Medizin - Open Access LMU - Teil 05/22 Archiv

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 pM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 pM (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 pM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 pM and after addition of 35 mM K+ a dissociation constant of 170 pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 pM and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+ for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+, points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate- polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed spectral changes were unaffected by the presence of Ca2+ (100 pM free), the sites interacting with the dye and Ca2+ must be regarded as different.

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+- dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha- toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.

Two modes of assembly of murine cytomegalovirus (MCMV) were observed in cultured mouse embryo fïbroblasts, generating two morphologically different types of viral particles: monocapsid virions and multicapsid virions. The assembly of nucleocapsids appeared to be the same for both types of morphogenesis. Three successive stages of intranuclear capsid formation could be distinguished: capsids with electron-lucent cores, coreless capsids, and capsids with dense cores. Some of the capsids were enveloped at the inner nuclear membrane to form monocapsid virions, which were first detectable in the perinuclear cisterna. Other capsids left the nucleus via nuclear pores and usually entered cytoplasmic capsid aggregates that received an envelope by budding into extended cytoplasmic vacuoles, thereby forming multicapsid virions. Since the formation of multicapsid virions is not restricted to cell culture conditions and also occurs in vivo in immunosuppressed mice, multicapsid virions may play a role in the pathogenesis of cytomegalovirus infection.

This paper presents the first case of extensive, diffuse, somatostatin- immunoreactive D-cell hyperplasia in the human stomach and duodenum. It occurred in a 37-yr-old woman, who showed clinical signs of dwarfism, obesity, dryness of the mouth, and goiter. The density of the distribution of D cells was increased 39-fold in the stomach fundus, 23- fold in the proximal antrum, 25-fold in the distal antrum, and 31-fold in the upper duodenum in comparison with normal values. At the same time, the gastrin-immunoreactive cells were increased 2.3-fold in the antrum. Although the range in size of the D cells was within normal limits in all regions examined, the G cells showed pronounced hypertrophy of up to 127%. A possible relationship between the immuno- histochemical findings and the clinical picture is discussed.

The aim of this study was to establish a morphologic time scheme with which cases of cerebral contusion with unknown survival periods can be dated. Our study of 275 cases was limited to qualitative and quantitative changes in macrophages. The appearance of macrophages and their distribution as well as their content of neutral fat, esterified cholesterol, erythrocytes, siderin, hematoidin, and ceroid were correlated with the survival period. For each cytologic criterium, the observation period, distribution-free limits of tolerance, and relative frequency of identification in different survival periods were determined, and the limits of confidence calculated. The findings permit the dating of trauma in cases with unknown survival periods. Moreover, the probability of this dating was calculated.