55-0.91%. The method has been adopted as official first action.


A convenient thin layer chromatographic cleanup procedure for screening several mycotoxins in oils.

A thin layer chromatographic cleanup development with benzene-hexane (3+1) effectively removed lipids and some contaminants from mixtures of mycotoxins in corn oil, olive oil, peanut oil, soybean oil, and seed extracts. A second development in the same direction as the first, using toluene-ethyl acetate-formic acid (6+3+1) or benzene-acetic acid (9+1), separated the mycotoxins. Satisfactory separation was achieved for commercial oils spiked with sterigmatocystin, zearalenone, ochratoxins A, B, and C, and aflatoxins B1, B2, G1, and G2. This technique permits detection of 5 ppb aflatoxin B1 in corn.


Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.


Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.

Pyridoxal compounds can either activate or inactivate horse liver alcohol dehydrogenase in differential labeling experiments. Amino groups outside of the active sites were modified with ethyl acetimidate, while the amino groups in the active sites were protected by the formation of the complex with NAD-plus and pyrazole. After removal of the NAD-plus and pyranzole, the partially acetimidylated enzyme was reductively alkylated with pyridoxal and NaBH4, with the incorporation of one pyridoxal group per subunit of the enzyme. The turnover numbers for the reaction of NAD-plus and ethanol increased by 15-fold, and for NADH and acetaldehyde by 32-fold. The Michaelis and inhibition constants increased 80-fold or more. Pyridoxal phosphate and NaBH4 also modified one group per subunit, but the turnover numbers decreased by 10-fold and the kinetic constants were intermediate between those obtained for pyridoxyl alcohol dehydrogenase and the partially acetimidylated enzyme. With native enzyme, the rates of dissociation of the enzyme-coenzyme complexes are rate-limiting in the catalytic reactions. The pyridoxyl enzyme is activated because the rates of dissociation of the enzyme-coenzyme complexes are increased. The rates of binding of coenzyme to phosphopyridoxyl enzyme have decreased due to the introduction of the negatively charged phosphate. The size of the group is not responsible for this decrease since these rates are not greatly decreased by the incorporation of pyridoxal. For both pyrodoxal and phosphopyridoxyl alcohol dehydrogenases, the interconversion of the ternary complex is at least partially rate-limiting. Chymotryptic-tryptic digestion of pryidoxyl enzyme produced a major peptide corresponding to residues 219 to 229, in which Lys 228 had reacted with pyridoxal. The same lysine residue reacted with pyridoxal phosphate.


Inhibition of initiation of protein synthesis in mammalian tissue culture cells by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

Incorporation of amino acids into proteins in HeLa cells, virus-transformed 3T3 mouse fibroblasts, and mouse plasmacytoma cells is inhibited after the addition of L-1-tosylamido-2-phenylethyl chloromethyl ketone, an alkylating agent and chymotrypsin-specific protease inhibitor. Addition of this drug to tissue culture cells at concentrations of 20 to 30 mug per ml results in an irreversible inhibition of the incorporation of amino acids into cellular proteins, and a rapid and complete breakdown of polyribosomes. A comparative study examining the effects of L-1-tosylamido-2-phenylethyl chloromethyl ketone and several known inhibitors of in vivo protein synthesis, with known mechanisms of action, revealed that an optimal concentration of L-1-tosylamido-2-phenylethyl chloromethyl ketone: (a) immediately and selectively inhibits initiation of protein synthesis, (b) does not significantly affect normal elongation rates, and (c) does not promote a premature release of nascent peptides. L-1-Tosylamido-2-phenylethyl chloromenthyl ketone may prove to be a useful tool in investigating the initiatior of protein synthesis in eukaryotic cells.


Lipid specificity of beta-hydroxybutyrate dehydrogenase activation.

