Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures.

Antibody against tubulin from the outer doublets of sea urchin sperm flagella reacts with tubulin-containing structures in mammalian cells. Thus cytoplasmic microtubules, vinblastine-induced paracrystals and the full spectrum of mitotic figures can be visualized by immunofluorescence. These results show that the tubulin structure has been highly conserved during evolution.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

Morphogenesis of bacteriophage lambda deletion mutants. II. Escherichia coli mutants which prevent maturation of short genomes.

We have isolated mutants of Escherichia coli which severely reduce the growth of bacteriophage lambda carrying the b221 deletion. Some of the bacterial strains also cause a moderate reduction in the growth of wild-type phage. In the mutant hosts tested, the growth of λb221 is restored by chromosomal alterations producing a non-specific increase in genome length. Thus the defect in growth can be attributed to the physical size of the genome, rather than a genetic effect of the b221 deletion. Our experiments show that the failure to grow results from a block to head morphogenesis and that growth can be restored by mutations in at least two phage head genes. In the accompanying paper we have shown that even in the normal bacterium, the process of packing and cutting the λb221 genome is perturbed as a result of its small size. The block to morphogenesis in the bacterial mutant we have studied most extensively appears to result from an enhancement of the same effect. The experiments described support the hypothesis that there is host participation in the cutting of encapsulated lambda DNA, although it is not yet clear if this involves the direct participation of a host gene product.     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

Separation of hyaluronate, chondroitin sulfate, and heparin by adsorption-desorption technique

An interesting method for separation of the three important mucopolysaccharides, hyaluronate, chondroitin sulfate, and heparin by adsorption to and elution from three inorganic salts Ca₃(PO₄)₂, BaSO₄, and Al₂O₃ has been described in details. Alumina has to be washed with dilute HCl before it can adsorb the mucopolysaccharides, and on treating with alkalies the mucopolysaccharides can be desorbed from it. Calcium phosphate and barium sulfate can adsorb the mucopolysaccharides without any pretreatment. The specific eluents for each of the polysaccharides depend on the nature of the adsorbants also. The recoveries of the mucopolysaccharides are quite satisfactory.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

Differential assay for choline acetyltransferase

A rapid and sensitive radiochemical assay for choline acetyltransferase (EC 2.3.1.6) is reported. The assay allows for the fact that during incubation of [¹⁴C]acetyl-CoA and choline with a cell homogenate, at least one product is formed besides [¹⁴C]acetylcholine, which passes an anion exchange column. In contrast to [¹⁴C]acetylcholine, this major contaminant ([¹⁴C]acetylcarnitine) is not hydrolyzed apparently by Electrophorus acetylcholinesterase. Therefore, two types of assays are performed, the one in the presence of an acetylcholinesterase inhibitor, the other in the presence of acetylcholinesterase from Electrophorus. After passing the reaction mixtures over anion exchange columns, the radioactivities of the effluents are determined. Their difference is proportional to the choline acetyltransferase activity.     

Topography of cyclodextrin inclusion complexes : Part II. The iodine-cyclohexa-amylose tetrahydrate complex; its molecular geometry and cage-type crystal structure

Iodine-cyclohexa-amylose tetrahydrate [(C₆H₁₀O₅)₆ ·I₂·d4H₂O] crystallizes in the orthorhombic space-group P2₁2₁2₁, a  14.240 Å, b  36.014 Å, c  9.558 Å. The structure was solved by heavy-atom techniques and refined by least-squares methods to a conventional discrepancy index R  0.148 for the 2872 observed data. The six d-glucose residues are in the C1 chair conformation; the conformational angles vary in magnitude from 45 to 66°, the angles O(5)-C(5)-C(6)-O(6) are close to · 70°, and the six O(4) atoms are almost coplanar (r.m. s. displacement 0.13 Å). Only four of the six O(2) ?O(3) intramolecular hydrogen bonds have formed, which renders the molecule less symmetrical and more conical-shaped than in the previously determined α-cyclodextrin-potassium acetate complex. The iodine molecule is coaxial with the cyclohexa-amylose molecule. The I-I distance is a conventional 2.677 Å. Close interactions between the iodine atoms and the host molecule comprise carbon atoms C(5) and C(6) and oxygen atoms O(4), with interatomic distances all equal to or greater than van der Waals contacts. Intermolecular, almost-linear, short contacts O ? I-I?O with I?O distances of 3.22 and 3.07 Å indicate attractive interaction.The molecules are arranged in herring-bone “cage-type” fashion, with the four water molecules as space-filling mediators; the structure is held together by an intricate network of hydrogen bonds.     

