Identification of genes involved in programmed cell death

Three modes of activation of apoptosis are described: induction, in which new gene expression occurs after the stimulus is applied; transduction, in which gene expression is unnecessary at the time of stimulation; and release, in which apoptosis is activated by the inhibition of gene expression. Genes activated in the induction mechanism were identified by a process of subtractive hybridization, whereby newly transcribed messenger RNAs could be isolated. Progress in characterizing some of these genes is described. There are many difficulties and conceptual problems associated with such a gene cloning approach, but the results will be worth the effort.     

Bone Morphogenetic Protein 2 (BMP-2) Enhances BMP-3, BMP-4, and Bone Cell Differentiation Marker Gene Expression During the Induction of Mineralized Bone Matrix Formation in Culturesof Fetal Rat Calvarial Osteoblasts

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling. Received: 27 November 1995 / Accepted: 19 July 1996     

Diurnal Rhythm in Proopiomelanocortin mRNA in the Arcuate Nucleus of the Male Rat

We tested the hypotheses that in the male rat, expression of proopiomelanocortin (POMC) mRNA in cells of the arcuate nucleus displays a diurnal fluctuation and that expression of this rhythm is dependent upon the secretory products of the testis. To accomplish this, we sacrificed groups of testes-intact and castrated adult male rats throughout the day and compared levels of POMC mRNA in individual cells of the arcuate nucleus across time and between groups. Adult male rats were housed on a 12–12 L D cycle with lights on a 0600 h and were divided into groups that were either castrated or left intact. Four days later, pairs from these groups were sacrificed at 0600 h, 1200 h, 1800 h, 2400 h, and again at 0600 h (n = 4 per group at each time point). We used in situ hybridization and a computerized image analysis system to measure cellular levels of POMC mRNA, as reflected by the number of autoradiographic grains over individual cells in the rostral quarter of the arcuate nucleus (counting ～ 30 cells per animal). Using cosinor analysis, we observed that in intact male rats, POMC mRNA levels varied significantly over the 24 h day with a nadir value at 1800 h. In contrast, there was no significant diurnal variation in POMC mRNA levels in castrated animals. POMC mRNA levels were significantly greater in the intact compared with castrated animals at every time point (P<0.01), except at 1800 h, when the groups did not differ significantly from one another. We conclude that adult male rats display at diurnal rhythm in cellular POMC mRNA levels in the arcuate nucleus, and we infer that testosterone or some other secretory product of the testis is a prerequisite for expression of this rhythm.     

Gi-mediated muscarinic adenylyl cyclase inhibition in timolol-treated stunned porcine myocardium

The Gi-mediated muscarinic receptor-adenylyl cyclase system was examined in stunned myocardium induced by either three or five brief ischaemic periods after β-adrenoceptor blockade by timolol (0.1 mg kg^-1). The mid-left anterior descending coronary artery was occluded for 2, 10 and 2 min in four pigs, and for 2, 2, 5, 10 and 2 min in four other pigs. All the ischaemic periods were separated by 30 min of reperfusion and the biopsies were obtained 60 min after the last ischaemic period. Segment length function was measured in the ischaemic region and in the control region supplied by the left circumflex artery. In the two groups, the percentage systolic shortening was reduced equally, to 59±9 and 58±10% of control in the region subjected to ischaemia and only minimally in the control region. The biopsies from the stunned region from both groups showed: (1) no change in either the affinity for carbachol or the number of binding sites of the muscarinic receptors; (2) no alterations in messenger RNA encoding for the α subunit-2 of the inhibitory guanine nucleotide binding protein, as demonstrated by northern blot and solution hybridization; (3) no change in membrane-bound inhibitory guanine nucleotide binding protein, as shown by enzyme immunoassay utilizing a specific anti-peptide antibody, and (4) unchanged inhibition of stimulated adenylyl cyclase activity. These results suggest that there is an intact inhibitory guanine nucleotide binding protein-mediated muscarinic receptor adenylyl cyclase system in the stunned porcine myocardium.     

