Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization

We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehyde-fixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase antigenicity was better preserved in cryostat sections of PLP-fixed materials than in formaldehyde-fixed, paraffin-embedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.     

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.     

Detection of immunoreactive human placental oxytocin and its contractile effect on the uterine muscle

Freshly obtained human placentas from various periods of gestation were quantitatively analysed for their immunoreactive oxytocin (OT) content and its biological activity was examined in a Magnus apparatus by utilizing rat uterus. The mean values for placental immunoreactive OT per gram tissue increased from the first to the second trimester, maintaining its high level to term. The total content of placental OT also increased continually from the beginning of pregnancy to term. Blood levels of estrogen stimulated neurophysin (ESN) and OT were concomitantly enhanced through gestation. Placental extract and synthetic OT showed similar peaks in the elution pattern of ion-exchange chromatography through a carboxymethyl cellulose column. Synthetic OT and placental extract induced marked uterine contraction in diestrous rats. However placental extract previously incubated with OT antiserum failed to induce this effect. Though detection of immunoreactive OT by immunoassay alone does not provide definite identification of pituitary and placental OT, the present study suggests that placental immunoreactive OT could have a contracting effect on the uterine muscle.     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.     

Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.     

Inhibition of Thiamine Pyrophosphate Utilization by Thiamine or Its Monophosphate in Escherichia coli

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.