Saturn ring particles as dynamic ephemeral bodies.

Although Saturn's rings are within the Roche zone, the accretion of centimeter-sized particles into large aggregates many meters in diameter occurs readily, on a time scale of weeks. These aggregates are disrupted when tidal stresses exceed their very low strengths; thus most of the mass of the ring system is continually processed through a population of large "dynamic ephemeral bodies," which are continually forming and disintegrating. These large aggregates are not at all like the idealized ice spheres often used in modeling Saturn's ring dynamics. Their coefficient of restitution is low, hence they form a monolayer in the ring plane. The optically observable characteristics of the rings are dominated by the swarm of centimeter-sized particles.

First images of asteroid 243 ida.

The first images of the asteroid 243 Ida from Galileo show an irregular object measuring 56-kilometers by 24 kilometers by 21 kilometers. Its surface is rich in geologic features, including systems of grooves, blocks, chutes, albedo features, crater chains, and a full range of crater morphologies. The largest blocks may be distributed nonuniformly across the surface; lineaments and dark-floored craters also have preferential locations. Ida is interpreted to have a substantial regolith. The high crater density and size-frequency distribution (-3 differential power-law index) indicate a surface in equilibrium with saturated cratering. A minimum model crater age for Ida-and therefore for the Koronis family to which Ida belongs-is estimated at 1 billion years, older than expected.

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells.

BACKGROUND: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3 alpha, GSK-3 beta and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3 alpha and GSK-3 beta can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3. RESULTS: Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3 alpha or GSK-3 beta decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies. CONCLUSIONS: Our data indicate that GSK-3 alpha and/or GSK-3 beta, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.

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.

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.

Stabilization of the anticodon stem-loop of tRNALys,3 by an A+-C base-pair and by pseudouridine.

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNALys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters derived from temperature dependent UV measurements. The pseudouridine-containing hairpin is thermodynamically more stable than the unmodified hairpin by 5 degreesC, and this corresponds with increased base stacking on the 3' side of the tRNA anticodon loop. An A+38-C32 base-pair also forms at the base of the anticodon stem with an approximate pKa of 6 for A38. Formation of the A+-C base-pair increases the Tm of both pseudouridine modified and unmodified RNA hairpins by 5-6 degreesC, and decreases the DeltaG degrees for hairpin formation by 1 kcal/mol. Solution structures were determined for both psi39 and unmodified hairpins under limiting pH conditions at pH 5 and pH 7 to assess the structural effects of both psi modification and the additional A+-C base-pair on tRNALys,3 structure. The A+38-C32 base-pair strengthens the 31-39 base-pair, and induces formation of a dynamic U33-A37 base-pair that effectively reduces the normal seven nucleotide anticodon loop to a three nucleotide UUU loop. These undermodified tRNALys,3 anticodon loops are distinctly different from those seen for other tRNAs exemplified by tRNAPhe. The conformation of the tRNA loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNALys,3 by HIV-1 as the native reverse transcriptase primer.

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.

Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Addison's disease and Cushing's syndrome: basal concentrations.

We measured basal plasma concentrations of the immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta END), and alpha MSH in 160 normal dogs, 32 dogs with Addison's disease, 42 dogs with adrenocortical tumors causing Cushing's syndrome, and 169 dogs with pituitary-dependent Cushing's disease. In normal dogs, plasma IR-POMC peptide levels were similar to those in man, except that IR-alpha MSH, a pars intermedia POMC product, was readily detected. In Addisonian dogs, plasma cortisol was decreased, and the IR-POMC peptides were increased, except for IR-alpha MSH, which was normal. In 7 Addisonian dogs given dexamethasone, elevated plasma IR-ACTH, beta LPH, and beta END levels fell dramatically. In dogs with Cushing's syndrome due to adrenal tumors, plasma IR-ACTH, beta LPH, and beta END were decreased, and cortisol was increased, but IR-alpha MSH was normal. Dogs with Cushing's disease due to pars distalis tumors had elevated plasma IR-ACTH, beta LPH, beta END, and cortisol, but normal IR-alpha MSH; their plasma cortisol was suppressed by dexamethasone. There appeared to be 2 types of pars intermedia tumors causing Cushing's disease: 1 dexamethasone nonsuppressible and with disproportionately high plasma IR-alpha MSH levels, the other relatively dexamethasone suppressible and with normal to slightly elevated IR-alpha MSH levels. These 2 pars intermedia tumor types may arise from 2 distinct normal canine pars intermedia cell types. Canine Cushing's disease may provide a useful model for variants of the disorder in man.

Lithium reduces tau phosphorylation: effects in living cells and in neurons at therapeutic concentrations.

