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PubMed2006

F-box proteins: more than baits for the SCF?

Regulation of protein stability through the ubiquitin proteasome system is a key mechanism underlying numerous cellular processes. The ubiquitin protein ligases (or E3) are in charge of substrate specificity and therefore play a pivotal role in the pathway. Among the several different E3 enzyme families, the SCF (Skp1-Cullin-F box protein) is one of the largest and best characterized. F-box proteins, in addition to the loosely conserved F-box motif that binds Skp1, often carry typical protein interaction domains and are proposed to recruit the substrate to the SCF complex. Strikingly, genomes analysis revealed the presence of large numbers of F-box proteins topping to nearly 700 predicted in Arabidopsis thaliana. Recent evidences in various species suggest that some F-box proteins have functions not directly related to the SCF complex raising questions about the actual connection between the large F-box protein family and protein degradation, but also about their origins and evolution.

Editorial↗

PubMed2000

Identification of a new missense mutation (Gly95Glu) in a highly conserved codon within the high-mobility group box of the sex-determining region Y gene: report on a 46,XY female with gonadal dysgenesis and yolk-sac tumor.

Leydig cells and Sertoli cells of the testes produce hormones that cause male differentiation, if receptors are present. The Y chromosomal SRY gene (sex determining Region Y gene) acts as TDF and is required for regular male sex determination. SRY represents a transcription factor belonging to the superfamily of genes sharing the HMG-box motif(high-mobility group-box), which acts as DNA binding region. Here, we describe a nonmosaic XY sex-reversed female with pure gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, absence of Wolffian ducts, streak gonads) who harbored a yolk-sac tumor and was referred for the assessment of primary amenorrhea. Using genomic PCR analysis, a 423-bp PCR product, encompassing the HMG-box of the SRY gene, was amplified from the proposita, her father, and her three brothers, whereas no band was visible in the patient's mother and her three sisters. The PCR products were sequenced for mutations subsequently. A new de novo missense mutation within the HMG-box of the SRY gene was discovered in the proposita. A G is replaced by an A in codon 95 at position +284, resulting in the replacement of the nonpolar aminoacid glycine by the polar amino acid glutamate. The glycine at codon 95 is highly conserved between the family of HMG-box proteins and between species. This point mutation has not been described earlier and brings the total number of SRY mutations described so far to 36, each mutation being unique. This mutation was not detected in the patient's father and her male siblings. The present data provide further evidence to support the functional importance of the putative DNA binding activity of the SRY HMG-box domain.

Adult↗

PubMed2004

A regulatory network of T-box genes and the even-skipped homologue vab-7 controls patterning and morphogenesis in C. elegans.

T-box genes form a large family of conserved transcription factors with diverse roles in animal development, but so far functions for only a few have been studied in detail. Here we show that four Caenorhabditis elegans T-box genes and the even-skipped-like homeobox gene vab-7 function within a regulatory network to control embryonic patterning and morphogenesis. tbx-8 and tbx-9 have functionally redundant roles in the intercalation of posterior dorsal hypodermal cells, in muscle cell positioning and in intestinal development. Inhibiting tbx-9 alone using RNA interference (RNAi) produces worms that have a thickened, 'bobbed tail' phenotype, similar to that seen in mutants of vab-7, which itself has been shown to pattern posterior muscle and hypodermal cells. In support of the view that these genes function in the same pathway, we find that tbx-8 and tbx-9 are both necessary and sufficient for vab-7 expression. In addition, a third T-box gene, tbx-30, acts to repress vab-7 expression in the anterior of embryos. We further show that vab-7 itself represses the T-box gene mab-9 in posterior cells. Thus, during posterior patterning in C. elegans, there are multiple interactions between T-box genes and the vab-7 homeobox gene. Evolutionary parallels in other organisms suggest that regulatory interactions between T-box genes and even-skipped homologues are conserved.

Animals↗

PubMed1998

Operant studies to determine the strength of preference in laboratory rats for nest-boxes and nesting materials.

Previous work has shown that laboratory rats preferred to use nest-boxes and nesting materials rather than empty parts of the cage. In preference tests, they chose opaque or semi-opaque nest-boxes and long strips of soft paper nesting material. Choice tests to demonstrate a preference between nest-boxes and nesting material were not possible because nesting materials were carried into the nest-boxes. Furthermore, preference tests did not show how important these items were to the animals. Accordingly, operant tests were conducted, in which the rats had to lift a weighted door in order to gain access to an empty cage, or one containing a nest-box, nesting material or both items. By progressively increasing the weight of the door in subsequent trials, it was shown that the rats would carry out more work to reach a nest-box, with or without nesting material, than to reach an empty cage.

