Studies on human uterine cervix and rat uterus using S-, X- and Q-band electron-spin-resonance spectroscopy.

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.     

Gas-liquid chromatography for the determination of acetylcholinesterase activity

Method has been modified and used for quantitative gas chromatographic determination of microtraces in a large volume of test mixture under study. The method can be applied for the differentiated determination of genuine and false blood cholinesterase by final products of specific substrate hydrolysis.     

Effect of flap modifications on human FEN1 cleavage.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.     

Plasmacytoma of the breast. Unusual initial presentation of myeloma: report of two cases and review of the literature

Two patients who presented initially with breast masses proven to be plasmacytomas, are described. In both cases the breast masses led to the diagnosis of multiple myeloma, which was retrospectively already present at the time of the breast biopsy. One patient with IgG lambda-type myeloma developed plasma cell leukemia and amyloidosis and failed to respond to radiochemotherapy. The second patient was in complete remission, four years after therapy, but has subsequently relapsed and died. All cases of breast plasmacytoma, described in the medical literatures are reviewed, and guidelines for future therapy are suggested.     

Measurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli. Studies on the allosteric binding site of acetyl-coenzyme A

Binding of the feedback inhibitor acetyl-coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin-labelled acetyl-CoA analogue 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-CoA-thioester. The spin-labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl-CoA, while CoA has no effect. AMP and fructose 1,6-bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin-labelled acetyl-CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl-CoA. The binding site for these activators seems to overlap with the acetyl-CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8-anilino-1-naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component. The spin-labelled acetyl-CoA analogue binds also to the product binding site of acetyl-CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl-CoA as well as by CoA.     

H-2-linked genetic control of the plaque-forming cell response to sheep red blood cells

The role of genes linked to the H-2 locus in effecting an immune response to SRBC was examined using strains of mice which differ in the classes of antibodies produced following multiple injections with SRBC. While H-2-linked gene action appeared to be at the level of regulating the number of plaque-forming cells (PFC) present in the spleens of different strains following two injections with SRBC, non-H-2-linked immune response genes seemed to determine whether an IgM-IgG switch occurred as well as how much of each antibody class was produced by the number of PFC available as a result of H-2-linked gene intervention. Mapping studies showed that the H-2-linked genetic effects were due to either the requirement for two genes or the presence of genes located between I-B and H-2D.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. I. Hypertrophy and change of orientation of the Golgi apparatus.

The ultrastructure of cytolytic T lymphocytes adhered to the surface of target cells was investigated at different periods after start of interaction. Fifteen-minute incubation led to increase of number of Golgi apparatus cisternae and vacuoles. After 30 min incubation Golgi apparatus become oriented to the contact area. If several lymphocytes adhered to one target cell the Golgi apparatus of each of them was oriented toward the contact area. If one lymphocyte adhered simultaneously to two target cells its Golgi apparatus was oriented toward both target cells. Giant Golgi apparatus vacuoles were formed 30 to 60 min later and then moved to plasma membrane of lymphocyte and then the content of those vacuoles moved to the intercellular space between a cytolytic T lymphocyte and a target cell. The period required for the hypertrophy and change of orientation of Golgi apparatus is supposed to represent the “mobilization” step of a medium-sized and small killer lymphocyte.