Genetic control and evolution of sesquiterpene biosynthesis in Lycopersicon esculentum and L. hirsutum

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of beta-caryophyllene and alpha-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of alpha-santalene, alpha-bergamotene, and beta-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.     

Signal transduction of somatostatin receptors negatively controlling cell proliferation.

Somatostatin acts as an inhibitory peptide of various secretory and proliferative responses. Its effects are mediated by a family of G-protein-coupled receptors (sst1-5) that can couple to diverse signal transduction pathways such as inhibition of adenylate cyclase and guanylate cyclase, modulation of ionic conductance channels, and protein dephosphorylation. The five receptors bind the natural peptide with high affinity but only sst2, sst5 and sst3 bind the short synthetic analogues. Somatostatin negatively regulates the growth of various normal and tumour cells. This effect is mediated indirectly through inhibition of secretion of growth-promoting factors, angiogenesis and modulation of the immune system. Somatostatin can also act directly through sst receptors present on target cells. The five receptors are expressed in various normal and tumour cells, the expression of each receptor being receptor subtype and cell type specific. According to the receptor subtypes, distinct signal transduction pathways are involved in the antiproliferative action of somatostatin. Sst1, 4 and 5 modulate the MAP kinase pathway and induce G1 cell cycle arrest. Sst3 and sst2 promote apoptosis by p53-dependent and -independent mechanisms, respectively.     

Somatostatin and its receptors contribute in a tissue-specific manner to the sex-dependent metabolic (fed/fasting) control of growth hormone axis in mice

Somatostatin (SST) inhibits growth hormone (GH) secretion and regulates multiple processes by signaling through its receptors sst1-5. Differential expression of SST/ssts may contribute to sex-specific GH pattern and fasting-induced GH rise. To further delineate the tissue-specific roles of SST and sst1-5 in these processes, their expression patterns were evaluated in hypothalamus, pituitary, and stomach of male and female mice under fed/fasted conditions in the presence (wild type) or absence (SST-knockout) of endogenous SST. Under fed conditions, hypothalamic/stomach SST/ssts expression did not differ between sexes, whereas male pituitary expressed more SST and sst2A/2B/3/5A/5TMD2/5TMD1 and less sst1, and male pituitary cell cultures were more responsive to SST inhibitory actions on GH release compared with females. This suggests that local pituitary SST/ssts can contribute to the sexually dimorphic pattern of GH release. Fasting (48 h) reduced stomach sst2A/B and hypothalamic SST/sst2A expression in both sexes, whereas it caused a generalized downregulation of pituitary sst subtypes in male and of sst2A only in females. Thus, fasting can reduce SST sensitivity across tissues and SST input to the pituitary, thereby jointly contributing to enhance GH release. In SST-knockout mice, lack of SST differentially altered sst subtype expression levels in both sexes, supporting an important role for SST in sex-dependent control of GH axis. Evaluation of SST, IGF-I, and glucocorticoid effects on hypothalamic and pituitary cell cultures revealed that these hormones could directly account for alterations in sst2/5 expression in the physiological states examined. Taken together, these results indicate that changes in SST output and sensitivity can contribute critically to precisely define, in a tissue-dependent manner, the sex-specific metabolic regulation of the GH axis.     

Evidence that endogenous SST inhibits ACTH and ghrelin expression by independent pathways

