Search

In the yeast Saccharomyces cerevisiae , one determinant of aging or life span is the accumulation of extrachromosomal copies of rDNA circles in old mother cells [1]. The production of rDNA circles depends upon intrachromosomal recombination within the rDNA tandem array, a process regulated by the protein Sir2 (Sir2p). Together with Sir1p, Sir3p, Sir4p and Orc1p, Sir2p is also involved in transcriptional silencing of genes at the silent mating-type cassettes, in the rDNA array, and at telomeres. Using a ‘triple silencer' strain that can monitor an increase or decrease in gene expression at these three loci, we found that deletion of the ZDS1 gene caused an increase in silencing in the rDNA and at a silent mating-type cassette at the expense of telomere silencing. The zds1 deletion also resulted in an increase in life span and a decrease in Sir3p phosphorylation. In contrast, deletion of its paralog ZDS2 caused a decrease in rDNA silencing, a decrease in life span and an increase in Sir3p phosphorylation. As Zds2p, but not Zds1p, had strong two-hybrid interactions with Orc1p and the four Sir proteins, Zds1p might indirectly control Sir3p through a Sir3p kinase.

The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3) strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG(1-3). Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3' TG(1-3) overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG(1-3) extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3' single-stranded TG(1-3) extension present at yeast telomeres.

Motor vehicle travel is the primary means of transportation in the United States, providing freedom in travel and enterprise for many people. Motor vehicle crashes are the leading cause of death for the young and contribute to a high degree of morbidity and mortality for all ages. Motor vehicle crashes produce an enormous burden for society in terms of suffering, disability, death, and costs. Motor vehicle crash injury prevention is developing as a focused discipline to implement proven interventions involving technology and behavior known to prevent or reduce the severity of motor vehicle crash injury. Emergency physicians have an important role in advocating motor vehicle safety and injury prevention, both in the emergency department and within the community.

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.

Objective: The use of automatic external defibrillators (AEDs) by EMS initial responders is widely advocated. Evidence supporting the use of AEDs is based largely on the experience of one metropolitan area, with effect on survival in many systems not yet proved. We conducted this study to determine whether the addition of AEDs to an EMS system with a response time of 4 minutes for first-responder emergency medical technicians (FREMTs) and 10 minutes for paramedics would affect survival from cardiac arrest. Methods: This prospective, controlled, crossover study (AED versus no AED) of consecutive cardiac arrests managed by 24 FREMT fire companies took place from 1992 to 1995 in Charlotte, North Carolina, a city of 455,000. Patients were stratified using the Utstein criteria. The primary endpoint was survival to hospital discharge among patients with bystander-witnessed arrests of cardiac origin. Results: Of the 627 patients, 243 were bystander-witnessed arrests of cardiac origin. Survival to hospital discharge was accomplished in 5 of 110 patients (4.6%; 95% confidence interval [CI] 0.6% to 8.4%) with AED compared with 7 of 133 (5.3%, 95% CI 1.5% to 9.1%) without AED ( P =.8). Both groups were comparable with regard to age, gender, history of myocardial infarction, congestive heart failure or diabetes, arrest at home, bystander CPR, and whether or not ventricular fibrillation (VF) was the initial rhythm. For arrests of any cause, witnessed by bystanders or EMS personnel, with an initial rhythm of VF or ventricular tachycardia (VT), 5 of 77 (6.5%, 95% CI 1.0% to 12.0%) with AED survived compared with 8 of 105 patients (7.6%, 95% CI 2.5% to 12.7%) without AED ( P =.8). Statistically significant differences were noted in race and EMS response times between the two groups, which did not affect survival. Conclusion: Addition of AEDs to this EMS system did not improve survival from sudden cardiac death. The data do not support routinely equipping initial responders with AEDs as an isolated enhancement, and raise further doubt about such expenditures in similar EMS systems without first optimizing bystander CPR and EMS dispatching. [Sweeney TA, Runge JW, Gibbs MA, Raymond JM, Schafermeyer RW, Norton HJ, Boyle-Whitesel MJ: EMT defibrillation does not increase survival from sudden cardiac death in a two-tiered urban-suburban EMS system. Ann Emerg Med February 1998;31:234-240.]

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na+/H+ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H+ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na+/H+ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na+/H+ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity.

