Universal DNA methylation age across mammalian tissues.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

National probability samples in studies of low-prevalence diseases. Part I: Perspectives and lessons from the HIV cost and services utilization study.

OBJECTIVE: To examine the trade-offs inherent in selecting a sample design for a national study of care for an uncommon disease, and the adaptations, opportunities and costs associated with the choice of national probability sampling in a study of HIV/AIDS. SETTING: A consortium of public and private funders, research organizations, community advocates, and local providers assembled to design and execute the study. DESIGN: Data collected by providers or collected for administrative purposes are limited by selectivity and concerns about validity. In studies based on convenience sampling, generalizability is uncertain. Multistage probability sampling through households may not produce sufficient cases of diseases that are not highly prevalent. In such cases, an attractive alternative design is multistage probability sampling through sites of care, in which all persons in the reference population have some chance of random selection through their medical providers, and in which included subjects are selected with known probability. DATA COLLECTION AND PRINCIPAL FINDINGS: Multistage national probability sampling through providers supplies uniquely valuable information, but will not represent populations not receiving medical care and may not provide sufficient cases in subpopulations of interest. Factors contributing to the substantial cost of such a design include the need to develop a sampling frame, the problems associated with recruitment of providers and subjects through medical providers, the need for buy-in from persons affected by the disease and their medical practitioners, as well as the need for a high participation rate. Broad representation from the national community of scholars with relevant expertise is desirable. Special problems are associated with organization of the research effort, with instrument development, and with data analysis and dissemination in such a consortium. CONCLUSIONS: Multistage probability sampling through providers can provide unbiased, nationally representative data on persons receiving regular medical care for uncommon diseases and can improve our ability to accurately study care and its outcomes for diseases such as HIV/AIDS. However, substantial costs and special circumstances are associated with the implementation of such efforts.

Antioxidant capacity of oat (Avena sativa L.) extracts. 2. In vitro antioxidant activity and contents of phenolic and tocol antioxidants.

Oat milling fractions were examined for concentrations of total phenolics, tocols, and phenolic acids and in vitro antioxidant activity to determine their potential as dietary antioxidants. Methanolic extracts of pearling fractions, flour and aspirations from flaking, and trichomes had high, intermediate, and low antioxidant activities, respectively, evaluated by the beta-carotene bleaching method. Pearling fractions were also highest in total phenolics and tocols. p-Hydroxybenzoic acid, vanillic acid, caffeic acid, vanillin, p-coumaric acid, and ferulic acid were identified and quantified by HPLC. Three avenanthramides and an unidentified ferulate derivative were also detected. Total phenolic content was significantly correlated with antioxidant activity, and regression equations that predicted antioxidant activity from phenolic and tocol concentrations were calculated. Antioxidant activity, evaluated by beta-carotene bleaching, was correlated with measures of oxygen radical absorbance capacity and low-density lipoprotein oxidation. These data indicate a potential for oat products, especially those enriched in outer layers of the groat, to contribute to dietary intakes of antioxidant phytonutrients.

Transport characteristics of the apical anion exchanger of rabbit cortical collecting duct beta-cells.

To functionally characterize transport properties of the apical anion exchanger of rabbit beta-intercalated cells, the mean change in anion exchange activity, dpHi/dt (where pHi is intracellular pH), was measured in response to lumen Cl- replacement with gluconate in perfused cortical collecting ducts (CCDs). beta-Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl-. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl-. beta-Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, beta-cell apical anion exchange was inhibited by alpha-cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl- resulted in a similar rate of alkalinization. Conversely, pHi was unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the beta-cell apical anion exchanger. Taken together, the results demonstrate a distinct "fingerprint" of the rabbit CCD beta-cell apical anion exchanger that is unlike that of other known anion exchangers.

The care of HIV-infected adults in the United States. HIV Cost and Services Utilization Study Consortium.