Animals↗

PubMed2006

The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation.

Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.

Amino Acid Sequence↗

PubMed2004

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs.

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.

Blotting, Western↗

PubMed2004

Usefulness of tobacco check boxes on death certificates: Texas, 1987-1998.

OBJECTIVES: We compared reports of deaths in which tobacco use was a contributing factor ("tobacco-associated deaths") before and after the addition to death certificates in Texas of a check-box question asking whether tobacco use contributed to an individual's death. METHODS: We examined Texas vital statistics files from 1987 to 1998. We calculated differences in percentages of reported tobacco-associated deaths (and 95% confidence intervals [CIs]) for the periods 1987 to 1992, before the addition of the check-box question, and 1993 to 1998, after the addition of the check-box. RESULTS: Reports of tobacco-associated deaths were significantly less frequent before addition of the check-box question (0.7%; 95% CI = 0.4%, 1.0%) than after addition of the question (13.9%; 95% CI = 13.0%, 14.7%). From 1993 to 1998, percentages of tobacco-associated deaths reported on the check-box question increased steadily. CONCLUSIONS: The addition of a tobacco-associated-death check box on Texas death certificates significantly increased reporting of tobacco use contributions to mortality.

Cause of Death↗

PubMed2004

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization.

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.

Amino Acid Sequence↗

PubMed1989

The box plot: a simple visual method to interpret data.

Exploratory data analysis involves the use of statistical techniques to identify patterns that may be hidden in a group of numbers. One of these techniques is the "box plot," which is used to visually summarize and compare groups of data. The box plot uses the median, the approximate quartiles, and the lowest and highest data points to convey the level, spread, and symmetry of a distribution of data values. It can also be easily refined to identify outlier data values and can be easily constructed by hand. We apply box plots to tabular data from two recently published articles to show how readers can use box plots to improve the interpretation of data in complex tables. The box plot, like other visual methods, is more than a substitute for a table: It is a tool that can improve our reasoning about quantitative information. We recommend that the box plot be used more frequently.

Alcohol Drinking↗

PubMed2004

Pathology of aural abscesses in free-living Eastern box turtles (Terrapene carolina carolina).

Aural abscess or abscess of the middle ear is common in free-living Eastern box turtles (Terrapene carolina carolina) of Virginia (USA) and elsewhere. Although its etiology remains unknown, hypovitaminosis A has been suggested on the basis of similar lesions occurring in captive chelonians fed diets that are deficient in vitamin A. This hypothesis was supported by significantly greater body burdens of organochlorine compounds (reported disruptors of vitamin A metabolism) and a nonsignificant trend toward lower serum and hepatic vitamin A levels in free-living box turtles with this lesion. The tympanic epithelium was evaluated in 27 box turtles (10 with aural abscesses and 17 without). Lesions of the tympanic epithelium of box turtles with aural abscesses included hyperplasia, squamous metaplasia, hyperemia, cellular sloughing, granulomatous inflammation, and bacterial infection. These changes were more severe in turtles with aural abscesses than in those without and were more severe in tympanic cavities that had an abscess compared to those without when the lesion was unilateral. Organs from 21 box turtles (10 with aural abscesses and 11 without) from the study population were examined for microscopic lesions, and minimal histopathologic changes were found, none of which were similar to those found in the tympanic epithelium. Histopathologic changes in box turtles with aural abscesses were consistent with a syndrome that may involve hypovitaminosis A.

Abscess↗

PubMed1999

The myogenic regulatory circuit that controls cardiac/slow twitch troponin C gene transcription in skeletal muscle involves E-box, MEF-2, and MEF-3 motifs.

