Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.

Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage.

Acquisition of dwarf male "harems" by recently settled females of Osedax roseus n. sp. (Siboglinidae; Annelida).

After the deployment of several whale carcasses in Monterey Bay, California, a time-series analysis revealed the presence of a new species of Osedax, a genus of bone-eating siboglinid annelids. That species is described here as Osedax roseus n. sp. It is the fifth species described since the erection of this genus and, like its congeners, uses a ramifying network of "roots" to house symbiotic bacteria. In less than 2 months, Osedax roseus n. sp. colonized the exposed bones of a whale carcass deposited at 1018-m depth, and many of the females were fecund in about 3 months post-deployment. As with other Osedax spp., the females have dwarf males in their tube lumens. The males accrue over time until the sex ratio is markedly male-biased. This pattern of initial female settlement followed by gradual male accumulation is consistent with the hypothesis that male sex may be environmentally determined in Osedax. Of the previously described species in this genus, Osedax roseus n. sp. is most similar to O. rubiplumus, but it has several anatomical differences, as well as much smaller females, dwarf males, and eggs. Osedax roseus n. sp. is markedly divergent (minimally 16.6%) for mitochondrial cytochrome oxidase subunit I (mtCOI) sequences from any other Osedax species.

Mobile Stroke Unit Reduces Time to Image Acquisition and Reporting.

Timely administration of thrombolytic therapy is critical to maximizing the likelihood of favorable outcomes in patients with acute ischemic stroke. Although emergency medical service activation overall improves the timeliness of acute stroke treatment, the time from emergency medical service dispatch to hospital arrival unavoidably decreases the timeliness of thrombolytic administration. Our mobile stroke unit, a new-generation ambulance with on-board CT scanning capability, reduces key imaging time metrics and facilitates in-the-field delivery of IV thrombolytic therapy.

Modulation of renal sodium-potassium-adenosine triphosphatase by aldosterone. Effect of high physiologic levels on enzyme activity in isolated rat and rabbit tubules.

The purpose of this study was to determine the nephron site, time course, and mechanism of mineralocorticoid action on renal tubular Na-K-ATPase in rats and rabbits, without dietary manipulation and by using the natural mineralocorticoid aldosterone. Sustained, high physiologic levels of circulating aldosterone mimicking those produced endogenously during potassium loading or sodium deprivation were provided by constant delivery of the hormone in doses of 5 or 50 micrograms/100 g body wt per 24 h, respectively, from osmotic minipumps implanted subcutaneously. In adrenal-intact rats receiving the 5-microgram dose, aldosterone levels were similar to those seen in animals fed a high K diet and produced a time-dependent increase in Na-K-ATPase activity in the cortical-collecting tubule (CCT) to a level 103% higher than in controls after 7 d (2,007 +/- 178 vs. 989 +/- 72 pmol/mm per h, P less than 0.001); the enzyme activity in the proximal convoluted tubule, medullary thick ascending limb, and the inner stripe of the medullary-collecting tubule did not change significantly. The increment in CCT Na-K-ATPase was larger (142%) in animals receiving for the same period of time the 50-micrograms dose, which produced circulating aldosterone levels similar to those of sodium-deprived rats. A significant stimulation of Na-K-ATPase activity was seen in the CCT of adrenalectomized rats after 24 h of treatment with either dose of the hormone, and at 12 h only in animals receiving the 50 micrograms/100 g per 24 h regimen. To determine whether the enhanced Na-K-ATPase activity produced by aldosterone is due to synthesis of new enzyme units or to alteration in its kinetics, we examined the ouabain-binding capacity and the affinity for Na and K of the enzyme from CCT of rabbits treated with 5 micrograms/100 g body wt per 24 h aldosterone for 3 d. These experiments revealed a parallel increment on Na-K-ATPase activity and specific [3H]ouabain binding in aldosterone-treated rabbits, while the affinity of the enzyme for either sodium or potassium was unaltered. The results of this study indicate that high physiologic levels of aldosterone simulating those measured during K loading or Na deprivation lead to a segment-specific increase in Na-K-ATPase activity in the CCT. This effect was time-and dose-dependent and was due to an increase in the number of active enzyme units. The segmental specificity and time course of the increase in enzyme activity suggest that modulation of Na-K-ATPase by aldosterone plays a role in the chronic adaptation of the CCT to altered availability of sodium and potassium, and therefore in the homeostasis of these cations by the kidney.

