TorC1 and nitrogen catabolite repression control of integrated GABA shunt and retrograde pathway gene expression.

Despite our detailed understanding of how the lower GABA shunt and retrograde genes are regulated, there is a paucity of validated information concerning control of GAD1, the glutamate decarboxylase gene which catalyzes the first reaction of the GABA shunt. Further, integration of glutamate degradation via the GABA shunt has not been investigated. Here, we show that while GAD1 shares a response to rapamycin-inhibition of the TorC1 kinase, it does so independently of the Gln3 and Gat1 NCR-sensitive transcriptional activators that mediate transcription of the lower GABA shunt genes. We also show that GABA shunt gene expression increases dramatically in response to nickel ions. The α-ketoglutarate needed for the GABA shunt to cycle, thereby producing reduced pyridine nucleotides, derives from the retrograde pathway as shown by a similar high increase in the retrograde reporter, CIT2 when nickel is present in the medium. These observations demonstrate high integration of the GABA shunt, retrograde, peroxisomal glyoxylate cycle, and ß-oxidation pathways.

Recurrent MiscarriageGreen-top Guideline No. 17.

In this guideline, recurrent miscarriage has been defined as three or more first trimester miscarriages. However, clinicians are encouraged to use their clinical discretion to recommend extensive evaluation after two first trimester miscarriages, if there is a suspicion that the miscarriages are of pathological and not of sporadic nature. Women with recurrent miscarriage should be offered testing for acquired thrombophilia, particularly for lupus anticoagulant and anticardiolipin antibodies, prior to pregnancy. [Grade C] Women with second trimester miscarriage may be offered testing for Factor V Leiden, prothrombin gene mutation and protein S deficiency, ideally within a research context. [Grade C] Inherited thrombophilias have a weak association with recurrent miscarriage. Routine testing for protein C, antithrombin deficiency and methylenetetrahydrofolate reductase mutation is not recommended. [Grade C] Cytogenetic analysis should be offered on pregnancy tissue of the third and subsequent miscarriage(s) and in any second trimester miscarriage. [Grade D] Parental peripheral blood karyotyping should be offered for couples in whom testing of pregnancy tissue reports an unbalanced structural chromosomal abnormality [Grade D] or there is unsuccessful or no pregnancy tissue available for testing. [GPP] Women with recurrent miscarriage should be offered assessment for congenital uterine anomalies, ideally with 3D ultrasound. [Grade B] Women with recurrent miscarriage should be offered thyroid function tests and assessment for thyroid peroxidase (TPO) antibodies. [Grade C] Women with recurrent miscarriage should not be routinely offered immunological screening (such as HLA, cytokine and natural killer cell tests), infection screening or sperm DNA testing outside a research context. [Grade C] Women with recurrent miscarriage should be advised to maintain a BMI between 19 and 25 kg/m2 , smoking cessation, limit alcohol consumption and limit caffeine to less than 200 mg/day. [Grade D] For women diagnosed with antiphospholipid syndrome, aspirin and heparin should be offered from a positive test until at least 34 weeks of gestation, following discussion of potential benefits versus risks. [Grade B] Aspirin and/or heparin should not be given to women with unexplained recurrent miscarriage. [Grade B] There are currently insufficient data to support the routine use of PGT-A for couples with unexplained recurrent miscarriage, while the treatment may carry a significant cost and potential risk. [Grade C] Resection of a uterine septum should be considered for women with recurrent first or second trimester miscarriage, ideally within an appropriate audit or research context. [Grade C] Thyroxine supplementation is not routinely recommended for euthyroid women with TPO who have a history of miscarriage. [Grade A] Progestogen supplementation should be considered in women with recurrent miscarriage who present with bleeding in early pregnancy (for example 400 mg micronised vaginal progesterone twice daily at the time of bleeding until 16 weeks of gestation). [Grade B] Women with unexplained recurrent miscarriage should be offered supportive care, ideally in the setting of a dedicated recurrent miscarriage clinic. [Grade C].

Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage.

