Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.     

An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.     

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta,Hymenoptera, Sphecidae): Structural and biochemical aspects

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 μm (∅ 125 μm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.     

Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers

The response of the glutathione S-transferase (GST, EC 2.5.1.18) and related proteins of Mytilus edulis to environmental pollution load was assessed. Mussels were reciprocally transplanted between an industrial estuary (Douglas), a rural estuary (Youghal) and a m arine site (Bantry). In addition, m ussels were sam pled along a pollution gradient in an estuary receiving leather tannery effluent (Colligan). These latter m ussels were previously shown to be subject to oxidative stress resulting from the discharges. GST specific activity of cytosolic extracts from the gill and digestive gland tissues was determ ined for all anim als. Specific activity was shown to vary significantly in anim als from different sites, with highest specific activity always observed in sam ples (local and transplanted) taken from the industrial site. By com parison, the m ussels exposed to tannery discharges displayed no significant alteration in GST specific activity. Total intracellular glutathione (GSH) was also determ ined for sam ples taken from the Douglas and Youghal estuaries but no correlation with pollution load was observed. Using FPLC analysis, we observed no specific effect on the relative levels of the GST and the individual GST related proteins in gill or digestive gland sam ples from local or reciprocally transplanted anim als from Douglas or Youghal. The increase in GST specific activity observed in samples from the industrial estuary are indicative of a possible, specific inductive agent at this site. The results from the tannery site, by com parison, indicate that general oxidative stress does not result in elevated G ST specific activity in M. edulis.     

Suppression of somatotrophs in growth hormone injected rats

Summary The response of somatotrophs to large doses of purified bovine growth hormone was examined in uninephrectomized rats drinking 1% sodium chloride in an attempt to prolong the action of the hormone. Growth hormone produced a triphasic response in the ultrastructure of somatotrophs. Uninephrectomy and salt alone induced no changes in somatotrophs as compared to unoperated animals consuming tap water. In the first phase (3 days) the cross sectional area of the cell increased, the size of the largest granule per cell as well as the number of granules per cell increased significantly. All these parameters then reached control level after 7 days of injections during a second phase. Suppression of somatotrophs by exogenous growth hormone was evident by 16 days (third phase) when the area of the cell, the size of the largest secretory granule as well as the number of granules per cell all decreased significantly. Somatotrophs apparently did not react to suppression by a lysosomal mechanism inasmuch as no increase in the number of lysosome-derived structures was seen even at 16 days.Supported by research grant HL 06975 from the National Heart and Lung Institute. Dr. Nakayama is the recipient of a Bertha P. and Henry C. Buswell fellowship. The authors are grateful to Mrs. Neonile Fylipiw, Mrs. Elisabeth Lawson, Mr. Luther Joseph, Mrs. Geneva Joseph and Mr. Robert Linsmair for skilled technical assistance.     

Effects of surgical denervations on the autonomic nerves in parotid glands of dogs

Summary The innervation of the dog's parotid has been studied by cholinesterase staining and catecholamine fluorescence. In normal glands cholinergic and adrenergic nerves are plentiful around acini, muscular blood vessels, and to a lesser extent striated ducts. The main ducts, although surrounded by many cholinesterase-positive nerves, are associated with few adrenergic nerves. Severance of the classical parasympathetic post-ganglionic nerve to the gland, the auriculo-temporal, caused a moderate loss of cholinesterase-positive nerves. When this procedure was combined with section of the nerves on the internal maxillary artery there was a greater loss. Fewest cholinesterase-positive nerves remained when, in addition to these two procedures, the facial nerve was cut. These findings support the concept that all three sets of nerves contain some post-ganglionic parasympathetic fibres for the dog's parotid. The source of the remaining nerves is unknown. Preganglionic parasympathetic denervation by section of the tympanic branch of the glossopharyngeal nerve did not reduce the number of cholinesterase-positive nerves. None of these parasympathetic denervations caused reduction of adrenergic nerves, indicating that they do not travel to the gland with the parasympathetic nerves. After superior cervical ganglionectomy a few scattered fluorescent nerves remained in the gland; their origin is unknown.     

