A polypeptide conjugate of bilirubin from human bile.

An alkali-stable bilirubin conjugate has been obtained from human T-tube bile as its phenylazo derivative. The conjugate consists of a polypeptide probably of molecular weight 7000 to which the azo pigment of bilirubin is linked covalently through its carboxyl group. It thus constitutes the first biliprotein found in mammals. It is not known whether both carboxyl groups of native bilirubin participate in the binding of the conjugating protein, nor has it been possible to determine the number of pigment moieties occurring on a single polypeptide chain. The isolation makes use of the tendency of the conjugate to form large aggregates and involves the following steps: azo coupling of the native bile, (NH4)2S04 precipitation of macromolecules and aggregates, removal of low molecular weight contaminants by dialysis and gel filtration (first on Sepharose 6B IN 6 M guanidine, then on Sephadex LH-20 in 50% acqueous 2-chloroethanol) and a concluding purification by chromatography on p-aminobenzyl cellulose using a PH gradient. The final preparation appeared to be homogeneous on polyacrylamide electrophoresis.

V(D)J recombination is regulated similarly in RAG-transfected fibroblasts and pre-B cells.

Here we compare the regulation of V(D)J recombination in the fibroblast cell line L4 and the pre-B cell line 38B9. The former has been rendered recombination-competent by stable transfection of a genomic fragment comprising the recombination activating genes, RAG-1 and RAG-2, along with some of their flanking sequences. We show that V(D)J recombination is similarly regulated in these two cell lines. Activating signals are transmitted through the protein kinase A (PKA) pathway, and inhibitory signals through the protein kinase C (PKC) and the calcium signalling pathways. In both cell lines, recombinational activity reflects steady state levels of mRNA transcribed from the RAG-1 and RAG-2 genes. This suggests that transcription of the RAG genes is a major determinant regulating V(D)J recombination. A comparison of RAG-1 and RAG-2 mRNA levels within each cell line reveals almost identical response patterns indicating that RAG-1 and RAG-2 transcription is coordinately regulated. Together, these results imply that the RAG-containing fragment in L4 fibroblasts carries most, if not all, control regions regulating the transcription of the RAG-1 and RAG-2 genes.

High mobility group proteins 1 and 2 bind preferentially to brominated poly(dG-dC).poly(dG-dC) in the Z-DNA conformation but not to other types of Z-DNA.

Three proteins from bull testis, previously thought to be Z-DNA-binding proteins but recently found to recognize brominated poly(dG-dC). poly(dG-dC) by criteria different from the Z-conformation, were partially sequenced. Of these, the 31 kDa protein was identified as a member of the high mobility group 2 protein family, and the 33 kDa protein as a member of the high mobility group 1 protein family. Both proteins had molecular weights approximately 30% higher than expected, indicating considerable posttranslational modification. In contrast, the 58 kDa protein remained unidentified for lack of any significant homology with known protein sequences.

Two-dimensional DNA-fingerprinting in animals.

DNA-fingerprinting has become, during the last five years, an important method of genetic analysis in medicine, veterinary medicine and other disciplines. The power of this technique, especially for genetic linkage analysis, may be enhanced in humans by using the two dimensional DNA-fingerprinting method. Here we show that this procedure can successfully be applied to different animal species, e.g. pig, dog and mouse. Optimal conditions, however, have to be determined for each species tested. With the use of marker systems as well as computer programs it will be possible to evaluate complex two-dimensional spot patterns in a short time and with high reliability.

Enzymology of DNA replication and repair in the brain.

A number of enzymes thought to be involved in DNA replication have been identified in the brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase alpha, a protein that binds Ap4A and might be classified as a DNA polymerase alpha accessory protein, RNase H, DNA polymerase beta, DNA ligase, an endo- and an exonuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil-DNA glycosylase, DNA polymerase beta, DNA polymerase alpha (which in neurons is present only at immature stages), DNA ligase, poly(ADPR) synthase, and O6-alkylguanine-DNA alkyltransferase. In addition, an exonuclease acting on depurinated single-stranded DNA (tentatively listed here as 3'----5' exonuclease), an endonuclease of unknown function as well as ill-defined acid and alkaline deoxyribonucleases also occur in brain.

The DNA content of cerebral cortex neurons. Determinations by cytophotometry and high performance liquid chromatography.

