Strong labeling of the cells in the subependymal layer was produced by stereotaxic injection of 5 PCi of 3H-thymidine into the left lateral ventricle of the brain of one and a quarter month old rats weighing about 100 gm.These animals were sacrificed by glutaraldehyde perfusion from two hours to 21 days later. Blocks of corpus callosum with adjacent subependymal and ependyma1 layers were excised from the injected and non-injected sides, and embedded in Epon; 0.5 P thick sections were radioautographed and stained with toluidine blue.In the subependymal region, on both injected and non-injected sides, there was an immediate uptake of label by many cells followed by an increase and later a decrease in the percent cells labeled. In the corpus callosum while at first the percent labeling of glial cells was rather low, it did increase slowly with time and, after seven days, exceeded that in the subependymal region. These results were interpreted as indicating that cells arising in the subependymal layer had migrated into the corpus callosum.Up to four days after injection, most of the label in corpus callosum was present in immature-looking cells resembling the cells of the subependymal layer and referred to as free subependymal cells. With time, the percent labeling decreased in these cells while increasing in some of the glial cells. A labeling peak was observed for light oligodendrocytes at four to seven days and for dark oligodendrocytes at 21 days, whereas labeling of medium shade oligodendrocytes occurred at intermediate times. The succession of labeling peaks indicated a sequence of development from free subependymal cells through light and medium shade to dark oligodendrocytes. Few astrocytes carried label at any time; those which did seemed to have arisen from the transformation of labeled free subependymal cells. Microglia were unlabeled at two hours, but their percent labeling was high at 4-14 days. While the labeling of other glial cells reflected their physiological behavior, the labeling of microglia was a consequence of the trauma produced by the injection of tracer into the ventricle.In conclusion, cells coming from the subependymal layer appear to migrate into the corpus callosum where, in 100 gm rats, many of them transform into oligodendrocytes and a few into astrocytes.

The staining of half-micron thick Epon sections with toluidine blue provides a reliable method for the identification of glial cells. The diagnostic features of these cells observed in the corpus callosum of one-month-old rats are as follows: (1) Astrocytes have a very pale nucleus and cytoplasm; the nuclear envelope is sharply outlined by a thin chromatin lining with occasional chre matin beads; ( 2 ) Microglia have a small nucleus in which large, dark chromatin masses contrast with the nucleoplasm; the nucleus is round or elongated, and may be somewhat angular. The pericytes, which have a similar but usually crescentic nucleus, may be related to microglia. ( 3 ) Oligodendrocytes vary from pale to very dense and may be arbitrarily classified into three subgroups: The light oligodendrocytes are large pale cells with round to ovoid nucleus containing a prominent nucleolus and little or no condensed chromatin; the cytoplasm is extensive and appears pale, although less so than the nucleus. The medium shade oligodendrocytes are smaller cells that have an ovoid nucleus carrying small chromatin clumps and appearing moderately basophilic throughout; the cytoplasm is less extensive and somewhat darker than in the "light" subgroup. The dark oligodendrocytes are usually smaller than medium shade cells and often have an indented or angular nucleus with large chromatin masses; both nucleoplasm and cytoplasm are densely stained; the cytoplasm is often scanty and accumulated on one side of the nucleus.Semithin sections of the cerebral cortex show glia with similar features, with the exception of the astrocytes, which have a smaller but more basophilic cytoplasm than in corpus callosum.The cells of the subependymal layer of the lateral ventricle, when examined in semithin sections in the neighborhood of the corpus callosum, display a patchy, irregular nucleus and a scanty cytoplasm. Similar cells with a more regular nucleus are found outside the layer and in the corpus callosum; they are designated free subependymal cells.In the past metallic stains have been the method of choice for the study of neuroglia in sections. Thus, astrocytes were identified by the gold sublimate method of Ramon y Cajal ('13) and microglia, by the weak silver carbonate method of Del Rio Hortega ('19 In the hope of identifying microglia in the electron microscope, it was reasoned that the silver deposited on these cells by the weak silver carbonate method of Del Rio Hortega ('19) should make them opaque tcl the electron beam and, indeed, they were thus recognized in the corpus callosum; they showed a high contrast between chromatin and nuclear sap as well as a scanty cytoplasm frequently containing dense bodies, furthermore, since pericytes also took up silver, it was suggested that they may be a type of microglia (Mori and Leblclnd, '69a; Baron and Gallego, '72). It was further pointed out by the first named authors that the features of microglia and pericytes may be recognized in semithin, toluidine blue-stained Epon sections.Later, the opaci...

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PC1 and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, proprotein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis. Int.

The corpus callosum of young rats was examined to clarify the behavior of the three subtypes of oligodendrocytes (the large organelle-rich "light oligodendrocytes," the smaller and more densely stained cells referred to as "medium oligodendrocytes," and the even smaller and denser "dark oligodendrocytes"). It was hoped to find out whether cells of the three subtypes undergo division and how they are related to one another. 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to a first group of 19- to 20-day old rats weighing about 40 g, and to a second group of 25-day old rats weighing about 80 g. The animals were sacrificed at various time intervals from 2 hours to 35 days after 3H-thymidine administration. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. Two hours after 3H-thymidine injection, label is virtually absent from light, medium and dark oligodendrocytes, from microglia, and probably from astrocytes, but is present in about 10% of the immature glial cells, which include the poorly differentiated glioblasts and the partially differentiated oligodendroblasts and astroblasts. Hence, the cells undergoing DNA synthesis and mitosis in the corpus callosum are these three types of immature cells. During the week that follow the administration of 3H-thymidine, label appears in oligodendrocytes and astrocytes, which presumably have arisen from the initially labeled immature cells. The oligodendrocytes acquire label in a sequential manner: the light cells show label first and their labeling index reaches a peak at the seven-day interval; the medium oligodendrocytes become labeled next with a labeling peak toward the 14- and 21-day intervals and, finally, the dark oligodendrocytes with a peak around the 28-day interval. Analysis by the method of Zilversmit et al. ('42-'43) provides precise details on the sequence: immature cells presumed to be oligodendroblasts give rise to light oligodendrocytes which, after four to seven days, transform into medium oligodendrocytes which, after another 11 to 18 days, transform into dark oligodendrocytes. The dark cells may persist indefinitely or turn over at a very slow rate. It is concluded that oligodendrocytes arise from the last division of oligodendroblasts and develop in three main periods: a light stage lasting less than a week, a medium stage lasting about two weeks, and a very long lasting dark stage.

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PC1 and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, proprotein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis. Int.