Negative Regulatory System Research Articles

The MinCDE Cell Division System Participates in the Regulation of Type III Secretion System (T3SS) Genes, Bacterial Virulence, and Motility in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice, which is one of the most severe bacterial diseases in rice in some Asian countries. The type III secretion system (T3SS) of Xoo encoded by the hypersensitive response and pathogenicity (hrp) genes is essential for its pathogenicity in host rice. Here, we identified the Min system (MinC, MinD, and MinE), a negative regulatory system for bacterial cell division encoded by minC, minD, and minE genes, which is involved in negative regulation of hrp genes (hrpB1 and hrpF) in Xoo. We found that the deletion of minC, minD, and minCDE resulted in enhanced hrpB1 and hrpF expression, which is dependent on two key hrp regulators HrpG and HrpX. The minC, minD, and minCDE mutants exhibited elongated cell lengths, and the classic Min system-defective cell morphology including minicells and short filamentations. Mutation of minC in Xoo resulted in significantly impaired virulence in host rice, swimming motility, and enhanced biofilm formation. Our transcriptome profiling also indicated some virulence genes were differentially expressed in the minC mutants. To our knowledge, this is the first report about the Min system participating in the regulation of T3SS expression. It sheds light on the understanding of Xoo virulence mechanisms.

Production of the Invasive Aspergillosis Biomarker Bis(methylthio)gliotoxin Within the Genus Aspergillus: In Vitro and in Vivo Metabolite Quantification and Genomic Analysis.

Gliotoxin (GT) is a fungal secondary metabolite that has attracted great interest due to its high biological activity since it was discovered by the 1930s. An inactive derivative of this molecule, bis(methylthio)gliotoxin (bmGT), has been proposed as an invasive aspergillosis (IA) biomarker. Nevertheless, studies regarding bmGT production among common opportunistic fungi, including the Aspergillus genus, are scarce and sometimes discordant. As previously reported, bmGT is produced from GT by a methyl-transferase, named as GtmA, as a negative feedback regulatory system of GT production. In order to analyze the potential of bmGT detection to enable identification of infections caused by different members of the Aspergillus genus we have assessed bmGT production within the genus Aspergillus, including A, fumigatus, A. niger, A. nidulans, and A. flavus, and its correlation with gtmA presence. In order to validate the relevance of our in vitro findings, we compared bmGT during in vitro culture with the presence of bmGT in sera of patients from whom the Aspergillus spp. were isolated. Our results indicate that most A. fumigatus isolates produce GT and bmGT both in vitro and in vivo. In contrast, A. niger and A. nidulans were not able to produce GT or bmGT, although A. niger produced bmGT from a exogenous GT source. The frequency and amount of bmGT production in A. terreus and A. flavus isolates in vitro was lower than in A. fumigatus. Our results suggest that this defect could be related to the in vitro culture conditions, since isolates that did not produce bmGT in vitro were able to synthetize it in vivo. In summary, our study indicates that bmGT could be very useful to specifically detect the presence of A. fumigatus, the most prevalent agent causing IA. Concerning A. terreus and A. flavus a higher number of analyses from sera from infected patients will be required to reach a useful conclusion.

TNFAIP3, a negative regulator of the TLR signaling pathway, is a potential predictive biomarker of response to antidepressant treatment in major depressive disorder

Inflammation and abnormalities in Toll-like receptor (TLR) expression and activation have been linked to major depressive disorder (MDD). However, negative regulators of TLR pathways have not been previously investigated in this context. Here, we sought to investigate the association of depression severity, measured by the 17-item Hamilton Depression Rating Scale (HAMD-17), with mRNA expression levels of negative regulators of the TLR pathway, including SOCS1, TOLLIP, SIGIRR, MyD88s, NOD2 and TNFAIP3, in peripheral blood mononuclear cells (PBMCs) from 100 patients with MDD and 53 healthy controls, before and after treatment with antidepressants. Positive regulators of the TLR4 pathway, including Pellino 1, TRAF6 and IRAK1, were also investigated. Among all patients, MyD88s, and TNFAIP3 mRNAs were expressed at lower levels in PBMCs from patients with MDD. Multiple linear regression analyses revealed that TNFAIP3 mRNA expression before treatment was inversely correlated with severity of depression and effectively predicted improvement in HAMD-17 scores. Among 79 treatment-completers, only TNFAIP3 mRNA was significantly increased by treatment with antidepressants for 4 weeks. Treatment of human monocytes (THP-1) and mouse microglia (SIM-A9) cell lines with fluoxetine significantly increased TNFAIP3 mRNA expression and suppressed IL-6 levels. The suppressive effect of fluoxetine on IL-6 was attenuated by knockdown of TNFAIP3 expression. These findings suggest that both dysfunction of the negative regulatory system in patients with MDD and antidepressant treatment exert anti-inflammatory effects, at least in part through increased expression of the TNFAIP3 gene. They also indicate that modulating expression of the TNFAIP3 gene to rebalance TLR-mediated inflammatory signaling may be potential therapeutic strategy for treating MDD.

