Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.     

Desmocalmin: a calmodulin-binding high molecular weight protein isolated from desmosomes

A unique high molecular weight protein (240,000 mol wt) has been purified from isolated desmosomes of bovine muzzle epidermis, using low-salt extraction at pH 9.5-10.5 and gel-filtration followed by calmodulin-affinity column chromatography. This protein was shown to bind to calmodulin in a Ca2+-dependent manner, so we called it desmocalmin here. Desmocalmin also bound to the reconstituted keratin filaments in vitro in the presence of Mg2+, but not to actin filaments. By use of the antibody raised against the purified desmocalmin, desmocalmin was shown by both immunoelectron and immunofluorescence microscopy to be localized at the desmosomal plaque just beneath the plasma membrane. Judging from its isoelectric point and antigenicity, desmocalmin was clearly distinct from desmoplakins I and II, which were identified in the desmosomal plaque by Mueller and Franke (1983, J. Mol. Biol., 163:647-671). In the low-angle, rotary-shadowing electron microscope, the desmocalmin molecules looked like flexible rods approximately 100-nm long consisting of two polypeptide chains lying side by side. The similar rodlike structures were clearly identified in the freeze-etch replica images of desmosomes. Taken together, these findings indicate that desmocalmin could function as a key protein responsible for the formation of desmosomes in a calmodulin-dependent manner (Trinkaus-Randall, V., and I.K. Gipson, 1984, J. Cell Biol., 98:1565-1571).     

Isolation of cell-to-cell adherens junctions from rat liver

A new isolation procedure for cell-to-cell adherens junctions has been developed using rat liver. From the bile canaliculi-enriched fraction obtained by homogenization of the liver and sucrose gradient centrifugation, the fraction rich in adherens junction was recovered by detergent treatment followed by sucrose gradient centrifugation. Light and electron microscopy revealed that this final fraction was mainly composed of the belt-like adherens junctions with their associated short actin filaments. Biochemical and immunological analyses have shown that vinculin is highly enriched in this fraction. Considering that vinculin is known to be localized in the cell-to-cell adherens junctions, we can conclude that we have succeeded in isolating the cell-to-cell adherens junctions. Furthermore, the constituents of the undercoat (dense layer underlying the membrane) of adherens junctions were selectively extracted from the fraction rich in junctions. Upon SDS electrophoresis of this extract, 10 polypeptides including vinculin, alpha-actinin, and actin were dominant. The results obtained are discussed with special reference to the molecular organization of the undercoats of cell-to-cell adherens junctions.     

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining

In northwest European countries maternal age is increasing. This will lead to an increase of the prevalence of Down syndrome conceptuses. Meanwhile, the increased use of prenatal cytogenetic diagnosis (PCD) will lead to a decrease in the prevalence of Down syndrome among livebirths. We were interested to know what the result of these two opposite developments would be in the near future, and we describe here a model to quantify these processes and the resulting livebirth prevalence of Down syndrome. The model is demonstrated for The Netherlands from 1992 to 2001. The predicted livebirth prevalence for The Netherlands in 1992 is 1.36 per 1000. Demographic factors will cause an increase to 1.76 per 1000 in 2001 with present indications for PCD and a utilization ratio of 50%. An increase of the utilization ratio to 90% in 2001 will lead to a prevalence of 1.22 per 1000, a little less than the present prevalence. Alternative screening programs, including maternal serum screening, could lead to a further decrease of the livebirth prevalence. The model described here can be used for evaluation of the consequences of alternative forms of Down syndrome screening.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)