Objectives: To profile all patients presenting to an urban ED with any injury, and to determine whether the rate of subsequent injury treated in the ED varied by demographic and E-code (external mechanism of injury) category. The hypothesis that young black males were disproportionately at risk for re-injury was addressed. Methods: A cohort of consecutive patients presenting to an urban ED with any injury between January 1, 1991, and November 31, 1992, were followed prospectively for 1 year from their index visit dates. Any repeat ED visits due to injury were sought. The mean number of injury visits per year (the total number of ED injury visits for each patient divided by 1 year) was computed for the overall population and by race, age, gender, and E-code. Results: The sample consisted of 34,378 patients who made 44,813 visits to the ED for injury. Of these patients, 22% had a repeat injury in 1 year, with a cohort mean of 1.30 injury visits per year. This mean did not vary appreciably by race (black 1.33, white 1.27), age (1–17 yr, 1.21; 18–24 yr, 1.32; 25–64 yr, 1.34; >65 yr, 1.23), gender (males 1.33, females 1.27), or E-code category. Having a prior injury visit in the preceding year was the best predictor of future injury (mean repeat visit rate = 2.08). Conclusions: When examining patients presenting with any injury to an urban ED, the mean numbers of injury visits are remarkably similar across demographic and E-code categories. Although there are factors that place patients at risk for recurrent injury, those factors are not demographic—all patients presenting to an ED with injury should be considered at risk for re-injury.

In the field of rheumatology, conventional diagnostic methods permit the detection only of advanced stages of the disease, which is at odds with the current clinical demand for the early diagnosis of inflammatory rheumatic diseases. Prompted by current needs, we developed a finger joint phantom that enables the optical and geometrical simulation of an early stage of rheumatoid arthritis (RA). The results presented here form the experimental basis for an evaluation of new RA diagnostic systems based on near infrared light. The early stage of RA is characterised mainly by a vigorous proliferation of the synovial membrane and clouding of the synovial fluid. Using a double-integrating-sphere technique, the absorption and scattering coefficients (mua, mus') are experimentally determined for healthy and pathologically altered synovial fluid and capsule tissue. Using a variable mixture of Intralipid Indian ink and water as a scattering/absorption medium, the optical properties of skin, synovial fluid or capsule can be selected individually. Since the optical and geometrical properties of bone tissue remain constant in early-stage RA, a solid material is used for its simulation. Using the finger joint phantom described herein, the optical properties of joint regions can be adjusted specifically, enabling an evaluation of their effects on an optical signal--for example, during fluorography--and the investigation of these effects for diagnostically useful information. The experimental foundation for the development of a new optical system for the early diagnosis of RA has now been laid.

Tumors create an extracellular pH (pHe) barrier and form an isolated microenvironment roughly 0.5 pH units lower than physiological pH, while the intracellular pH (pHi) is maintained within 0.2 pH ...

Model organisms such as budding yeast, worms and flies have proven instrumental in the discovery of genetic determinants of aging, and the fission yeast Schizosaccharomyces pombe is a promising new system for these studies. We devised an approach to directly select for long-lived S. pombe mutants from a random DNA insertion library. Each insertion mutation bears a unique sequence tag called a bar code that allows one to determine the proportion of an individual mutant in a culture containing thousands of different mutants. Aging these mutants in culture allowed identification of a long-lived mutant bearing an insertion mutation in the cyclin gene clg1(+). Clg1p, like Pas1p, physically associates with the cyclin-dependent kinase Pef1p. We identified a third Pef1p cyclin, Psl1p, and found that only loss of Clg1p or Pef1p extended lifespan. Genetic and co-immunoprecipitation results indicate that Pef1p controls lifespan through the downstream protein kinase Cek1p. While Pef1p is conserved as Pho85p in Saccharomyces cerevisiae, and as cdk5 in humans, genome-wide searches for lifespan regulators in S. cerevisiae have never identified Pho85p. Thus, the S. pombe system can be used to identify novel, evolutionarily conserved lifespan extending mutations, and our results suggest a potential role for mammalian cdk5 as a lifespan regulator.

