Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes.

Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C-reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

Accumulation of Cd and Pb in various body parts,organs and tissues of Lymantria dispar asiatica (Lepidoptera: Erebidae)

To investigate the accumulation patterns of Cd and/or Pb in various body parts, organs and tissues of the Asian gypsy moth (Lymantria dispar) larvae under Cd and/or Pb stress, Cd and Pb treated artificial diets were used to feed the larvae in the current study. These larval body parts/organs/tissues included the heads, integuments (body walls), alimentary canals, fat bodies and hemolymphs. Our results showed that under Cd and/or Pb stress, their accumulations in larval body parts/organs/tissues were significantly higher than those in the control, with the amounts of tested metals in the fat bodies and hemolymphs and alimentary canals being significantly higher than those in the heads and integuments. Under the single Cd (0, 7.5, 10.5 mg/kg) or Pb stress (0, 55, 90, 125 mg/kg), the accumulations of these heavy metals were positively correlated with their concentrations in diets. Under the combined stress (Cd × Pb), the Cd accumulation at the lower Pb concentrations was higher than that at higher Pb concentrations for the body parts/organs/tissues, similar effects of Cd concentrations on Pb accumulations were also recorded. These results clearly showed that Cd and/or Pb were accumulated in various body parts/organs/tissues at different levels under the heavy metal stress. And accumulations of Pb/Cd were positively correlated with their concentrations in the diets under the single stress. Under the combined stresses, Cd and Pb had synergistic effects at low concentrations whereas antagonistic effects at high concentrations. The accumulations of Cd and/or Pb in the gypsy moth larvae affects normal physiological and biochemical functions, and thus affects their growth and development.     

Accumulation of cadmium by the fourth instar larva of the fly Chironomus thummi

Accumulation and effects of cadmium were investigated in Chironomus thummi larvae exposed to 10, 100 and 250 mug radiolabeled Cd/1 for up to 4 days. (1) After 4 days, average cadmium accumulation was 6.6 ng Cd/mg dry weight (10 mug Cd/1 exposure) and 177 ng Cd/mg dry weight (250 mug Cd/1 exposure). (2) Dissection studies showed that by 32 hr of exposure to both cadmium concentrations, 63.5-81.4% of accumulated cadmium was confined to the posterior midgut epithelium. Light microscope autoradiography similarly showed accumulations of cadmium in posterior midgut epithelium and smaller amounts in fat body and muscle. Little cadmium was associated with Malphigian tubules, haemocoel, anterior midgut or exoskeleton. (3) After exposure to 10 or 250 mug Cd/1, 60-75% of cadmium in ultracentrifuged homogenates of whole animals or dissected guts was associated with the resulting supernatant. When supernatants were further analyzed by gel chromatography, cadmium eluted with both a high and low molecular weight peak. The relative proportions of cadmium in the two peaks varied with concentration and length of exposure. (4) Transmission electron microscopy of posterior midgut cells from animals exposed to cadmium demonstrated frequent mitochondrial lesions. Exposure to high cadmium concentrations caused some posterior midgut cells to undergo generalized structural degeneration.     

Substitutions in the glycoprotein (GP) of the Candid#1 vaccine strain of Junin virus increase dependence on human transferrin receptor 1 for entry and destabilize the metastable conformation of GP

Candid#1 (Cd1) is an attenuated vaccine strain of Junin virus, the causative agent of Argentine hemorrhagic fever. Although several substitutions are present in Cd1, their importance for attenuation has not been established. We functionally characterized the substitutions present in the Cd1 glycoprotein (GP) and identified F427I in the transmembrane domain of the GP2 subunit as reducing infectivity in a reconstituted viral system. We further showed that this phenotype derives from the destabilization of the GP metastable conformation. Lastly, we identified an increased dependence of Cd1 GP on human transferrin receptor type 1 (hTfR-1) for entry, which may affect the tropism of the attenuated strain in vivo.     

Yolk polypeptide synthesis in the fat body of Sarcophaga bullata: Localization,hormonal induction and cell-free translation

The fat body of adult Sarcophaga bullata consists of different cell-types. The yolk polypeptides (YPs) are localized in secretory granules in the cytoplasm of female trophocyte fat body cells while the oenocytes and larval fat body cells are immunonegative. An antiserum against the larval serum protein 1 of Drosophila crossreacts on immunoblotting with several polypeptide bands in the haemolymph with mol. wt ∼80 kD. This antiserum specifically reacts with some storage granules of the persisting larval fat body cells and not with the other fat body cell types. The trophocyte fat body cells of male Sarcophaga treated with 20-OH-ecdysone, display a similar granular type of immunoreaction with an anti-YP antiserum as in vitellogenic females. Moreover, 20-OH-ecdysone induced in the fat body of males, in contrast to methoprene, synthesis of mRNA coding for YPs to a level as high as that in vitellogenic females, as shown in the reticulocyte lysate cell-free system.     