Corticosterone and total ghrelin levels are increased in somatostatin (SST) knockout mice (Sst-/-) compared with SST-intact controls (Sst+/+). Because exogenous ghrelin can increase glucocorticoids, the question arises whether elevated levels of ghrelin contribute to elevated corticosterone levels in Sst-/- mice. We report that Sst-/- mice had elevated mRNA levels for pituitary proopiomelanocortin (POMC), the precursor of adrenocorticotropic hormone (ACTH), whereas mRNA levels for hypothalamic corticotropin-releasing hormone (CRH) did not differ from Sst+/+ mice. Furthermore, SST suppressed pituitary POMC mRNA levels and ACTH release in vitro independently of CRH actions. In contrast, it has been reported that ghrelin increases glucocorticoids via a central effect on CRH secretion and that n-octanoyl ghrelin is the form of ghrelin that activates the GHS-R1a and modulates CRH neuronal activity. Consistent with elevations in total ghrelin levels, Sst-/- mice displayed an increase in stomach ghrelin mRNA levels, whereas hypothalamic and pituitary expression of ghrelin was not altered. Despite the increase in total ghrelin levels, circulating levels of n-octanoyl ghrelin were not altered in Sst-/- mice. Because glucocorticoids and ghrelin increase in response to fasting, we examined the impact of fasting on the adrenal axis and ghrelin in Sst+/+ and Sst-/- mice and found that endogenous SST does not significantly contribute to this adaptive response. We conclude that endogenous SST inhibits basal ghrelin gene expression in a tissue specific manner and independently and directly inhibits pituitary ACTH synthesis and release. Thus endogenous SST exerts an inhibitory effect on ghrelin synthesis and on the adrenal axis through independent pathways.     

Somatostatin receptors in GH-secreting pituitary adenomas--their relationship to the response to octreotide

Twenty pituitary adenomas, surgically removed from patients suffering from acromegaly, were studied. The tumours were immunostained with anti-GH and anti-PRL antibodies and with antibodies raised against particular subtypes of somatostatin receptors (rsst1-5). Expression of rsst immunoreactivity was scored using the following scale: 0--negative reaction, 1--weak reaction, 2--moderate reaction, 3--strong reaction. In 15 patients in whom the GH response to the acute administration of 200 ug octreotide was tested the correlation between the expression of rsst and the percentage of GH drop was estimated. All the tumours were GH-immunopositive and in the majority (14/20) co-expression of PRL was also found. All the adenomas examined expressed rsst2A (20/20) and rsst5 (12/12) receptor proteins. Receptors sst2B and rsst3 were found in all but one of the tumours examined (19/20 and 11/12, respectively). None of tumours investigated presented rsst4 immunopositivity. The mixed (GH/PRL) adenomas showed a tendency to a higher expression of rsst2A + + rsst2B and a greater response to octreotide administration. A significant positive correlation was found between rsst2A + rsst2B expressions and a drop in GH after octreotide. To conclude, the GH-inhibiting effect of octreotide depends on the intensity of expression of both rsst2A and rsst2B. Both isoforms of rsst2 mediate the same biological response (inhibition of GH secretion) in GH-secreting and GH/PRL-secreting adenomas.     

The N terminus of Saccharomyces cerevisiae Sst2p plays an RGS-domain-independent, Mpt5p-dependent role in recovery from pheromone arrest.

The Saccharomyces cerevisiae RGS protein Sst2p is involved in desensitization to pheromone and acts as a GTPase-activating protein for the Galpha subunit Gpa1p. Other results indicate that Sst2p acts through Mpt5p and that this action occurs downstream of Fus3p and through Cln3p/Cdc28p. Our results indicate that the interaction of Sst2p with Mpt5p requires the N-terminal MPI (Mpt5p-interacting) domain of Sst2p and is independent of the C-terminal RGS domain. Overexpression of the MPI domain results in an Mpt5p-dependent increase in recovery from pheromone arrest. Overexpression of either intact Sst2p or the MPI domain leads to partial suppression of a gpa1 growth defect, and this suppression is dependent on Mpt5p, indicating that MPI function occurs downstream of Gpa1p and through Mpt5p. Combination of an mpt5 mutation with the GPA1(G302S) mutation, which uncouples Gpa1p from Sst2p, results in pheromone supersensitivity similar to the sst2 mutant, and promotion of recovery by overexpression of Sst2p is dependent on both Mpt5p and the Gpa1p interaction. These results indicate that Sst2p is a bifunctional protein and that the MPI domain acts through Mpt5p independently of the RGS domain. RGS family members from other fungi contain N-terminal domains with sequence similarity to the Sst2p MPI domain, suggesting that MPI function may be conserved.     