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4 + marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Yeasts are powerful model systems to examine the evolutionarily conserved aspects of eukaryotic aging because they maintain many of the same core cellular signaling pathways and essential organelles as human cells. We constructed a strain of the budding yeast Saccharomyces cerevisiae that could monitor the distribution of proteins involved in heterochromatic silencing and aging, and isolated mutants that alter this distribution. The largest class of such mutants cause defects in mitochondrial function, and appear to cause changes in nuclear silencing separate from the well-known Rtg2p-dependent pathway that alters nuclear transcription in response to the loss of the mitochondrial genome. Mutants that inactivate the ATP2 gene, which encodes the ATPase subunit of the mitochondrial F(1)F(0)-ATPase, were isolated twice in our screen and identify a lifespan extending pathway in a gene that is conserved in both prokaryotes and eukaryotes. The budding yeast S. cerevisiae S. cerevisiae has been used with great success to identify other lifespan-extending pathways in screens using surrogate phenotypes such as stress resistance or silencing to identify random mutants, or in high throughput screens that utilize the deletion strain set resource. However, the direct selection of long-lived mutants from a pool of random mutants is more challenging. We have established a new chronological aging assay for the evolutionarily distant fission yeast Schizosaccharomyces pombe that recapitulates aspects of aging conserved in all eukaryotes. We have constructed a novel S. pombe S. pombe DNA insertion mutant bank, and used it to show that we can directly select for a long-lived mutant. The use of both the budding and fission yeast systems should continue to facilitate the identification and validation of lifespan extending pathways that are conserved in humans.

We isolated mutants of Saccharomyces cerevisiae that lose a 100 kb linear yeast artificial chromosome (YAC) at elevated rates. Mutations in two of these LCS (linear chromosome stability) genes had little or no effect on the loss rate of a circular YAC that had the same centromere and origin of replication as present on the linear YAC. Moreover, mutations in these LCS genes also increased the loss rate of an authentic linear yeast chromosome, chromosome III, but had only small effects on the loss rate of a circular derivative of chromosome III. As these mutants preferentially destabilize linear chromosomes, they may affect chromosome stability through interactions at telomeres. Telomeres are thought to be essential for the protection and complete replication of chromosome ends. The cytological properties of telomeres suggest that these structures may play additional roles in chromosome function. The lengths of the terminal C1-3A repeats at the ends of yeast chromosomes were unaltered in the linear preferential lcs mutants, suggesting that these mutants do not affect the replication or protection of telomeric DNA. Thus, the linear-preferential lcs mutants may identify a role for telomeres in chromosome stability that is distinct from their function in the replication and protection of chromosomal termini.

The public health problem of injury has an enormous impact on individuals and society, both as a health and economic issue. Prior to the last decade, little attention has been paid to the costs generated by injury, much of which is preventable. By looking at aggregate economic costs for different injuries and population groups, the physician can understand where the problems occur that need to be addressed to curb this staggering burden on society.

Study purpose: Little information exists on the cause of injury for patients who are treated and discharged from emergency departments; these patients comprise approximately 90% of all injured patients requiring medical care. A method is described to assign external cause of injury codes (E codes) prospectively to all injured patients seen in a large-volume ED. Methods: E code assignment was performed by the ED triage nurses on entrance to the ED. A checklist was used that contained frequently occurring codes as identified in a pilot study. E codes were entered into the patients' records on the hospital mainframe computer by a medical records technician. These were acquired for a nine-month period to determine feasibility, accuracy, and ease of use. Accuracy was verified retrospectively. Results: During the nine-month period, 67,495 patients were treated. Acute injury accounted for 16,186 of the visits, and 2,085 were return visits for a previously treated injury. The majority of patients with any injury type were treated and discharged and would not have been included in traditional data sets of injured patients. Overall admission rate for injury was 13%. Accuracy of prospectively assigned E codes was 98%, and this method of assigning E codes resulted in no additional effort by the triage nurse. Conclusion: E coding is a valuable method for injury surveillance, easily performed in EDs with high volume. Its value is essential for injury prevention research on injuries from any cause.

[Runge JW. Linking data for injury control research. Ann Emerg Med . June 2000;35:613-615.]

