乙型肝炎病毒表面抗原的微团化及其免疫刺激复合物抗原的理化特性和免疫原性

本文用哺乳动物细胞系表达的乙型肝炎病毒表面抗原(HBsAg),制备了HBsAg的微团化(Micelle)和免疫剂激复合物(Immune-stimulating Complexes,简称ISCOMS)两种形式的抗原。在电镜下观察,微团化抗原是由球形亚单位颗粒组成直径100～150nm的较原颗粒大得多的大颗粒,在蔗糖中的浮力密度为1.24g/ml;而ISCOMS在电镜下为直径30～40nm左右稍大于原颗粒的多面体形态颗粒。SDS-PAGE分析表明,这两种形式的颗粒都是由HBsAg的P23和GP27蛋白所组成。 小鼠免疫接种结果显示,ISCOMS的免疫原性优于微团化抗原,后者又优于原22nm HBsAg颗粒。在抗体产生的速度和强度上,ISCOMS组显著优于微团化抗原组,而微团化抗原组略优于22nm HBsAg组。 ISCOMS的免疫性强,抗体产生早,强度高,又易于制备,而且不需要使用氢氧化铝胶佐剂,有可能发展成为一种新一代的乙型肝炎疫苗。     

Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee,Scaptotrigona postica depilis

Summary Juvenile hormone III content and ecdysteroid titer were analyzed for larval and pupal development of the stingless bee,Scaptotrigona postica depilis. Castespecific differences in juvenile hormone III content were detected at three developmental phases: at the transition from the fourth to the fifth larval stadium, in the spinning phase of the fifth larval stadium, and shortly after the imaginal moult. During the fifth larval stadium, juvenile hormone content closely reflects corpora allata activity. Juvenile hormone synthesis may thus be responsible for the elevated hormone titer in spinning-phase queen larvae, a phase of known sensitivity for induction of queen characters by exogenous juvenile hormone. For ecdysteroids, two phases of caste-specific differences were found: in the pre-pupal phase, and shortly after the imaginal moult. In both periods the titer in queens is distinctly higher compared to workers.Abbreviations Im imago 1 day after eclosion - L3, L4, L5 larval instars 3, 4, and 5 - L5F1, L5F2 substages of feeding phase in fifth larval instar - L5S1, L5S2, L5S3 substages of spinning phase in fifth larval instar - PP1, PP2 substages of prepupal phase - Pw white eyed pupa - Pp pink eyed pupa - Pr red eyed pupa - Pd dark eyed pupa - Pdl, Pdm, Pdd dark eyed pupa with progressive tanning of cuticle - RIA radioimmunoassay     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth,Manduca sexta

Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

烟草愈伤组织器官发生过程中外源激素的作用

近年来,利用愈伤组织系统在与器官发生有关的形态学、生理学和生物化学研究方面已经取得了一些进展(Thorpe 1980,刘涤 1983)。对烟草愈伤组织系统的研究明确了外源激素对器官发生类型的调节作用(Skoog 1971,Engelke等1973,刘涤等1980)。但是,这些研究仅考虑到外源激素的作用,而对器官发生过程中的其它变化并不了解。最近,Kamada和Harada(1981)比较了胡萝卜体细胞胚发育过程中内源IAA和ABA含量的变化,Noma等(1982)证实形成胚的和不形成胚的细胞间GA_3含     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.     

Separation of Neuropeptides by HPLC: Evaluation of Different Supports, with Analytical and Preparative Applications to Human and Porcine Neurophysins, β-Lipotropin, Adrenocorticotropic Hormone, and β-Endorphin

The separation of 38 hypophyseal or hypothalamic peptides and proteins by reversed-phase HPLC is described. The efficacy of several supports is discussed: large-pore reversed-phase silica supports turned out to be most effective in the separation of neuropeptides and proteins. The use of high sodium phosphate and phosphoric acid in the eluent resulted in high yields and better separation. On the basis of this procedure, beta-lipotropin, beta-endorphin, and adrenocorticotropic hormone were prepared from porcine pituitary glands, and human neurophysins from human pituitary posterior lobes. Moreover, this system is used for the analysis of the perifusate of pituitary posterior lobes, showing the release of micro-heterogeneous neurophysins.