Regulation of renal Na-K-ATPase in the rat. Role of the natural mineralo- and glucocorticoid hormones.

Both mineralo- and glucocorticoids stimulate renal Na-K-ATPase, but their relative role in the regulation of the enzyme remains controversial. In this study we measured Na-K-ATPase activity in the cortical collecting tubule (CCT) of adrenalectomized rats replaced with either the native mineralocorticoid (aldosterone) or glucocorticoid (corticosterone) in doses calculated to yield previously determined physiologic concentrations of these hormones (5 ng X dl-1 and 5 micrograms X dl-1, respectively). This was achieved by continuous delivery of aldosterone (1 microgram X 100 g-1 X d-1) from an osmotic minipump or of corticosterone (2 pellets of 20 mg each), implanted subcutaneously either at adrenalectomy or 7 d later, when Na-K-ATPase activity reached its nadir. Adrenalectomized rats not receiving hormone replacement and adrenal-intact animals served as controls. The CCT was chosen because it contains the highest concentration of binding sites for both hormones. Na-K-ATPase activity declined 52% in the CCT of untreated adrenalectomized rats after 7 d, and remained unchanged thereafter. Physiologic replacement doses of aldosterone prevented this decline and restored the activity of the enzyme after it had been allowed to decrease maximally following adrenal ablation, whereas similar replacement of corticosterone was without effect. These observations suggest that under physiologic conditions Na-K-ATPase in the CCT, a probable target nephron segment of both hormones, is under mineralocorticoid rather than glucocorticoid control.

Metabolism and excretion of the antiepileptic/antimigraine drug, Topiramate in animals and humans.

The metabolism and excretion of 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (TOPAMAX, topiramate, TPM) have been investigated in animals and humans. Radiolabeled [14C] TPM was orally administered to mice, rats, rabbits, dogs and humans. Plasma, urine and fecal samples were collected and analyzed. TPM and a total of 12 metabolites were isolated and identified in these samples. Metabolites were formed by hydroxylation at the 7- or 8-methyl of an isopropylidene of TPM followed by rearrangement, hydroxylation at the 10-methyl of the other isopropylidene, hydrolysis at the 2,3-O-isopropylidene, hydrolysis at the 4,5-O-isopropylidene, cleavage at the sulfamate group, glucuronide conjugation and sulfate conjugation. A large percentage of unchanged TPM was recovered in animal and human urine. The most dominant metabolite of TPM in mice, male rats, rabbits and dogs appeared to be formed by the hydrolysis of the 2,3-O-isopropylidene group.

PCR primers for an aldolase-B intron in acanthopterygian fishes.

BACKGROUND: Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. RESULTS: Aldolase intron 'G' was amplified from four fish species using previously described primer sets that target several loci indiscriminately. Phylogenetic analyses were used to group these fragments and other full-length aldolase proteins from teleost fishes into orthologous clades and a primer set was designed to target specifically an intron within the aldolase-B locus in acanthopterygian fishes. DNA amplifications were tried in a variety of acanthopterygian fishes and amplification products, identifiable as aldolase-B intron 'G', were observed in all atherinomorph and percomorph taxa examined. Sequence variation within this locus was found within and among several species examined. CONCLUSIONS: Using 'universal' primer sets coupled with phylogenetic analyses it was possible to develop a genetic assay to target a specific locus in a variety of fish taxa. Sequence variation was observed within and among species suggesting that this targeted assay might facilitate interspecific and intraspecific comparisons.

Validating the Questionnaire for Verifying Stroke-Free Status (QVSFS) by neurological history and examination.