Restriction endonuclease Sst 12I from a strain of Streptomyces sp. recognizing the nucleotide sequence 5'-CTGCAG-3'

A new restriction endonuclease Sst12I belonging to the II type and recognizing the sequence 5'-CTGCAG-3' was isolated from the bacterial strain Streptomyces sp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 degrees and 55 degrees C and remains active after long-term storage.     

Expression of genes coding melatonin and serotonin receptors in rodent skin

Targeted search for expression of melatonin and serotonin receptors genes in the skin of C57BL/6J mice showed expression of MT1B (but not MT1A). Mouse skin and hamster melanomas also expressed 5HT2B and 5HT7. We identified two novel isoforms of MT1A and 5HT7.     

Saccharomyces cerevisiae Mpt5p interacts with Sst2p and plays roles in pheromone sensitivity and recovery from pheromone arrest.

SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest. Recently, a family of Sst2p homologs that act as GTPase-activating proteins (GAPs) for G alpha subunits has been identified. We have identified an interaction between Sst2p and the previously identified Mpt5p by using the two-hybrid system. Loss of Mpt5p function resulted in a temperature-sensitive growth phenotype, an increase in pheromone sensitivity, and a defect in recovery from pheromone-induced G1 arrest, although the effects on pheromone response and recovery were mild in comparison to those of sst2 mutants. Overexpression of either Sst2p or Mpt5p promoted recovery from G1 arrest. Promotion of recovery by overexpression of Mpt5p required Sst2p, but the effect of overexpression of Sst2p was only partially dependent on Mpt5p. Mpt5p was also found to interact with the mitogen-activated protein kinase homologs Fus3p and Kss1p, and an mpt5 mutation was able to suppress the pheromone arrest and mating defects of a fus3 mutant. Because either mpt5 or cln3 mutations suppressed the fus3 phenotypes, interactions of Mpt5p with the G1 cyclins and Cdc28p were tested. An interaction between Mpt5p and Cdc28p was detected. We discuss these results with respect to a model in which Sst2p plays a role in pheromone sensitivity and recovery that acts through Mpt5p in addition to a role as a G alpha GAP suggested by the analysis of the Sst2p homologs.     

Expression of serotonin receptors in bone

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.     

Neuregulin 4 (Nrg4) cooperates with melatonin to regulate the PRL expression via ErbB4/Erk signaling pathway as a potential prolactin (PRL) regulator

Neuregulin-4 (Nrg4) and melatonin play vital roles in endocrine diseases. However, there is little discussion about the function and potential mechanism of Nrg4 and melatonin in prolactin (PRL) regulation. The human normal pituitary data from Gene Expression Profiling Interactive Analysis (GEPIA) database was used to explore the correlation between NRG4 and PRL. The expression and correlation of NRG4 and PRL were determined by Immunofluorescence staining (IF) and human normal pituitary tissue microarray. Western Blot (WB) was used to detect the expression of PRL, p-ErbB2/3/4, ErbB2/3/4, p-Erk1/2, Erk1/2, p-Akt and Akt in PRL-secreting pituitary GH3 and RC-4B/C cells treated by Nrg4, Nrg4-small interfering RNA, Erk1/2 inhibitor FR180204 and melatonin. The expression of NRG4 was significantly positively correlated with that of PRL in the GEPIA database and normal human pituitary tissues. Nrg4 significantly increased the expression and secretion of PRL and p-Erk1/2 expression in GH3 cells and RC-4B/C cells. Inhibition of Nrg4 significantly inhibited PRL expression. The increased levels of p-Erk1/2 and PRL induced by Nrg4 were abolished significantly in response to FR180204 in GH3 and RC-4B/C cells. Additionally, Melatonin promotes the expression of Nrg4, p-ErbB4, p-Erk1/2, and PRL and can further promote the expression of p-Erk1/2 and PRL in combination with Nrg4. Further investigation into the function of Nrg4 and melatonin on PRL expression and secretion may provide new clues to advance the clinical control of prolactinomas and hyperprolactinemia.     