BACKGROUND: Recurrent pregnancy loss, (RPL) affecting 1%-2% of couples, is defined as ≥3 consecutive pregnancy losses before 20-week' gestation. Women with RPL are routinely screened for etiological factors, but routine screening of male partners is not currently recommended. Recently it has been suggested that sperm quality is reduced in male partners of women with RPL, but the reasons underlying this lower quality are unclear. We hypothesized that these men may have underlying impairments of reproductive endocrine and metabolic function that cause reductions in sperm quality. METHODS: After ethical approval, reproductive parameters were compared between healthy controls and male partners of women with RPL. Semen reactive oxygen species (ROS) were measured with a validated inhouse chemiluminescent assay. DNA fragmentation was measured with the validated Halosperm method. RESULTS: Total sperm motility, progressive sperm motility, and normal morphology were all reduced in the RPL group vs controls. Mean ±SE morning serum testosterone (nmol/L) was 15% lower in RPL than in controls (controls, 19.0 ± 1.0; RPL, 16.0 ± 0.8; P < 0.05). Mean ±SE serum estradiol (pmol/L) was 16% lower in RPL than in controls (controls, 103.1 ± 5.7; RPL, 86.5 ± 3.4; P < 0.01). Serum luteinizing hormone and follicle-stimulating hormone were similar between groups. Mean ±SE ROS (RLU/sec/106 sperm) were 4-fold higher in RPL than in controls (controls, 2.0 ± 0.6; RPL, 9.1 ± 4.1; P < 0.01). Mean ±SE sperm DNA fragmentation (%) was 2-fold higher in RPL than in controls (controls, 7.3 ± 1.0; RPL, 16.4 ± 1.5; P < 0.0001). CONCLUSIONS: Our data suggest that male partners of women with RPL have impaired reproductive endocrine function, increased levels of semen ROS, and sperm DNA fragmentation. Routine reproductive assessment of the male partners may be beneficial in RPL.

A Randomized Trial of Progesterone in Women with Recurrent Miscarriages.

BACKGROUND: Progesterone is essential for the maintenance of pregnancy. However, whether progesterone supplementation in the first trimester of pregnancy would increase the rate of live births among women with a history of unexplained recurrent miscarriages is uncertain. METHODS: We conducted a multicenter, double-blind, placebo-controlled, randomized trial to investigate whether treatment with progesterone would increase the rates of live births and newborn survival among women with unexplained recurrent miscarriage. We randomly assigned women with recurrent miscarriages to receive twice-daily vaginal suppositories containing either 400 mg of micronized progesterone or matched placebo from a time soon after a positive urinary pregnancy test (and no later than 6 weeks of gestation) through 12 weeks of gestation. The primary outcome was live birth after 24 weeks of gestation. RESULTS: A total of 1568 women were assessed for eligibility, and 836 of these women who conceived naturally within 1 year and remained willing to participate in the trial were randomly assigned to receive either progesterone (404 women) or placebo (432 women). The follow-up rate for the primary outcome was 98.8% (826 of 836 women). In an intention-to-treat analysis, the rate of live births was 65.8% (262 of 398 women) in the progesterone group and 63.3% (271 of 428 women) in the placebo group (relative rate, 1.04; 95% confidence interval [CI], 0.94 to 1.15; rate difference, 2.5 percentage points; 95% CI, -4.0 to 9.0). There were no significant between-group differences in the rate of adverse events. CONCLUSIONS: Progesterone therapy in the first trimester of pregnancy did not result in a significantly higher rate of live births among women with a history of unexplained recurrent miscarriages. (Funded by the United Kingdom National Institute of Health Research; PROMISE Current Controlled Trials number, ISRCTN92644181.).

Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms.

Nitrogen catabolite repression (NCR)-sensitive transcription is activated by Gln3 and Gat1. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc(13) exhibited two constitutively produced species. Investigating this paradox, we demonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc(13) tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UASGATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61.

A domain in the transcription activator Gln3 specifically required for rapamycin responsiveness.

Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.

gln3 mutations dissociate responses to nitrogen limitation (nitrogen catabolite repression) and rapamycin inhibition of TorC1.

The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, "good" nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, "poor" nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.

Genetic analysis of phage Mu Mor protein amino acids involved in DNA minor groove binding and conformational changes.

Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, P(m), requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a -10 hexamer, but no -35 hexamer. Instead P(m) has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing -35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a ß-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the ß-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the ß-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A(3) demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.

N- and C-terminal Gln3-Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization.

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656-666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3-Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3-Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3-Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3-Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3-15-fold with three of them by 10-15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3-Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

Prevalence and pattern of congenital heart disease in school children of eastern Uttar Pradesh.