We describe a new chronological lifespan (CLS) assay for the yeast Schizosaccharomyces pombe. Yeast CLS assays monitor the loss of cell viability in a culture over time, and this new assay shows a continuous decline in viability without detectable regrowth until all cells in the culture are dead. Thus, the survival curve is not altered by the generation of mutants that can grow during the experiments, and one can monitor the entire lifespan of a strain until the number of viable cells has decreased over 10(6)-fold. This CLS assay recapitulates the evolutionarily conserved features of lifespan shortening by over nutrition, lifespan extension by caloric restriction, increased stress resistance of calorically restricted cells and lifespan control by the AKT kinases. Both S. pombe AKT kinase orthologs regulate CLS: loss of sck1(+) extended lifespan in over nutrition conditions, loss of sck2(+) extended lifespan under both normal and over nutrition conditions, and loss of both genes showed that sck1(+) and sck2(+) control different longevity pathways. The longest-lived S. pombe cells showed the most efficient cell cycle exit, demonstrating that caloric restriction links these two processes. This new S. pombe CLS assay will provide a valuable tool for aging research.

The linear chromosomes of eukaryotes contain specialized structures to ensure their faithful replication and segregation to daughter cells. Two of these structures, centromeres and telomeres, are limited, respectively, to one and two copies per chromosome. It is possible that the proteins that interact with centromere and telomere DNA sequences are present in limiting amounts and could be competed away from the chromosomal copies of these elements by additional copies introduced on plasmids. We have introduced excess centromeres and telomeres into Saccharomyces cerevisiae and quantitated their effects on the rates of loss of chromosome III and chromosome VII by fluctuation analysis. We show that (i) 600 new telomeres have no effect on chromosome loss; (ii) an average of 25 extra centromere DNA sequences increase the rate of chromosome III loss from 0.4 x 10(-4) events per cell division to 1.3 x 10(-3) events per cell division; (iii) centromere DNA (CEN) sequences on circular vectors destabilize chromosomes more effectively than do CEN sequences on 15-kb linear vectors, and transcribed CEN sequences have no effect on chromosome stability. We discuss the different effects of extra centromere and telomere DNA sequences on chromosome stability in terms of how the cell recognizes these two chromosomal structures.

An oncofetal protein (OFP) studied in our laboratory associated with embryogenesis, carcinogenesis and tumorigenesis has as its known biological function the modification of RNA release from isolated nuclei. In the present study, we have developed and investigated the use of monoclonal antibodies against OFP. Six hybridoma cell lines (A-F) were isolated by screening the hybridoma culture media for anti-OFP antibodies (MOFP) with an indirect ELISA and by testing the ability of these antibodies complexed with anti-mouse IgG-agarose to bind to rat OFP and remove its associated RNA transport activity from solution (Immunobioassay). An inhibition ELISA developed to measure OFP gave a linear response up to 20 ng of plasma protein from a tumor-bearing rat. Western blot analysis using these monoclonals showed that OFP from a rat tumor (H7777) cytosol that shed to the blood consisted of two species exhibiting molecular weights of 50 and 55 kD respectively. In order to show the usefulness of our assays, a preliminary study showing the ability of the immunobioassay to detect the expression of OFP in the plasma of carcinogen treated rats in a dosage dependent manner has been presented. Since OFP is produced in the target organ of rats shortly after treatment with carcinogens and persists in the preneoplastic foci and subsequent tumors, these monoclonal antibodies will be valuable in studying its involvement in chemical carcinogenesis and tumorigenesis.

National Highway Traffic Safety Administration: Child safety seat distribution: What works? Ann Emerg Med September 1999;34:403-404.]

Sublethal doses of organophosphate anticholinesterases cause acute pancreatitis in dogs within 2 h. In vitro studies using canine pancreatic fragments have also demonstrated that the peak of amylase release in response to acetylcholine is shifted far to the left after incubation with the organophosphates echothiophate (10(-4) M) or tetraisopropyl pyrophosphoramide (iso-OMPA) (10(-3) M), indicating an increased sensitivity of response. The present in vitro study examined whether there was also an increased susceptibility to acinar cell damage at the electron microscopic level after acetylcholine or cholecystokinin. Minced pieces of whole fresh canine pancreas 2-3 mm in size were placed in buffered Eagle's solution and gassed with 100% O2. After pretreatment 1 h with echothiophate or iso-OMPA, they were then incubated with acetylcholine (10(-5) M). Other tissues preincubated with echothiophate were stimulated with cholecystokinin (10(-9) M). These are submaximal doses for untreated canine pancreatic fragments. After acetylcholine and echothiophate or acetylcholine and iso-OMPA, there was extensive acinar damage with the appearance of large vacuoles and lakes, and interstitial edema. There was evidence of intense supramaximal stimulation and lateral exocytosis. Similar destructive changes were seen after echothiophate and cholecystokinin. In control sections from tissues stimulated with acetylcholine (10(-5) M) or cholecystokinin (10(-9) M, there were lumenal exocytotic patterns typical of submaximal stimulation. Other controls, organophosphate alone and unstimulated basal conditions, showed only minor changes. It is concluded that the increased sensitivity to acetylcholine after organophosphate incubation correlates with an increased susceptibility to acinar ultrastructural damage from acetylcholine and cholecystokinin.