Beef heart mitochondrial beta-hydroxybutyrate dehydrogenase forms a catalytically active complex with lecithin and is inactive in the absence of lecithin. The specificity of the activation process was probed by studying the interaction of the enzyme with phospholipids and other compounds. The compounds were tested for their ability to form active complexes with the enzyme, for the stability of the complex formed, and for the correlation between the activator concentration and the level activation. The phospholipids tested were synthetic lecithins varying in the length (C2 to C18) and degree of unsaturation of the aliphatic chains and in the stereochemistry and type of linkage from the aliphatic chain to the glycerol moiety, synthetic and egg yolk lysolecithins, stearylphosphorylcholine, egg yolk phosphatidylethanolamine, egg yolk phosphatidyl-O-serine, and synthetic cardiolipins. Lecithins, lysolecithins, and stearylphosphoryl-choline form active complexes with the enzyme; the L-alpha-diC4:0 is the smallest lecithin forming an active complex and L-alpha-C12:0 is the smallest lysolecithin. Glycerophosphorycholine, mytistoylcholine, N-trimethyl-n-dodecylamine, decamethonium, sodium dodecyl sulfate, Triton X-100, and Lubrol do not activate the enzyme. A hydrophobic chain followed sequentially by a negative and a positive charge, as in stearylphosphorylcholine, is the minimal structural requirement of an activator. However, the stability of the enzyme-activator complex depends strongly on the aggregation state of the activators, complexes of appreciable stability being formed only with those phospholipids which exist in bilayer membrane-like structures. Thus, lecithins with long aliphatic chains (C9 to C18) form active and stable complexes with the enzyme. The maximal activity and the strength of the lipid-protein interactions depend on the nature of the aliphatic chains of the lipids. Lecithins with saturated and unsaturated fatty acid chains activate the enzyme, but the latter form somewhat more stable complexes. The enzyme-activator interactions in the bilayers can be qualitatively understood in terms of competition between lipid-lipid and lipid-protein interactions: the strength of the interaction between the protein and phosphatidylcholines decreases as the crystalline to amorphous phase transition temperature, which is a measure of the strength of lipid-lipid interactions, increases...


Purification, properties, and crystallographic data for a principal nontoxic lectin from seeds of Abrus precatorius.

Nontoxic abrus lectin has been prepared by a new purification procedure. The method is accomplished by 45% saturation ammonium sulfate fractionation from a 5% acetic acid extract of the seeds of Abrus precatorius followed by diethylaminoethyl-Sephadex A-50 and Sepharose 4B affinity chromatography. The abrus lectin appeared homogeneous as judged by electrophoresis, analytical ultracentrifugation, and isoelectric focusing. The lectin molecule has a weight of 126,000 as determined by sedimentation equilibrium. It is composed of four subunits, of which two pairs have either identical or closely similar molecular sizes (33,800 and 32,200 daltons) as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of amino acid analyses are given; none of the cysteic acid appears to arise from cysteine. An electrofocusing experiment indicated the isoelectric point to be 5.0. Crystals large enough for x-ray investigation were obtained by a vapor diffusion technique. X-ray precession photographs revealed that abrus lectin crystallizes in a tetragonal unit cell of symmetry P41212 and dimensions a = 140 and c = 210 A. The asymmetric unit contains 2 protein molecules of molecular weight 126,000 and has a solvent content of approximately 41% by volume.


Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly.

The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoué could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.


Inhibition by colchicine of the mitogenic stimulation of lymphocytes prior to the S phase.

Colchicine, vinblastine, and vincristine inhibit the mitogenic stimulation of lymphocytes by concanavalin A as measured by the incorporation of [3H]thymidine and the appearance of blast cells. The inhibitory effect of colchicine could not be accounted for by diminution in cell viability or by metaphase arrest of mitosis in the stimulated cells. Moreover, the inhibition of [3H]thymidine incorporation was not due to blockage of thymidine transport or inhibition of DNA synthesis inasmuch as addition of colchicine had no effect on cells in the S phase of the cell cycle. The time of inhibition was correlated with the kinetics of cellular commitment to lectin activation and the kinetic data indicated that colchicine blocks stimulation early in the sequence of events following addition of the mitogen. These findings support the hypothesis that cytoplasmic microtubular function plays a role in the commitment of resting cells to undergo mitotic division.


Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle.  Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals.  Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis.  Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations.  Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm.  Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h.  A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection.  This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.


Effects of serum on mammary epithelial proliferation in vitro during mammary gland development.

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.


Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.


Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.


Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts.

Fibroblast growth factor (FGF), a polypeptide that has been shown to stimulate division in 3T3 cells, was tested for mitogenic effects on diploid, early-passage cells from human and murine sources. The quantitative assay of [3H]thymidine incorporation into acid-insoluble material showed that FGF at low concentrations (10 minus 9 M) was more effective than additional serum for provoking the initiation of DNA synthesis in human foreskin fibroblasts or mouse fibroblasts maintained in 5 or 10% serum, respectively. The growth of the human fibroblasts was twice as fast in the presence of FGF plus 10% calf serum as it was in the presence of 10% calf serum or 20% fetal calf serum alone. The addition of FGF to primary cultures of mouse fibroblasts in 0.4% serum resulted in a twofold increase in cell number  compared to controls. In contrast to results obtained with 3T3 cells, neither insulin nor a glucocorticoid potentiated the effects of FGF on either human or mouse cells.


Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.


Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.


Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.


Ultrastructural localization of phosphohydrolases in gametes, zygotes and zoospores of Ulva mutabilis Foyn.

Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization.


Some evidence for replication-transcription coupling in Physarum polycephalum.

Hydroxyurea, at concentrations of 40-60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5-10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.


Studies on the metabolic clearance rate and production rate of human luteinizing hormone and on the initial half-time of its subunits in man.

The metabolic clearance rate (MCR) of human luteinizing hormone (hLH) has been determined in 10 normal men, 3 normal women, and in 12 women with ovulatory disorders resulting in oligomenorrhea or amenorrhea. The MCR was determined by the constant infusion technique using either iodinated or unlabeled highly purified hLH, and these results were compared to MCR determined by using crude pituitary preparations containing both follicle-stimulating hormone and hLH. Both preparations produced essentially similar results for the MCR of hLH and virtually identical results were obtained when complete or incomplete immunoprecipitation of the infused material was achieved. The MCR/body surface area of hLH was significantly greater in normal men (25.6 plus or minus 3.6 ml/min-m-2) than in normal premenopausal (19.2 plus or minus 0.9 ml/min-m-2) or postmenopausal women (17.4 plus or minus 1.9 ml/min-m-2). No difference was noted in the MCR of hLH in women with oligomenorrhea or amenorrhea. Production rates (PRs) were calculated by using a pituitary standard, the values being 85.1 plus or minus 21.5 IU/24 h in normal men, 39.9 plus or minus 12.6 IU/24 h in normal premenopausal women, and 294.6 plus or minus 61.9 IU/24 h in normal postmenopausal women. The initial half-times of disappearance of the alpha- and beta-subunits of hLH were measured in two normal men and found to be 15-18 min, respectively. The half-time of intact hLH was twice as great.


Utilization of Starea, urea, or soybean meal in complete rations for lactating dairy cows.

Three complete rations containing 15.8% of the dry matter as crude protein were compared to a 14.5% crude protein basal ration (dry matter basis) through eight Holstein cows in a replicated 4 times 4 Latin square design. The high protein rations contained supplemental nitrogen totally from soybean meal, urea, or Starea, the latter two providing 16 and 22% of total ration nitrogen, respectively. Concentrations were mixed with wilted alfalfa-bromegrass silage and complete rations fed ad libitum. Data were collected during the last week of each 28 days. Ration had no effect on milk yield, composition of fat or protein, or apparent digestibility of dry matter, crude protein, or acid detergent fiber. Weight gains and dry matter intake were greatest with the soybean meal ration. Intake of the low protein ration was depressed. There were no significant differences between the high protein rations for efficiency of nitrogen utilization.


