A cross reactive sensor array to probe divalent metal ions.

A simple sensing ensemble was designed to discriminate structurally similar divalent metal chlorides utilizing multivariate data analysis. The system features the binding of four synthesized coumarin-enamine probes to a series of ten metal chlorides. Linear discriminant analysis (LDA) achieves what univariate data analysis alone cannot i.e., full analyte discrimination and differentiation.

Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.

We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes.

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery.

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.

An important 2'-OH group for an RNA-protein interaction.

We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Synthesis and biophysical characterization of tRNA(Lys,3) anticodon stem-loop RNAs containing the mcm(5)s(2)U nucleoside.

[reaction: see text] Phosphoramidite reagents of the naturally occurring modified nucleosides mcm(5)s(2)U and mcm(5)U were synthesized and along with pseudouridine were incorporated into 17-nucleotide lysine tRNA anticodon stem-loop domains. Standard RNA phosphoramidite coupling chemistry allowed us to systematically investigate the thermodynamic effects of nucleoside modification and to correlate thermodynamic trends with qualitative structure effects seen by NMR spectroscopy.

Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U-turn structure.

Modified nucleosides in the anticodon domain of Escherichia coli tRNA(Lys) are necessary for high-affinity codon recognition and reading frame maintenance. Human tRNA(Lys,3) is the specific primer for HIV-1 reverse transcriptase and also requires nucleoside modification for proper function. We now present NMR solution structures for the fully modified 17-nucleotide E. coli tRNA(Lys) anticodon stem-loop domain (ASL). NMR data were also collected for several partially modified ASLs, revealing the contributions each modified nucleoside (mnm(5)s(2)U34, t(6)A37, and psi39) makes in transforming the disordered, unmodified tRNA ASL into the highly ordered native structure. The solution structure of the native ASL domain provides insight into longstanding questions regarding both wobble position modification and the nearly ubiquitous t(6)A37 found in tRNAs with an adjacent U at position 36. Native tRNA(Lys) has a U-turn structure similar to the yeast tRNA(Phe) crystal structure, unlike previously proposed "unconventional" anticodon structures characterized by stable interactions between mnm(5)s(2)U-34 and t(6)A-37.

Multiplex detection and quantitation of proteins on western blots using fluorescent probes.

The uses of multiplex detection methodologies are dramatically increasing as a means to increase sample throughput and to demonstrate quantitative differences between multiple targets in gene or protein expression analysis. In this study, we investigate the application of multiplex fluorescent detection for three proteins on the same Western blot using a laser-scanning imaging system, the Bio-Rad Molecular Imager FX. We show that independent detection and quantitation of multiple targets is achievable with little or no correction for fluorescent crosstalk by using fluorescent tags preferentially excited with different laser lines and detected at wavelengths that minimize fluorescence crosstalk. We demonstrate that the use of fluorescent detection methods can provide a tenfold greater quantifiable range but with two- to fourfold less sensitivity than chemiluminescent detection methodologies. Two examples of three-color multiplex detection using FITC-, Cy3- and Cy5-conjugated probes on Western blots are provided to demonstrate applications of this approach.

Laparoscopic secondary port conversion using a reusable blunt conical trocar.

BACKGROUND: During operative laparoscopy, large (10 mm or more) ancillary ports are often used for instrumentation and tissue removal. Although sharp pyramidal trocars can be used to place these ports, their use appears to increase the risk of vessel injury and herniation. We describe a simple and cost-effective technique for converting a 5-mm port to a 10- or 12-mm port using a blunt conical trocar. TECHNIQUE: When a larger port is required, a previously placed 5-mm port is removed, and the skin incision is lengthened. A reusable 10- or 12-mm blunt conical trocar with a threaded sleeve is placed through the incision. The fascial defect is located by probing and is dilated gently with the blunt tip. Once the tip is through the fascia, it is advanced through the peritoneal defect with a clockwise, twisting motion. Afterwards, the fascial defect is closed with a single, interrupted absorbable suture. EXPERIENCE: We have had no complications or difficulty when using this technique in 26 cases, either during or after surgery. CONCLUSION: A reusable blunt conical trocar is a simple, safe, and cost-effective instrument for converting a 5-mm laparoscopic port into a 10- or 12-mm port.