Current models of telomere formation and replication involve either telomerase, a novel ribonucleoprotein, or recombination between the ends of DNA molecules. However, present models will have to be modified to explain recent data on telomere formation in yeast. An understanding of the mechanisms of telomere maintenance in yeast may reveal how other organisms with heterogeneous telomeric repeats replicate their chromosomal termini.

[National Highway Traffic Safety Administration: Safe mobility for older people. Ann Emerg Med April 1999;33:469-470.] [Jolly BT: NHTSA Notes commentary: Older drivers: Growth industry for the future. Ann Emerg Med April 1999;33:470.]

In many organisms, telomeric DNA consists of long tracts of short repeats. Shorter tracts are preferentially lengthened by telomerase, suggesting a conserved mechanism that recognizes and elongates short telomeres. Tel1p, an ATM family checkpoint kinase, plays an important role in telomere elongation, as cells lacking Tel1p have short telomeres and show reduced recruitment of telomerase components to telomeres. We show that Tel1p association increased as telomeres shortened in vivo in the presence or absence of telomerase and that Tel1p preferentially associated with the shortest telomeres. Tel1p association was independent of Tel1p kinase activity and enhanced by Mre11p. Tel1p overexpression simultaneously stimulated telomerase-mediated elongation and Tel1p association with all telomeres. Thus, Tel1p preferentially associates with the shortest telomeres and stimulates their elongation by telomerase.

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends on reduced vitamin K generated by vitamin K oxidoreductase (VKOR) and a redox protein that regenerates VKOR activity. VKD protein carboxylation is inefficient in mammalian cells, and to understand why carboxylation becomes saturated, we developed an approach that directly measures the extent of intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow egress of fIX from the endoplasmic reticulum and preferential secretion of the carboxylated form contribute to secreted fIX being more fully carboxylated. The analysis also revealed the first reported in vivo VKD protein turnover, which was 14-fold faster than that which occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and the extent of secreted carboxylated fIX approximately 2-fold, which shows that carboxylation is the rate-limiting step in fIX turnover and which was surprising because turnover in vitro is limited by release of carboxylated fIX. Interestingly, the increases were significantly smaller than the amount of VKOR overexpression (15-fold). However, when cell extracts were tested in single-turnover experiments in vitro, where redox protein is functionally substituted with dithiothreitol, VKOR overexpression increased the fIX carboxylation rate 14-fold, showing r-VKORC1 is functional for supporting fIX carboxylation. These data indicate that the effect of VKOR overexpression is limited in vivo, possibly because a carboxylation component like the redox protein becomes saturated or because another step is now rate-limiting. The studies illustrate the complexity of carboxylation and potential importance of component stoichiometry to overall efficiency.

Telomeres are the physical ends of linear eukaryotic chromosomes. In many organisms, including humans and yeasts, telomeres are composed of short repeated DNA sequences and their associated proteins. These repeats are lost gradually due to incomplete DNA synthesis of the chromosome end, or by degradation from nucleases, and can be resynthesized by the enzyme telomerase. Many human somatic cells do not express sufficient telomerase activity to prevent telomere repeat loss, resulting in cell senescence or death when telomeres shorten to a critical length. This telomere length checkpoint for aging and cell growth is also seen in yeast whose genes for telomerase components have been deleted, allowing yeast to serve as a model for telomere-linked senescence and aging in human cells. Telomeres also serve as a reservoir of bound proteins that are released as the telomere DNA shortens, and work in yeast has shown that these telomere-associated proteins play important roles in yeast replicative aging. The advantages of yeast as an aging model include rapid growth and aging, a small well-defined and well-annotated genome, powerful molecular genetics for modifying this genome, simple but robust assays for the activity and location of telomeric proteins, publicly available collections of mutants in every gene, and multiple assays for replicative and chronological aging. This chapter will describe how the budding yeast Saccharomyces cerevisiae has contributed to our knowledge of telomeres and aging, and the emerging role of the evolutionarily divergent fission yeast Schizosaccharomyces pombe as another important model system. The powerful genetics of these two different yeast systems should identify central evolutionarily conserved processes that control cellular aging.