Transitory changes in plasma progestins, estradiol, and luteinizing hormone approaching ovulation in the bovine.

Progestins, estradiol, and luteinizing hormone were measured by radioimmunoassay in bovine plasma samples collected from indwelling jugular catheters daily on days minus 6 to minus 4, every 6 h on days minus 3 and minus 2, and every 2 h from day minus 1 to ovulation. Least squares analyses characterized time trends within animals and interrelationships between these hormones. Plasma progestins decreased from 5.7 ng/ml at day minus 6 to .07 ng at the peak of luteinizing hormone. No preovulatory peripheral increase in plasma progestins was detected. Estradiol increased from 2 pg/ml at day minus 4 to 6 pg at minus 12 h and then increased abruptly to 7.4 pg at the peak of luteinizing hormone. This matter increase in estradiol was synchronous with the acute preovulatory surge of luteinizing hormone. Association was positive between estradiol and luteinizing hormone, but negative between progestins and estradiol. Estradiol decreased 50% by 5 h following the peak of luteinizing hormone with a return to base (2 pg) at 14 h. An increase of .1 ng luteinizing hormone/ml plasma per day was linear from day minus 6 to 8 h prior to the luteinizing hormone peak. Luteinizing hormone increased to a peak of 13.5 ng/ml and remained elevated for 10.5 h. These results are consistent with the hypothesis that an increase at proestrus in estradiol rather than progestins triggers the preovulatory surge of luteinizing hormone in the bovine.


Histology of induced bovine lactogenesis.

Nine cycling nonlactating dairy cows were treated hormonally to induce lactation with progesterone, estradiol-17beta, and dexamethasone. Mammary tissue biopsies were taken on days 18, 21, and 23 after initiation of treatment (different three cows for each period). First milking was initiated on day 21 in the latter two groups. Mean differences in tissue development (percent area) between day 18 and 23 were, epithelium + 11.1%, stroma -25.9%, alveolar lumen + 13.9%, adipose + 1.58%, duct - 974%, and + 15 nuclei/alveolar section. On day 18 epithelia containing small lipid droplets predominated but were replaced with mixed lipid droplets by day 23. Epithelia day 18 had elongated dark staining nuclei. Epithelia on day 23 had spherically shaped nuclei often containing several nucleoli and an increased cytoplasmic/nuclear ratio. Marked histological development occurred from 18 to 23 days after initiation of treatment. Mammary tissue development in nongravid hormonally treated animals was equal to or greater than 2-wk-prepartum tissue but less than 2-day-prepartum tissue for natural lactations.


Comprehensive evaluation of fatty acids in foods. II. Beef products.

An estensive survey of the literature on lipids of beef covering the past fouteen years revealed that a number of factors, such as sex, age, and diet of animal, and analytical method, can affect the lipid content and fatty acid composition of beef. The feeding regimen of the animal was a factor considered in evaluating data for the food table. Extraction method was important in assessing information on lean tissue lipids. Statistical analysis showed that fatty acid patterns for the separable fat and separable lean of various meat cuts were not significantly different at the 5 per cent level. Cooking by dry or moist heat has little effect on relative fatty acid composition. Methods for converting methyl ester data into per cent composition of fatty acids in the meat cut are presented. A comprehensive table of fatty acids per 100 gm. separable lean, separable fet, and total edible portions of Choice grade cuts of beef is given, as well as a table acids per 100 gm. fat for lean and adipose tissues.


Vitamin B 6 in reheated, held, and freshly cooked turkey breast.