BACKGROUND AND AIM: The prevalence of congenital heart disease (CHD) is not known in our country. The aim of present study was to find out the prevalence of CHD in school children of eastern Uttar Pradesh. METHOD: A team consisting of a cardiologist, physicians and junior residents visited schools in the area. All the children were examined for presence of cardiac murmur or history of heart disease or any intervention. Those with murmurs or previous history of heart disease were called to the Medical College Hospital for evaluation by ECG, chest X-ray and echocardiography for confirmation of the lesion. RESULTS: Out of 118,212 children examined, 142 were found to have CHD. The prevalence was 1.3 per 1000 children and the commonest lesions were ventricular and atrial septal defects, aortic stenosis with or without regurgitation, and pulmonary stenosis. CONCLUSION: CHD prevalence is 1.3 per 1000 school children that is nearly two and a half times more than that of RHD. Knowing it is important for development of facilities for CHD care in our setup.

More than One Way in: Three Gln3 Sequences Required To Relieve Negative Ure2 Regulation and Support Nuclear Gln3 Import in Saccharomyces cerevisiae.

Gln3 is responsible for Nitrogen Catabolite Repression-sensitive transcriptional activation in the yeast Saccharomyces cerevisiae In nitrogen-replete medium, Gln3 is cytoplasmic and NCR-sensitive transcription is repressed. In nitrogen-limiting medium, in cells treated with TorC1 inhibitor, rapamycin, or the glutamine synthetase inhibitor, methionine sulfoximine (Msx), Gln3 becomes highly nuclear and NCR-sensitive transcription derepressed. Previously, nuclear Gln3 localization was concluded to be mediated by a single nuclear localization sequence, NLS1. Here, we show that nuclear Gln3-Myc13 localization is significantly more complex than previously appreciated. We identify three Gln3 sequences, other than NLS1, that are highly required for nuclear Gln3-Myc13 localization. Two of these sequences exhibit characteristics of monopartite (K/R-Rich NLS) and bipartite (S/R NLS) NLSs, respectively. Mutations altering these sequences are partially epistatic to a ure2Δ. The third sequence, the Ure2 relief sequence, exhibits no predicted NLS homology and is only necessary when Ure2 is present. Substitution of the basic amino acid repeats in the Ure2 relief sequence or phosphomimetic aspartate substitutions for the serine residues between them abolishes nuclear Gln3-Myc13 localization in response to both limiting nitrogen and rapamycin treatment. In contrast, Gln3-Myc13 responses are normal in parallel serine-to-alanine substitution mutants. These observations suggest that Gln3 responses to specific nitrogen environments likely occur in multiple steps that can be genetically separated. At least one general step that is associated with the Ure2 relief sequence may be prerequisite for responses to the specific stimuli of growth in poor nitrogen sources and rapamycin inhibition of TorC1.

Prevalence of rheumatic heart disease in school-going children of Eastern Uttar Pradesh.

BACKGROUND: Rheumatic heart disease is a major health problem in our country. There is evidence from South India that its prevalence is declining. This study attempts to confirm whether this is so in North India as well. METHODS AND RESULTS: A total of 118,212 (68,357 males, 49,855 females) schoolchildren in the age group of 4-18 years were examined for the presence of heart disease. Evaluation, including echocardiography, confirmed that of a total of 389 suspected to have heart disease, 61 had rheumatic heart disease. Thus, the prevalence of rheumatic heart disease was found to be approximately 0.5 per 1000 children. CONCLUSION: In a fairly large school survey conducted by us, the prevalence of rheumatic heart disease turned out to be approximately 0.5 per 1000 children. This is the lowest figure reported from our country so far and confirms the decline of this disease in our country.

General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization.

Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1 In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3 However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc13 localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2 Bmh1/2 are also required for nuclear Gln3-Myc13 localization and appear to function downstream of Ure2 Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2 Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.

Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration.

A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc(13) sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477-493) region required for effective cytoplasmic Gln3-Myc(13) sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc(13) to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc(13) sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477-493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.

PROMISE: first-trimester progesterone therapy in women with a history of unexplained recurrent miscarriages - a randomised, double-blind, placebo-controlled, international multicentre trial and economic evaluation.