Mating relationships and breeding suppression in the dwarf mongoose

Dwarf mongooses live in packs containing a dominant breeding pair. The alpha female produces litters at regular intervals, usually three times per year. Other sexually mature females come into oestrus in synchrony with the alpha female and occasionally become pregnant but may not raise their offspring. Some females which had not been visibly pregnant nursed the young of other females. Early in the alpha female's oestrous cycle the alpha male maintains proximity and copulates with her exclusively, attacking any other adult males which approach. Later he also copulates with other adult females and the beta male mates with the alpha female. The alpha pair are likely to be the parents of the great majority of young born in the pack.     

An improved method for the quantitative determination of hexosamines according to Elson and Morgan

An improved method for performing the Elson-Morgan reaction in the microliter range is described, wherein the proceeding hydrolysis of the sample and the heating of the reaction components is improved. This is accomplished with help of a covered rack. The cover exerts pressure on the plugs of the reaction vials preventing them from bursting open during heating or hydrolysis and preventing water from seeping into the reaction mixture during cooling in a water bath. The reaction vials are readily available from most laboratory suppliers. The simultaneous cooling of all reaction vials is accomplished by immersion of the closed rack in ice-cold water. The rack is also applicable in all reactions, where many samples must be heated and/or cooled at the same time.     

5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices : Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.     

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1.Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Opioid-specific recognition sites of the mu- and the delta-type in rat striatum after lesions with kainic acid

The binding of 3H-dihydromorphine (3H-DHM) and of 3H-D-ala2, D-leu5-enkephalin (3H-DADL), which are regarded as relatively selective ligands for mu- or delta-type opioid receptors, respectively, was estimated in total particulate fraction of the striatum of rats in vitro, either in tissue of rats after striatal chemolesions with kainic acid or in control rats (not operated or saline injected into the striatum). Kainate lesions reduced the Bmax values of 3H-DHM by about 78 - 88% depending on the method of calculation, and of 3H-DADL by greater than 90%. Furthermore they lowered the Kd-values, suggesting an increase in affinity. The results are discussed with regard to recent hypotheses on the structure and function of opioid receptors.     

Activation of wheat chloroplast sedoheptulose bisphosphatase: a continuous spectrophotometric assay

A new continuous spectrophotometric assay for sedoheptulose 1,7-bisphosphatase, applied to studies of the activation and steady-state kinetics of the wheat enzyme, is described. The assay enzyme sequence couples the formation of sedoheptulose 7-phosphate to the oxidation of NADH. The recycling of the reaction substrate enables measurements to be made at essentially constant substrate concentrations. Activation of wheat chloroplast sedoheptulose 1,7-bisphosphatase required a reducing agent and could be described by a first-order rate constant. The rate of activation was greatly increased in the presence of Mg²⁺ and sedoheptulose 1,7-bisphosphate. The K_m of the activated enzyme for sedoheptulose 1,7-bisphosphate. and its S_0.5 for Mg²⁺ were found to be 13.3 μm and 1.6 mm respectively. A high recovery method for purifying wheat chloroplast sedoheptulose 1,7-bisphosphatase is also detailed.     

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.     

A modified preparative gel electrophoresis apparatus with special application to the separation of long polypeptide chains

An apparatus for preparative electrophoresis is described which modifies earlier designs substantially. The apparatus is applicable to both continuous and discontinuous buffer systems. Its efficiency is demonstrated for the separation of the three components of the pyruvate dehydrogenase complex. The modifications are discussed with respect to earlier designs.