Vitamin B6 content, percentage total moisture, and ether extract of freshly roasted turkey breast muscle; muscle reheated in either an electric or microwave oven; and muscle sliced and held after roasting were evaluated. Reheating times were recorded, and losses were calculated. Reheating time was less (p smaller than 0.01) and total reheating loss greater (P smaller than 0.05) for quarters reheated in the microwave oven. Muscle reheated in the microwave oven had greater volatile (P smaller 0;05) but less drip (P smaller than 0.05) loss than did that reheated in the electric oven. Moisture content was greater (P smaller than 0.05) in freshly roasted muscle. When vitamin B6 was calculated on the basis of cooked weight, no significant differences were observed among treatments. However, when calculation was on a moisture-free and fat-free basis, freshly roasted samples contained the most (P smaller than 0.05) vitamin B6. Although we found significant differences due to treatment variation among birds was greater than among treatments.


A simple and sensitive micro-haemagglutination inhibition assay for the semi-quantitation of the third component of complement.

C3-coated cells have been prepared by incubating sensitised erythrocytes with the appropriate dilutions of serum. Inhibition of agglutination of these cells by an anti-C3 serum provides a sensitive and specific assay for C3 detection and semi-quantitation.


The inhibition of the primary interaction between 125I-labeled human hemoglobin and rabbit anti-human hemoglobin sera: a sensitive radioimmunoassay technique.

A rapid and sensitive radioimmunoassay technique for hemoglobin based upon a modification of the Farr test is described. Since hemoglobin is soluble in ammonium sulfate whereas antibody is precipitated, the amount of free and antibody-bound radiolabeled human hemoglobin can be quantitatively measured after ammonium sulfate precipitation. Nanogram quantities of hemoglobin can be detected by the addition of unlabelled human hemoglobin to the antiserum before the labeled hemoglobin, thus establishing a competition reaction between labeled and unlabeled antigen for antibody. If sufficient quantities of unlabeled human hemoglobin are present, all of the antibody combining sites become saturated and the primary interaction between 125-I-labeled hemoglobin and antibody is completely inhibited. The method can be employed to discriminate various normal human hemoglobins, non-human primate hemoglobins as well as sickle-cell hemoglobin (HbS). Other possible applications of the technique are discussed.


Collagen biosynthesis in liver disease of the alcoholic.

Percutaneous liver biopsies obtained from patients with a history of chronic alcoholism and normal liver, fatty liver, alcoholic hepatitis, or active cirrhosis were incubated with tritiated proline to determine the pattern of collagen biosynthesis in these conditions. Incorporation of labeled proline and hydroxyproline into salt-soluble and insoluble fractions of collagen was evaluated by radiochemical analysis and tissue localization documented by autoradiography. Biopsy specimens of alcoholic hepatitis and cirrhosis exhibit a significant increase in the amount of radioactive proline and hydroxyproline in salt-soluble and insoluble collagen. Marked accumulation of radioactivity occurred over bile ducts, fibroblasts, and collagen fibers in the portal area and over hepatocytes, fibroblasts, and collagen fibers in the centrilobular area. Fatty liver is associated with an increase in uptake of proline and hydroxyproline in the salt-soluble fraction of collagem; silver grains appear in the periphery of fat-laden cells and in areas of focal inflammation. Digestion by collagenase indicates that labeling over fibroblasts and collagen reflects active synthesis, whereas, entry of proline into the cell protein pool is responsible for accumulation of radioactivity in other sites. In vitro ethanol causes a significant increase in the incorporation of proline and hydroxyproline into collagen in biopsy specimens of alcoholic hepatitis or active cirrhosis, but has no effect on collagen synthesis by normal or fatty liver.


Distribution of zinc in skeletal muscle and liver tissue in normal and dietary controlled alcoholic rats.

