Synthesis Of Secretory Proteins Research Articles

Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts

Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit. Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation. In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact. Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes. The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H. Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain. Metabolic labelling in hepatocytes cultured with [35S]methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium. This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell. Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not. The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum [Robbi & Beaufay (1986) Eur. J. Biochem. 158, 187-194; (1987) Biochem. J. 248, 545-550] indistinguishable from those occurring early in the synthesis of secretory proteins. Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

Alpha‐ and gamma‐interferon (IFNα, IFNγ) but not interleukin‐1 (IL‐1) modulate synthesis and secretion of β2‐microglobulin by hepatocytes

Soluble serum beta 2-microglobulin has been thought to result from membrane shedding by activated T-lymphocytes. This hypothesis could explain the increase of beta 2-microglobulin serum levels during virally induced mononucleosis, but not elevated levels as observed in other virally induced and in malignant diseases. In this paper we demonstrate that beta 2-microglobulin is a true secretory protein, and that its synthesis in hepatocytes is modulated by IFNs but not by IL-1. While the 45,000 MW HLA antigen can be found only in cell lysates, beta 2-microglobulin is shown to be secreted also into the culture medium like other secretory proteins (e.g. albumin-factor B-complement C3). Furthermore, interferon alpha (IFN alpha) as well as interferon gamma (IFN gamma) directly stimulate, in a dose- and time-dependent manner, beta 2-microglobulin synthesis by human hepatoma cells (Mz-Hep-1 and PLC/PRF5) and murine hepatocyte primary cultures. The increase of beta 2-microglobulin production induced by interferons is demonstrated at both the protein and the RNA level, indicating that interferon acts at a pretranslational level. The interferon effect on beta 2-microglobulin synthesis is specific since synthesis of secretory proteins like complement C3 or albumin, and of a structural protein like actin, remains unchanged. In contrast to IFN, IL-1, the main mediator of acute phase response, does not change beta 2-M biosynthesis rate. These data indicate that (i) beta 2-microglobulin is a secretory protein, (ii) IFNs but not IL-1 can mediate increased beta 2-M serum levels, and (iii) the liver may be its primary source.

Secretory Protein Biosynthesis in Snail Hemocytes: In vitro Modulation by Larval Schistosome Excretory-Secretory Products

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.

Ultrastructure of odontogenic cells during enameloid matrix synthesis in tooth buds from an elasmobranch, Raja erinacae

The ultrastructure of the inner dental epithelial cells (IDE) and odontoblasts in elasmobranch (Raja erinacae) tooth buds was investigated by transmission electron microscopy to determine what contribution each cell type makes to the forming enameloid matrix. Row II, early stage, IDE cells contained few organelles associated with protein synthesis, whereas preodontoblasts appeared competent to initiate extracellular matrix production. Row III IDE cells are also devoid of organelles related to secretory protein synthesis, although these IDE cells accumulated large pools of intracellular glycogen. The glycogen appeared to be packaged into vesicles and exocytosed into the lateral extracellular space toward the forming enameloid matrix. Row III odontoblasts had a morphology consistent with an active protein secretory cell. No procollagen granules were present within the odontoblasts, however, nor were many collagen fibers observed in the enameloid matrix. Instead, non-collagenous "giant" fibers having 17.5-nm periodic cross striations were associated with the invaginations of odontoblast cell processes. Giant fibers, which spanned a clear zone adjacent to the odontoblasts, terminated within the enameloid matrix. Smaller 25-nm-wide "unit" fibers emanated from the giant fiber tips to form the bulk of the enameloid matrix. The clear zone, which separated the odontoblasts from the enameloid matrix at early stages, diminished in size at later stages until the odontoblast processes were completely embedded in the enameloid matrix. Nascent enameloid crystallites were observed only after a layer of unmineralized predentin was deposited beneath fully formed enameloid matrix. The results suggest that the major constituent of the enameloid matrix in skates is a non-collagenous protein derived from the odontoblasts. The inner dental epithelial cells appear to contribute large quantities of carbohydrates to the forming enameloid matrix.

Ultrastructural observations on human sublingual gland.

