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Biomedical subjects

B Wittig

Publications and source records attributed to B Wittig.

107 records · Page 6Linked to original sources
PubMed1982

A new acrosin inhibitor from boar spermatozoa.

A new proteinase inhibitor (Mr 7500) was isolated to apparent homogeneity from boar spermatozoa by repeated gel filtration on Sephadex G-50 and affinity chromatography on concanavalin-A--Sepharose 4B. The inhibitor strongly inhibits boar acrosin in a competitive 1:1 stoichiometric reaction with Kass = 7 x 10(10)1 mol-1. The inhibitor is a glycoprotein and represents a first member of a new class of proteinase inhibitor with a rather short polypeptide backbone of only 42 amino acid residues and a low cystine content. The basic protein (isoelectric point 9.4) contains a single disulfide loop, which is easily reducible by sodium borohydride. Upon reduction the inhibitory activity is lost, but rapidly regained after air reoxidation of the corresponding half-cystine residues. The reactive site residue was established to be arginine by inhibition with 2,3-butanedione. The inhibitor is rather specific for acrosin and inhibits bovine trypsin only to a limited extent. However, incubation with catalytic amounts of trypsin (or acrosin) at acid pH (pH2-3) rapidly leads to a limited proteolysis at the reactive site with formation of 67% modified (reactive site hydrolysed), but still active inhibitor. The equilibrium constant was established to be Khyd = 2.0.

Acrosin↗
PubMed1980

R-loop hybridization of collagen DNA: separation in Cs2SO4 gradients.

High molecular weight poly(A)RNA (26-35 S) from chicken embryo trunks which is enriched for collagen mRNA was iodinated and hybridized to DNA under conditions of R-loop formation. The R-loops were separated in Cs2SO4 gradients from the bulk of DNA yielding double stranded DNA enriched for collagen genes.

Animals↗
PubMed1979

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences.

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.

Animals↗
PubMed1979

A phase relationship associates tRNA structural gene sequences with nucleosome cores.

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.

Animals↗
PubMed1978

Purification of class A, B, and C DNA-dependent RNA polymerases from chicken embryos.

Crude nuclei were isolated from trunks of 13-day-old chicken embryos under conditions which prevent leakage of RNA polymerases from nuclei. RNA polymerases were solubilized by subsequent incubation in alkaline buffer and sonication at high salt concentration. Purification of RNA polymerases A, B, and C was achieved by conventional column chromatographic procedures. RNA polymerase B was freed from an UTP:polynucleotidyl exotransferase by chromatography on a tRNA-Sepharose column. Purified RNA polymerase A contained six putative subunits with molecular weights 190 000 (A1), 117 000 (A2), 57 000 (A3), 50 000 (A4), 25 000 (A5), 19 000 (A6); RNA polymerase B contained eight putative subunits with molecular weights 98 000 (B2'), 86 000 (B2''), 155 000 (B3), 44 000 (B4), 31 000 (B5), 28 000 (B6), 26 000 (B7), 19 000 (B8); RNA polymerase C contained nine putative subunits with molecular weights 170 000 (C1), 117 000 (C2), 84 000 (C3), 60 000 (C4), 49 000 (C5), 36 000 (C6), 33 000 (C7), 22 000 (C8), 19 000 (C9).

Amanitins↗
PubMed1978

Reverse transcription of tRNA.

The 3' terminus of tRNA was enzymatically elongated by an oligo(A) tail. A fragment of DNA polymerase I (E. coli) was used in the presence of manganese to phase and synthesize a cleavable primer at the oligo(A)-tRNA template. When the threedimensional structure of oligo(A)-tRNA is being unfolded under conditions where the primer is still hybridized at the oligo(A) tail, the DNA polymerase I fragment transcribes oligo(A)-tRNA into DNA. Reverse transcription is slowed down and its fidelity suspended by the 1-methyladenine in oligo(A)-tRNAPhe(yeast). The reaction is stopped by the highly modified Y-base present in this template. Approximately full length transcripts can be obtained from oligo(A)-tRNA3Gly(E.coli). The transcription products were characterized by sequence analysis.

Adenine Nucleotides↗
PubMed1977

Nucleosome mono, di, tri-, and tetramers from chicken embryo chromatin.

The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0-1 M KC1 that decreases with increasing repeat number. Less histone H1 is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same Tm of 78 degrees C has been determined for the KC1-soluble nucleosomes and for the KC1-insoluble monomer. The Tm of the KC1-insoluble oligomers is 79.8 degrees C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4 degrees C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KC1) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the sedimentation and molecular weight data of KC1-soluble nucleosome mono- and tetramers.

Animals↗
PubMed1977

Comparative characterization of four purified lysine-specific transfer ribonucleic acids from chicken embryos.

Four purified tRNALys species from 13-day-old chick embryo muscle have been characterized with respect to the following properties: qualitative oligoribonucleotide composition (polyacrylamide gel electrophoresis after RNase T1 digestion), anticodon response towards AAG and AAA (equilibrium dialysis and polylysine synthesis), strength of the aminoacyl bond (de-esterification kinetics), sedimentation coefficient, and temperature-dependent double helix-to-coil transition. The results confirm the existence of four molecularly independent lysine-specific tRNA's in this eukaryotic system.

Animals↗
PubMedDate not supplied

Intratumoral adoptive immunotherapy with tumor infiltrating lymphocytes (TIL) in a melanoma patient leading to regression of local tumor mass. A case report.

We describe a 52-year old man with a confined massive subcutaneous and intramuscular tumor metastasis on his back. He was treated with tumor-infiltrating lymphocytes (TIL) which were gained from the tumor of his back 3 months previously. Between 2.5 x 10(7) and 2 x 10(8) TIL were administered, five times at 2-week-intervals on day 2 in combination with a continuous subcutaneous infusion of interleukin(IL)-2 (1.8 x 10(6) IE/24 hours) over 3 days and a single intratumoral injection of a low dose of interferon(IFN)-alpha (1 x 10(6) IE) on day 1. There were no severe side effects. Immunotherapy led to a consistent regression of the tumor mass. After 3 months, treatment was discontinued due to the development of intracranial metastases. In parallel, due to an erythematous induration at the infusion site, it became increasingly difficult to infuse IL-2 subcutaneously. Additionally, high titers of anti-IL-2-antibodies were detected in serum. Following cessation of the immunotherapy and brain irradiation, the patient's clinical course has remained stable over the past 5 months, suggesting that small amounts of TIL with a low-dosed IL-2 may be helpful in the treatment of local tumor masses.

Drug Administration Schedule↗