Biomedical subjects
A Jacobson
Publications and source records attributed to A Jacobson.
Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay.
The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD). A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified. The HUpf1 has also been shown to play a role in NMD in mammalian cells. Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved. The yeast Upf1p demonstrates RNA-dependent ATPase and 5' --> 3' helicase activities. In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein. We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5'--> 3' helicase activity. Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP. Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.
The perception of optimal profile in African Americans versus white Americans as assessed by orthodontists and the lay public.
This study was designed to assess the perceived optimal profiles of African Americans versus white Americans. A survey was conducted using profile silhouettes of 30 African American and 30 white patients, ranging in age from 7 to 17 years. Twenty white orthodontists, 18 African American orthodontists, 20 white laypersons, and 20 African American laypersons evaluated the profiles. The preference of each rater for each of the 60 profiles was scored on an attached visual analog scale. Eighteen cephalometric variables were measured for each profile, and statistical analyses were performed on the profiles that had a mean rating of 60 or greater from an analog scale of 0 to 100. The results show the following 6 cephalometric variables were significant: Z-angle, skeletal convexity at A-point, upper lip prominence, lower lip prominence, nasomental angle, and mentolabial sulcus. All raters preferred the African American sample to have a greater profile convexity than they preferred for the white sample. The raters preferred the African American sample with upper and lower lips that were more prominent compared with the white sample. However, only the choice of the African American orthodontists for the African American sample was significantly different for this parameter. The white orthodontists gave the highest mean scores for the profile chosen, whereas the African American laypersons gave the lowest scores.
Upf1p control of nonsense mRNA translation is regulated by Nmd2p and Upf3p.
Upf1p, Nmd2p, and Upf3p regulate the degradation of yeast mRNAs that contain premature translation termination codons. These proteins also appear to regulate the fidelity of termination, allowing translational suppression in their absence. Here, we have devised a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100), and used it to determine the regulatory roles of the UPF/NMD gene products. Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy-number can1-100 expression in wild-type cells caused an increase in mRNA abundance similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. Our findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay. Consistent with these relationships, Upf1p, Nmd2p, and Upf3p were shown to be present at 1, 600, 160, and 80 molecules per cell, levels that underscored the importance of Upf1p but minimized the likelihood that these proteins were associated with all ribosomes or that they functioned as a stoichiometric complex.
mRNA stability and polysome loss in hibernating Arctic ground squirrels (Spermophilus parryii).
All small mammalian hibernators periodically rewarm from torpor to high, euthermic body temperatures for brief intervals throughout the hibernating season. The functional significance of these arousal episodes is unknown, but one suggestion is that rewarming may be related to replacement of gene products lost during torpor due to degradation of mRNA. To assess the stability of mRNA as a function of the hibernation state, we examined the poly(A) tail lengths of liver mRNA from arctic ground squirrels sacrificed during four hibernation states (early and late during a torpor bout and early and late following arousal from torpor) and from active ground squirrels sacrificed in the summer. Poly(A) tail lengths were not altered during torpor, suggesting either that mRNA is stabilized or that transcription continues during torpor. In mRNA isolated from torpid ground squirrels, we observed a pattern of 12 poly(A) residues at greater densities approximately every 27 nucleotides along the poly(A) tail, which is a pattern consistent with binding of poly(A)-binding protein. The intensity of this pattern was significantly reduced following arousal from torpor and undetectable in mRNA obtained from summer ground squirrels. Analyses of polysome profiles revealed a significant reduction in polyribosomes in torpid animals, indicating that translation is depressed during torpor.
An internal open reading frame triggers nonsense-mediated decay of the yeast SPT10 mRNA.
Yeast cells containing a temperature-sensitive mutation in the PRT1 gene were found to selectively stabilize mRNAs harboring early nonsense codons. The similarities between the mRNA decay phenotypes of prt1-1 cells and those lacking the nonsense-mediated mRNA decay (NMD) factor Upf1p led us to determine whether both types of mutations cause the accumulation of the same mRNAs. Differential display analysis and mRNA half-life measurements demonstrated that the HHF2 mRNA increased in abundance in prt1-1 and upf1Delta cells, but did not manifest a change in decay rate. In both mutant strains this increase was attributable to stabilization of the SPT10 transcript, an mRNA encoding a transcriptional regulator of HHF2. Analyses of chimeric mRNAs used to identify the cis-acting basis for NMD of the SPT10 mRNA indicated that ribosomes scan beyond its initiator AUG and initiate at the next downstream AUG, resulting in premature translation termination. By searching a yeast database for transcripts with sequence features similar to those of the SPT10 mRNA, other transcripts that decay by the NMD pathway were identified. Our results demonstrate that mRNAs undergoing leaky scanning are a new class of endogenous NMD substrate, and suggest the existence of a novel cellular regulatory circuit.