That part of the human sublingual gland that corresponds in morphology to the conventional description of this organ presented in most histology texts (probably the major sublingual gland, in contradistinction to the aggregated small glands that compose the minor sublingual glands) was studied by electron microscopy. The gland is mixed, with slightly more mucous elements than seromucous ones. The mucous cells are arranged in tubules that usually are capped by seromucous demilunes. Seromucous cells also form occasional acini or may be scattered in the walls of the mucous tubules. The appearance of the mucous cells varies with the stage of the secretory cycle that they may be in. Their secretory droplets increase in number and progressively compress cytoplasmic organelles. Filamentous bodies also may be present. Based on secretory-granule substructure, four different kinds of seromucous cells can be recognized; these may be a morphological expression of asynchronous synthesis of different secretory proteins. The duct system is an abbreviated one compared to the other major salivary glands. The first duct segments, into which the mucous tubules drain, are similar to intercalated ducts. Larger ducts contain mitochondria-rich cells but lack the basal striations that characterize striated ducts. The paucity of typical striated ducts may be correlated with the elaboration of sodium-rich saliva by the human sublingual gland.

Direct effects of endotoxin on hepatocytes. Synthesis of a specific secretory protein.

The synthesis of acute-phase proteins by the liver during sepsis has been thought to be induced primarily by monokines released from activated macrophages, although glucocorticoid hormones may also stimulate this process to a lesser degree. According to this concept, synthesis of these proteins following administration of bacterial endotoxins would be an indirect effect and would not reflect a direct interaction of the endotoxin molecule with the hepatic parenchymal cell. We observed, however, that the synthesis of a 23-kilodalton protein was stimulated directly by the addition of lipopolysaccharide to cultures of primary mouse hepatocytes. The synthesis of this protein was also stimulated by glucocorticoids and interleukin 1. These findings demonstrate that certain hepatic proteins are subject to complex regulation by several factors thought to be important mediators of sepsis; in addition, they suggest that hepatic parenchymal cells may have the intrinsic capacity to respond directly to bacterial endotoxins.

Gene expression in male accessory glands of interspecific hybrids of Drosophila

Electrophoretic analysis of the male accessory-gland proteins of the three sibling species Drosophila melanogaster, D. simulans and D. mauritiana shows that the secretory proteins have a larger diversity than the epithelial proteins. In the male hybrids produced by crossing either simulans or mauritiana females to melanogaster males, genes coding for proteins specific to one or the other parent species are expressed normally in the hybrid cells. The rate of synthesis and accumulation of species-specific secretory proteins in interspecific hybrids is half of that observed in the homozygous parent species, corresponding thus to the gene dose. Injection experiments provide evidence that the efficiency of receptivity inhibition and ovulatory stimulation of the hybrid secretion are comparable to that of both homologous and heterologous secretions between the three sibling species.

Identification and Characterization of an Estrogen-Inducible Glycoprotein (Uterine Secretory Protein-1) Synthesized and Secreted by Rat Uterine Epithelial Cells*

An estrogen-inducible 97-kilodalton (kDa) secretory glycoprotein, designated as uterine secretory protein (USP)-1, synthesized by rat uterine epithelial cells was identified and characterized. Uterine luminal fluid (ULF) proteins were labeled by direct administration of [35S]methionine into uterine lumen of rats. Incorporation of [35S]methionine into ULF proteins was negligible in ovariectomized rats. However, when 17 beta-estradiol (E2) was administered in the ovariectomized rat as sc paraffin pellets, a marked increase of [35S]methionine incorporation was noted after 2 days of treatment, showing that the de novo synthesis and secretion of ULF proteins were induced by E2. Six estrogen-inducible polypeptides (130, 110, 97, 65, 42, and 39 kDa) were identified in the analysis of the labeled ULF proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Four of these polypeptides (110, 65, 42, and 39 kDa) were adsorbed by the immobilized antibody against rat serum proteins, indicating that these polypeptides are antigenically similar to serum proteins. Two polypeptides (130 and 97 kDa) not adsorbed by the antibody column were suggested to be uterine-specific secretory proteins. The 97-kDa protein (USP-1) was further purified by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis and electroelution. Reactivity of purified USP-1 to various lectins and carbohydrate composition analysis suggested that USP-1 possesses biantennary N-linked complex-type carbohydrate chain with fucose. Rabbit polyclonal antibody which can specifically immunoprecipitate [35S]methionine-labeled USP-1 was developed. Dot blot enzyme immunoassay showed that 4-day E2-treated ULF contains 78.3 +/- 24.1 (+/- SE) micrograms USP-1/mg protein. Immunohistochemical staining of rat uterine tissue showed that this protein localized only in the epithelial cells treated with estrogen (E2 and diethylstilbestrol). Testosterone, progesterone, and dexamethasone failed to induce synthesis and secretion of USP-1 as assessed by dot blot enzyme immunoassay, immunoprecipitation of [35S]methionine-labeled ULF proteins, and immunohistochemical staining of uterine tissue. The present result, for the first time, revealed that estrogen can induce synthesis and secretion of specific secretory protein which could be the useful marker to analyze the molecular mechanism of estrogen action in rat uterine epithelial cells.

