Immunochemical distinctions among histones and their variants in a solid-phase radioimmunoassay

A solid-phase radioimmunoassay allows detection of small structural differences in histones. In this assay, histones are absorbed to plastic tubes; the coated tubes bind antibody from the IgG fraction of antihistone rabbit antiserum, and the bound rabbit IgG is detected with ¹²⁵I-labeled purified goat anti-rabbit IgG. H2a-anti-H2a and H2b-anti-H2b systems showed no cross-reactivity with each other. H2a variants (H2a.1 and H2a.2) showed some cross-reaction but were distinguished reciprocally by corresponding antisera even though they differ in only two amino acids. This quantitative distinction was detected with a wide range of IgG concentrations, whereas only low concentrations revealed the differences in complement fixation assays. Histones in solution competed with the tube-absorbed histone for binding of the IgG.     

Two methods for the separation of human alpha-fetoprotein and albumin. (A) Affinity chromatography using Blue Sepharose CL-6B and (B) ampholyte displacement chromatography.

Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method.     

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that >80% of those cells' DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,000–200,000 daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3′-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells' soluble cytoplasm. The second polymerase, comprising 1.5–3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfhydryl-blocking reagents and has an apparent molecular weight of 49,000. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.     

Studies on the reaction mechanism of DT diaphorase. Action of dead-end inhibitors and effects of phospholipids.

The relationship between the inhibition of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC.1.6.99.2] by 4-hydroxycoumarin and the 1,3-indandione derivates was investigated. Evidence is presented that these two classes of anticoagulants, although both acting as competitive inhibitors with respect to NAD(P)H, bind to different sites of the enzyme in a synergistic fashion. These findings are interpreted as indicative of a cooperativity between different substrate-binding sites of the enzyme.Neutral phospholipids exert effects on partially purified rat-liver DT diaphorase similar to those earlier obtained with nonionic detergents. The effects concern several kinetic parameters of the enzyme, including V, K_m for electron donor and acceptor, and K_i for various inhibitors. The changes in the kinetic parameters vary in extent and direction according to the individual phospholipids.     

Increased central serotonergic activity associated with nocturnal anorexia induced by Walker 256 carcinoma

The role of brain serotonin levels in Walker 256 tumor induced anorexia was investigated. Total and free plasma tryptophan, regional brain serotonin and 5-hydroxyindoleacetic acid were determined at night, and their relationship to nocturnal anorexia assessed by linear regression analysis. No significant difference in tryptophan, serotonin, or 5-hydroxyindoleacetic acid levels was detected between pair fed and tumor bearing rats exhibiting a 20% reduction of nighttime food intake. Tumor bearing rats with a 40% reduction in food intake had higher nighttime plasma free tryptophan and regional 5-hydroxyindoleacetic acid levels than their pair fed malnourished controls. These results indicate that increased plasma free tryptophan and elevated serotonin metabolism may not be the initial dysfunction responsible for nocturnal anorexia. However, it may contribute to the decreasing nocturnal food intake in severely anorexic tumor rats.     

Separation of substituted pteroyl monoglutamates and pteroyl oligo-gamma-L-glutamates by high pressure liquid chromatography.

Rapid analysis of substituted and unsubstituted pteroyl-oligo-γ-l-glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.     

Interactions between an alpha-helix and a beta-sheet. Energetics of alpha/beta packing in proteins

Conformational energy computations have been carried out to determine the favorable ways of packing a right-handed alpha-helix on a right-twisted antiparallel or parallel beta-sheet. Co-ordinate transformations have been developed to relate the position and orientation of the alpha-helix to the beta-sheet. The packing was investigated for a CH3CO-(L-Ala)16-NHCH3 alpha-helix interacting with five-stranded beta-sheets composed of CH3CO-(L-Val)6-NHCH3 chains. All internal and external variables for both the alpha-helix and the beta-sheet were allowed to change during energy minimization. Four distinct classes of low-energy packing arrangements were found for the alpha-helix interacting with both the parallel and the anti-parallel beta-sheet. The classes differ in the orientation of the axis of the alpha-helix relative to the direction of the strands of the right-twisted beta-sheet. In the class with the most favorable arrangement, the alpha-helix is oriented along the strands of the beta-sheet, as a result of attractive non-bonded side-chain-side-chain interactions along the entire length of the alpha-helix. A class with nearly perpendicular orientation of the helix axis to the strands is also of low energy, because it allows similarly extensive attractive interactions. In the other two classes, the helix is oriented diagonally relative to the strands of the beta-sheet. In one of them, it interacts with the convex surface near the middle of the saddle-shaped twisted beta-sheet. In the other, it is oriented along the concave diagonal of the beta-sheet and, therefore, it interacts only with the corner regions of the sheet, so that this packing is energetically less favorable. The packing arrangements involving an antiparallel and a parallel beta-sheet are generally similar, although the antiparallel beta-sheet has been found to be more flexible. The major features of 163 observed alpha/beta packing arrangements in 37 proteins are accounted for in terms of the computed structural preferences. The energetically most favored packing arrangement is similar to the right-handed beta alpha beta crossover structure that is observed in proteins; thus, the preference for this connectivity arises in large measure from this energetically favorable interaction.     

Solid-phase tuftsin (Thr-Lys-Pro-Arg) synthesis: deprotection and resin cleavage with trifluoromethane sulfonic acid.

A method is described for the synthesis of the naturally occurring tetrapeptide tuftsin (Thr-Lys-Pro-Arg). This stimulates phagocytosis of granulocytes and macrophages. Trifluoromethanesulfonic acid is used to cleave the tetrapeptide from its supporting resin in solid-phase synthesis. This reagent also causes deprotection of several protecting groups in bifunctional residues. The most significant is the complete removal of the tosyl group from N^G-tosyl-arginine-resin ester.     