Tools for turnover: methods for analysis of mRNA stability in eukaryotic cells.
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Linking mRNA turnover and translation: assessing the polyribosomal association of mRNA decay factors and degradative intermediates.
mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated.
Reduction of cannula-related laparoscopic complications using a radially expanding access device.
STUDY OBJECTIVE: To determine the safety of a new radially expanding access device compared with complication rates associated with sharp laparoscopic cannulas. DESIGN: Prospective, multicenter study (Canadian Task Force classification II-1). SETTING: Free-standing and hospital-based ambulatory surgery centers. PATIENTS: Two hundred twelve women undergoing various laparoscopic procedures and followed over 44 months. INTERVENTION: Five hundred forty-one radially dilating access devices were used exclusively for laparoscopic abdominal wall access. MEASUREMENTS AND MAIN RESULTS: No major vascular injury, abdominal wall bleeding, intestinal injury, bladder or ureteral injury, liver trauma, or postoperative incisional hernia occurred. One patient developed a postoperative mesenteric hematoma probably caused by a venous injury from the Veress needle. Of the 541 radially expanding access cannulas placed, only 6 (1%) slipped, despite absence of fascial anchoring devices. CONCLUSION: Radially dilating abdominal access devices may reduce laparoscopic complications, lessen a surgeon's exposure to liability, and improve patient outcomes while reducing facility costs. (J Am Assoc Gynecol Laparosc 6(1):79-84, 1999)
Overexpression of truncated Nmd3p inhibits protein synthesis in yeast.
The yeast NMD3 gene was identified in a two-hybrid screen using the nonsense-mediated mRNA decay factor, Upf1p, as bait. NMD3 was shown to encode an essential, highly conserved protein that associated principally with free 60S ribosomal subunits. Overexpression of a truncated form of Nmd3p, lacking 100 C-terminal amino acids and most of its Upf1p-interacting domain, had dominant-negative effects on both cell growth and protein synthesis and promoted the formation of polyribosome half-mers. These effects were eliminated by truncation of an additional 100 amino acids from Nmd3p. Overexpression of the nmd3delta100 allele also led to increased synthesis and destabilization of some ribosomal protein mRNAs, and increased synthesis and altered processing of 35S pre-rRNA. Our data suggest that Nmd3p has a role in the formation, function, or maintenance of the 60S ribosomal subunit and may provide a link for Upf1p to 80S monosomes.
Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.
In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.
New reference levels for CA125 in pre- and postmenopausal women.
Objective: CA125 is a glycoprotein commonly produced by the endometrium. Serum CA125 is widely useful as a marker for ovarian cancer screening, with 35 U/mL the generally accepted upper limit of normal. Since CA125 is known to fluctuate with vaginal bleeding and menopausal status, the purpose of this study was to develop different reference ranges for normal women of differing physiologic states.Methods: We retrospectively analyzed 1,407 patients with serum CA125 levels taken at Cleveland Clinic Florida between 1992 and 1997. Patients with obvious elevation of CA125 from gynecologic cancers were excluded. Student t test was used to determine statistical significance.Results: Among 1,407 patients studied, 256 were premenopausal, 46 perimenopausal, 957 postmenopausal, and 148 excluded due to known gynecologic cancers. There was a significant difference between the mean CA125 values of premenopausal (19.3 +/- 15.6) and postmenopausal women (11.7 +/- 9.2) with P.7 x 10(-6). Among premenopausal women, there was a statistically significant difference between CA125 levels during menses (21.4 +/- 19.3) and luteal phase (14.0 +/- 9.1) with P =.03. Among the subgroup of postmenopausal women with known bleeding history, the mean CA125 values for women with vaginal bleeding was 12.49 +/- 11.5 compared to those without bleeding, 9.62 +/- 4.6 (P =.017).Conclusions: For normal premenopausal women, the overall upper limit of CA125 should be 50 U/mL. However, if menstrual status is known, the upper normals should be: 62 during menses, 51 for proliferative phase, and 32 for luteal phase. For postmenopausal women, the CA125 levels should be no more than 35 for those with vaginal bleeding and 20 for those without bleeding. The stratified CA125 values may help provide more specific ovarian cancer screening.
A single amino acid substitution in yeast eIF-5A results in mRNA stabilization.
Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors, approximately 4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted alpha-helical segment of the protein. ts1159 also exhibited an approximately 30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping.
Obesity, health services use, and health care costs among members of a health maintenance organization.
