SUMMARYThe expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.

S U M M A R Y VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans -Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

purified from certain rat and humantissues and characterized (12, 13, 23). The enzyme isolated from these sources has a molecular mass of 31 kDa, shows maximal activity around neutral pH, requires divalent metal cations (Ca2+ and Mg2+) for activation, and hydrolyzes native DNAto produce oligonucleotides with 5'-phosphoryl and 3'-hydroxy termini (13, 23). Therefore, DNase I differs from deoxyribonuclease II (DNase II), which has an optimal pH of approximately 5.0 and does not require cations for enzymaticactivity. The primary structure of human DNase I slightly differs from that of the rat one. As DNaseI is secreted from exocrine glands such as the pancreas and parotid gland, this enzyme was originally considered to have a digestive function. According to a number of previous reports (12, 22, 23, 30, 31), DNase I is present in a wide variety of tissues, including the kidney, lymph node, small intestine, heart, liver and epididymis, and serum and urine contain appreciable amounts of DNase I (7, 14). Although the enzymatic activity of DNase I is inhibited by monomeric actin (3, 15), the function of this enzyme remains obscure.In this study, the expression and distribution of DNaseI in human and rat pituitary cells were examined by the DNase I activity assay, and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization and immunoelectron microscopy. As a result, we could demonstrate the presence of considerable amounts of DNase I in the hormone-secreting cells of the anterior and intermediate lobes of humanand rat pituitary glands. This is, to our knowledge, the first evidence that endocrine cells synthesize and secrete DNase I exocytotically. MATERIALS AND METHODS Tissue preparationHumantissue samples were obtained from 10 male human bodies during postmortemexaminations with the consent of the bereaved families. The pathological patients, aged 37 to 66 years, had died within 2 weeks after having suffered from myocardial infarction (n = 6), angina (1), subarachonoideal hemorrhage (2), or anaphylactic shock (1). Animal samples were taken from young adult Wistar rats, weighing approximately 180 g, which had been killed by decapitation under ether anesthesia.Antibo dies For the humansamples, we used three different polyclonal antibodies which were kindly donated by Drs. K. Kishi and T. Yasuda (Gunma University School of Medicine) and one monoclonal antibody prepared in our laboratory. The characteristics of the polyclonal antibodies used were reported previously (33, 34). The antibodies were as follows: (1) a polyclonal antibody obtained from a rabbit immunized with DNase I pun-* To whomcorrespondence should be addressed.

Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.