Synthesis and characterization of the native anticodon domain of E. coli TRNA(Lys): simultaneous incorporation of modified nucleosides mnm(5)s(2)U, t(6)A, and pseudouridine using phosphoramidite chemistry.

The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1. 8 mg of purified RNA from a 1 micromol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.

Physical activity and peptic ulcers. Does physical activity reduce the risk of developing peptic ulcers?

BACKGROUND: Although Helicobacter pylori has been identified as a major cause of chronic gastritis, not all infected patients develop ulcers, suggesting that other factors such as lifestyle may be critical to the development of ulcer disease. OBJECTIVE: To investigate the role physical activity may play in the incidence of peptic ulcer disease. METHODS: The participants were men (n = 8,529) and women (n = 2,884) who attended the Cooper Institute for Aerobics Research, Dallas, Texas, between 1970 and 1990. The presence of gastric or duodenal ulcer disease diagnosed by a physician was determined from a mail survey in 1990. Participants were classified into 3 physical activity groups according to information provided at the baseline clinic visit (before 1990): active, those who walked or ran 10 miles or more a week; moderately active, those who walked or ran less than 10 miles a week or did another regular activity; and the referent group consisting of those who reported no regular physical activity. RESULTS: With the use of gender-specific proportional hazards regression models that could be adjusted for age, smoking, alcohol use, body mass index, and self-reported tension, active men had a significantly reduced risk for duodenal ulcers (relative hazard [95% confidence interval] for the active group, 0.38 [0.15-0.94], and 0.54 [0.30-0.96] for the moderately active group). No association was found between physical activity and gastric ulcers for men or for either type of ulcer for women. CONCLUSION: Physical activity may provide a nonpharmacologic method of reducing the incidence of duodenal ulcers among men.

Physical activity and self-reported, physician-diagnosed osteoarthritis: is physical activity a risk factor?

This prospective study evaluated regular physical activity and self-reported physician-diagnosed osteoarthritis of the knee and/or hip joints among 16,961 people, ages 20-87, examined at the Cooper Clinic between 1970 and 1995. Among those aged 50 years and older, osteoarthritis incidence was higher among women (7.0 per 1000 person-years) than among men (4.9 per 1000 person-years, P = 0.001), while among those under 50 years of age, osteoarthritis incidence was similar between men (2.6) and women (2.7). High levels of physical activity (running 20 or more miles per week) were associated with osteoarthritis among men under age 50 after controlling for body mass index, smoking, and use of alcohol or caffeine (hazard ratio = 2.4, 95% CI: 1.5, 3.9), while no relationship was suggested among women or older men. These findings support the conclusion that high levels of physical activity may be a risk factor for symptomatic osteoarthritis among men under age 50.

Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification.

Surface plasmon resonance (BIACORE) was used to determine the kinetic values for formation of the HIV TAR-TAR* ('kissing hairpin') RNA complex. The TAR component was also synthesized with the modified nucleoside 2-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation constants compared with the unmodified TAR hairpin. The BIACORE data show an equilibrium dissociation constant of 1.58 nM for the complex containing the s(2)U modified TAR hairpin, which is 8-fold lower than for the parent hairpin (12.5 nM). This is a result of a 2-fold faster k(a) (4.14x10(5) M(-1) s(-1) versus 2.1x10(5) M(-1) s(-1)) and a 4-fold slower k(d) (6.55x10(-4) s(-1) versus 2.63x10(-3) s(-1)). (1)H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s(2)U-modified complex. Magnesium has been reported to significantly stabilize the TAR-TAR* complex and we found that Mn(2+) and Ca(2+) are also strongly stabilizing, while Mg(2+) exhibited the greatest effect on the complex kinetics. The stabilizing effects of 2-thiouridine indicate that this base modification may be generally useful as an antisense RNA modification for oligonucleotide therapeutics which target RNA loops.

Does physical activity reduce the risk of developing peptic ulcers?