BACKGROUND: Obesity is an independent risk factor for a variety of chronic diseases and is therefore a potential source of avoidable excess health care expenditures. Previous studies of obesity and health care costs have used group level data, applying estimates of population-attributable risks to estimates of US total costs of care for each obesity-related disease. OBJECTIVE: To quantify the association between body mass index (BMI) and health services use and costs stratified by age and use source at the patient level, a level of detail not previously reported. METHODS: In 17,118 respondents to a 1993 health survey of members of a large health maintenance organization, we ascertained through computerized databases all hospitalizations, laboratory services, outpatient visits, outpatient pharmacy and radiology services, and the direct costs of providing these services during 1993. RESULTS: There was an association between BMI and annual rates of inpatient days, number and costs of outpatient visits, costs of outpatient pharmacy and laboratory services, and total costs (P < or = .003). Relative to BMI of 20 to 24.9, mean annual total costs were 25% greater among those with BMI of 30 to 34.9 (rate ratio, 1.25; 95% confidence interval, 1.10-1.41), and 44% greater among those with BMI of 35 or greater (rate ratio, 1.44; 95% confidence interval, 1.22-1.71). The association between BMI and coronary heart disease, hypertension, and diabetes largely explained these elevated costs. CONCLUSION: Given the high prevalence of obesity and the associated elevated rates of health services use and costs, there is a significant potential for a reduction in health care expenditures through obesity prevention efforts.
An in vivo comparison between a visible light-cured bonding system and a chemically cured bonding system.
Direct bonding of brackets has become a routine procedure in clinical orthodontics. Many techniques and materials are currently advocated and used, the most recent being light-cured composites. Advantages of the light-cured systems are their relative ease of use, improved bracket placement, and more rapid set of the composite. For a new system to be clinically viable, it must possess properties that are at least as reliable as existing systems. The purpose of this longitudinal clinical study was to evaluate and compare the rate of success and/or failure between a visible light-cured bonding material (Sequence) and a chemically cured bonding material (System 1+), using both systems in every patient. Contralateral quadrants were bonded with each system respectively. A total of 32 patients were followed for a mean period of 11 months (range of 3 to 21 months), with a total of 531 brackets bonded, 265 with visible light-cured and 266 with chemically cured resins. Failures for each system were recorded and failure rates calculated. The failure rate of the visible light-cured composite was 11.3% and that of the chemically cured composite was 12%. A Chi-squared (chi2) test did not reveal any statistically significant differences between the failure rates of the two systems, (chi2 = 0.014, df-1, P > 0.9).
Viral escape from antisense RNA.
RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.
Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation.
The poly(A) tail of an mRNA is believed to influence the initiation of translation, and the rate at which the poly(A) tail is removed is thought to determine how fast an mRNA is degraded. One key factor associated with this 3'-end structure is the poly(A)-binding protein (Pab1p) encoded by the PAB1 gene in Saccharomyces cerevisiae. In an effort to learn more about the functional role of this protein, we used a two-hybrid screen to determine the factor(s) with which it interacts. We identified five genes encoding factors that specifically interact with the carboxy terminus of Pab1p. Of a total of 44 specific clones identified, PBP1 (for Pab1p-binding protein) was isolated 38 times. Of the putative interacting genes examined, PBP1 promoted the highest level of resistance to 3-aminotriazole (>100 mM) in constructs in which HIS3 was used as a reporter. We determined that a fraction of Pbp1p cosediments with polysomes in sucrose gradients and that its distribution is very similar to that of Pab1p. Disruption of PBP1 showed that it is not essential for viability but can suppress the lethality associated with a PAB1 deletion. The suppression of pab1Delta by pbp1Delta appears to be different from that mediated by other pab1 suppressors, since disruption of PBP1 does not alter translation rates, affect accumulation of ribosomal subunits, change mRNA poly(A) tail lengths, or result in a defect in mRNA decay. Rather, Pbp1p appears to function in the nucleus to promote proper polyadenylation. In the absence of Pbp1p, 3' termini of pre-mRNAs are properly cleaved but lack full-length poly(A) tails. These effects suggest that Pbp1p may act to repress the ability of Pab1p to negatively regulate polyadenylation.
Fission yeast rad12+ regulates cell cycle checkpoint control and is homologous to the Bloom's syndrome disease gene.
The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning of rad12+, which is the fission yeast homolog of BLM and is identical to the recently reported rhq1+ gene. We showed that rad12 null cells are sensitive to DNA damage induced by UV light and gamma radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12+ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically that rad12+ acts upstream from rad9+, one of the fission yeast G2 checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12+ overproducer implicate rad12+ as a key coupler of chromosomal integrity with cell cycle progression.