RESUMO
PURPOSE: To correlate changes in urinary patient-reported outcomes including the International Prostate Symptom Score (IPSS), acute urinary retention and urethral stricture with urethral dose in those treated with low dose rate (LDR) prostate brachytherapy. MATERIALS AND METHODS: Patients treated with prostate LDR between 2012 and 2019 (n=117) completed IPSS urinary symptom assessments prior to treatment and at each follow-up. CT simulation was obtained with urinary catheter 1-month post-implant for dosimetric analysis. 113 patients with pre- and ≥1 post-LDR IPSS score available were analyzed. Urethral dosimetric parameters including U75, U100, U125, U150 and U200 were abstracted from post-implant dosimetry and assessed for association with urinary toxicity using bivariate logistic regression and Spearman correlation. Outcomes included clinically significant change (CSC, defined as 4 or more points or 25% rise above baseline) in IPSS score at 6 and 12 months, acute urinary retention (AUR), and urethral stricture (US). RESULTS: 89 (79%) patients were treated with LDR monotherapy (145 Gy) and 24 (21%) with LDR boost (110 Gy) with external beam radiation therapy. Twenty (18%) had baseline IPSS ≥15. Median IPSS scores were: baseline 6 (3-12; n=113), 1-month 17 (10-25; n=110), 6 months 12 (7-18; n=77), 1 year 8 (5-14; n=52). CSC-6 was observed in 59 (77%), CSC-12 in 26 (50%), AUR in 12 (11%), and US in 4 (4%). No association was identified between urethral dose parameters and CSC-6, CSC-12, AUR, or US. No correlation between urethral dose and IPSS at 6- and 12-months was identified. The IPSS ≥15 group exhibited lower rates of CSC-12 (13% v. 57%, p=0.05) but not CSC-6 (55% v. 80%, p=0.12). CONCLUSIONS: We did not find a relationship between urethral dose and IPSS elevation, AUR or US. We did identify a significantly lower change in IPSS at 12 months for those with baseline IPSS ≥15 compared to those with low baseline scores.
Assuntos
Braquiterapia , Sintomas do Trato Urinário Inferior , Neoplasias da Próstata , Braquiterapia/métodos , Humanos , Sintomas do Trato Urinário Inferior/diagnóstico , Sintomas do Trato Urinário Inferior/etiologia , Masculino , Próstata , Antígeno Prostático Específico , Neoplasias da Próstata/radioterapiaRESUMO
Hexaploid wheat, one of the world's most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.
Assuntos
Flores/genética , Técnicas de Transferência de Genes , Plantas Geneticamente Modificadas/genética , Transformação Genética , Triticum/genética , Agrobacterium tumefaciens/genética , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , RNA de Plantas/genética , Transgenes , Zea mays/genéticaRESUMO
This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.
Assuntos
Arabidopsis/fisiologia , Fitosteróis/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Germinação/efeitos dos fármacos , Giberelinas/metabolismo , Giberelinas/farmacologia , Fitosteróis/metabolismoRESUMO
Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GA-insensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/enzimologia , Arabidopsis/genética , Giberelinas/farmacologia , Fosfoproteínas Fosfatases/biossíntese , Fosfoproteínas Fosfatases/genética , Supressão Genética , Ácido Abscísico/genética , Ácido Abscísico/fisiologia , Alelos , Arabidopsis/crescimento & desenvolvimento , Resistência a Medicamentos/genética , Repressão Enzimática/efeitos dos fármacos , Repressão Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/genética , Giberelinas/biossíntese , Fenótipo , Supressão Genética/efeitos dos fármacosRESUMO
The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Proteínas Fúngicas/genética , Cinética , Meiose , Regiões Promotoras Genéticas , Recombinação Genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Esporos Fúngicos , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes. UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors. This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster. In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein. At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149). The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone. DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence.
Assuntos
DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Meiose/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Zinco/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Fenômenos Químicos , Físico-Química , Cromatografia em Gel , Dicroísmo Circular , Cisteína/análise , Pegada de DNA , DNA Fúngico/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Proteínas Fúngicas/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Zinco/análiseRESUMO
This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.