Fluorescent labeling of fragments of high molecular weight RNA.

We describe a method to fluorescently label microgram quantities of high molecularweight RNA with acriflavine. The method involves hydrolyzing the RNA with HCl at pH 1.0 for 10 min to obtain segments of about 80 nucleotides. The 3′-terminal phosphate is removed from the ribose with alkaline phosphatase, and the terminal ribose is oxidized with periodate to form dialdehydes. Acriflavine is bound to the dialdehyde by the formation of a Schiff's base, and unbound acriflavine is removed by dialysis followed by chromatography on a Sephadex G-25 column eluted with phosphate buffered guanidine-HCl. Human 18 S rRNA bound 0.94 acriflavine molecules per 100 nucleotides and had a fluorescence excitation maximum at 460 nm and an emission maximum at 508 nm. If the hydrolysis step was omitted, this RNA bound only 0.12 acriflavine molecule per 100 nucleotides. Acriflavine-labeled high molecular weight yeast RNA showed a fluorescent intensity which was proportional to RNA concentration to a 1000-fold dilution.     

Fertility on synchronized estrus in crossbred (Bos taurus x Bos indicus) heifers

This study was performed in Western Maharastra (India). The 272 crossbred heifers were randomly allocated within herds to one of the four following groups: 1) silastic coils, 10 days treatment (n = 65); 2) Norgestomet implants, 10 days treatment (n = 71); 3) prostaglandin F(2alpha) two injections 11 days apart (n = 70); and 4) control (n = 66). Almost all heifers in the treated groups were detected in heat during the four days following treatment (88-100%) vs 26% in the control group in the first 21 days. Mean conception rates at first AIs were respectively 62,48 and 60% in groups 1,2 and 4 (p > 0.05) and only 29% in group 3. By 90 days after treatment, 66, 59, 46 and 33 per cent of the females were pregnant for groups 1 to 4, respectively (p < 0.001). In conclusion, progestogen treatments seemed to be highly satisfactory both in terms of conception rates and intervals from treatment to pregnancy.     

Kidney model for study of electromagnetic thawing.

The rationale for using electromagnetic heating techniques, such as in the microwave frequency range (2450 MHz), to thaw large organs after cryopreservation is that this technique, in contrast to numerous others, has been shown to yield functioning dog kidneys which had been frozen to ?20 °C and even to ?80 °C. The development of equipment specifically designed to thaw dog and human kidneys must be based upon knowledge of the fundamental interaction of the biomaterial (volume and shape), the radiation frequency, and the electrical properties (dielectric constant and loss tangent) of the biomaterial. The electrical properties of the organ will be partially determined by the choice of cryoprotectant and its concentration. The electrical properties will change with temperature and with the ratio of liquid to solid water. A model which predicts the influence of these parameters is given. The values of the electrical properties of tissue generally were up to an order of magnitude greater in the thawed state than in the frozen state.     

Modified two-dimensional electrophoresis for identification and autoradiography of ribosomal proteins reacted withp-chloromercuribenzoate

Ribosomal proteins labelled with [¹⁴C]PCMB, a reversibly reacting sulfhydryl reagent, lose most of their radioactivity when separated by the conventional two-dimensional polyacrylamide gel electrophoresis. Therefore, the two-dimensional method was modified to insure stability of the PCMB-protein complex during the procedure. Proteins ofEscherichia coli ribosomes were separated by the modified method, and those which reacted with [¹⁴C]PCMB were identified by counting of stained spots and by autoradiography of dried gels.     

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis.

A-Side (4-R)-(4-²H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d₆ to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final $\text{A}{}_{260}\text{A}{}_{340}$ ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.     

New methods for the purification of vertebrate vitellogenin.

Two procedures are described for the isolation of vitellogenin from plasma. Vitellogenin can be selectively precipitated from the plasma of estrogen-treated animals by Mg²⁺ in the presence of EDTA. The vitellogenin thus obtained is >99% pure and is relatively undegraded. In the second procedure, vitellogenin can also be isolated by DEAE-cellulose chromatography, resulting in preparations that contain <2% albumin. In combination, these two isolation procedures yield vitellogenin preparations of very high purity.     

A rapid sampling method for measuring efflux from erythrocytes.

A method is described for obtaining kinetic data using a water-jacketed Hamilton gas-tight syringe as a reaction vessel and delivering aliquots of the reaction mixture to a quench solution at intervals as small as 2 sec apart by means of a repeating dispenser attachment. This apparatus has been used to measure the rate of [¹⁴C]uridine equilibrium exchange efflux from human erythrocytes at 15°C and at uridine concentrations near the K_m for the transport process. It should be useful for kinetic studies of any reaction having a half time of the order of 4 sec or more, provided a method of rapidly quenching the reaction is available.     

The activity of guanylate cyclase and cyclic GMP phosphodiesterase during synchronous growth of the acellular slime mould Physarum polycephalum.

Guanylate cyclase (EC 4.6.1.2.) and cyclic GMP phosphodiesterase (EC 3.1.4.-.) activity were measured in three subcellular fractions of Physarum polycephalum macroplasmodia isolated at intervals during synchronous growth. In a particulate fraction prepared by high-speed centrifugation guanylate cyclase activity was twice to ten times that of other fractions and highest in mid S and late G2. Two-thirds of the cyclic GMP phosphodiesterase activity was in a soluble fraction but there was no significant change in enzyme activity or distribution during the mitotic cycle.