Human cells arrest in S phase in response to adenovirus 12 E1A

In the present investigation, nuclei of endodermal cells, primary and secondary mesenchyme cells (PMCs and SMCs), and small micromere descendants (SMDs) of the sea urchin Lytechinus variegatus were counted and mapped at five developmental stages, ranging from primary invagination to pluteus larva. The archenteron and its derivatives were measured three dimensionally with STERECON analytical software. For the first time SMC production is included in the kinetic analysis of archenteron formation. While the archenteron lumen doubled in length during secondary invagination, the number of archenteron cells increased by at least 38% (over 50% when SMCs that emigrated from the tip of the archenteron were included). The volume of the archenteron epithelial wall plus the volume of 17 new SMCs increased by 40% over the equivalent volumes at the end of primary invagination. Because secondary invagination involves the addition of archenteron cells and an increase in volume of the archenteron epithelium, we conclude that secondary invagination is not accomplished simply by the rearrangement and reshaping of the primary archenteron cells. Both archenteron cell number and wall volume continued to increase at the same rates from the end of secondary invagination until the 27-h prism stage, although the lumen lengthened more slowly. SMCs were also produced at a constant rate from primary invagination until the prism stage. Because the production of both endodermal and mesodermal cells continues until the late prism stage, we conclude that gastrulation (defined as the establishment of the germ layers) also extends into the late prism stage.     

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.     

Effects of cyproterone acetate on FSH and testosterone influenced spermatogenesis, steroidogenesis and epididymis in the Indian wall lizard, Hemidactylus flaviviridis (Ruppell)

The effects of FSH, testosterone and either cyproterone acetate (CPA) alone or in combination with FSH or testosterone on testis and epididymis in male lizards were studied histologically and histochemically during recrudescent phase to find out whether the onset of spermatogenesis is androgen dependent or FSH dependent. The testes of control lizards consisted of mainly spermatogonia, a few primary spermatocytes and secondary spermatocytes rarely. The interstitial or Leydig cells were atrophied. FSH treatment induced spermatogenic activity substantially as indicated by increase in number of primary and secondary spermatocytes and transformation of secondary spermatocytes into spermatids and into spermatozoa in a few cases. Besides, steroidogenic activity was also remarkably stimulated as evidences by considerable depletion of sudanophilic lipid and an increase in Delta5-3beta-hydroxysteroid dehydrogenase enzyme activity in Leydig cells. However, testosterone treatment resulted in the inhibition of spermatogenesis. A significant inhibition of spermatogenesis was noted in lizards treated either with CPA alone or in combination with FSH. The inhibitory effect of CPA on spermatogenesis was increased when it was given in combination with testosterone. The results indicate that onset of spermatogenic activity is dependent on FSH (or FSH-like protein), but not on the androgen. The ductus epididymidis in control lizards was regressed with low cuboidal epithelium. The lumen of the tubules was totally devoid of secretory material and spermatozoa. FSH treatment induced a marked hypertrophy in epididymidis. The lumen became filled with secretory material mixed with spermatozoa. The hypertrophy in epididymidis was also recorded after the treatment with testosterone, but the degree of induction was not to that extent as noted in FSH treated ones. However, CPA injected either with FSH or with testosterone resulted in the profound atrophy in epididymidis.     

Role of the brachial somites in the development of the appendicular musculature in rat embryos

