Dipeptidyl aminopeptidase IV and aminopeptidase P, two proline specific enzymes from the cytoplasm of guinea-pig brain: their role in metabolism of peptides containing consecutive prolines.

In this study the majority of dipeptidyl aminopeptidase IV and aminopeptidase P activities of guinea-pig brain are reported to reside in the cytoplasm. Both activities were purified and soluble dipeptidyl aminopeptidase IV was found to have a relative molecular mass of 194000 and to be comprised of two equal subunits of relative molecular mass 93000 while native soluble aminopeptidase P had a relative molecular mass of 140000. Both activities require proline or alanine in the penultimate position from the N-terminus. Dipeptidyl aminopeptidase IV removed the N-terminal dipeptide whereas aminopeptidase P removed only the N-terminal amino acid. Dipeptidyl aminopeptidase IV was inactive if proline was also present in the third position from the N-terminus whereas aminopeptidase P was unable to remove the N-terminal glycyl, pyroglutamyl or prolyl residues even though proline was present in the second position. Soluble dipeptidyl aminopeptidase IV was differentiated from the previously reported particulate form by its sensitivity to p-chloromercuribenzoate, N-ethyl maleimide and puromycin. The metabolism of Leu-Pro-Pro-Ser by guinea-pig cytoplasm was investigated in the presence of inhibitors to evaluate the contribution by dipeptidyl aminopeptidase IV and aminopeptidase P to the hydrolysis of a peptide containing two consecutive proline residues. The results indicated that either dipeptidyl aminopeptidase IV or prolyl oligopeptidase were required along with aminopeptidase P and prolidase to achieve complete hydrolysis of this tetrapeptide.

Intratubular germ cell neoplasia of the testis: identification by placental alkaline phosphatase immunostaining and argyrophilic nucleolar organizer region quantification.

We assessed the value of placental alkaline phosphatase (PLAP) immunostaining and argyrophilic nucleolar organizer region (AgNOR) quantification as techniques for the identification of intratubular germ cell neoplasia (ITGCN), and compared them with hematoxylin-eosin and periodic acid-Schiff staining. We examined 46 malignant testicular germ cell tumors for the presence of ITGCN; 43 had sufficient tubules available for assessment. We also examined 16 cryptorchid testes, 16 testicular biopsies from 10 subfertile men, and 12 normal adult intrascrotal testes. In tubules adjacent to invasive tumors, hematoxylin-eosin staining identified 30 cases (70%) of ITGCN, while PLAP and AgNOR staining identified 36 cases (84%). All the seminomas (18) and 22 of 28 nonseminomatous germ cell tumors were PLAP-positive and had high AgNOR counts. Intratubular germ cell neoplasia was not identified in the other groups examined; germ cells in these groups were PLAP-negative and had low AgNOR counts. Cells of ITGCN showed cytoplasmic block positivity with periodic acid-Schiff staining but this was not a consistent finding. We conclude that ITGCN is present adjacent to most invasive germ cell tumors, and is reliably identified by hematoxylin-eosin staining when fully developed. Periodic acid-Schiff staining was not helpful as normal spermatogonia were also positive. Staining with PLAP and AgNOR were useful diagnostic adjuncts, but results with PLAP were easier to interpret.

Immunohistochemical staining of bromodeoxyuridine-labelled cells in the mouse embryo.

The aim of this study was to compare two immunohistochemical methods, avidin-biotin peroxidase and immunogold silver staining (IGSS), in the detection of 5-bromodeoxyuridine incorporation in mouse embryo tissues. In addition, two fixation schedules, formal-saline and a mixture of formaldehyde and glutaraldehyde (4FIG), and two embedding procedures, paraffin wax and the acrylic resin L.R. White, were also compared. Pregnant mice were injected with 600 mg per kilogram body weight on days 10 or 15 (plug day = day 1) of gestation and the embryos recovered 2 h after treatment and fixed in formal-saline or 4FIG. Fixed material was then processed into wax blocks or L. R. White resin. After sectioning, the antigen-antibody reaction was visualized using either the avidin-biotin peroxidase or IGSS methods. All methods tested gave a well-contrasted, highly specific reaction, but IGSS in combination with 4FIG and L. R. White gave a clearer and more sensitive signal than other combinations.

Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores.

This study assessed the value of argyrophilic nucleolar organizer region (AgNOR) staining as a potential technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Using a silver staining technique and immunohistochemistry, the authors correlated the numbers of argyrophilic nucleolar organizer regions (AgNORs) and Ki67 scores in 70 breast carcinomas and 27 benign breast lesions. Epithelial cells in fibrocystic disease and fibroadenomas contained a mean of 2.65-6.8 small uniform AgNORs per cell, whereas malignant cells contained 4.6-26.9 frequently highly irregular AgNORs. In benign tissue, Ki67 scores ranged from 0 to 4%; in malignant tumors, Ki67 scores ranged from 3.0 to 98%. The correlation between AgNOR counts and Ki67 scores was highly significant (P less than 0.001). The authors concluded that AgNOR counts performed on routine formalin-fixed paraffin sections furnish significant kinetic information. Furthermore, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic value.

Desmosomal plaque proteins are preserved in all grades of breast cancer. An immunohistochemical study utilizing monoclonal antibodies to desmoplakin.

Using a monoclonal antibody specific for desmoplakin, we evaluated 52 breast carcinomas, 25 normal tissues, 10 benign breast lesions, and 14 nonepithelial tumors. Carcinomas were classified as ductal or lobular, and they were graded histologically according to degree of malignancy and differentiation. Nonepithelial tumors were negative for desmoplakin. All carcinomas stained positively. Desmosomal staining occurred along epithelial cell borders as discrete, punctate granules. Staining intensity was similar in infiltrating carcinomas, in situ carcinomas, benign breast lesions, and normal breast epithelium. Our study demonstrates that most infiltrating breast carcinomas retain abundant desmoplakin, regardless of tumor grade, degree of differentiation, or tumor type (duct or lobular). Although altered desmosomal structure may be important, our findings suggest that loss of desmosomes is not necessary for tumor invasion or metastasis. Desmosomes have a characteristic staining pattern that is easy to interpret, and monoclonal antibodies to desmoplakin may prove useful as markers for distinguishing undifferentiated carcinomas from nonepithelial cancers.