Polymorphism of the AHSG gene is associated with increased adipocyte beta2-adrenoceptor function

The alpha(2) Heremans-Schmid glycoprotein (AHSG) gene is implicated in the regulation of body fat and insulin sensitivity. The Met/Met genotype of the common single-nucleotide polymorphism (SNP), rs4917, in the AHSG gene has been shown to be associated with reduced plasma levels as well as lower body fat. Here, we studied the association of this variation with subcutaneous adipocyte lipolysis. Ninety-three obese and nonobese healthy men were genotyped for Thr230Met, and subcutaneous adipose tissue biopsies were analyzed for lipolysis characteristics. The Met/Met genotype was associated with a marked increase of 1.5 log units in the lipolytic sensitivity to the beta2-adrenoceptor agonist terbutaline (P=0.0008) as compared with the Thr/Thr and Thr/Met genotypes. This corresponds to an approximately 35-fold increase in beta2-adrenoceptor function. The genotype effect was independent of body mass index and waist circumference. In contrast, lipolytic sensitivity to both the beta1-adrenoceptor agonist dobutamine (P=0.25) and the alpha2A-adrenoceptor agonist clonidine (P=0.54) was unaffected by the Thr230Met variation. Moreover, no difference in either maximal stimulation or inhibition of lipolysis was found between genotypes. We conclude that a common variation (Thr230Met) in the AHSG gene is associated with a marked increase in beta2-adrenoceptor sensitivity in subcutaneous fat cells, which may be of importance in body weight regulation.     

Growth inhibition of BALB/c 3T3 cells by a high-molecular-weight mucin-like glycoprotein of human milk fat globule membrane

PAS-O is a high-molecular-weight mucin-like glycoprotein of the human milk fat globule membrane, and originates from the lactating mammary epithelial cell membrane. The cell proliferation rate of BALB/c 3T3 cells was markedly reduced by addition of PAS-O to their culture medium, this inhibition being concentration-dependent (0-10 microgram/ml) and reversible. The inhibitory activity was removed by periodate or protease treatment of the glycoprotein. PAS-O may interact with 3T3 cells to mimic the effect of high cell density on growth. PAS-O, which has been recognized as a glycoprotein molecule carrying a differentiation antigen expressed on the mammary epithelial cell surface, may play a role in regulating the growth of the mammary cells.     

Control of pupal fat body disappearance in the female black blow fly,Phormia regina (Meigen) by the brain and the corpus allatum

Neck-ligation, brain implantation, allatectomy, methoprene treatment, and ovariectomy indicated that the disappearance of pupal fat body cells in newly emerged adult female blow fly, Phormia regina, is controlled by the brain and the corpus allatum (CA). Absence of brain or CA greatly lowered the rate of fat body cell disappearance (i.e. death). Dependency on the CA decreased from 0 to 36h post-emregence, indicating that the CA was active during the earlier part of this timespan. Methoprene treatment enhanced pupal fat body cell disappearance in allatectomized females. Brain implantation restored the rate of pupal fat body cell disappearance in neck-ligated flies. Brains from day 1 sugar-fed flies proved to be more effective than those from day 2 sugar-fed flies, indicating that there may be a window after adult emergence that allows the brain to act directly or indirectly on the death of pupal fat body cells. Ovariectomy did not alter the rate of pupal fat body cell death in test animals. Dying pupal fat body cells were smaller in size, less dense (i.e. did not sink in saline like normal pupal fat body cells), and stickier (i.e. attached to other tissues tighter) than the healthy cells. A possible role played by ecdysteroids is also discussed.     

Accumulation of cadmium and induction of its binding protein in the digestive tract of fleshfly (Sarcophaga peregrina) larvae

The larva of Sarcophaga peregrina ( fleshfly ) was fed with cadmium (Cd)-containing diet and the distribution of Cd among tissues was determined by separating each organ. Approximately 90% of Cd accumulated in the larva was found in the digestive tract, the fat body and the Malpighian tube being less effective tissues in its accumulation. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.     

The fate and possible role of arylphorin during the metamorphosis of Mamestra brassicae.

1. Arylphorin, one of the storage proteins has been isolated from the hemolymph of Mamestra brassicae. 2. It has been established that Mamestra arylphorin is the most similar to manducin from among the known storage proteins of other species. 3. A rabbit polyclonal antibody has been developed against arylphorin, and its concentration changes have been determined quantitatively by ELISA in the hemolymph and fat body from the 1st day of the last larval instar to the 3rd day of the imago stage. 4. Histological sections were made on each day during the investigated period and it was shown by immunohistochemical methods that the main quantity of arylphorin was accumulated in the storage protein granules of the fat body and it could be detected even in the imaginal fat body. 5. The uptake of arylphorin by the fat body is induced by 20-hydroxyecdysone. 6. During differentiation of the imaginal cuticle arylphorin is incorporated first in the epidermal cells and it is built in the endocuticular layer of the integument thereafter.     