BACKGROUND AND OBJECTIVES: Progesterone is essential to maintain a healthy pregnancy. Guidance from the Royal College of Obstetricians and Gynaecologists and a Cochrane review called for a definitive trial to test whether or not progesterone therapy in the first trimester could reduce the risk of miscarriage in women with a history of unexplained recurrent miscarriage (RM). The PROMISE trial was conducted to answer this question. A concurrent cost-effectiveness analysis was conducted. DESIGN AND SETTING: A randomised, double-blind, placebo-controlled, international multicentre study, with economic evaluation, conducted in hospital settings across the UK (36 sites) and in the Netherlands (nine sites). PARTICIPANTS AND INTERVENTIONS: Women with unexplained RM (three or more first-trimester losses), aged between 18 and 39 years at randomisation, conceiving naturally and giving informed consent, received either micronised progesterone (Utrogestan(®), Besins Healthcare) at a dose of 400 mg (two vaginal capsules of 200 mg) or placebo vaginal capsules twice daily, administered vaginally from soon after a positive urinary pregnancy test (and no later than 6 weeks of gestation) until 12 completed weeks of gestation (or earlier if the pregnancy ended before 12 weeks). MAIN OUTCOME MEASURES: Live birth beyond 24 completed weeks of gestation (primary outcome), clinical pregnancy at 6-8 weeks, ongoing pregnancy at 12 weeks, miscarriage, gestation at delivery, neonatal survival at 28 days of life, congenital abnormalities and resource use. METHODS: Participants were randomised after confirmation of pregnancy. Randomisation was performed online via a secure internet facility. Data were collected on four occasions of outcome assessment after randomisation, up to 28 days after birth. RESULTS: A total of 1568 participants were screened for eligibility. Of the 836 women randomised between 2010 and 2013, 404 received progesterone and 432 received placebo. The baseline data (age, body mass index, maternal ethnicity, smoking status and parity) of the participants were comparable in the two arms of the trial. The follow-up rate to primary outcome was 826 out of 836 (98.8%). The live birth rate in the progesterone group was 65.8% (262/398) and in the placebo group it was 63.3% (271/428), giving a relative risk of 1.04 (95% confidence interval 0.94 to 1.15; p = 0.45). There was no evidence of a significant difference between the groups for any of the secondary outcomes. Economic analysis suggested a favourable incremental cost-effectiveness ratio for decision-making but wide confidence intervals indicated a high level of uncertainty in the health benefits. Additional sensitivity analysis suggested the probability that progesterone would fall within the National Institute for Health and Care Excellence's threshold of £20,000-30,000 per quality-adjusted life-year as between 0.7145 and 0.7341. CONCLUSIONS: There is no evidence that first-trimester progesterone therapy improves outcomes in women with a history of unexplained RM. LIMITATIONS: This study did not explore the effect of treatment with other progesterone preparations or treatment during the luteal phase of the menstrual cycle. FUTURE WORK: Future research could explore the efficacy of progesterone supplementation administered during the luteal phase of the menstrual cycle in women attempting natural conception despite a history of RM. TRIAL REGISTRATION: Current Controlled Trials ISRCTN92644181; EudraCT 2009-011208-42; Research Ethics Committee 09/H1208/44. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 41. See the NIHR Journals Library website for further project information.

Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae.

A leucine, leucyl-tRNA synthetase-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine.

Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell's overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64-73 are required for nuclear Gln3 export.

GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation.

The TorC1 protein kinase complex is a central component in a eukaryotic cell's response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g., proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor-dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3-Myc(13) localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells' nitrogen environment.

Ammonia-specific regulation of Gln3 localization in Saccharomyces cerevisiae by protein kinase Npr1.

Events directly regulating Gln3 intracellular localization and nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae are interconnected with many cellular processes that influence the utilization of environmental metabolites. Among them are intracellular trafficking of the permeases that transport nitrogenous compounds and their control by the Tor1,2 signal transduction pathway. Npr1 is a kinase that phosphorylates and thereby stabilizes NCR-sensitive permeases, e.g. Gap1 and Mep2. It is also a phosphoprotein for which phosphorylation and kinase activity are regulated by Tor1,2 via Tap42 and Sit4. Npr1 has been reported to negatively regulate nuclear localization of Gln3 in SD (ammonia)-grown cells. Thus we sought to distinguish whether Npr1: (i) functions directly as a component of NCR control; or (ii) influences Gln3 localization indirectly, possibly as a consequence of participating in protein trafficking. If Npr1 functions directly, then the ability of all good nitrogen sources to restrict Gln3 to the cytoplasm should be lost in an npr1Delta just as occurs when URE2 (encoding this well studied negative Gln3 regulator) is deleted. We show that nuclear localization of Gln3-Myc(13) in an npr1Delta occurred only with ammonia as the nitrogen source. Other good nitrogen sources, e.g. glutamine, serine, or asparagine, restricted Gln3-Myc(13) to the cytoplasm of both wild type and npr1Delta cells. In other words, the npr1Delta did not possess the uniform phenotype for all repressive nitrogen sources characteristic of ure2Delta. This suggests that the connection between Gln3 localization and Npr1 is indirect, arising from the influence of Npr1 on the ability of cells to utilize ammonia as a repressive nitrogen source.