A Negative Feedback Loop Controlling bHLH Complexes Is Involved in Vascular Cell Division and Differentiation in the Root Apical Meristem

Controlling cell division and differentiation in meristems is essential for proper plant growth. Two bHLH heterodimers consisting of LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5)/TMO5-LIKE1 (T5L1) regulate periclinal cell division in vascular cells in the root apical meristem (RAM). In this study, we further investigated the functions of LHW-T5L1, finding that in addition to controlling cell division, this complex regulates xylem differentiation in the RAM via a novel negative regulatory system. LHW-T5L1 upregulated the thermospermine synthase gene ACAULIS5 (ACL5), as well as SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3), which encodes a bHLH protein, in the RAM. The SACL3 promoter sequence contains a conserved upstream open reading frame (uORF), which blocked translation of the main SACL3 ORF in the absence of thermospermine. Thermospermine eliminated the negative effect of uORF and enhanced SACL3 production. Further genetic and molecular biological analyses indicated that ACL5 and SACL3 suppress the function of LHW-T5L1 through a protein-protein interaction between LHW and SACL3. Finally, we showed that a negative feedback loop consisting of LHW-T5L1, ACL5, SACL3, and LHW-SACL3 contributes to maintain RAM size and proper root growth. These findings suggest that a negative feedback loop regulates the LHW-T5L1 output level to coordinate cell division and differentiation in a cell-autonomous manner.

E2F1 acts as a negative feedback regulator of c-Myc‑induced hTERT transcription during tumorigenesis.

Since induction of hTERT expression and subsequent telomerase activation play a critical role in the multistep process of tumorigenesis, a better understanding of hTERT regulation may provide not only a rationale for the molecular basis of cancer progression but also a path to the development of cancer prevention. The c-Myc oncoprotein can function effectively in activating the transcriptional expression of hTERT through E-box elements on its promoter. E2F transcription factor1(E2F1) was found to be a repressor of hTERT transcription by directly binding to its promoter, thereby inhibiting hTERT protein expression. For the extensively crosstalk between c-Myc and E2F1 signals, which is now known to be vital to cell fate, we speculated that E2F1 may play a negative regulatory role in c-Myc-induced hTERT transcription. In the present study, we chose to use human embryonic fibroblast cells as an experimental model system, and present evidence that the E2F1 transcription factor constitutes a negative regulatory system to limit c-Myc transcriptional activation of hTERT in normal cells. Furthermore, we demonstrated that upregulation of the miR-17-92 cluster (miR-20a/miR-17-5p) is involved in the regulation of E2F1-mediated negative feedback of the c-Myc/hTERT pathway. Our results not only reveal novel insights into how normal cells control the transmission of c-Myc-mediated oncogenic signals, but also further establish E2F1 as an important molecular target for cancer therapy.

Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4(-/-) OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4(-/-) or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4(-/-) OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.

Osteocyte Network; a Negative Regulatory System for Bone Mass Augmented by the Induction of Rankl in Osteoblasts and Sost in Osteocytes at Unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.

ABA signaling mediated by PYR/PYL/RCAR in plants

Abscisic acid (ABA) is a phytohormone that plays critical roles in numerous developmental stages as well as in adaptive responses to biotic and abiotic stresses. Recent breakthroughs in the field of ABA signaling have indicated that there are three major components, PYR/PYL/RCAR (an ABA receptor), type 2C protein phosphates (PP2C, a negative regulator), and SNF1-related protein kinase 2 (SnRK2, a positive regulator). Further results show that these three proteins construct a double negative regulatory system, PYR/PYL/RCAR-| PP2C-| SnRK2, to regulate ABA signal responses in plant cells. Moreover, the combination patterns of these components in vivo are restricted by spatio-temporal and biochemical determinants and the combinational variation in the ABA signalosome is specific to different ABA signal responses. This review summarizes recent advances of study on the molecular basis and regulatory mechanism of PYR/PYL/RCAR-mediated ABA signaling pathway and PYR/PYL/RCAR-PP2C-SnRK2 complex-mediated ABA signal regulation network in plants. The perspectives related to this study are proposed.

