Immunohistochemical localization of cytochrome P-450]

Distribution of different forms of cytochrome P-450 (forms C, D and B) in human placenta by immunohistochemical methods on light microscopy level with colloid gold was studied. Positive reaction with cytoplasm of endothelial cells, called the "epithelioid" plates of chorionic villi was found. Trophoblastic and stromal cells were not stained.     

Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase

The depletion of an essential amino acid (aa), tryptophan, caused by interferon-gamma (IFN-gamma)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection. To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-gamma, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt2cAMP). A cDNA library was prepared from poly(A)+ RNA isolated from CMT-93 cells treated with IFN-gamma/Bt2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an Mr of 45,639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-gamma induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt2cAMP, while Bt2cAMP itself had no effect on mRNA induction.     

Cervicovaginal and endometrial cytology in ovarian cancer

The clinical significance of cytologic examination was studied in 114 patients with ovarian cancer who had received preoperative cytologic examinations. The overall positive rate of the cytologic examinations was 26.3% (30 of 114): 22 (19.3%) of the 114 cases had positive cervicovaginal smears while 13 of 31 endometrial aspiration smears (41.9%) were positive. The positive rate was not related to the volume of ascites but rather to its presence or absence. Thus, if ascites was observed, the positive rate was about 2.1 times higher than if it was absent. In two of four cases of ovarian cancer with no endometrial invasion but a positive cytologic examination of ascitic fluid, fallopian tube specimens contained cancer cells; this suggests that ovarian cancer cells may reach the cervix and/or vagina by passing through the fallopian tube, particularly if ascites is present. Since cytologic examination, especially of endometrial aspiration smears, shows a high positive rate if ovarian cancer cells are observed in the abdominal cavity, cytology should be used as an important ancillary method for the assessment of ovarian cancer.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Trypanosoma brucei sspp.: cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.     

Correlation between serum enzymes and serum unsaturated vitamin B12 binding proteins in primary liver carcinoma

The serum unsaturated vitamin B12-binding capacity (UBBC), unsaturated transcobalamin (UTC) I, UTC II, UTC III levels, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities and bilirubin concentration were estimated in 61 patients with liver diseases (31 with hepatoma, 30 with viral hepatitis). The levels of serum cobalamin, UTC I, UTC III, UBBC, alanine and aspartate aminotransferases, and bilirubin were raised in both hepatoma and viral hepatitis patients. Serum UTC II was reduced in both conditions. Alkaline phosphatase activity was significantly increased in hepatoma. Four significant correlations were observed among these parameters in the hepatoma patients while only one significant correlation was observed in viral hepatitis.     

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.     

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF1 by high-resolution 1H-NMR spectroscopy

Escherichia coli translational initiation factor IF1 was studied by 1H-NMR spectroscopy at 400 MHz. IF1 displays a very well resolved spectrum in both aromatic and aliphatic regions. Other spectral characteristics include relatively narrow resonance lines and lack of relevant cross-relaxation phenomena. The resonances of the aromatic residues, in particular of the two His and two Tyr, were assigned by selective chemical modifications and spectroscopic techniques to individual residues in the protein sequence. The relative mobility of various residues of IF1 has been evaluated on the basis of the spin-lattice relaxation times which are rather short and homogeneous. Overall the factor appears to have a complex secondary and tertiary structure and to be a flexible protein whose residues have a high degree of internal mobility.