Zinc deficiency is a concomitant of both alcoholism and cirrhosis, as indicated by plasma and tissue measurements in man. The intracellular sites of zinc distribution, the site-specific nature of alcohol/cirrhosis-related depletion, and the alcohol exposure-zinc depletion time function have not been reported. Spague-Dawley rats (16) at 5 to 6 weeks were given normal chow and 20 per cent ethanol as sole water source. Control animals (14) had tap water. In rats killed at 2, 5, 9, and 14 weeks, zinc levels were measured by atomic absorption spectroscopy in plasma (p); muscle tissue (MT), cell sap (MCS) cell sap-free (MCSF), and mitochondria (MM); liver tissue (LT), cell sap (MCS), cell sap-free fraction (LCSF), And mitochondria (LM). Control zinc levels were stable in all tissues over the 14-week study; p = 108, plus or minus 10 mug per 100 ml.,  MT = 125 plus or minus 18, MCS = 30.3 plus or minus 3, MCSF = 70 plus or minus 6, MM = 209 plus or minus 17, LT = 198 plus or minus 29, LCS = 125 plus or minus 11.0, LCSF = 79.5 plus or minus 11.2, and LM = 291 plus or minus 30 mug per gram of dry tissue. Ethanol-fed rats showed marked decrease in all liver zinc fractions from the earliest (2 weeks) time, with the greatest depletion in LM to 35 per cent of control.  MT and p zinc showed monotonic gradual declines at the rate of 3 per cent per week, becoming statistically different from control at 9 weeks in both tissues. Normal weight gain occurred in control animals: alcohol rats gained 52 per cent of control to 5 weeks, and showed no subsequent gain, weighing 62 per cent of control levels at 14 weeks. Liver mitochondria contain the highest zinc concentration, and are most rapidly depleted. MT and p declines follow hepatic zinc loss.


Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.


Specific tritium labeling of cerebrosides at the 3-positions of erythro-sphingosine and threo-sphingosine.

Cerebrosides containing either threo- or erythro-[3-3-H]sphingosine were synthesized by a new procedure. Glucopyranosyl or galactopyranosyl ceramides were converted to their 3-keto derivatives with 2,3-dichloro-5,6-dicyanobenzoquinone and reduced with 3-H-labeled sodium borohydride. The resulting tritiated cerebrosides, which contained erythro- and threo-sphingosines in the ratio of 84:16, were deacylated with butanol-KOH, and the erythro- and threo-psychosines were separated by silica gel column chromatography and reacylated with lignoceroyl chloride.


Fixed-ratio escape and avoidance-escape from naloxone in morphine-dependent monkeys: effects of naloxone dose and morphine pretreatment.

Lever pressing by rhesus monkeys was maintained by morphine injections during four equally spaced sessions each day. During other periods, lever pressing was maintained by timeout from a continuous naloxone infusion (escape), or by timeout from a stimulus that preceded naloxone injections, or termination of the injections (avoidance-escape). As naloxone dose increased in the escape procedure, response rate increased to a maximum and then decreased. In the avoidance-escape procedure, response rate generally increased as naloxone dose increased, but the changes in rate were small compared to the excape procedure. Substitution of saline for naloxone in the escape procedure led to a very low response rates within three sessions. In the avoidance-escape procedure, rate decrements produced by saline substitution appeared to be related to the behavioral history of the monkey. Previous escape experience led to more rapid decreases in responding when saline was introduced, whereas responding was maintained for 15 sessions in a monkey without prior escape conditioning. Morphine pretreatment produced comparable, dose-dependent decreases in response rates in both procedures. The rate-decreasing effects of morphine were exacerbated when no naloxone was delivered in the escape procedure.


Effects of previous housing conditions on shock-induced aggression.

This study investigated shock-induced aggression as a function of housing rats in single or communal cages for varying periods of time before testing. Rates of fighting were unaffected by housing conditions when rats spent seven or 14 days in either of these settings before testing. Placing rats in these settings for 21 or 28 days before testing, however, did affect rates of fighting. Communal caging of subjects for 28 days before testing was particularly deleterious to shock-induced aggression. How rats are housed outside the experimental chamber is a variable affecting shock-induced aggression and should be considered both in designing future studies and as a topic for future investigation.