BACKGROUND AND PURPOSE: The Questionnaire for Verifying Stroke-Free Status (QVSFS) is an 8-item structured interview designed to identify stroke-free individuals. Previously, the QVSFS was validated with medical record review in a cohort with a low prevalence (7.1%) of stroke or transient ischemic attack (TIA). The objective of this study was to evaluate the validity of the QVSFS by comparing it with stroke status as determined by neurological history and examination in a population with a higher prevalence of stroke. METHODS: A research assistant administered the QVSFS to outpatients from Veterans Administration stroke and general medicine clinics. Subjects were defined as QVSFS negative if responses to all 8 questions were negative. Questions requiring rephrasing or clarification were noted. Neurologists, blinded to QVSFS scores, interviewed and examined all subjects to determine stroke-free status, defined as no history or examination findings of previous stroke and/or TIA. RESULTS: One hundred fifty-five subjects were examined; mean age was 70 years; 98.1% were male. Seventy-eight subjects were determined to be stroke free by the neurologist. The negative predictive value of the QVSFS was 0.96, with positive predictive value of 0.71. No question required rephrasing or clarification >5 times. Twenty-two subjects (14.2%) required rephrasing or clarification of at least 1 question. CONCLUSIONS: The QVSFS can effectively identify stroke-free individuals with a high degree of accuracy, even in a population with a large proportion of patients with prior stroke or TIA. Accuracy for identifying subjects with stroke and/or TIA is lower, but the QVSFS may still be useful as a screening tool in that regard.

Genomic organization of the mouse double minute 2 gene.

Transfection of the mouse double minute 2 (Mdm2) oncogene has been found to induce immortalization of primary cells and to transform cultured cells. Amplification and/or overexpression of human MDM2 has been documented in a large percentage of human cancers. Mouse and human Mdm2 cDNA have been cloned from transformed cells and the cDNA sequence of both genes have been reported previously. In this report, we present the gene structure of mouse Mdm2. Comparison of the coding sequences of the Mdm2 gene with the previously reported cDNA sequence and with Mdm2 sequences obtained from an Mdm2-bearing cosmid clone capable of inducing transformation revealed that the reported cDNA sequence was in error, and that Mdm2-induced transformation of cells does not require an activating mutation in Mdm2. Ligation-anchor PCR analysis of transcripts produced from the P1 and P2 promoters indicates that transcription initiates at sites upstream of those reported previously for both promoters.

In vivo microdialysis and liquid chromatography/thermospray mass spectrometry of the novel anticonvulsant 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (topiramate) in rat brain fluid.

The concentration of a novel anticonvulsant, 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (topiramate), was determined in the extracellular fluid of rat brain by in vivo microdialysis combined off-line with liquid chromatography/thermospray mass spectrometry. A microdialysis probe was stereotaxically implanted in the nucleus accumbens region of the rat brain. The maximum concentration of topiramate in the brain dialysate for a dose of 50 mg kg-1 i.v. was approximately 10 microM and occurred 45 min post-injection. The detection limit of topiramate in the extracellular fluid of rat brain was in the 0.1 microM range using selected ion monitoring techniques. The base peak, which was the ammonium adduct ion [M + NH4]+, was used for detection. An internal standard of d12-labeled topiramate was utilized for quantitation by isotope dilution analysis.

Comparison of micellar electrokinetic chromatography (MEKC) with capillary gas chromatography in the separation of phenols, anilines and polynuclear aromatics potential field-screening applications of MEKC.

Capillary electrophoresis (CE) is known to be complementary to liquid chromatography, but comparison of CE with capillary gas chromatography (GC) for applicable analytes has not been extensive. Capillary GC has been the preeminent separation technique for environmental analysis, but CE has yet to be applied systematically to the determination of environmental analytes. We present data on separations of three classes of semivolatile analytes of interest to environmental analysis: phenols, anilines and polynuclear aromatic hydrocarbons (PNAs). Standard GC conditions were used to illustrate typical separations observed on 30-m and 40-m columns. Rapid analyses were addressed using a high-temperature 15-m column of thinner film. CE separations employed borate buffer with sodium cholate as the micellar agent in micellar electrokinetic chromatography (MEKC). The effects of organic additives were studied using methanol, acetone and tetrahydrofuran. gamma-Cyclodextrin was also used in MEKC to enhance the separation of polynuclear aromatic hydrocarbons and to examine its effects on separations of phenols and anilines. Short capillaries effected very rapid (< 3 min) compound-class characterization, an approach which has potential use in site characterization/remediation (field-screening) studies.

Analysis of chlorobutanol in ophthalmic ointments and aqueous solutions by reverse-phase high-performance liquid chromatography.