We have characterized the specific DNA regulatory elements responsible for the function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in myogenic cells. We used functional transient transfection assays with deletional and site-specific mutagenesis to evaluate the role of the conserved sequence elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the ability of the functional sites to interact with nuclear proteins. We demonstrate that three distinct transcription activator binding sites commonly found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least four redundant E-box sites) all contribute to full enhancer activity but a CArG box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the E-boxes specifically bind, respectively, to MEF-2 and myogenic determination factors derived from nuclear extracts. EMSA assays using a MEF-3 containing oligonucleotide revealed indistinguishable separation patterns with extracts from myogenic cells and nonmyogenic cells. These data suggest that expression of the cTnC gene in slow-twitch skeletal muscle is sustained through complex interactions at the 3'Ile enhancer between muscle-specific and nontissue-specific transcription factors: either a myogenic bHLH complex or MEF-2 can activate transcription but only in the presence of a third transcriptional activator that appears not to be muscle specific. We conclude from these observations that the cTnC 3'Ile element is a composite enhancer that functions through the combined interactions of at least five regulatory elements and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a MEF-3 site. The data support the notion that all of these sites contribute to enhancer function in cell systems in an additive way but that none are absolutely required for enhancer activity. The data imply that the levels of transcription of cTnC in myogenic tissues in which the activities of one of the transcriptional factors is lacking would be partially but not wholly suppressed. Our data support the critical role of E-box sites in conjunction with the adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary" rather than to the "novel" category of transcriptional regulation during skeletal myogenesis.

Base Sequence↗

PubMed2001

MADS-box genes are involved in floral development and evolution.

MADS-box genes encode transcription factors in all eukaryotic organisms thus far studied. Plant MADS-box proteins contain a DNA-binding (M), an intervening (I), a Keratin-like (K) and a C-terminal C-domain, thus plant MADS-box proteins are of the MIKC type. In higher plants most of the well-characterized genes are involved in floral development. They control the transition from vegetative to generative growth and determine inflorescence meristem identity. They specify floral organ identity as outlined in the ABC model of floral development. Moreover, in Antirrhinum majus the MADS-box gene products DEF/GLO and PLE control cell proliferation in the developing flower bud. In this species the DEF/GLO and the SQUA proteins form a ternary complex which determines the overall "Bauplan" of the flower. Phylogenetic reconstructions of MADS-box sequences obtained from ferns, gymnosperms and higher eudicots reveal that, although ferns possess already MIKC type genes, these are not orthologous to the well characterized MADS-box genes from gymnosperms or angiosperms. Putative orthologs of floral homeotic B- and C-function genes have been identified in different gymnosperms suggesting that these genes evolved some 300-400 million years ago. Both gymnosperms and angiosperms also contain a hitherto unknown sister clade of the B-genes, which we termed Bsister. A novel hypothesis will be described suggesting that B and Bsister might be involved in sex determination of male and female reproductive organs, respectively.

Evolution, Molecular↗

PubMed2006

[Sequencing the Rhesus boxes and determining the homozygosity of RHD gene in Han Chinese with RhD negative].

OBJECTIVE: To analyze the sequences of Rhesus boxes of RhD gene, and explore the genetic mechanism of RhD negative phenotype in Chinese Han population. Meanwhile the PCR product of Rhesus boxes is analyzed for determining RHD gene homozygosity. METHODS: DNA of 74 RhD negative samples were firstly analyzed with multiplex PCR-sequence specific primer(SSP). The further analysis was given to Rhesus boxes specific sequencing and RHD gene homozygosity determined by PCR-restriction fragment length polymorphism(RFLP) analysis to Rhesus boxes. RESULTS: In DNA samples of 74 RhD negative individuals, 46 samples(62%) showed the absence and homozygous negative of RHD gene; 22 samples(30%) all showed the existence of RHD specific exons, of which 19 were RHD gene heterozygous and 3 were homozygous; regardless of PCR-RFLP analysis showing no RHD specific exons, but further analysis of RHD specific PCR revealed one RHD gene, at least RHD gene exon 1 and 10 existing in 5 DNA samples(7%); 1 sample(1%) was lacking RHD exon 6 although the multiplex PCR showed the RHD gene to be positive. Analyzing the hybrid Rhesus box of 27 RhD negative samples revealed the Han Chinese population to have the same DNA sequence of hybrid Rhesus box as Caucasians. CONCLUSION: The RHD gene deletion is the main molecular mechanism of causing RhD negative formed in Han Chinese population, who have had the RHD gene deletion taken place within the defined breakpoint region as Caucasians.

Asian People↗

PubMedDate not supplied

[Yielding of xenodiagnosis, according to the number of boxes used in 1,181 persons with chronic chagasic infection diagnosed with indirect hemagglutination reaction].