BACKGROUND: Although Helicobacter pylori has been identified as a major cause of chronic gastritis, not all infected patients develop ulcers, suggesting that other factors such as lifestyle may be critical to the development of ulcer disease. AIM: To investigate the role physical activity may play in the incidence of peptic ulcer disease. METHODS: The subjects were men (8529) and women (2884) who attended the Cooper Clinic in Dallas between 1970 and 1990. The presence of gastric or duodenal ulcer disease diagnosed by a doctor was determined from a mail survey in 1990. Subjects were classified into three physical activity groups according to information provided at the baseline clinic visit (before 1990): active, those who walked or ran 10 miles or more a week; moderately active, those who walked or ran less than 10 miles a week or did another regular activity; the referent group consisting of those who reported no regular physical activity. RESULTS: With the use of gender specific proportional hazards regression models that could be adjusted for age, smoking, alcohol use, body mass index, and self reported tension, active men were found to have a significant reduction in risk for duodenal ulcers (relative hazard (95% confidence interval) for the active group was 0.38 (0.15 to 0.94) and 0.54 (0.30 to 0.96) for the moderately active group). No association was found between physical activity and gastric ulcers for men or for either type of ulcer for women. CONCLUSIONS: Physical activity may provide a non-pharmacological method of reducing the incidence of duodenal ulcers among men.

Muscular strength and physical function.

PURPOSE: The purpose of this study was to evaluate the potential association of muscular strength and endurance at baseline with the prevalence of functional limitations at follow-up. METHODS: Study participants were 3,069 men and 589 women (30-82 yr) who received a clinical examination including a strength evaluation at the Cooper Clinic between 1980 and 1989 and responded to a 1990 mail-back survey. Participants also had to achieve at least 85% of their age-predicted maximal heart rate on a maximal exercise treadmill test and have no history of heart attack, stroke, diabetes, high blood pressure, cancer, or arthritis at their first visit. A strength index composite score (0-6) was calculated using age- and sex-specific tertiles from bench press, leg press, and sit-up tests. Those scoring 5 or 6 were categorized in the high strength group. Functional health status was assessed by responses to questions about the participant's ability to perform light, moderate, and strenuous recreational, household, daily living, and personal care tasks. RESULTS: After an average follow-up of 5 yr, 7% of men and 12% of women reported at least one functional limitation. A logistic regression model including age, aerobic fitness, body mass index, and new health problems at follow-up found that, relative to those with lower levels of strength, the odds of reporting functional limitations at follow-up in men and women categorized as having higher levels of strength were 0.56 (95%CI = 0.34, 0.93) and 0.54 (95%CI = 0.21, 1.39), respectively. CONCLUSIONS: These findings, if replicated in other populations, suggest that maintenance of strength throughout the lifespan may reduce the prevalence of functional limitations.

Highly regulated cell type-restricted expression of human renin in mice containing 140- or 160-kilobase pair P1 phage artificial chromosome transgenes.

We generated transgenic mice with two P1 artificial chromosomes, each containing the human renin (HREN) gene and extending to -35 and -75 kilobase pairs, respectively. HREN protein production was restricted to juxtaglomerular cells of the kidney, and its expression was tightly regulated by angiotensin II and sodium. The magnitude of the up- and down-regulation in HREN mRNA caused by the stimuli tested was identical to the endogenous renin gene, suggesting tight physiological regulation. P1 artificial chromosome mice were mated with transgenic mice overexpressing human angiotensinogen to determine if there was a chronic compensatory down-regulation of the transgene. Despite a 3-fold down-regulation of HREN mRNA, plasma angiotensin II and blood pressure was modestly elevated in the double transgenic mice. Nevertheless, this elevation was significantly less than a different double transgenic model containing a poorly regulated HREN transgene. The increase in blood pressure, despite the decrease in HREN mRNA, suggests that the HREN gene can partially, but not completely, compensate for excess circulating angiotensinogen. These data suggest the possibility that increases in circulating or tissue angiotensinogen may cause an increase in blood pressure in humans, even in the presence of a functionally active servo-mechanism to down-regulate HREN expression.

Nucleoside modifications affect the structure and stability of the anticodon of tRNA(Lys,3).

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNA(Lys,3). The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNA(Phe) and provides an explanation of the critical role of modification in this tRNA.

The value of strength training for older adults.

Health in older adults can best be measured in terms of functional status. Skeletal muscle strength has been reported to be a determinant of functional status in older individuals. Two major contributors to the decline in muscle function as a person ages are disuse and physical inactivity. Declining muscle function through a loss of muscular strength may decrease functional independence and mobility and increase the risk for falls and injuries, physical frailty, and disability. Older individuals lacking an appropriate amount of muscular strength may not be able to perform various activities of daily living, which are important indicators of independence.