Enhanced HIV-1 Gag-specific immunity induced by a sPD-1-based vaccine directed to dendritic cells (53.23)

Abstract HIV/AIDS is a serious disease worldwide. The development of a safe, effective and affordable HIV-1 vaccine remains the ultimate solution. This study is based on the discovery of a negative immune regulatory system, the PD-1/PD-L pathway, which exists on both human and other animals. The PD-1 ligands (PD-L1 and PD-L2) are expressed on DCs. So we hypothesize to utilize the soluble PD-1 parts to intervene in the negative signal transmission of the PD-1 pathway and to target the antigen to DCs as a mean to indentify vaccine strategy to enhance the virus specific immune responses. Here we constructed the novel vaccine and controls by inserting soluble PD-1 gene and/or HIV-1 p24 gene into the pVAX1 vector with fusing of rabbit Fc fragment named mspd1-p24-fc, and controls named mspd1-IgVΔ-p24-fc and p24-fc. When compared with control vaccines, we discovered that mspd1-p24fc can significantly enhance HIV-1 Gag-specific immune responses by measuring the number of IFN-γ expressed CD4 and CD8 T cells using Elispot assays and CD8 T cell tetramer staining. Furthermore, the mspd1-p24-fc can significantly enhance humoral immune responses by anti-Gag antibody titer analysis. Importantly, this novel vaccine protects mice against recombinant vaccinia virus-gagpol challenges. This novel vaccine design may be used as DNA vaccine model against other infectious disease and cancer which need eliciting significant antigen specific humoral and cellular immune responses.

Molecular Basis of the Core Regulatory Network in ABA Responses: Sensing, Signaling and Transport

ABA is a major phytohormone that regulates a broad range of plant traits and is especially important for adaptation to environmental conditions. Our understanding of the molecular basis of ABA responses in plants improved dramatically in 2009 and 2010, banner years for ABA research. There are three major components; PYR/PYL/ RCAR (an ABA receptor), type 2C protein phosphatase (PP2C; a negative regulator) and SNF1-related protein kinase 2 (SnRK2; a positive regulator), and they offer a double negative regulatory system, [PYR/PYL/RCAR—| PP2C—| SnRK2]. In the absence of ABA, PP2C inactivates SnRK2 by direct dephosphorylation. In response to environmental or developmental cues, ABA promotes the interaction of PYR/PYL/RCAR and PP2C, resulting in PP2C inhibition and SnRK2 activation. This signaling complex can work in both the nucleus and cytosol, as it has been shown that SnRK2 phosphorylates basic-domain leucine zipper (bZIP) transcription factors or membrane proteins. Several structural analyses of PYR/PYL/RCAR have provided the mechanistic basis for this ‘core signaling’ model, by elucidating the mechanism of ABA binding of receptors, or the ‘gate–latch–lock’ mechanism of interaction with PP2C in inhibiting activity. On the other hand, intercellular ABA transport had remained a major issue, as had intracellular ABA signaling. Recently, two plasma membrane-type ABC transporters were identified and shed light on the influx/efflux system of ABA, resolving how ABA is transported from cell to cell in plants. Our knowledge of ABA responses in plants has been greatly expanded from intracellular signaling to intercellular transport of ABA.

Identification of a calcium‐controlled negative regulatory system affecting Vibrio cholerae biofilm formation

Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is calcium (Ca(+2)). In this study, we investigated the response of V. cholerae to changes in extracellular Ca(2+) levels. Whole-genome expression profiling revealed that Ca(2+) decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca(2+) (LBCa(2+)) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca(2+) led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR), was decreased in cells grown in LBCa(2+). Analysis of null and overexpression alleles of carS and carR revealed that expression of vps (Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression.

