Electron microscope studies on salivary gland cells

Summary A study of the ultrastructure of the nuclear envelope of the salivary glands cells in the sciarid Bradysia suggests that it probably consists of four membranes, instead of two as found in most cells. The fine structure associated with the pores closely resembles that described in amphibian oocytes by Wischnitzer 1958.British Empire Cancer Campaign.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

Histochemical studies on pseudocysts in adenoid cystic carcinoma of the human salivary gland

Summary Pseudocysts are unique structures found in adenoid cystic carcinomata of human salivary glands. They were studied in 13 such cases by histochemical and immunohistochemical means. The pseudocysts contained an abundance of mucoid materials which reacted strongly with both Alcian Blue and dialysed iron ferrocyanide. The mucoid material was digested with chondroitinase ABC and heparitinase, but was resistant toStreptomyces hyaluronidase. The inner surfaces of the pseudocysts were strongly reactive for laminin, whereas the interface between the tumour cell nests and the outer stromal area was intensely reactive for fibronectin. Numerous fibronectin-reactive fibrils and blood coagulation factor XIII (F-XIII)-positive cells were distributed extensively in the outer stromal area. The F-XIII-positive cells were also found within some pseudocysts. The results obtained in the present study have shown that the pseudocysts represent a peculiar structure consisting of basement membrane components; laminin, fibronectin, heparan sulphate and chondroitin sulphate.     

Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans

Summary The ultrastructural localization of acid phosphatase (AcPase) activity in regressing salivary gland cells of Chironomus tentans was studied with Gomori's lead method. In last instar intermolt larvae AcPase activity is restricted to Golgi vesicles, to small electrondense bodies of about 0.25 diameter, and to larger, more electron-lucid bodies which are considered to be lysosomes. The smaller bodies apparently arise from Golgi vesicles. The average frequency of lysosomes increases as development proceeds. Until the end of the pupal molt, only very few of them contain degenerating fragments of other cellular components.Overt cell regression begins in young pupae. At this stage practically all lysosomes contain degenerating cell components. In addition, cellular breakdown seems to occur outside of these organelles. Regressing cellular areas show in addition free AcPase reaction products (lead deposits), the amount of which closely parallels the degree of regression of the particular area.Possible genetic relationships between the various AcPase-containing cell organelles and the role of lysosomes in the control of gland cell breakdown are discussed.Supported by NSF Grant GB-2639 to U. Clever. The technical assistance of Mr. Hermann Bultmann in part of these studies is gratefully acknowledged.     

Further studies of the nucleolar material in salivary gland nuclei of Sciara coprophila

The nucleolar-like bodies or micronucleoli of Sciara coprophila salivary gland nuclei have been studied with phase contrast, the Nomarski optics, Azure B staining, and electron microscopy. In the late fourth instar the main nucleolus formed at the X-chromosome may become extensively fragmented and may appear as a large aggregate of micronucleoli. At about the same time large numbers of micronucleoli in a more peripheral location are also found. Studies in partially squashed and stained nuclei, as well as in unfixed glands have shown that, at a time when the nucleolar material is abundant, the X-NOR is highly ramified with its branches permeating much of the nuclear space. These observations make it appear probable that most or all of the nucleolar material, even the more peripherally located, is actually in contact with the main nucleolar organizer or its branches. On the other hand, many chromosomal bands are also in close association with micronucleoli. At the level of electron microscopy some of the associations between chromo somal bands and micronucleoli are very intimate with the nucleolar material often found deep within the band. In other instances there seems to be physical continuity between extensions of band chromatin and certain areas of the fibrillar component. The bands in question could be the sites of secondary nucleolar organizers. In the electron microscope a large aggregate of micronucleoli, interspersed with portions of chromatin can often be seen in an approximately central location. This is interpreted as the main nucleolus with portions of its NOR. Both the main nucleolus and the more peripheral micronucleoli are indistinguishable in their fine structure and show the components typically found in nucleoli, i.e., fibrils and granules. On the other hand the fine structure of both RNA and DNA puffs is strikingly different.Supported by funds from Public Service Grants GM 12191 from the National Institute of General Medical Sciences and 5 RO 1 AM 10016-06 from the National Institute of Arthritis and Metabolic Diseases (to Dr. A. M. Garcia).     

Drosophila salivary gland proteins and pupation

Pulse-labeling experiments of salivary glands from the prepupal stages of development showed selectively high rates of synthesis of a set of low molecular weight proteins (6K–12K). These proteins are stably maintained in the salivary glands during prepupal development and are subsequently transported to the pupation fluid (found between the pupal case and the prepupal cuticle) when pupation occurs. These small polypeptides are very basic with the major components having isoelectric points of 8.6–8.7 and the minor components having isoelectric points of 9.1–9.5. This study shows the continuing function of the salivary glands—specifically, the synthesis and secretion of a set of proteins with a putative role in pupation.     

Biochemical studies on muscarinic receptors

Muscarinic receptors have been characterized in smooth muscle and brain by the binding of reversible (e.g. atropine, quinuclidinylbenzylate) or irreversible (benzilylcholine or propylbenzilylcholine mustards) ligands. There is a close correlation between affinity constants derived from binding experiments and the affinities of muscarinic ligands for these sites obtained in pharmacological experiments on smooth muscle. Whereas atropine shows a single high affinity binding component (in subcellular preparations) several other ligands (QNB, ACh, oxotremorine) show multiple affinity binding. This indicated the existence of several types of binding sides which show selectivity toward certain cholinergic effectors. Most detergents inhibit the binding of ligands to the receptor site and therefore cannot be used to solubilize the receptor protein from the membrane. Treatment of brain subcellular membrane preparations with high salt concentrations (2M NaI) solubilize proteins which possess the muscarinic ligand binding properties observed in the membrane preparation. The affinities for muscarinic antagonists however are decreased, which suggests that a conformational change occurs in the protein upon solubilization.