Growth of rat seminal vesicle epithelial cells in culture: neurotransmitters are required for androgen-regulated synthesis of tissue-specific secretory proteins.

Epithelial cells from enzymically dispersed seminal vesicle tissue of castrated adult rats were grown on collagen-coated surfaces in serum-containing medium for up to 6 weeks. Androgens were neither required for growth nor were they mitogenic. They also failed to induce the synthesis of tissue-specific secretory proteins that serve as markers of androgen action in vivo. A variety of potential growth factors and culture procedures (such as feeder layers, extracellular matrices, collagen gels, and growth from tissue explants) were examined for their ability to support androgen action. Testosterone and 5 alpha-dihydrotestosterone were only able to induce seminal vesicle secretory protein synthesis in the presence of neurotransmitters (acetylcholine, noradrenaline, or serotonin). The mechanism of this permissive effect of neurotransmitters on androgenic induction has been examined. It is not related to their mitogenic activity since other mitogens (epidermal growth factor and corticosterone) did not facilitate androgen action. Agents that raise intracellular cAMP were also ineffective so cAMP is probably not involved, but the phosphatidyl inositol pathway may be important.

Secretory function of the ovine uterus: effects of gestation and steroid replacement therapy.

In Exp. 1, changes in uterine fluid content of protein, calcium and prostaglandin F2 alpha (PGF) were examined between d 30 to 144 of gestation. Volume of uterine fluid (mean +/- SE) in the nongravid uterine horn of unilaterally pregnant ewes increased (P less than .05) between d 30 (8 +/- 1 ml) and d 144 (749 +/- 46 ml) of gestation. Protein concentration and total protein in uterine fluid also increased (P less than .05) between d 30 (2.3 +/- .4 mg/ml; 19 +/- 7 mg) and d 144 (23.5 +/- 3.3 mg/ml; 17.4 +/- 2.0 g). Total recoverable calcium (mg) in secretions increased from .1 mg (d 30) to over 1.4 g (d 144) due to day of gestation effects (P less than .05). Total PGF also increased (P less than .01) from 7 ng on d 30 to 15.7 g on d 144. In Exp. II, ovariectomized (OVX) ewes were treated with either corn oil, estrone (E), progesterone (P) or P+E (PE) for 30 d. Treatment with P or PE increased calcium concentration and content (P less than .01) in secretions, but differences in uterine fluid volumes and concentrations of protein and PGF were not significant. Treatment of OVX ewes with P stimulated (P less than .05) in vitro synthesis of secretory proteins by endometrium, whereas treatment with E enhanced release of unlabeled proteins (P less than .05). The major endometrial secretory proteins were identified in allantoic fluids from d 60 to term and were detected along the mesenchymal border of the chorioallantois using immunohistochemistry. Results from this study indicate that P may be a primary hormone regulating accumulation of fluid, protein, calcium and specific endometrial proteins in the uterine lumen during gestation, and that uterine milk proteins gain access to the fetal-placental unit.

Androgen-regulated expression of secretory protein synthesis in mouse ventral prostate

Two proteins of molecular weights 25 and 12 kDa (p25 and p12 respectively), whose expression is regulated by testosterone, were identified in mouse ventral prostate. An antiserum raised to mouse ventral prostate secretion was used to demonstrate that p25 corresponds to the major secretory glycoprotein in mouse prostatic fluid. This antiserum does not cross-react with the major secretory proteins of the rat ventral prostate. Western blot analysis of mouse ventral prostate proteins using the prostatic secretion antiserum demonstrates that p12 and p25 are detectable at 3 weeks of age, but the maximum level of both proteins is not attained until 5 weeks of age. In addition, synthesis of p25 was also observed in prostate tissue derived from differentiated embryonic urogenital sinus tissue growing as implants under the renal capsule of syngeneic male hosts.

Secretory proteins from adrenal medullary cells are carboxyl-methylated in vivo and released under their methylated form by acetylcholine.

The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon.

Down-regulation of interleukin 1 production by macrophages of sarcoma-bearing mice.