In Aplysia californica enterocytes, alanine-stimulated Na+ absorption increases both apical membrane exocytosis and fractional capacitance (fCa; a measure of relative apical membrane surface area). These increases are thought to reduce membrane tension during periods of nutrient absorption that cause the enterocytes to swell osmotically. In the absence of alanine, exocytosis and fCa are constant. These findings imply equal rates of constitutive endocytosis and exocytosis and constitutive recycling of the apical plasma membrane. Thus, the purpose of this study was to confirm and determine the relative extent of constitutive apical membrane recycling in Aplysia enterocytes. Biotinylated lectins are commonly used to label plasma membranes and to investigate plasma membrane recycling. Of fourteen biotinylated lectins tested, biotinylated wheat germ agglutinin (bWGA) bound preferentially to the enterocytes apical surface. Therefore, we used bWGA, avidin D (which binds tightly to biotin), and the UV fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated avidin D to assess the extent of constitutive apical membrane recycling. A temperature-dependent (20 vs. 4°C) experimental protocol employed the use of two tissues from each of five snails and resulted in a ˜60% difference in apical surface fluorescence intensity. Because the extent of membrane recycling is proportional to the difference in surface fluorescence intensity, this difference reveals a relatively high rate of constitutive apical membrane recycling in Aplysia enterocytes. J. Exp. Zool. 301A:857–866, 2004. © 2004 Wiley-Liss, Inc.

Recent advances in the discovery of new functions for vitamin K-dependent (VKD) proteins and in defining vitamin K nutriture have led to a substantial revision in our understanding of vitamin K physiology. The only unequivocal function for vitamin K is as a cofactor for the carboxylation of VKD proteins which renders them active. While vitamin K was originally associated only with hepatic VKD proteins that participate in hemostasis, VKD proteins are now known to be present in virtually every tissue and to be important to bone mineralization, arterial calcification, apoptosis, phagocytosis, growth control, chemotaxis, and signal transduction. The development of improved methods for analyzing vitamin K has shed considerable insight into the relative importance of different vitamin K forms in the diet and their contribution to hepatic vs. non-hepatic tissue. New assays that measure the extent of carboxylation in VKD proteins have revealed that while the current recommended daily allowance for vitamin K is sufficient for maintaining functional hemostasis, the undercarboxylation of at least one non-hemostatic protein is frequently observed in the general population. The advances in defining VKD protein function and vitamin K nutriture are described, as is the potential impact of VKD proteins on atherosclerosis. Many of the VKD proteins contribute to atherogenesis. Recent studies suggest involvement in arterial calcification, which may be influenced by dietary levels of vitamin K and by anticoagulant drugs such as warfarin that antagonize vitamin K action.

Vitamin K-dependent (VKD) proteins require carboxylation for diverse functions that include hemostasis, apoptosis, and Ca 2+ homeostasis, yet the mechanism of carboxylation is not well understood. Combined biochemical and chemical studies have led to a long-standing model in which a carboxylase Cys catalytic base deprotonates vitamin K hydroquinone (KH 2 ), leading to KH 2 oxygenation and Glu carboxylation. We previously identified human carboxylase Cys-99 and Cys-450 as catalytic base candidates: Both were modified by N -ethylmaleimide (NEM) and Ser-substituted mutants retained partial activity, suggesting that the catalytic base is activated for increased basicity. Mutants with Cys-99 or Cys-450 substituted by Ala, which cannot ionize to function as a catalytic base, were therefore analyzed. Both single and double mutants had activity, indicating that Cys-99 and Cys-450 do not deprotonate KH 2 . [ 14 C]NEM modification of C99A/C450A revealed one additional reactive group; however, Ser-substituted mutants of each of the eight remaining Cys retained substantial activity. To unequivocally test, then, whether any Cys or Cys combination acts as the catalytic base, a mutant with all 10 Cys substituted by Ala was generated. This mutant showed 7% wild-type activity that depended on factor IX coexpression, indicating a VKD protein effect on carboxylase maturation. NEM and diethyl pyrocarbonate inhibition suggested that the catalytic base is an activated His. These results change the paradigm for VKD protein carboxylation. The identity of the catalytic base is critical to understanding carboxylase mechanism and this work will therefore impact both reinterpretation of previous studies and future ones that define how this important enzyme functions.