DiI, a fluorescent lipophilic dye, was micro-injected into the brachial somites of 10.5 day rat embryos to determine whether these somites can contribute cells to the development of the fore-limb bud. The injected embryos were cultured and harvested at the 20-25-somite stage. The dye did not interfere with somitogenesis because, at the injection site, the DiI-labelled somites were able to differentiate into dermomyotome and sclerotome. We have analyzed cryo-sections of 20-21-somite stage embryos and were unable detect the presence of DiI-labelled cells in the fore-limb buds. However, at the 22-somite stage, a few DiI-positive cells were found in the proximal region of the limb bud. These labelled cells had migrated into the limb from the lateral border of the dermomyotome. From the 23-somite stage onwards, there were even more DiI-positive cells inside the limb. We have performed an additional set of experiments to confirm that the somitic cells do have the ability to invade and colonize the limb bud. This was achieved by first labelling newly formed somites isolated from the caudal region of 10.5 day embryos with DiI and then grafting them into corresponding regions in 8-11-somite stage hosts. The donor somites were not orientated when they were implanted into the host. However, this did not disrupt their ability to undergo normal somitogenesis. We have detected the presence of DiI-positive cells in the limb buds of approximately 71% of the 19-30-somite stage embryos that have been examined. This is similar to what we obtained for the injected embryos. Nevertheless, there is one slight difference and that is the stage the somitic cells begin their invasion of the limb. For the injected embryos, migration began at the 22-somite stage but in the transplanted embryos, it commenced as early as the 18-somite stage. We have also investigated the myogenic potential of the fore-limb bud at various stages of development to ascertain whether there is a correlation between the stage the somitic cells first appear in the limb bud and the stage the bud acquires the capacity to form skeletal muscles. This was realized by culturing fore-limb buds excised from 18-30-somite stage embryos conventionally and in the kidney capsules of adult rats. In both methods, bone and cartilage were present in all of the cultures whereas skeletal muscles were only present in cultured explants older than the 21-22-somite stage.(ABSTRACT TRUNCATED AT 400 WORDS)     

The bovine tubouterine junction: general organization and surface morphology

The bovine tubouterine junction is composed of three parts (terminal tubal segment, transition region proper, uterine apex) and follows a sigmoidal course displaying a tubal and an uterine curvature. In the terminal tubal segment, 4-8 primary longitudinal folds and a system of lower secondary folds, ridges and chords project into the centrally located lumen. The transition region proper possesses a slit-like lumen because of the existence of a thick mucosal pad containing the first uterine glands. The longitudinal primary folds of the tube broaden, flatten and start to diverge when they reach the transition region proper. The mucosal pad and broadened folds are heavily vascularized. A system of lateral outpocketings with blind ends pointing in an ampullary direction develops between the primary and secondary folds, the ridges and chords of the terminal tubal segment and transition region proper. From the bottom of these outpocketings, short tubulo-alveolar crypts originate. The mucosa of the uterine apex forms low transversal ridges. The musculature of the bovine tubouterine junction is divided into a continuous circular or spiral intermediate layer, flanked by inner and outer longitudinal layers. The outer longitudinal layer is incomplete in the terminal tubal segment but increases in thickness to form a continuous stratum in the uterine apex. An inner longitudinal layer occurs only in the terminal tubal segment where it is best developed in the bases of the primary longitudinal folds. The simple columnar surface epithelium of the tubouterine junction contains ciliated and non-ciliated cells. The former undergo cyclical changes, and increase during estrus and postestrus. During proestrus, groups of non-ciliated cells display bulbous apical protrusions. During proestrus and estrus, circumscribed epithelial lesions expose the underlying basal lamina.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

Morphology and function of rooster efferent ductule epithelial cells in culture

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5-6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.     

A correlative histofluorescence and light microscopic study of the formation of the sympathetic trunks in chick embryos

The appearance and development of the primary and secondary sympathetic trunks in staged chick embryos was studied using the Falck-Owman histochemical method for the demonstration of primary monoamines. The earliest appearance of catecholamine (stage 20) was in individual fluorescent cells located in the region of the dorsal root ganglia about two stages prior to the formation of primary trunk aggregates. These cells are believed to be sympathetic precursor cells and correspond to formaldehyde-induced fluorescent cells observed in recent explantation experiments. Aggregates of fluorescent cells had formed bilaterally dorsolateral to the aorta at stage 22. These aggregates became continuous to form primary trunks by stage 24. The secondary sympathetic trunks were first seen in stage 25 and appeared to form at least partially by dorsal migration of cells from the primary trunks. Fluorescent cell processes were first observed at this stage. Secondary trunk formation was essentially complete by stage 28, and the primary trunks had become small and discontinuous. Definite rami communicantes could be observed by the early part of stage 28 in silver preparations. The significance of the development of two successive trunks in avians is discussed.     

Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity

Recognition of an avirulent pathogen stimulates an oxidative burst generating O2- and H2O2, and these reactive oxygen intermediates (ROIs) cue the induction of defense genes and cell death in the development of a restricted lesion. This localized hypersensitive response (HR) is accompanied by the development of systemic acquired resistance to virulent pathogens. Here we show that inoculation of Arabidopsis leaves with avirulent Pseudomonas syringae induces secondary oxidative bursts in discrete cells in distant tissues, leading to low-frequency systemic micro-HRs. The primary oxidative burst induces these systemic responses, and both the primary burst and the secondary microbursts are required for systemic immunity. Hence, ROIs mediate a reiterative signal network underlying systemic as well as local resistance responses.     

Intraperitoneal transplantation of isolated hepatocytes of the pig: the implantable bioartificial liver]

To elucidate the histopathological features of pancreatic ischemia, we examined postmortem pancreases in which cholesterol emboli were present. Cholesterol emboli were detected in 17 pancreases (6 of 36 cases of aortic aneurysm and 11 of 223 control cases). Two of the 17 pancreases had well-demarcated patchy lesions composed of degenerating acinar cells showing deeply eosinophilic cytoplasm and pyknotic nuclei, indicating fresh ischemia. In the marginal zone of the larger lesions and in the small lesions, the intralobular ductules had avoided the ischemic changes. Five of the 17 pancreases had patchy fibrotic foci containing small ductules with slightly retraction features. These ductules are considered to be the remnant intralobular ductules that have avoided the previous ischemic damage. We conclude that these patchy fibrotic foci are the healed ischemic lesions. The current findings suggest that the healed ischemic lesions can be differentiated from common pancreatic fibrosis. The existence of remnant intralobular ductules and the patchy retraction features may be useful histological markers for the determination of healed ischemic lesions.     

More informative abstracts revisited

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.     

Genetic analysis of the essential components of the immunoprotective response to infection with Eimeria vermiformis

The immune responses generated after infection with Eimeria spp. are complex, include both cellular and humoral components, and lead to protection against re-infection. To facilitate the rational development of the next generation of anticoccidial vaccines it is important that the nature of the immunoprotective response against infection with Eimeria spp. is determined. In this brief report we discuss results that were obtained using a combination of genetic and cellular approaches to dissect the essential immune effector components that operate against infection with Eimeria vermiformis. Mice rendered deficient of immune function by targeted gene disruption at a variety of immune loci represent an integral component of our studies and include those with targeted gene disruption at loci that encode the B- and T-cell receptors (BCR, TCR), antigen presentation molecules and immune-effector molecules. Our studies demonstrated that TCR-alpha-beta + T cells are essential for immunoprotection during both primary and secondary infection. Moreover, during primary infection the major effector cell type is a population of major histocompatibility complex class II-restricted, interferon-gamma-producing TCR-alpha-beta T cell consistent with a T helper 1 phenotype. In addition, there is a supplementary role for another class of cells (presumably T cells) that are restricted to either non-classical antigen presentation molecules or classical major histocompatibilty complex class I loaded via an atypical pathway. Mice with a deficiency in interleukin-6 were slightly more susceptible to primary infection than intact animals, consistent with the reported effects of interleukin-6 upon the generation of T helper 1-type responses in vivo. In terms of the host response to re-infection, TCR-alpha-beta T cells were essential for immunity, but the requirement for specific cell subsets and effector mechanisms was much less stringent. Mice deficient in gamma-delta T cells, classical major histocompatibility complex class I, non-classical antigen presentation pathways, the cytokines interferon-gamma, interleukin-4, interleukin-6 and the cytolytic effector molecules perforin or FasL were completely immune to secondary infection. Moreover, major histocompatibility complex class II-deficient I-A-beta-/- mice were capable of mounting a substantial response to secondary infection, manifest by a 95% reduction in oocyst output compared with primary infection. These data have important consequences for the development of immune intervention strategies and indicate that vaccine development may be targeted toward the generation of a wider range of effector mechanisms than those that operate during primary infection.     