Acute heat stress brings down milk secretion in dairy cows by up-regulating the activity of the milk-borne negative feedback regulatory system

BackgroundThe objective of this study was to determine if acute heat stress (HS) decreases milk secretion by activating the milk-borne negative feedback system, as an emergency physiological response to prevent a life-threatening situation. To induce HS, summer acclimatized dairy cows were exposed to full sun under mid-summer Mediterranean conditions, with and without conventional cooling procedures.ResultsExposure to HS induced a rapid and acute (within 24 h) reduction in milk yield in proportion to the heat load. This decrease was moderated by cooler night-time ambient temperature. The reduction in milk yield was associated with corresponding responses in plasminogen activator/plasminogen-plasmin activities, and with increased activity (concentration) of the (1–28) N-terminal fragment peptide that is released by plasmin from β-casein (β-CN (1–28)). These metabolites constitute the regulatory negative feedback system. Previously, it has been shown that β-CN (1–28) down-regulated milk secretion by blocking potassium channels on the apical aspects of the mammary epithelial cells.ConclusionHere we demonstrate that the potassium channels in mammary tissue became more susceptible to β-CN (1–28) activity under HS. Thus, the present study highlighted two previously unreported features of this regulatory system: (i) that it modulates rapidly in response to stressor impact variations; and (ii) that the regulations of the mammary epithelial potassium channel sensitivity to the inhibitory effect of β-CN (1–28) is part of the regulatory system.

Impairment in a negative regulatory system for TCR signaling in CD4 + T cells from old mice

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4 + T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4 + T cells have a defect in a negative SFK regulatory system.

Gonadotropin-Releasing Hormone Type II Antagonists Induce Apoptotic Cell Death in Human Endometrial and Ovarian Cancer Cells In vitro and In vivo

In human endometrial and ovarian cancers, gonadotropin-releasing hormone type I (GnRH-I), GnRH-II, and their receptors are parts of a negative autocrine regulatory system of cell proliferation. Based on a tumor-specific signal transduction, GnRH-I and GnRH-II agonists inhibit the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in down-regulation of cancer cell proliferation. Induction of apoptosis is not involved. In this study, we show that treatment of human endometrial and ovarian cancer cells with GnRH-II antagonists results in apoptotic cell death via dose-dependent activation of caspase-3. The antitumor effects of the GnRH-II antagonists could be confirmed in nude mice. GnRH-II antagonists inhibited the growth of xenotransplants of human endometrial and ovarian cancers in nude mice significantly, without any apparent side effects. Thus, GnRH-II antagonists seem to be suitable drugs for an efficacious and less toxic endocrine therapy for endometrial and ovarian cancers.

Long-distance signaling to control root nodule number

Symbiotic nitrogen fixation is beneficial to legumes. Excessive nodule development, however, disturbs the host growth by over-consuming energy from the plant. To keep a balance, legumes possess a systemic negative feedback regulatory system called 'autoregulation of nodulation', which controls the nodule number and the nodulation zone through long-distance signaling. Plants that are deficient in autoregulation display a hypernodulating phenotype. Recently, genes encoding a CLAVATA1-like receptor-like kinase that mediates autoregulation of nodulation have been identified from several legumes, such as Lotus japonicus and soybean. Other hypernodulation mutants that are regulated by shoot or root genotypes have also been isolated.

MinC Mutants Deficient in MinD- and DicB-Mediated Cell Division Inhibition Due to Loss of Interaction with MinD, DicB, or a Septal Component

The min locus encodes a negative regulatory system that limits formation of the cytokinetic Z ring to midcell by preventing its formation near the poles. Of the three Min proteins, MinC is the inhibitor and prevents Z-ring formation by interacting directly with FtsZ. MinD activates MinC by recruiting it to the membrane and conferring a higher affinity on the MinCD complex for a septal component. MinE regulates the cellular location of MinCD by inducing MinD, and thereby MinC, to oscillate between the poles of the cell, resulting in a time-averaged concentration of MinCD on the membrane that is lowest at midcell. MinC can also be activated by the prophage-encoded protein DicB, which targets MinC to the septum without recruiting it first to the membrane. Previous studies have shown that the C-terminal domain of MinC is responsible for the interaction with MinD, DicB, and the septal component. In the present study, we isolated mutations in the C-terminal domain of MinC that affected its interaction with MinD, DicB, and the septal component. Among the mutations isolated, R133A and S134A are specifically deficient in the interaction with MinD, E156A is primarily affected in the interaction with DicB, and R172A is primarily deficient in the interaction with the septum. These mutations differentiate the interactions of MinC with its partners and further support the model of MinCD- and MinC-DicB-mediated cell division inhibition.

Natriuretic peptides and myocardial ischaemia.