BACKGROUND: The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. METHODS: Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. RESULTS: Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concentrations did not alter neuronal morphology. Green fluorescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. CONCLUSIONS: Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders.

Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase Mos.

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.

Modulation of PHF-like tau phosphorylation in cultured neurones and transfected cells.

Two cellular systems have been used to investigate the modulation of tau hyperphosphorylation. In the first system, the effects of the excitatory amino acid glutamate, the microtubule destabilising agent colchicine, and beta 25-35-amyloid peptide on tau phosphorylation were studied in rat cortical neurones in primary culture. Using immunocytochemistry and western blot analysis, we demonstrated that tau in these cultures is normally highly phosphorylated, but a proportion becomes rapidly dephosphorylated following treatment of the cultures with glutamate or colchicine. These changes in tau phosphorylation occurred prior to cell death. In the second system, the ability of p42 MAP and p44 MAP kinases, glycogen synthase kinases 3 alpha and 3 beta (GSK-3 alpha and GSK-3 beta) to phosphorylate tau in transfected COS cells was investigated. Both GSK-3 alpha and GSK-3 beta phosphorylated tau to produce a PHF-like state of phosphorylation but the MAP kinases failed to induce such a transformation in tau. These results suggest that aberrant regulation of GSK-3 alpha/beta may be a pathogenic mechanism in Alzheimer's disease.

AIDS/HIV risk behavior among the chronic mentally ill.

OBJECTIVE: There is growing concern that chronic mentally ill adults living in the community have a high risk for HIV infection. The purpose of this study was to identify risk knowledge, high-risk behaviors, and risk-related encounters of chronic psychiatric outpatients. METHOD: Detailed information on high-risk behaviors and risk-related situations during the past 12 months was collected from 60 outpatients appearing for regular visits at inner-city community mental health clinics. RESULTS: Of the 60 outpatients, 37 (62%) had been sexually active during the past year, and 42% of the men and 19% of the women reported multiple sexual contacts and infrequent use of condoms during intercourse. Assessments of the patients' knowledge of AIDS risks revealed substantial deficits in their practical understanding of AIDS and risk reduction measures. Although use of intravenous drugs was uncommon in this group, many subjects reported histories of 1) trading sex for money, drugs, or a place to stay, 2) coercion to engage in unwanted sex, 3) causal sexual encounters, and 4) sexual activity after use of drugs or intoxicants. Twenty percent of the subjects had met their sexual partners on the streets, in parks, or in other public places. One-third had been treated for sexually transmitted diseases other than AIDS. CONCLUSIONS: These findings underscore the need for AIDS risk assessment, counseling, and prevention programs for the chronic mentally ill.

Tau isoform expression and phosphorylation state during differentiation of cultured neuronal cells.

The axonal microtubule-associated protein, tau, is thought to play an important role in axonal growth and in the establishment of neuronal polarity. In adult human brain there are six alternatively spliced tau isoforms, which have different microtubule binding affinities in vitro. The tubulin-tau interaction is further modified by phosphorylation of tau and, compared to adult brain tau, both foetal brain tau and paired helical filament (PHF) tau, characteristic of Alzheimer's disease, are hyperphosphorylated. In vivo both the expression of tau isoforms and their phosphorylation states are developmentally regulated. In order to establish the correlation between the expression of tau isoforms and their pattern of phosphorylation, we have characterised these two features in several in vitro models of neuronal differentiation, including the human neuroblastoma cell lines, SK-N-SH, SH-SY5Y and IMR32 cells, rat PC12 cells and primary rat cortical neurones. Sensitive RT-PCR analysis revealed a different complement of tau isoforms in the different cell lines and neuritogenesis was associated mainly with an increase in the overall tau protein level with no apparent phosphorylation changes. A switch in tau isoform expression occurred only at the terminal stages of neuronal development, when it may be important in reinforcing the previously established axonal cytoarchitecture.

Potentially useful criteria for judging nutritional adequacy.

Four different diets designed to be of differing quality were fed to large groups of male weanling rats. These diets were: diet C consisting of commercial Rat Chow: diet CG, the same diet diluted with 70% glucose calories, diet A, a simulated "American" diet made up of 25 widely used foods, diet AS, the same diet supplemented with small amounts of 25 vitamins and minerals. The rats on these four diets were observed for 69 days under different circumstances and treatments to ascertain the existence of hitherto unused criteria which might advantageously be applied in nutritional studies. Among the less traditional criteria found to be significantly affected by the diet were: 1) voluntary consumption of food, 2) sleeping time after anesthesia, 3) weight gains after surgery, 4) healing time after surgery, 5) hair growth after clipping, 6) voluntary sugar consumption, and 7) recovery time after cyanide poisoning. These findings suggest that there are probably many other unexplored criteria which could be used advantageously in nutritional experimentation.