A reverse-phase high-performance liquid chromatographic assay for chlorobutanol was developed and found suitable for the routine analysis of ophthalmic ointments and aqueous solutions. The method utilized a column packed with 10-microns octadecylsilane with a mobile phase of methanol-water (50:50). Peak detection was by UV absorption at 210 nm. In this chromatographic system, chlorobutanol had a capacity factor (K') of 4.1. Standard curves obtained in the presence of ointment vehicle containing an aminoglycoside were linear, intercepted at zero, and averaged 99.4% recovery. Degradation studies indicated that the method was stability indicating. The analytical results for a complete experimental ophthalmic ointment and an aqueous ophthalmic diluent are presented. This high-performance liquid chromatographic method of analysis represents an alternative to GC procedures for determining chlorobutanol.

Trauma management therapy: a preliminary evaluation of a multicomponent behavioral treatment for chronic combat-related PTSD.

The development and initial evaluation of a new, comprehensive and multicomponent behavioral treatment (Trauma Management Therapy, or TMT) for chronic combat-related Post-Traumatic Stress Disorder (PTSD) is described. The program utilizes elements of intensive exposure therapy, programmed practice, and structured social and emotional skills training to target the multiple aspects of chronic combat-related PTSD. The treatment was found to be effective in alleviating a broad spectrum of difficulties in combat veterans with chronic PTSD, most of whom had co-occurring Axis I and/or Axis II disorders. The results are discussed with respect to the implementation of the new treatment and the general need for a comprehensive approach to treating combat-related PTSD. Implications for the potential cost-effectiveness of the treatment program also are discussed.

Analyses of nuclear ldhA gene and mtDNA control region sequences of Atlantic northern bluefin tuna populations.

There has been considerable debate about whether the Atlantic northern bluefin tuna exist as a single panmictic unit. We have addressed this issue by examining both mitochondrial DNA control region nucleotide sequences and nuclear gene ldhA allele frequencies in replicate size or year class samples of northern bluefin tuna from the Mediterranean Sea and the northwestern Atlantic Ocean. Pairwise comparisons of multiple year class samples from the 2 regions provided no evidence for population subdivision. Similarly, analyses of molecular variance of both mitochondrial and ldhA data revealed no significant differences among or between samples from the 2 regions. These results demonstrate the importance of analyzing multiple year classes and large sample sizes to obtain accurate estimates when using allele frequencies to characterize a population. It is important to note that the absence of genetic evidence for population substructure does not unilaterally constitute evidence of a single panmictic population, as genetic differentiation can be prevented by large population sizes and by migration.

Assessment and support of sexual functioning in 458 men seeking genitourinary reconstructive surgery.

Horton and associates focused on an aspect of genital reconstructive surgery: restored or improved sexual functioning. The availability a physician skilled in the application of modern sexual counseling has been demonstrated in a group of 458 men to assist in maximizing the benefit of such surgery on patients' sexual and coital success. The sexual therapist was a valued member of the team approach to patient care. The necessity of psychiatric assessment for some was apparent, as it would be in any population of patients seeking plastic or corrective surgery. The association of the sexual therapist with various mental health professionals was important. The recognition of mental and emotional difficulties was primary, but the further skills the sexual therapist brought by application of sexual therapy techniques was also important. Men disadvantaged sexually by genital abnormality gained from a sensitive and educational approach, one that included their partners in the context of open communication and offered specific suggestions for their own specific needs and situations. Sexual therapy techniques and skills instruction added a vital dimension to the complete care of these men seeking correction of genital abnormalities. The techniques and approaches themselves were fairly straightforward and simple, as well as fairly standardized. Described by Masters and Johnson, Jones and others, they were clearly effective in enhancing comfort with sexuality and less pressured approaches to sexual behavior, and ultimately in enhancing sexual functioning itself.

Softgels: consumer perceptions and market impact relative to other oral dosage forms.