急性酒精暴露对人原代肝细胞HO-1 mRNA表达的影响

目的 :　研究急性酒精暴露对人原代肝细胞 HO- 1 m RNA表达的影响。方法 :　经体外灌流、分离培养人原代肝细胞 ,观察酒精对人原代肝细胞上清液中天冬氨酸氨基转移酶(AST)的释放及 GSH含量的变化 ,用 RT- PCR方法检测酒精对人原代肝细胞 HO- 1 m RNA的表达。结果 :　急性酒精暴露导致人原代肝细胞上清液中释放的 AST增加 ,并呈明显的剂量效应和时间效应关系 ;此外 ,在 1 0 0 mmol/L乙醇 2 4 h暴露下 ,肝细胞中的 GSH明显降低 ,HO- 1 m RNA表达开始明显增加 ,3～ 9h之间达到最高峰 ,随后开始降低。结论 :　在 2 5～ 1 0 0 mmol/L范围内 ,急性酒精暴露导致人原代肝细胞明显的氧化损伤 ,且影响 HO- 1 m RNA表达。     

Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation

Surges of luteinizing hormone (LH) that result In luteinizationbut occur prematurely with respect to the diameter of the leadingfolilde, prevent attempts to induce multiple follicular maturationfor in-vitro fertilization (IVF) in a significant number ofwomen. We examined the possibility of blocking premature LIIsurges by the administration of Cetrorelix, a potent antagonistof gonadotrophin-releasing hormone (GnRH), in a study Including20 patients, some of whom had previously shown premature LHsurges. All patients were treated with human menopausal gonadotrophins(HMG) starting on day 2. From day 7 until the induction of ovulationby human chorionic gonadotrophin (HCG) the GnRII antagon Cetrorelixwas given daily. HCG was injected when the dominant fofficlehad reached a diameter of >18 mm and oestradlol concentrationwas >300 pg/ml for each follicle having a diameter of >15mm. Oocyte collection was performed 36 h later by transvaginalultrasound puncture, followed by IVF and embryo transfer. Thehormone profiles of these patients and the results of IVF andembryo transfer are comparable to those treated with GnRH agonistsand HMG. However, less time and especially less HMG Is neededin comparison to patients stimulated with a long agonist protocol.Hence, treatment with Cetrorelix proved to be much more comfortablefor the patient. In this study we showed that combined treatmentwith gonadotrophins and the GnRH antagonist Cetrorelix is apromising method for ovarian stimulation in patients who frequentlyexhibit premature LH surges and therefore fall to complete treatment.     

Dopamine-β-Hydroxylase Activity is Necessary for Hypothalamo-Pituitary-Adrenal (HPA) Responses to Ether, and Stress-Induced Facilitation of Subsequent HPA Responses to Acute Ether Emerges as HPA Responses are Inhibited by Increasing Corticosterone (B)

To determine a role of norepinephrine (NE) in stress-induced HPA function, young male rats were treated with diethyldithiocarbamide (DDC) which inhibits dopamine-β-hydroxylase, the enzyme that synthesizes NE from dopamine (DA). DDC injected 5 h prior to ether stress stimulated ACTH and corticosterone (B) during this time, and there was no further HPA response to ether. To control for elevated B feedback in DDC effects on HPA responses to ether, rats were adrenalectomized (Adx) and replaced with no (0% B), moderate (40% B) and high (80% B) levels of steroid 5 d prior to DDC or saline with ether stress 5 h later; Sham-Adx rats were included. In Adx rats increasing B inhibited thymus weight, median eminence CRF content, pituitary and plasma ACTH. In saline-treated rats, ether 5 h later caused increased CRF content and plasma ACTH in Sham-Adx and Adx, 0%B, increased ACTH in Adx, 40%B, and no response in Adx, 80% B. B treatment did not alter catecholamine content, and DDC treatment reduced NE content in the paraventricular nuclei by 50–60% in all groups. 5 h after DDC, pituitary ACTH was decreased in all rats with B and plasma ACTH was increased in sham-Adx and Adx, 40%B; thus DDC caused significant, prolonged stress which should facilitate subsequent HPA responses to acute stress. There was no HPA response to ether in Sham-Adx, Adx, 0% or 40% B groups, but there was a marked ACTH response to ether in the Adx, 80%B group treated with DDC. We conclude that: 1) the HPA response to ether stress is probably mediated by catecholamines; 2) DDC does not stimulate responses in the HPA axis in the absence of B; and, 3) facilitation of HPA responses to acute stress depends on increased steady-state B signals. Facilitated responses are probably not mediated by catecholamines. The consequence of facilitation is that under conditions of chronic stress and elevated B concentrations, as in depression or anorexia nervosa in man, or adjuvent-induced arthritis in rats, the HPA axis is continually responsive to new stimuli.     

老年大鼠弓状核内生长抑素mRNA的表达

采用原位杂交组织化学技术观察了老年大鼠弓状核内生长抑素信使核糖核酸的表达。和年青大鼠相比，老年大鼠弓核内生长抑素ｍＲＮＡ阳性胞体大多染色浅淡，与背景反差小，数量上明显减少。结果提示：在衰老过程中，弓状核内生长抑素减少可能对机体内分泌和神经内分泌功能的进行性衰退有重要作用。     

The Combined Effects of High Temperature and Carbon Monoxide on Heat Stress Response

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