Together we can save lives and reduce suffering Road traffic crashes are not just a highway safety problem—they are a public health problem. With over a million people killed each year on the world’s roads, and tens of millions more injured, road traffic crashes are a leading cause of death and the ninth leading cause of disability adjusted life years (DALYs) lost worldwide. By 2020, road traffic injuries are projected to become the third leading cause of DALYs. This is all the more tragic because we could prevent so many of these deaths, so many of these injuries, and so much of this suffering. In the United States, road traffic injuries accounted for more than 42 000 deaths in 2002 and almost three million non-fatal injuries.1 They are the leading cause of death for people ages 1–34 years and the leading cause of injury related death. The cost of motor vehicle crashes exceeded $230 billion in 2000.2 The United States has the most motor vehicles per capita of any country in the world (765 motor vehicles per 1000 population).3 Therefore, we had to begin addressing the problem of road traffic safety many …

The Association for the Advancement of Automotive Medicine (AAAM) sponsored an international conference, April 23-24, 2001, in Washington, D.C., to promote scientifically sound public policy on child booster seats in motor vehicles. This commentary lists 12 recommendations drafted by the conference planning committee, and also includes a brief summary of relevant scientific evidence presented at the conference related to each recommendation.

We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na+ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by approximately 30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (ISC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na+ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na+ and alanine entry because increments in alanine-stimulated ISC as small as 1 microA/cm2 stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated ISC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by approximately 65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.

Increased customer demand for state-of-the-art vehicles means that the vehicle manufacturer must integrate new, more complex electronic functions. Therefore, an increasing number of systems in passenger cars are electronically-controlled, and the complexity of existing systems increases out of all proportion. Electronics is beginning to determine functions and value, and has therefore become a considerable factor with regard to competitiveness.

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG(1-3) sequence for the most internal approximately 100 bp of the 250-350 bp TG(1-3) repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG(1-3) sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70 and tel1 mutants, whose entire telomeres are only approximately 100 bp, were examined by analyzing 5.1 kb of cloned TG(1-3) sequences from telomeres formed during transformation of wild type, yku70 and tel1 cells. The internal domains were 97-137 bp in wild type cells, 27-36 bp in yku70 cells and 7-9 bp in tel1 cells. These data suggest that the majority of the tel1 cell TG(1-3) repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70 cell telomeres are protected. TG(1-3) sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG(1-3) repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG(1-3) repeats, only those consistent with telomere hybridization to the ACACAC in the 3'-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40-60% of the TG(1-3) sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3'-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG(1-3) sequences that mimic this process.

[Runge JW. NHTSA Notes Commentary: Screening for alcohol use disorders—barriers and excuses. Ann Emerg Med. December 2000;36:629-630.]

[Runge JW. NHTSA Notes Commentary: Antihistamines and driving performance—an underrecognized issue in traffic safety. Ann Emerg Med. October 2000;36:389-390.]

We have developed a simple laboratory exercise that uses an inexpensive dialysis membrane (molecular weight cutoff = 100) to illustrate the generation of membrane potentials (Vm) across plasma membranes of animal cells. A piece of membrane approximately 2.0 cm2 is mounted in an Ussing-like chamber. One chamber half is designated cytosol and the other half external. Chamber sidedness helps students relate their findings to those of real cells. As in real cells, outward directed K+ concentration gradients [high cytosolic K+ concentration ([K+]c) and low extracellular K+ concentration] generate cytosol electrically negative Vm with a slope of approximately -45 mV/decade change in [K+]c. The polarity of Vm reflects the outward flow of potassium ions because flow of the larger counterion, H2PO4-, is restricted to the pores in the membrane. A slope less than Nernstian (