BACKGROUND AND METHODS: In order to elucidate the medical care of patients with human immunodeficiency virus (HIV) infection in the United States, we randomly sampled HIV-infected adults receiving medical care in the contiguous United States at a facility other than military, prison, or emergency department facility during the first two months of 1996. We interviewed 76 percent of 4042 patients selected from among the patients receiving care from 145 providers in 28 metropolitan areas and 51 providers in 25 rural areas. RESULTS: During the first two months of 1996, an estimated 231,400 HIV-infected adults (95 percent confidence interval, 162,800 to 300,000) received care. Fifty-nine percent had the acquired immunodeficiency syndrome according to the case definition of the Centers for Disease Control and Prevention, and 91 percent had CD4+ cell counts of less than 500 per cubic millimeter. Eleven percent were 50 years of age or older, 23 percent were women, 33 percent were black, and 49 percent were men who had had sex with men. Forty-six percent had incomes of less than $10,000 per year, 68 percent had public health insurance or no insurance, and 30 percent received care at teaching institutions. The estimated annual direct expenditures for the care of the patients seen during the first two months of 1996 were $5.1 billion; the expenditures for the estimated 335,000 HIV-infected adults seen at least as often as every six months were $6.7 billion, which is about $20,000 per patient per year. CONCLUSIONS: In this national survey we found that most HIV-infected adults who were receiving medical care had advanced disease. The patient population was disproportionately male, black, and poor. Many Americans with diagnosed or undiagnosed HIV infection are not receiving medical care at least as often as every six months. The total cost of medical care for HIV-infected Americans accounts for less than 1 percent of all direct personal health expenditures in the United States.

A national survey of physician-assisted suicide and euthanasia in the United States.

BACKGROUND: Although there have been many studies of physician-assisted suicide and euthanasia in the United States, national data are lacking. METHODS: In 1996, we mailed questionnaires to a stratified probability sample of 3102 physicians in the 10 specialties in which doctors are most likely to receive requests from patients for assistance with suicide or euthanasia. We weighted the results to obtain nationally representative data. RESULTS: We received 1902 completed questionnaires (response rate, 61 percent). Eleven percent of the physicians said that under current legal constraints, there were circumstances in which they would be willing to hasten a patient's death by prescribing medication, and 7 percent said that they would provide a lethal injection; 36 percent and 24 percent, respectively, said that they would do so if it were legal. Since entering practice, 18.3 percent of the physicians (unweighted number, 320) reported having received a request from a patient for assistance with suicide and 11.1 percent (unweighted number, 196) had received a request for a lethal injection. Sixteen percent of the physicians receiving such requests (unweighted number, 42), or 3.3 percent of the entire sample, reported that they had written at least one prescription to be used to hasten death, and 4.7 percent (unweighted number, 59), said that they had administered at least one lethal injection. CONCLUSIONS: A substantial proportion of physicians in the United States report that they receive requests for physician-assisted suicide and euthanasia, and about 7 percent of those who responded to our survey have complied with such requests at least once.

Halide transport patterns of apical anion exchange in rabbit cortical collecting duct intercalated cells.

To investigate halide selectivity patterns of cortical collecting duct (CCD) intercalated cells (ICs), intracellular pH (pHi) was measured in perfused rabbit outer CCD ICs in Na(+)-free, HCO-3-buffered, 115 mM Cl solutions. Apical anion exchange activity was measured as the dpH/dt after lumen Cl- replacement with gluconate. The perfusate was randomly changed to equimolar Br-, F-, or I-. Addition of Br- to the lumen in place of gluconate caused a brisk acidification similar to that with Cl- return. The acidification rate with I- replacement was only approximately 25% of that seen with Cl- readdition, and F- substitution resulted in a small alkalinization. In a separate protocol, each halide was substituted for lumen Cl-. Replacement of lumen Cl- with Br- resulted in intracellular acidification, whereas F- substitution caused an increase in pHi similar to that with gluconate. A slower rate of alkalinization was seen with I-. Separate tubules were perfused and bathed in Cl(-)-free gluconate solutions for 10 min, and then either Cl- or Br- was returned to the lumen. Similar rates of acidification were found with either Cl- or Br- return. Taken together, the results show that, at a halide concentration of 115 mM, the halide selectivity transport pattern of the apical anion exchanger of CCD ICs is Cl- = Br- > I- > F-.

Evidence for tissue-associated alpha(2) macroglobulin in mouse skeletal muscle.

Alpha(2)-Macroglobulin (alpha(2)M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum alpha(2)M. In release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity persisted after an extensive prewash with PBS (up to 5-6 h), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfusion procedure was effective in removing serum alpha(2)M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated alpha(2)M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of alpha(2)M in skeletal muscle was hypothesized.

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.

Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status.

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct.