The gas bladder of Argentina silus L., with special reference to the ultrastructure of the gas gland cells and the countercurrent vascular bundles

Summary The orifice between the two chambers of the gas bladder in Argentina silus is surrounded by a sphincter muscle. Gas analyses of the gas bladder contents of fish from 400 meters depth give 0–1% carbon dioxide and 9–72% oxygen. Micro-retia mirabilia form a countercurrent vascular system, and the arterial component has peripherally a sphincter mechanism. The function of the glandular layer of the anterior chamber remains uncertain, but the structure indicates secretion into blood capillaries. The lining epithelium of the anterior chamber may secrete some substance into blood or directly into the lumen, which may be involved in a secretory mechanism. This conclusion is not supported by our histochemical tests. The posterior chamber has no micro-retia and the blood vessels have a different origin from those of the anterior chamber. The blood vessels form a plexus of capillaries or sinuses in contact with the flat lining epithelium, thus allowing gases to pass freely by diffusion. — The muscular layers of both chambers are innervated by catecholamine-containing nerve fibres.The investigation was supported by grants from the Swedish Natural Research Council (No. 99-35) and by the Faculty of Mathematics and Science, University of Lund.     

Presence of AVT-, α-MSH-, LHRH- and somatostatin-like compounds in the rat pineal gland and their relationship with the UMO5R pineal fraction

Using antibodies against AVT, alpha-MSH, LHRH and somatostatin, immunoreactive cells were detected in the rat pineal gland. All of these antibodies stain the same cells, which also react immunocytochemically when an antibody against the UMO5R sheep pineal fraction, a fraction that presents antigonadotropic properties in vivo, is used. Relatively more immunoreactive cells are present in the pineals of young rats than in the pineals of adult animals. Comparison of the results obtained with different potent antibodies against each of the peptides, and a study of the staining properties of the antibodies in the pineal after solid phase absorption to different peptides or to different sheep pineal fractions, led to the proposal that the immunoreactivity found in the rat pineal is not due to the presence of AVT, alpha-MSH, LHRH or somatostatin, but to a cross-reaction of each of these antibodies with (an) unidentified compound(s). This compound is synthetized in the pineal gland, as was demonstrated using cultured pineals. The UMO5R and the Prot. 4 fractions of the sheep pineal seem to be chemically related to this unknown compound, the possible endocrine nature of which is discussed.     

鸡新城疫副眼和副鼻器官微环境局部免疫机理的研究——Ⅰ.血清学和免疫荧光研究

本文研究了用新城疫B_1系疫苗免疫后的SPF雏鸡的泪液、唾液,气管粘液和血液中的血凝抑制(HI)抗体的性质和动态变化规律;同时采取哈德泪腺、气管、肺脏、脾脏、胸腺和法氏囊,作低温组织切片间接免疫荧光试验。结果证明:泪液、唾液和气管粘液中存在着HI抗体.经过两次免疫后,这种抗体至少可持续71天,而且在免疫初期(泪液在一免后的第26天前,唾液和气管粘液在一免后的第29天前)这些外分泌液中的HI抗体滴度要比血液中相应的抗体滴度高.说明这些抗体主要是由局部免疫组织产生的,而不是由血液中机械性渗透来的。哈德泪腺是以B淋巴细胞为主的外周免疫器官,它是在局部产生抗体的主要部位,同时气管、肺脏的局部淋巴组织也参与抗体的合成。参与局部免疫的抗体主要属于IgA。局部免疫反应以体液免疫为主,并由体液免疫和细胞免疫共同完成。     

Inhibin α-subunit N terminus interacts with activin type IB receptor to disrupt activin signaling

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin β-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LβT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LβT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.