Nuclear interactions of retinoic acid-binding protein in chemically induced mammary adenocarcinoma.

Cellular retinoic acid-binding protein (CRABP) was detected in the nuclear fraction of N-methyl-N-nitrosourea-induced mammary cancers after the incubation of cytosol containing [3H]retinoic acid (RA)-bound CRABP with isolated nuclei. CRABP extracted from the nuclei in buffer containing 0.4 M-KCl sedimented as a 2 S component when subjected to sucrose-density-gradient analysis. [3H]RA-CRABP was found to be a prerequisite for the detection of nuclear binding, since the incubation of isolated nuclei or 0.4 M-KCl extract of the nuclei with [3H]RA did not result in any significant binding. Incubation of [3H]RA-CRABP at 25 or 30 degrees C before incubation with the nuclei neither altered the sedimentation coefficient nor enhanced the nuclear binding compared with 0 degrees C incubation. The tumour nuclei contained a saturable number of binding sites with a dissociation constant of 1.6 x 10(-9) M. These results indicate that the action of retinoic acid in the target organ may be mediated by its interaction with the nuclei.     

Amino acid transport and interconversions in tissues of freshly caught and food-deprived plaice, Pleuronectes platessa L.

Distribution ratios (intracellular/extracellular concentration) of α-amino isobutyric acid (AIB) and activities of the transaminases of alanine (Ala AT), aspartate (Asp AT), leucine (Leu AT) and glutamate dehydrogenase (GDH) were determined in tissues of freshly caught plaice, Pleuronectes platessa , deprived of food for three months. Liver, red muscle and white muscle demonstrated increased distribution ratios for AIB, while the ratios decreased in kidney and gill with deprivation. Values of the distribution ratio were below 1.0 only in white muscle, with kidney and liver having the highest absolute values. Transaminase activities generally rose with deprivation in all tissues, although the absolute increases were greatest in liver and for the two enzymes Ala AT and GDH. These data indicate that with food deprivation, the role of the liver in amino acid metabolism is maintained and slightly enhanced, while that of the kidney is reduced. Both muscle types (red and white) showed increased water content implicating protein mobilization which could enhance the availability of amino acids for liver gluconeogenesis and metabolite sparing in other tissues during conditions of depletion.     

Preferred conformations and flexibility of aminoacyl side chain of penicillins

Possible conformations of penicillin G; d and l isomers of ampicillin; α-amino-α-methyl-benzyl penicillins and 3- pyridyl methyl penicillin have been studied by an energy minimization procedure using empirical potential functions. The preferred conformations of these antibiotics have been correlated with their biological activity. The conformational requirement of the antibiotic to be active against Gram-positive and Gram-negative (β-lactamase-negative) bacterial strains seems to be the same. The reduced activity of penicillin G against Gram-negative bacteria has been attributed to its lower ability to permeate the outer membrane. The flexibility of the sidechains of these antibiotics is also shown to be important for the desired biological activity.     

Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.     

Progression of Epiphytic Microflora in Wheat and Alfalfa Silages as Observed by Scanning Electron Microscopy

Wheat and alfalfa silages were examined by scanning electron microscopy and standard methods of microbial enumeration. Epiphytic microflora were present at levels of 10⁶ to 10⁸/g in the fresh-cut plants. This flora was initially observed microscopically primarily on the surfaces. After 4 days of fermentation, lactic acid bacteria were observed on the surface in high concentrations near open stomata and throughout the interior mesophyll air sac spaces. At 4 days, populations on interior surfaces were restricted to the exterior surfaces of the air sacs. After 8 days the mesophyllic cells showed marked deterioration, and bacteria were observed on their inner surfaces. At 32 days, the end of the fermentation, vascular bundles and epidermal cells remained intact whereas stomata and mesophyllic cells were collapsed and often contained microorganisms. It is concluded that the interior of the leaves offers substantial nutritional and environmental advantages to epiphytic flora and is an important if not major deterioration site in fermented products. Since little deterioration of exterior surfaces was observed, these sites may play a minor role in supplying nutrients for microbial growth.     

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei

Summary A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.     

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.