The distribution of Na(+)-independent Cl(-)-HCO3- exchange was studied in individual intercalated cells from in vitro perfused rabbit outer CCDs using dual excitation laser scanning confocal microscopy by measuring the pHi response to sequential removal of Cl- from both sides of the tubule. Three patterns of intracellular pH (pHi) response were observed. 39% of intercalated cells had only apical Cl(-)-HCO3- exchange (beta cell), 4% had only basolateral Cl(-)-HCO3- exchange (alpha cell), and 57% had both apical and basolateral Cl(-)-HCO3- exchange (gamma cell). Valinomycin-high K+ voltage clamping had no effect on the pHi response of intercalated cells with bilateral Cl(-)-HCO3- exchange. Although the mean rates of dpHi/dt following apical Cl- removal were similar in beta cells compared to gamma cells, a wide range of apical rates was seen among individual beta and gamma intercalated cells. Neither the apical nor the basolateral Cl(-)-HCO3- exchanger in gamma cells was inhibited by 0.5 mM H2DIDS. Binding of apical peanut lectin was seen both in beta cells and in gamma cells. In 41% of CCDs with four to seven intercalated cells studied, all intercalated cells were of the same subtype. We conclude that the majority of intercalated cells from the rabbit outer CCD have both apical and basolateral Na(+)-independent Cl(-)-HCO3- exchangers (gamma cells), which are stilbene-insensitive. Intercalated cells with only basolateral Cl(-)-HCO3- exchange are very uncommon in the rabbit outer CCD. There is a tendency for all intercalated cells in a given rabbit outer CCD to be of the same subtype (either all beta cells or all gamma cells), suggesting the presence of CCD intertubule heterogeneity at the same cortical level. This finding may account for intertubule differences in transepithelial H(+)-base transport.

H+/base transport pathways in the cortical collecting duct.

The CCD possesses the unique ability to alter not only the magnitude but also the direction of transepithelial H+/base and Cl- transport during changes in systemic acid-base balance. The single cell transport processes which are responsible for mediating these changes are presently incompletely understood. Functional evidence is emerging that the subdivision of intercalated cells into 2 subtypes is overly simplistic. The complexity of CCD H+/base transport is further demonstrated by the species differences which exist between the rat and rabbit. With the finding of a third CCD intercalated cell subtype (gamma cell), it is expected that future studies will focus on the transport changes which occur in this cell during systemic acid-base disorders.

Axial heterogeneity of rabbit cortical collecting duct.

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.

Effects of HIV infection, perceived health and clinical status on a cohort at risk for AIDS.

Data from a general population sample of 621 healthy homosexual men are used to evaluate the social and emotional effects of HIV antibody status, clinical signs detected by medical examination, and subjectively perceived symptoms. Participants are unaware of their serologic status at the time of data collection, thus allowing the effects of the virus to be separated from reactions to the knowledge of serologic status. The data show that seropositivity for HIV is not associated with elevated levels of social or emotional impairment. Clinical signs lead to impairment in baseline data, but these effects do not persist at a second wave. This weakening suggests that the effects are mediated by psychological pathways rather than biologic ones. This suspicion is confirmed in further analyses, which show that the effects of clinical signs are mediated by subjectively perceived symptoms. These results show that neither social nor emotional impairment is likely to be a prodromal sign of HIV infection in otherwise healthy homosexual men. The substantial levels of distress found among these men is more directly influenced by psychological determinants than biologic ones. This suggests that physicians should be aware of the psychological toll imposed on gay men who develop health problems in the current atmosphere of uncertainty regarding risk of AIDS.

A comparison of the symptoms of medical and psychiatric patients matched on the Beck Depression Inventory.

The goals of this study were to examine, in greater detail, the experience of depression in the medically ill, and to compare their experience with that of depressed psychiatric patients. Medical and psychiatric inpatients were matched in terms of total scores on the Beck Depression Inventory (BDI). In addition to the BDI, all patients completed a self-report symptom battery. No difference was found between the two groups in terms of total BDI scores, but psychiatric patients scored significantly higher on the affective BDI items, and medical patients scored significantly higher on the somatic BDI items. Discriminant analysis was used to compare their responses to the symptom battery. Depression in the psychiatric patients was characterized primarily by suicidal ideation and loss of interest, whereas in medical patients a lack of energy and worry were the predominant symptoms. The implications of these findings for assessing depression in the medically ill are discussed.