Is misonidazole neurotoxicity altered by the use of phenytoin and/or dexamethasone in RTOG 79-18 and RTOG 79-16?

An analysis of Misonidazole (MISO) neurotoxicity in RTOG 79-16 and RTOG 79-18 was undertaken to evaluate the incidence of neurotoxicity relative to dexamethasone dose and phenytoin use. MISO was administered as follows: 79-16 arm A, 1 gm/m2 5 days a week for a total of 10 gm/m2 in 2 weeks; 79-16 arm B, 2 gm/m2 twice weekly for a total of 12 gm/m2 in 3 weeks; and 79-18, 2.5 gm/m2 once a week for a total of 15 gm/m2 in 6 weeks. Practically all patients were on dexamethasone, and 240 out of 550 were on phenytoin for seizures. CNS toxicity and ototoxicity rates were no different between treatment groups with overall rates of 2.7 and 1.1%, respectively. Peripheral neuropathy (PN) was 5.1% in 79-16 arm A, 5.9% in 79-16 arm B, and 8.7% in 79-18. Phenytoin did not significantly alter CNS and PN toxicity rates. All ototoxicities occurred in patients not on phenytoin. There was no correlation between dexamethasone dose and incidence of neurotoxicity within each study. However, the incidence of (PN) for the combined studies was 6.4% (35/550) which is lower than 18.9% (85/449) for non-brain Phase III protocols where patients are rarely, if ever, on dexamethasone or other corticosteroids. Four hour and 24 hour plasma MISO levels, and 24 hour/4 hour MISO ratios did not correlate with toxicity.

M235-->T polymorphism of the angiotensinogen gene predicts hypertension in the elderly.

OBJECTIVE: To determine whether the M235-->T polymorphism (exon 2) of the angiotensinogen gene is associated with hypertension in elderly patients with isolated systolic hypertension [ISH: systolic blood pressure (SBP) > or = 160 mmHg, diastolic blood pressure (DBP) < 90 mmHg) or systolic-diastolic hypertension (SDH: DBP > or = 90 mmHg, SBP > or = 160 mmHg) compared with normotensive controls (SBP < 160 mmHg, DBP < 90 mmHg). DESIGN: A case-control study in 769 non-institutionalized, elderly (aged > or = 60 years; female:male ratio 0.85) residents of Dubbo, New South Wales. METHODS: Individuals were classified as having ISH (n = 171), having SDH (n = 218) and being normotensive controls (n = 366) with age and sex matching. MM, TT and MT genotypes were determined by a nested polymerase chain reaction strategy using DNA extracted from serum. The prediction of ISH or SDH by genotype or allele was examined in a multiple-logistic regression model that controlled for various confounders. RESULTS: SBP (mean +/- SD, mmHg)/DBP (mean +/- SD, mmHg) was 176 +/- 16/79 +/- 8 in the ISH group, 167 +/- 23/97 +/- 7 in the SDH group and 134 +/- 14/74 +/- 9 in the normotensive control group. The frequencies of M and T alleles in the normal population (0.69 and 0.31, respectively) were altered significantly in the ISH group (0.61 and 0.39, respectively; chi 2 = 6.0, P < 0.02) and the SDH group (0.62 and 0.38, respectively; chi 2 = 6.0, P < 0.02). The presence of the TT genotype predicted both ISH (odds ratio 1.9, 95% confidence interval 1.1-3.3) and SDH (1.7, 1.0-3.0) as did that of the T allele (ISH: 1.3, 1.0-1.7; SDH: 1.3, 1.0-1.7). CONCLUSIONS: The M235-->T polymorphism may be a marker for both forms of hypertension in the elderly. Whether the TT genotype represents a genetic risk factor for the development of hypertension in later life requires confirmation.

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.

Patterns of non-response to a mail survey.

This paper describes a basic investigation of possible non-response bias in a mail survey. We compare characteristics of responders and non-responders to a mail survey of health outcomes among participants of a longitudinal study of physical activity, physical fitness, and health. Results indicate that, at the first clinic visit, the responders were essentially the same as the non-responders on personal health history and laboratory measurements, while reporting significantly more family history of specific chronic diseases (cardiovascular disease, hypertension, stroke). The male responders were younger and reported more positive health behaviors as well as better weight and treadmill times at the first clinic visit. These results suggest that both response groups were equally healthy at entry, and that individuals who had family members with certain chronic conditions and who had positive health behaviors were more likely to respond (participate) in this health-related survey. Differences of this type could affect interpretation of future analyses. This work illustrates the importance of incorporating methods to examine non-response into any epidemiologic study.

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.

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.