Peritoneal macrophages from mice bearing a transplantable methylcholanthrene-induced sarcoma produced progressively less IL 1 as tumor burden increased. The loss of activity was not explained by the production of any inhibitor of the mouse thymocyte comitogen bioassay. Immune precipitation with a polyclonal antibody confirmed the decline in IL 1 appearance. Although tumor-bearing animals lost approximately 17% of their carcass mass, the reduced production of IL 1 was not satisfactorily explained by coexistent malnutrition, since similarly depleted non-tumor-bearing mice were capable of producing IL 1. In addition to an altered IL 1 production by macrophages of tumor-bearing mice, SDS-polyacrylamide gel electrophoresis and autoradiography revealed that the pattern of secretory protein synthesis from LPS-stimulated and unstimulated peritoneal macrophages differed between tumor-bearing and control animals. Administration of LPS to tumor-bearing mice early after tumor transplantation resulted in reduced tumor growth and prevented the down-regulation of in vitro IL 1 production by peritoneal macrophages. These findings demonstrate a specific defect in IL 1 production associated with increasing tumor burden. Further studies are required to determine whether this defect in IL 1 synthesis contributes to the increased tumor growth.

Nutrition support of the adult liver transplant candidate

Liver transplantation now provides a viable alternative to patients with end-stage hepatic failure. Because of the damaging effects of liver failure on other organ systems, transplant candidates frequently suffer from moderate to severe malnutrition. Chronic hepatic failure can result in deranged metabolism of not only macronutrients and micronutrients but also various hormones. Patients are frequently in a catabolic state. Wasting of the skeletal muscle mass occurs and synthesis of secretory proteins and clotting factors decreases. Hepatic encephalopathy and fluid/electrolyte imbalances often complicate the provision of appropriate nutrition support. Nutrition support may be provided by the oral route, tube feeding, parenteral nutrition, or a combination of those routes. A multidisciplinary approach to determine the appropriate nutrition support regimen is most optimal. To assess the efficacy of the support, monitoring of the nutritional/metabolic status is necessary on an ongoing basis. An aggressive nutrition support regimen can induce positive nitrogen balance, promote hepatic protein synthesis, and expand lean body mass, controlling the symptoms of malnutrition in end-stage liver disease.

Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages.

The concentrations of ATP and the ATP/AMP concentration ratios were maintained in thioglycollate-elicited mouse peritoneal macrophages incubated in vitro for 90 min in the presence or absence of added substrate: rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 60 min of incubation. The rate of oxygen consumption by macrophages was only increased above the basal rate (i.e. that in the absence of added substrate) by addition of succinate or pyruvate, or by addition of the uncoupling agent carboxyl cyanide m-chlorophenylhydrazone ('CCCP'); it was decreased by 75% by the addition of KCN. These findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period. The rates of glucose and glutamine utilization by incubated macrophages were approx. 300 and 100 nmol/min per mg of protein respectively. A large proportion of the glutamine that is utilized is converted into glutamate and aspartate, and very little (perhaps less than 10%) is oxidized. Similarly almost all of the glucose that is utilized is converted into lactate and very little is oxidized. This characteristic is similar to that of resting lymphocytes and rapidly dividing cells; in non-proliferating macrophages it may be a mechanism to provide precision in control of the rate of biosynthetic processes that utilize intermediates of these pathways, e.g. purines and pyrimidines for mRNA for the synthesis of secretory proteins and glycerol 3-phosphate for phospholipid synthesis for membrane recycling. No utilization of acetoacetate or 3-hydroxybutyrate by macrophages was detected. In contrast, both butyrate and oleate were oxidized. The rate of [14C]oleate conversion into 14CO2 (1.3 nmol/h per mg of protein) could account for most of the oxygen consumption by incubated macrophages, suggesting that long-chain fatty acids might provide an important fuel in situ. This may be one explanation for the secretion of lipoprotein lipase by these cells, to provide fatty acids for oxidation from the degradation of local triacylglycerol.

Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pancreatic secretory proteins.

The effect of ingesting isocaloric and isonitrogenous diets with increasing amounts of lipid (0-30%) and consequently decreasing amounts of carbohydrates (68.7-1.25%) on the exocrine pancreas was studied in adult male Wistar rats. Pancreatic contents of chymotrypsin, lipase and colipase activity, as well as synthesis of amylase, lipase, procarboxypeptidases and individual serine proteases were examined. Lipid-free diets and diets containing 1% lipid were found to have little effect on pancreatic proteins as compared with lipid-rich diets where two distinct patterns of response were observed. Ingestion of diets containing 3-20% lipid resulted in a progressive increase in the activity of lipase, colipase and chymotrypsin up to 2-fold in the first case and 1.6-fold in the two other cases when animals were fed the 20% fat diet. Under the latter conditions, the relative synthesis of secretory proteins, as expressed as percentage of the radioactivity incorporated into individual proteins compared to that incorporated into the total mixture of exocrine proteins, was unchanged for procarboxypeptidases, whereas it was stimulated for lipase (2-fold) and serine proteases (1.6-fold). Amylase relative synthesis progressively decreased as the lipid content of diets increased. Consumption of hyperlipidic diets containing 25% and 30% fat resulted in a further enhancement in the activity of lipase and colipase in the gland in contrast with chymotrypsin activity which was unchanged as compared to the control diet (3% lipid). As far as biosynthesis was concerned, a plateau in the relative synthesis of lipase and serine protease was reached. Amylase relative synthesis further decreased down to 2.2-fold when rats were fed the 30% fat-rich diet whereas that of procarboxypeptidases was markedly increased (about 1.7-fold). Absolute rates of synthesis of total pancreatic secretory proteins, as expressed with regard to the DNA content of the tissue, indicated that biosynthesis of all secretory pancreatic proteins was stimulated by hyperlipidic diets (at least 2-fold with the 30% lipid diet). Consequently, when such an increase was taken into consideration, the absolute synthesis of amylase was found to be unchanged throughout the dietary manipulations, whereas that of lipase, procarboxypeptidases and serine proteases were stimulated by 4.0-fold, 3.4-fold and 3.2-fold, respectively.

