Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells

We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.     

Octapeptide analogs of somatostatin exhibiting greatly enhanced in vivo and in vitro inhibition of growth hormone secretion in the rat

Analogs of a superactive somatostatin (SRIF) octapeptide (code named SMS 201-995 (1)) were synthesized using solid-phase synthetic methodology and assayed for their ability to inhibit growth hormone release from cultured rat anterior pituitary cells and in sodium pentobarbital-anesthetized rats. One analog: (Formula: see text) exhibited greatly enhanced in vitro inhibitory activity (greater than 1,000x) relative to both the parent octapeptide molecule and to the 14 amino acid SRIF molecule. This analog which was also very potent in vivo contains a tyrosine residue and, given its high in vitro activity, may be of investigative importance as a radioiodinated ligand in receptor assays. An octapeptide retro-inverso analog also exhibited significant SRIF-like activity. Several very low activity octapeptide analogs were synthesized and were found to be devoid of SRIF-antagonist activity. A dodecapeptide analog previously shown to be superactive in vivo also demonstrated high in vitro activity.     

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

白细胞介素－2中枢镇痛作用途径的探讨

抗ＩＬ－２受体α亚基的单克隆抗体不能阻断ＩＬ－２的中枢镇痛作用,以及丧失与ＩＬ－２受体β亚基结合能力的ＩＬ－２突变体仍具有提高大鼠痛阈的能力,这表明ＩＬ－２的中枢镇痛作用并不是通过ＩＬ－２受体所介导,亦表示ＩＬ－２的免疫和镇痛作用是通过不同的受体途径实现的。加之内源性阿片肽与ＩＬ－２分子有着共同的抗原决定基和结构相似性,提示ＩＬ－２可以与阿片受体直接结合产生中枢镇痛效应。从放射免疫法测定的ＩＬ－２侧脑室注射后不同时间大鼠脑内不同核团的内源性阿片肽含量,推测ＩＬ－２的中枢镇痛作用可能还与弓状核、室旁核、蓝斑等核团的β－ＥＰ和ＬＥＫ有关。     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.