Previous work from our laboratories has indicated that the DNA content of rat cerebral cortex neurons increases postnatally to a level of slightly above 3c, where 2c denotes the diploid DNA complement. We have re-evaluated this concept by using various cytophotometric assays and a novel high performance liquid chromatography (HPLC) technique. The latter consists of digesting the DNA in isolated neuronal nuclei by a mixture of DNA-degrading enzymes followed by analysis of the resulting deoxynucleosides by HPLC. We find that the various methods fall into two groups. The first gives evidence of a postnatal DNA (or histone) increase, while the second does not. The first group (DNA increase) comprises cytofluorometry for DNA following Schiff-type staining with fluorochromes 2,5-bis-(4-aminophenyl)-1,3,4-oxadiazole (BAO) and pararosaniline, ultraviolet absorption scanning for DNA and cytofluorometry for histones following staining with sulfaflavine at pH 8. The second group (no DNA increase) consists of cytofluorometry for DNA following staining with the DNA-complexing agents mithramycin, chromomycin A3, 4',6-diamidino-2-phenylindole (DAPI) and bisbenzimide (Hoechst 33258), as well as the newly developed HPLC technique. Since the HPLC technique measures DNA by a direct chemical approach without interference from other nuclear constituents or from higher order packaging in the chromatin, and detects at least 94-95% of the total DNA contained in neuronal nuclei independent of the developmental stage, we infer that the HPLC technique and, by consequence, the cytochemical assays of the second group reflect true DNA values. Therefore, we propose that cerebral cortex neurons retain a diploid DNA level throughout development.

Deoxyribonucleic acid turnover in immature neurons of the rat cerebral cortex.

To test for metabolic deoxyribonucleic acid (DNA) turnover in differentiating neurons, [methyl-3H]thymidine was injected into the lateral cerebral ventricles of newly born rats, and after 6, 24 and 96 h, neuronal nuclei were prepared from the immature cerebral cortex. Enzymatic treatment converted virtually all of the DNA into soluble deoxynucleosides which were fractionated by high-performance liquid chromatography for determination of specific activity. The specific activity of thymidine was found to decline rapidly with time. The rate of this loss correlated with the radioactivity initially incorporated into the DNA. This suggested that DNA was being replaced by DNA repair as a consequence of radiation damage, rather than by spontaneous metabolic DNA turnover.

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug.

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin.

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).

Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds.

N.m.r., i.r. and optical spectra of model compounds were recorded. These were to help in elucidating the structures of the phenylazo derivatives of bilirubin conjugates isolated from human bile. Model compounds included commercial and human bile bilirubin, mesobilirubin, bilirubin dimethyl ester, dimethoxybilirubin dimethyl ester and the corresponding phenylazo derivatives. The phenylazo derivative of vinylneoxanthobilirubinic acid was also investigated. All compounds were of the type IXalpha, and no other isomer could be detected with the spectroscopic methods employed. The compounds crystallize as the lactams, except for dimethoxybilirubin dimethyl ester and its phenylazo derivative, which are held in the lactim ether configuration. With all other compounds no tautomeric forms other than the lactams could be detected, although small proportions of bilirubin must exist as the lactim. Bilirubin does not form a betaine, a structure that has been proposed by von Dobeneck & Brunner (1965) to explain the bathochromic shift of its optical spectrum as compared with the expected position of the absorption maximum at 420nm. However, this shift to 453nm can be explained on the basis of internal hydrogen bonds occurring between the carboxylic protons and the pyrrole rings of bilirubin, as proposed by Fog & Jellum (1963), and new evidence for such a bonding has been accumulated. The bilirubin sulphate described by Watson (1958), which is formed by treatment of bilirubin with concentrated sulphuric acid and acetic anhydride, was also investigated. The main product of this reaction was isolated as its phenylazo derivative, and was shown to be 3,18-di(ethylidene sulphate)-2,7,13,17-tetramethylbiladiene-ac-8,12-dipropionic acid. The reaction leading to this compound is an addition of sulphuric acid to the vinyl side chains of bilirubin according to Markownikoff's rule.

Bilirubin conjugates of human bile. The excretion of bilirubin as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid, with a branched-chain hexuronic acid as one of the components of the hexuronosylhexuronide.

Structure elucidations have been performed on the bilirubin conjugates isolated from human hepatic bile as the phenylazo derivatives. The major bilirubin conjugates are excreted, not as was formerly thought in the form of glucuronides, but as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid. The isolated aldobiouronides are proposed to have the structures of an acyl 6-O-hexopyranosyluronic acid-hexopyranoside, an acyl 4-O-hexofuranosyluronic acid-d-glucopyranoside, and an acyl 4-O-beta-d-glucofuranosyluronic acid-d-glucopyranoside respectively, with the acyl radicals being those of the phenylazo derivative of bilirubin. The pseudoaldobiouronide is suggested to be the acyl 4-O-alpha-d-glucofuranosyl-beta-d -glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of vinylneoxanthobilirubinic acid. The hexuronosylhexuronide presumably is the acyl 4-O-(3-C-hydroxymethylribofuranosyluronic acid)-beta-d-glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of bilirubin. The 3-C-hydroxymethylriburonic acid, isolated as one of the components of the hexuronosylhexuronide, is the first natural branched-chain hexuronic acid to be detected, and the first branched-chain sugar ever detected in humans.

The chromatin repeat length of brain cortex and cerebellar neurons changes concomitant with terminal differentiation.

Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis. The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages. Administration of [3H]thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually. This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length. In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30. Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes. No changes were detected in liver nuclei during the same period. Non-astrocytic glia cells of the adult cortex had 200 bp repeats.