In both experimental and clinical myocardial ischaemia, release of BNP occurs rapidly from ventricular myocardium, prompting speculation that the early activation of the natriuretic peptide receptor/cGMP signalling system may be an important autocrine/paracrine response to ischaemia. Among the growing list of pleiotropic actions of natriuretic peptides is the attenuation of tissue susceptibility to ischaemic injury. BNP and other natriuretic peptides limit the extent of tissue infarction during ischaemia and reperfusion. The mechanism of cytoprotection is related to cGMP accumulation and opening of ATP-sensitive K(+) channels. The effects of longer-term upregulation of natriuretic peptide expression in the heart following myocardial infarction could include the suppression of growth and proliferative responses in myocytes and fibroblasts. Thus, chronic elevation of natriuretic peptide expression in infarcted myocardium probably represents a negative regulatory system to counter the pro-hypertrophic and pro-fibrotic signalling activated by other mediators such as angiotensin II and catecholamines. The acute and chronic actions of natriuretic peptides in myocardial ischaemia suggest a profile of activity that may be therapeutically beneficial in the management of patients with acute coronary syndromes and for the optimisation of post-infarction remodelling.

EXORDIUM--a gene expressed in proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis.

To identify new genes expressed in meristematic cells, a promoter trap insertional mutagenesis strategy was used in Arabidopsis thaliana. Transgenic line AtEM201 exhibits promoter trap GUS activity in embryos and in the regions of active cell division in the seedling, notably the apical meristems and young leaves. The tagged gene was named EXORDIUM (EXO). AtEM201 contains a single copy of the promoter trap T-DNA, located in the EXO gene promoter, resulting in a much reduced level of EXO transcription. Seedlings homozygous for the T-DNA insertion have no obvious mutant phenotype. The EXO gene, which forms part of a small gene family in Arabidopsis, is structurally related to the tobacco PHI-1 gene, which is re-activated in cultured cells following release from phosphate starvation-induced cell cycle arrest. Expression of both the EXO-GUS and the native EXO genes is downregulated by exogenous cytokinin. Expression studies using semisynchronised cells suggest that EXO mRNA is preferentially abundant during M phase of the cell cycle. Double mutant studies revealed that the exo mutation can suppress the defective root meristem phenotype of the hydra2 mutant, suggesting that EXO may be a component of a negative regulatory system for cell division.

Plasma leptin suppression by arginine vasopressin in normal women and men

Leptin inhibits appetite by activating several neuroendocrine systems, including the hypothalamo-pituitary-adrenal cortical (HPA) axis. In turn, elevated glucocorticoids can increase circulating leptin. We therefore measured plasma leptin in 12 normal women and eight normal men administered low-dose physostigmine (PHYSO) and arginine vasopressin (AVP) to stimulate the HPA axis. The subjects underwent four test sessions 5–7 days apart: PHYSO (8 μg/kg IV), AVP (0.08 U/kg IM), PHYSO + AVP, and saline control. Serial blood samples were taken before and after pharmacologic challenge and analyzed for leptin, adrenocorticotropin (ACTH)1–39, cortisol, and AVP. Estradiol and testosterone also were measured at each test session. PHYSO and AVP produced no side effects in about half the subjects and predominantly mild side effects in the other half, with no significant female-male differences. Correlations between side effects (absent or present) after PHYSO or AVP and the corresponding leptin responses were nonsignificant. Baseline plasma leptin concentrations were significantly higher in the women than in the men (p < 0.003). Leptin concentrations following PHYSO remained unchanged from baseline, indicating that the short-lived ACTH and cortisol increases produced by PHYSO did not affect leptin secretion. In contrast, AVP administration, while also increasing ACTH and cortisol, suppressed leptin, to a significantly greater degree in the women than in the men (p = 0.01). This significant suppression of leptin by AVP has not been previously described; physiologically, it may be part of a negative feedback regulatory system between central leptin and its activation of the HPA axis, by inhibition of leptin production or acceleration of its clearance.

Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally

Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose. S. cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S. cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration. In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression. Hxt2p was not expressed by wild-type S. cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added. An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources. Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression. Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system. High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally. In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed. Deletion of the HXT2 locus does not diminish this level of transport. However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose. Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions. Similarly, Hxt2p was found to be expressed under low-glucose conditions in an snf3 mutant which does not display high-affinity uptake. This finding suggests that SNF3 may be involved in the posttranslational regulation of Hxt2p.