APOC3基因SstⅠ单核苷酸多态性与冠心病、Ⅱ型糖尿病合并高甘油三酯血症的关联性研究

对许多人群研究表明 ,位于APOA1/C3/A4 /A5基因簇上的载脂蛋白C3基因 (APOC3)SstⅠ多态性与高甘油三酯血症 (Hypertriglyceridaemia ,HTG)密切相关 ,高甘油三酯是冠心病和糖尿病的独立危险因素。为探讨中国人群APOC3基因SstⅠ单核苷酸多态性与冠状动脉粥样硬化性心脏病 (coronaryatheroscleroticheartdisease,CHD)合并高甘油三酯血症 (HTG)、Ⅱ型糖尿病 (non insulin dependentdiabetesmellitus,NIDDM)合并高甘油三酯血症 (HTG)患者的相关性 ,应用聚合酶链反应 限制性片段长度多态性 (PCR RFLP)的方法 ,分析了 2 6 7例CHD患者、2 4 6例NIDDM患者及 4 91例健康对照APOC3基因SstⅠ位点 (S1/S2 )多态性。CHD组、NIDDM组和对照组的APOC3基因SstⅠ多态位点S2等位基因频率分别为 0 30 1、0 30 7和 0 2 86 ,其基因型频率和等位基因频率分布与对照组比较均无显著性差异 (P >0 0 5 )。以TG >1 90mmol/L为标准将CHD组、NIDDM组分为正常甘油三酯组 (NTG)和高甘油三酯组(HTG)发现 ,在CHD患者 ,HTG亚组S1S2基因型频率显著高于NTG亚组 (0 5 4 2 >0 35 7,χ2 =8 77,P =0 0 12 4 ) ;在NIDDM患者 ,HTG亚组S2S2基因型频率显著高于NTG亚组 (0 2 0 0 >0 0 5 5 ,χ2 =2 0 2 1,P =0 0 0 0 0 ) ,两亚组间等位基因频     

Cell surface expression of 5-hydroxytryptamine type 3 receptors is controlled by an endoplasmic reticulum retention signal

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R).5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.     

Effects of dietary equol on the pituitary of the ovariectomized rats.

The aim of our study was to evaluate the effects of dietary equol, metabolite of a phytoestrogen daidzein, on the secretion of prolactin (PRL) and lutenizing hormone (LH), as well as the expression of estrogen receptors (ERalpha, ERbeta and truncated estrogen receptor-1 (TERP-1) in the pituitary gland of ovariectomized (ovx) female Sprague-Dawley rats. Two doses of equol (50 mg/kg of chow and 400 mg/kg of chow) were used and the results were compared with the effects of estradiol 3-benzoate (E2B), also given at two doses (4.3 mg/kg of chow and 17.3 mg/kg of chow). Treatment period was 3 months. Dietary equol administration at the high dose increased significantly serum PRL levels. This effect was also observed in the E2B group but this difference did not reach statistical significance. Surprisingly, high dose dietary equol treatment also significantly increased serum LH levels, which was in contrast to E2B treatment where serum LH levels were significantly decreased at both doses. Serum LH levels in the equol low group were unaffected. Equol treatment had no effects on pituitary ERalpha or ERbeta gene expression. In contrast, high dose E2B treatment increased significantly pituitary ERalpha mRNA levels but decreased those of ERbeta. Both doses of E2B also increased significantly pituitary TERP-1 mRNA levels. This effect was also observed in the equol high group but at a much smaller magnitude. In conclusion, high dose dietary equol administration to ovx rats exerts estrogenic like effects on the lactotropes and anti-estrogenic on the gonadotropes.     

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.