Cytochemical and electrophoretic studies of haemoglobin synthesis in the fat body of a midge, Chironomus thummi

The fat body of developing mid- and late fourth instar larvae of a midge, Chironomus thummi, has been investigated by means of the benzidine reaction for the localization of haemoglobin within cells. In the subepidermal fat body the reaction deposits of the haemoglobin pseudo-peroxidase activity appear predominantly in the intracisternal cavities of ER and the Golgi, and later, in the pharate pupal stage, in small dense granules (0.5–1 μm in. diameter).All the major protein bands of fat body extracts, which are resolved in electrophoresis, give the benzidine reaction and show incorporation of ¹⁴C-amino levulinic acids, in this case a specific marker for haemoglobin synthesis. In addition, labelled proteins show identical electrophoretic mobility as the haemoglobins of the haemolymph, suggesting that haemoglobins are synthesized in the fat body. Two types of fat body cells seem to differ with respect to their rôle in haemoglobin metabolism.     

LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine‐rich‐alpha‐2‐glycoprotein 1 (LRG‐1), that could promote fat graft survival through RAB31‐mediated inhibition of hypoxia‐induced apoptosis. We showed that LRG‐1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG‐1‐treated groups. Furthermore, we discovered that LRG‐1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG‐1‐mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG‐1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.     

The distribution of the neural fat body sheath and the accessibility of the extraneural space in the stick insect, Carausius morosus

The fluid compartment delimited at the surface of the central nervous system by the closely applied fat body sheath has been shown to be accessible to molecules of horseradish peroxidase and Indian ink particles. Quantitative analysis indicates that leakage via the intercellular spaces between fat body cells would preclude the possibility of a significant involvement of the fat body cells in sodium regulation of the extraneural fluid. This possibility is further precluded by the absence of a fat body sheath from peripheral nerves.     

Biosynthesis of major plasma proteins in the primary culture of fat body cells from the silkworm, Bombyx mori

Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, Bombyx mori, in a sex- and stage-specific manner during larval development. We successfully established a primary culture of the fat body cells in order to investigate the regulatory mechanisms of plasma protein gene expression. The primary cultures of fat body cells contained at least two cell types: small oval cells, and large spherical cells. The cells adhered to and migrated on the cultured dish after plating. By the 7th day of cultivation, the cells clustered to form fat body-like structures, which were maintained for at least 3 months. Plasma proteins were actively synthesized in the primary cultures of the fat body cells isolated from the final instar larvae only when the cells tightly adhered to and clustered on the cultured dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system, indicating that the primary culture comprises heterogeneous cells that are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced their sex-dependency in vivo.     

Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family.     

Long lasting cadmium intake is associated with reduction of insulin receptors in rat adipocytes

The effects of chronic cadmium exposure on adipose tissue have not been extensively reported. In adult Wistar male rats we investigated in vivo effect of 6 weeks lasting cadmium intake in drinking tap water (CdCl₂ 9,7 mg/l). Insulin receptors in isolated adipocytes from epididymal fat and glucose transporter protein GLUT4 content in fat tissue plasma membranes were determined. Control and Cd treated rats had similar water intake with subsequent heavy augmentation of Cd content in liver of experimental animals. In comparison with controls, Cd intake did not influence body mass increment and fat cell size, but significantly increased serum glycemia and moderately elevated insulinemia. Cadmium intake significantly reduced (50%) both, total insulin receptors number and density of the receptors in fat cells. No differences in the content of GLUT4 in crude plasma membranes of adipose tissue were observed. Diminished insulin receptors in adipocytes could account for diabetogenic effect of long lasting cadmium intake.     

意大利蜜蜂工蜂脂肪体胚后发育过程中细胞的增殖和凋亡

脂肪体是昆虫体内物质贮备和中间代谢的重要组织。本研究通过显微形态观察、 BrdU免疫组织化学和原位末端转移酶标记(TUNEL)细胞凋亡检测技术, 对意大利蜜蜂Apis mellifera ligustica工蜂脂肪体胚后发育过程中细胞的增殖和凋亡特点进行了比较研究。结果表明： 意大利蜜蜂工蜂脂肪体细胞数量的快速增加集中在幼虫发育前期（1-3龄）, 而细胞的凋亡则集中在蛹发育早期的2-3 d（预蛹-2日龄蛹）时间之内。在变态发育中, 工蜂幼虫脂肪体凋亡降解后重新组建形成成虫的脂肪体。本研究为昆虫脂肪体的功能研究以及昆虫组织细胞自噬和凋亡的机制研究提供一定的证据。