Intravascular ultrasound predictors of restenosis after percutaneous transcatheter coronary revascularization

OBJECTIVES: This study sought to evaluate preintervention and postintervention intravascular ultrasound studies for potential predictors of angiographic restenosis and to use ultrasound predictors of restenosis to enhance our understanding of the pathophysiology of the restenosis disease process. BACKGROUND: Restenosis remains the major limitation of percutaneous transcatheter coronary revascularization. Although its mechanisms remain incompletely understood, numerous studies have identified some of the clinical, anatomic and procedural risk factors for restenosis. Intravascular ultrasound imaging of target lesions before and after catheter-based treatment consistently demonstrates more target lesion calcium, more extensive reference segment atherosclerosis, smaller final lumen dimensions, significant residual plaque burden and a greater degree of tissue trauma than is evident by angiography. METHODS: Intravascular ultrasound studies were performed in 360 nonstented native coronary artery lesions (final diameter stenosis 18 +/- 11%) in 351 patients for whom follow-up angiographic data were available 6.4 +/- 3.6 months later. Hospital charts were reviewed, and qualitative and quantitative coronary angiographic and intravascular ultrasound analyses were performed by independent core laboratories. Four dependent angiographic end points were tested: restenosis as a binary definition (> or = 50% diameter stenosis at follow-up) was the primary end point; follow-up diameter stenosis, late lumen loss and follow-up minimal lumen diameter were the secondary end points. RESULTS: Reference vessel size, the preintervention quantitative coronary angiographic assessment of lesion severity and the postintervention intravascular ultrasound cross-sectional measurements predicted the late angiographic results. In particular, the intravascular ultrasound postintervention cross-sectional narrowing (plaque plus media cross-sectional area divided by external elastic membrane cross-sectional area) predicted the primary end point (restenosis) and two of the three secondary end points (follow-up diameter stenosis and late lumen loss) and was therefore the most consistent predictor of restenosis. CONCLUSIONS: Intravascular ultrasound variables are more powerful and consistent predictors of angiographic restenosis than currently accepted clinical or angiographic risk factors.     

Differentiation of oxyntic cells and cell-matrix interactions during avian gastric gland morphogenesis

The relation between the expression of the oxyntic cell phenotype and the modifications of the extracellular matrix during development of the gastric glands, was studied in 10 to 21 day-old chick embryos. Cytodifferentiation of the oxyntic cells was established by ultrastructural methods, while the expression of pepsinogen, mitochondrial enzyme markers and apical secretory membranes was determined by histochemical and biochemical procedures. Results show that the morphogenesis of the glandular lobules occurs between days 8 and 15 of gestation. Later on, the lobules enlarge but maintain their basic morphology. Until day 13, the developing glands consist of primary tubes lined by a stratified columnar epithelium. The apical poles of the cells that contact the lumen show cytoplasmic processes, and Mg-ATPase activity and F-actin are concentrated at the apical cell borders. From day 13 on, the cells of the simple epithelium that lines secondary tubules budding from the primary tube, show all the features that define differentiated oxyntic cells. The synthesis of glycosaminoglycans during glandular morphogenesis was studied measuring the incorporation of radioactive sulfate into developing chick embryo proventriculi. An important increase in isotope incorporation was found between days 13 and 18 of development. Histochemical localization of these macromolecules shows that glycosaminoglycans are closely associated with the developing glandular lobules. Variations in the structure of epithelial cells undergoing morphogenesis and in the composition of the extracellular matrix are synchronous, suggesting that interactions between them may be significant in terms of the establishment and maintenance of the adult gastric gland phenotype.     