Softgels, which contain a liquid formulation of a drug, often provide clinical benefit over other solid oral dosage forms and may represent an attractive alternative to them. A consumer preference survey of softgels versus other solid forms investigated four areas: (1) identification of various dosage forms; (2) perception of therapeutic benefit (easiest to swallow, faster-acting, work longer); (3) impact of individual product characteristics on overall product selection; and (4) market impact in terms of premiums consumers would pay on the basis of dosage form. The 300 survey participants strongly preferred clear softgels over other dosage forms in virtually every area. Softgels were perceived as easy to swallow and fast-acting, with a duration of action second only to that of a two-piece capsule. Overall preference was driven by ease of swallowing, and softgels were rated first by the majority of respondents. Consumers would be interested in various products if these were available as softgels rather than in their current oral dosage forms and may be willing to pay a premium for softgel products. This survey confirms consumer preferences for particular dosage forms and for softgels over other solid forms. Pharmaceutical scientists and marketers should consider softgels as alternative dosage forms when developing new compounds or considering life-cycle management of existing products.

A priority queuing model to reduce waiting times in emergency care.

Investigates the increased waiting time costs imposed on society due to inappropriate use of the emergency department by patients, seeking non-emergency or primary care. Proposes a simple economic model to illustrate the effect of this misuse at a public or not-for-profit hospital. Provides evidence that non-emergency patients contribute to lengthy delays in the ER for all classes of patients. Proposes a priority queuing model to reduce average waiting times.

Norplant prescribing in family practice.

BACKGROUND: This study describes the Norplant prescribing experience, training, and attitudes of South Carolina family practice and general practice (FP/GP) physicians. METHODS: A survey was mailed to all FP/GP physicians licensed in South Carolina, with two follow-up mailings to nonrespondents. RESULTS: Responses were received from 520 physicians, representing 43% of all licensed FP/GP physicians and 73% of all board-certified FP physicians. Norplant had been inserted by 39% of the respondents, and fewer than 10% reported encountering complications during the insertion process. Norplant removal was reported by 135 physicians, averaging 4.4 removals per year. Complications during removal were reported by 52% of physicians, with the most common problem being difficulty finding the capsules. Some degree of training in Norplant insertion was reported by 82% of physicians; 69% reported having had some training, formal or informal, in removal; but only 57% reported having received formal training either during residency or at a workshop. Training reduced insertion time but did not affect removal time or the number of complications encountered. Formal training was more likely to prepare physicians to successfully manage procedural complications. CONCLUSIONS: FP/GP physicians are important providers of Norplant. Improved training is needed to ensure that insertion is performed properly and to disseminate effective removal techniques.

PIP: A descriptive study of Norplant prescribing experiences, training, and attitudes among South Carolina physicians revealed a need for improved training opportunities. A questionnaire mailed to all 1201 family and general practice physicians licensed in South Carolina was returned by 540 (43%) physicians. 200 (39%) of these respondents had prescribed Norplant for women seeking contraception. Female physicians, younger physicians, and those who listed their specialty as family rather than general practice were significantly more likely than their counterparts to prescribe this method. The mean number of Norplant insertions per month was 1.9; only 7 physicians performed 10 or more insertions per month. Only 13 physicians reported any insertion-related complications (primarily bruising). Norplant removal was reported by 135 (24%) physicians, with an average of 4.4 removals annually. Complications during removal, primarily difficulties locating the capsules, were reported by 52% of physicians. 82% of physicians had received some form of training in Norplant insertion, including observing another physician, and 69% had been trained in capsule removal; formal training (residency or workshop) in insertion and removal was reported by only 57% and 53%, respectively. Training both reduced insertion time and prepared physicians to manage procedural complications.

Preventing falls in hospitals. The roles of patient age and diagnostic status in predicting falls.

Patient falls are some of the most frequent accidents in hospitals, and they can lead to significant personal and financial costs for both patients and facilities. This study examines the relationship between two patients characteristics and patient falls at a large general hospital and a small psychiatric facility in Tennessee; its findings should be useful for hospital risk management.

Succeeding beyond the Year 2000: how to rise to the challenges of the new millennium. Interview by Jill L. Sherer.

Are you ready to take your organization into the 21st century? Do you fully grasp the implications of current and emerging trends in the field? Healthcare Executive talked with six healthcare experts and asked them what they saw as the greatest challenge for both executives and their organizations in the new millennium. Although the experts' opinions vary, their responses emphasize the importance of repairing old relationships and building new partnerships between those working in healthcare organizations, as well as bringing a consumer focus back to healthcare delivery.