A study for evaluation of the yielding of xenodiagnosis (XD) in 1,181 persons with a previous positive indirect hemagglutination test (IHAT) for Trypanosoma cruzi infection was carried out. The infection of these people was detected during epidemiological surveys performed in rural-periurban and urban sections of the endemo-enzootic area of Chagas' disease in Chile, which involves the first seven, out of the thirteen political-administrative regions of the country. The sex distribution was 75.0% females and 25.0% males, varying the ages between 2 and 80 years. According to individual and geographical possibilities each person was submitted to 1-8 XD, which consisted in cylindrical wooden boxes containing seven third instar nymphs, laboratory reared, of Triatoma infestans. The boxes, covered with a piece of tulle fixed with a rubber band were applied on the skin of the posterior side of the arm of the subject to be examined, held with a linen bracelet during 25-30 min. After the insects were fed the boxes were maintained in the laboratory at 27 degrees C and 85% relative environmental humidity. Posteriorly, all the nymphs of each box were examined at 30, 60 and 90 days after the application. A drop of abdominal content of each of them, homogenized with a drop of saline, was examined at the microscope looking for T. cruzi. XD resulted positive in 503 (42.6%) people. The positiveness of XD showed a trend of increasing according to the number of boxes used, from 11.4% with one to 51.6% with six. Under a practical point of view, the simultaneous application of four XD boxes seems to be advisable.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1989

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

Base Sequence↗

PubMed1988

Dermatitis from plastic tote boxes impregnated with an antistatic agent.

An outbreak of dermatitis occurred among employees of a microelectronics firm. In a cross-sectional epidemiologic investigation, we found that dermatitis of the hands or arms had occurred among 14 of 29 (48.3%) employees of the incoming inspection department where plastic tote boxes recently purchased from one manufacturer had been used, compared to only one case among 17 (5.9%) employees in another department which had not used these boxes. Affected workers could detect an oily film on the surfaces of these new boxes, but not on older ones. We identified the oily film to be a surface accumulation of bis-hydroxyethyl-tallow amine (BHETA), an antistatic agent with which the tote boxes had been impregnated. Subsequent toxicologic investigation established that BHETA could provoke both follicular and nonfollicular irritant dermatitis, and was also a potential skin sensitizer. Antistatic agents should be considered as potential causes of dermatitis among employees who handle electrical parts transported in plastic boxes, particularly when affected employees can detect an oily film on the box surfaces.

Amines↗

PubMed1978

[Construction and operation of an anaerobic glove box (author's transl)].

Construction and operation of an anaerobic glove box is described mainly built of commercially available material. The box is made of refined steel and has the dimensions of 150 cm in length, 62 cm in height and 68 cm in depth. It is equipped with one round vacuum tight steel airlock for transfer of material. Three Neopren gloves and an oblique glass window are installed into the frontside. The anaerobic box is continuously perfused by 35 l/h of O2-free gas. In the box the atmosphere is whirled round by a fan and hereby led over a layer of catalyst. The O2-content of the anaerobic atmosphere is less than 1 ppm-3 ppm to 4 ppm on a yearly average - measured by an oxygen trace analysor. All bacteriological work as cultivation and isolation of strictly anaerobic bacteria can be done within a continuous O2-free atmosphere by conventional technics. The anaerobic glove box described was used for three years without any failure. In glove boxes built in the same manner as above mentioned similar O2-values will be achievable.

Anaerobiosis↗

PubMed1994

Structure, chromosomal locus, and promoter analysis of the gene encoding the mouse helix-loop-helix factor HES-1. Negative autoregulation through the multiple N box elements.

HES-1 is a mammalian helix-loop-helix factor structurally related to the Drosophila hairy and Enhancer of split proteins. It binds more preferentially to the N box (CACNAG) than to the E box (CANNTG) and acts as a negative regulator. In this study, we have isolated and characterized the mouse HES-1 gene. This gene consists of four exons, and the positions of introns are well conserved when compared with those of the Drosophila hairy gene, except for the third intron. Southern blot and interspecies backcross analyses suggest that the mouse HES-1 gene is a single-copy gene and is located around position 26 on chromosome 16. The transcription initiation site, determined by the S1 nuclease and primer extension experiments, is located 31 nucleotides downstream of a TATA box. In the 5'-regulatory region, there are four N box sequences, and the DNase I foot-printing and gel mobility shift analyses show that HES-1 binds to these sequences. Transient transfection assays using C3H10T1/2 cells suggest that there are several positive regulatory regions in the HES-1 gene. However, cotransfection of the HES-1 expression vector leads to approximately 40-fold repression in promoter activity. Furthermore, when the N box sequences are disrupted, this negative regulation is severely impaired. These results raise the possibility that HES-1 gene expression may be negatively autoregulated through the N box sequences.

Amino Acid Sequence↗