Papillary cystadenoma of the epididymis. An ultrastructural and immunohistochemical study

Papillary cystadenoma of the epididymis is a rare neoplasm that is sometimes associated with von Hippel-Lindau's syndrome. Electron microscopic study of the present case revealed that neoplastic cells contained abundant glycogen granules and large lipid droplets, but a few organelles. On the apical surface there were numerous microvilli and a few single cilia, but no ciliated cells. Subepithelial basal lamina was noted, but it was occasionally disrupted. Furthermore, microvilli sprang from the circumference of the small tumor-cell nest and became associated with matrix components (microvillus-matrix associations). On immunohistochemical study, neoplastic cells showed epithelial characteristics, but positive reactivity for S-100 protein. These findings resembled those of the epithelial cells of the efferent ductules of the epididymis. In the stroma, prominent vasculature was characteristic and fenestrated-type capillaries were found in the peripheral portion of the tumor. Papillary cystadenoma of the epididymis may originate from non-ciliated epithelial cells of the efferent ductules.     

Potent differentiation-inducing properties of the antiretroviral agent 9-(2-phosphonylmethoxyethyl) adenine (PMEA) in the rat choriocarcinoma (RCHO) tumor cell model

We have compared the splenic responses following immunization with the T cell-independent (TI)-2 antigen native dextran B512 and with a thymus-dependent (TD) protein-dextran conjugate. Interestingly, primary immunization with native dextran induced germinal center (GC) formation in the spleen to the same extent as the protein-dextran conjugate. The GC were antigen-specific as characterized by the presence of peanut agglutinin (PNA)-positive areas that were also binding FITC-conjugated dextran. Dextran-binding B cells were also detected outside the GC as sites of antibody-producing cells. The secondary splenic response to native dextran was suppressed compared to the primary response, with almost no dextran-specific GC or extra-follicular sites with dextran-specific B cells present in the sections. This suppression could be reverted by using cholera toxin (CT) as an adjuvant for the dextran immunizations. Following native dextran immunization with CT adjuvant a secondary splenic GC response similar to a TD secondary splenic GC response was generated, with almost all the dextran-specific B cells located in the GC. Collectively, this indicates that the difference between TI and TD antigen responses is not due to different abilities in inducing GC development, rather the GC reaction is less productive for a TI antigen than for a TD antigen. CT can both increase secondary GC formation in particular for the TI form of dextran and ameliorate the GC reaction, as reflected by increased anti-dextran antibody levels.     

Preoperative treatment and survival of patients with pheochromocytomas

A 9-year-old male Shetland Sheepdog had a small mass in the left testis. Grossly, the round to oval cyst was present at the upper pole of the testicular parenchyma near the head of the epididymis. Histologically, the cyst was lined by a single layer of nonciliated and ciliated epithelial cells. Immunohistochemically, the epithelial cells of the cyst showed expression of the low- and high-molecular-weight cytokeratins, vimentin, and desmin similar to that of normal efferent ductules in the dog. The testicular cystic dysplasia was thought to originate from the efferent ductules.     

Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells

Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.     

Regulation by the H-2 gene complex of macrophage-lymphoid cell interactions in secondary antibody responses in vitro

The ability of antigen-bearing syngeneic and allogeneic peptone-induced peritoneal exudate macrophages to support development of primary and secondary antibody responses by murine lymphoid or spleen cells in vitro has been investigated. The antigen used was the terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Syngeneic and allogeneic macrophages supported development of comparable primary antibody responses to GAT, indicating that genetic restrictions do not limit efficient macrophage-lymphocyte interactions in primary responses. By contrast, immunized spleen or lymphoid cells developed secondary antibody responses preferentially when stimulated in vitro with GAT on macrophages syngeneic to the macrophages used to present GAT during in vivo immunization. Thus, genetic restrictions regulate efficient macrophage-lymphocyte interactions in secondary antibody responses. These restrictions have been demonstrated from 2 to 8 wk after a single immunization with limiting quantities of GAT and are controlled by the H-2 gene complex. The implications that immune lymphocytes selectively recognize and respond to antigen presented in the context of the macrophage membrane-antigen complex which sensitized the lymphocytes initially are considered.