Development of secretory activity in the long hyaline gland of the male migratory grasshopper, Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins. Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.

Hormonal regulation of rat uterine secretory protein synthesis.

Treatment of the rat uterus with estradiol results in many morphological and biochemical changes. In order to examine the biochemical mechanisms of these changes, we are interested in finding a protein which can be used as an end-point indicator or marker. Estradiol administration results in the increased synthesis and release of a 115,000 and 65,000 dalton protein into the incubation media. In this paper, we demonstrate that these proteins are actually subunits of a larger protein having a molecular weight of 180,000 daltons. This protein appears to be specific to the uterus and may be produced mainly or solely by the epithelial cells. In the normal estrous cycle, maximal production is seen at estrus with essentially no production at diestrus. In the immature rat, synthesis of the 180K dalton protein is blocked by the interaction of progesterone and estradiol. However, unlike some uterine responses, the 180K protein is increased by treatment with several antiestrogens. This protein is also present in sufficient quantities to allow for the production of monoclonal antibodies. Thus this protein has many attributes of an excellent "marker protein" and will be of value in future studies aimed at elucidating the molecular mechanism(s) of steroid hormone action in the uterus.

Tissue distribution, developmental profile and hormonal regulation of androgen-responsive secretory proteins of rat seminal vesicles studied by immunocytochemistry

The seminal vesicles of the rat synthesise large amounts of androgen-regulated secretory proteins. Indirect immunofluorescence cytochemistry and immunoblotting with monospecific polyclonal antibodies against three of the major secretory proteins (II, S and F) have been used to investigate the tissue distribution, subcellular localisation, androgen-regulation and developmental profile of secretory protein synthesis. There was no evidence for regional specialisation of the seminal vesicle epithelium; every epithelial cell synthesises all three proteins via a classical secretory route involving storage in secretory vesicles. Proteins S and II are contained within the same secretory vesicles. The time course of deinduction of proteins S and F after castration and their reinduction by testosterone closely followed that for their specific mRNAs described previously. During development, proteins S and F first appear between 10 and 15 days after birth. A protein immunologically related to seminal vesicle protein II is present in the lateral and dorsal lobes of the prostatic complex.

Modified low density lipoproteins suppress production of a platelet-derived growth factor-like protein by cultured endothelial cells.

Cultured endothelial cells (EC) produce a platelet-derived growth factor-like protein (PDGF-c) that stimulates the growth of cultured cells of mesenchymal origin. We have examined the effect of native plasma low density lipoprotein (LDL) and chemically modified LDL on production of PDGF-c by EC. Acetyl-LDL, but not native LDL, suppressed the production of PDGF-c by bovine aortic EC. Half-maximal inhibition was observed at a concentration of 25-75 micrograms of cholesterol per ml, and maximal inhibition (0-25% of control) at 150 micrograms of cholesterol per ml. EC treated with acetyl-LDL showed no morphological damage, there was no change in cell number, and the effect on production of PDGF-c was substantially reversed upon removal of the acetyl-LDL. The observed inhibition of PDGF-c production was specific, since total cellular and secreted protein synthesis were unaffected by acetyl-LDL. Acetyl-LDL suppressed PDGF-c production in both bovine aortic and human umbilical vein EC, but not in rat heart EC. This cell specificity correlated with the presence of scavenger receptors as measured by degradation of 125I-labeled acetyl-LDL and uptake of fluorescently labeled acetyl-LDL. Dimethylpropanediamine-LDL, a cationic modified lipoprotein, also inhibited PDGF-c production. The inhibition by both types of modified LDL was accompanied by significant intracellular cholesterol accumulation, suggesting a role for EC lipid composition in the regulation of production of PDGF-c.