Photosynthesis of previtamin D3 in human skin and the physiologic consequences.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.

Intergenic disease-associated regions are abundant in novel transcripts.

BACKGROUND: Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. RESULTS: To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. CONCLUSIONS: This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

1,25-dihydroxycholecalciferol stimulates osteoclasts in rat bones in the absence of parathyroid hormone.

Three experiments were carried out to test the time course of effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the ultrastructural morphometry of osteoclasts. The addition of lactose to a vitamin D-deficient diet with a high calcium and phosphate content, fed to weanling rats for 4 weeks, ensured normacalcemia and normophosphatemia and allowed thyroparathyroidectomy without ill effects. In these vitamin D-deficient thyroparathyroidectomized rats, iv injection of 50 ng 1,25-(OH)2D3 resulted in significant changes in the osteoclasts in the metaphysis of the tibiae compared to those in corresponding controls; the size of these cells, their nuclei, ruffled borders, and clear zones enlarged after 6 h and the number of osteoclasts increased after 48 h. Serum calcium and serum phosphate levels increased after 12 h in one experiment, but not in a second experiment. Serum 25-hydroxyvitamin D and serum immunoreactive parathyroid hormone levels were undetectable. Mineralization of metaphyseal bone matrix was normal, as quantified by histomorphometry. When, dependent on the mineral content in the diet, mineralization was impaired and the volume density of the osteoid seams was increased, activation of osteoclasts by 1,25(OH)2D3 was not seen until 12--24 h after injection. It is concluded that a physiological dose of 1,25-(OH)2D3 stimulates the activity of osteoclasts in the absence of parathyroid hormone.

The metabolism of [6-3H]1alpha, hydroxycholecalciferol to [6-3H]1alpha,25-dihydroxycholecalciferol in a patient with renal insufficiency.

To evaluate whether 1alpha-hydroxycholecalciferol is metabolized to 1alpha,25-dihydroxycholecalciferol in man, [6-3H]1alpha-hydroxycholecalciferol was given intravenously to a patient with renal failure who was maintained daily on 100,000 IU vitamin D and calcium supplements. Using Sephadex LH-20 and high-pressure liquid chromatography, it was clearly demonstrated that 1alpha-hydroxycholecalciferol rapidly disappears from the blood and is metabolized to 1alpha,25-dihydroxycholecalciferol.

Consistency between US dietary fat intake and serum total cholesterol concentrations: the National Health and Nutrition Examination Surveys.

The National Health and Nutrition Examination Surveys (NHANESs) are conducted periodically to assess the health and nutritional status of the US population by means of standardized interviews and physical examinations. Since the early 1970s there have been three national cross-sectional surveys: NHANES I, 1971-1974; NHANES II, 1976-1980; and NHANES III, phase 1, 1988-1991. During the 18 y between the midpoint of NHANES I (1972) and the midpoint of phase 1 of NHANES III (1990), the age-adjusted mean percentage of energy from fat declined from 36.4% to 34.1% for adults aged 20-74 y. Trend data are shown for dietary fat and cholesterol as well as for serum cholesterol from NHANES I (1971-1975) to NHANES III (1988-1991) by age, sex, and race-ethnicity. The results document a decline in dietary fat, saturated fat, dietary cholesterol, and serum cholesterol. The observed changes reflect those that are predicted by the classic Keys and Hegsted formulas. Changes in reported intake are matched by similar shifts in the food supply for sources of these nutrients. These changes suggest that the Healthy People 2000 goal of reducing the mean serum cholesterol concentration of US adults to < or = 200 mg/dL (5.17 mmol/L) is attainable. The changes in diet are promising, yet we are challenged to achieve greater reductions in the intake of total fat and saturated fatty acids.

Sudden death during ambulatory monitoring. Analysis of six cases.

The ambulatory electrocardiographic recordings of six patients with coronary artery disease who died during monitoring were analyzed. In four patients, sinus rhythm was interrupted by sinoatrial, atrioventricular, nodal, or infra-His conduction abnormalities leading to bradyarrhythmic sudden death. Two patients died of sustained ventricular tachycardia or ventricular fibrillation. These data emphasize that the arrhythmias involved in the sudden death syndrome may be more heterogenous than currently appreciated.

Long-term function of human cardiac allografts assessed by two-dimensional echocardiography.

Previous studies from our laboratory demonstrated increasing left ventricular mass in cyclosporine-treated cardiac allograft recipients over 30 days after transplantation, but the long-term evolution of this process and possible effects on allograft function are unknown. Accordingly, quantitative two-dimensional echocardiography was performed 2 and 23 days and 15 months postoperatively in 14 recipients treated with cyclosporine and prednisone. Changes in left ventricular ejection fraction, end-diastolic volume, mass, and end-systolic wall stress were analyzed. Comparison of studies at 2 and 23 days revealed significant (p less than 0.01) increases in ejection fraction (54% +/- 8% [standard deviation] to 62% +/- 4%), end-diastolic volume (84% +/- 32 ml to 96 +/- 31 ml), and left ventricular mass (118 +/- 45 gm to 136 +/- 41 gm). Comparison of studies at 23 days and 15 months revealed no significant change in end-diastolic volume or left ventricular mass, whereas ejection fraction decreased slightly (62% +/- 4% to 57% +/- 4%, p less than 0.01). End-systolic wall stress decreased when data at 2 days and 15 months were compared (83 +/- 24 gm/cm2 versus 66 +/- 18 gm/cm2, p less than 0.05), but no change in contractility was apparent from the ejection fraction/end-systolic stress relation. We conclude that left ventricular mass and end-diastolic volume increase early after transplantation in cyclosporine-treated cardiac allograft recipients, but these changes are not predictive of long-term results, which are characterized by no significant late variation in left ventricular mass, end-diastolic volume, or contractility.

A phase I study of paclitaxel for mobilization of peripheral blood progenitor cells.

We conducted a phase I trial to determine the dose and schedule of paclitaxel, when given together with filgrastim, which would optimally promote mobilization of stem cells with tolerable toxicity. Dose escalation began at 275 mg/m2 3 h infusion. Dose-limiting neuropathy was observed at the 300 mg/m2 dose level. A second dose escalation was conducted utilizing 24 h infusion schedules, beginning at 225 mg/m2. Dose escalation was continued by 25 mg/m2 increments to 300 mg/m2, at which dose neuropathy was again dose-limiting. The recommended dose and schedule of paclitaxel for the purpose of mobilization of stem cells, when given together with filgrastim, are 275 mg/m2 as a 24 h infusion. The median stem cell yield after this dose of paclitaxel was 6.6 x 10(6) CD34+ cells/kg/apheresis (range 3.6 x 10(6)-7.7 x 10(6)).

Effect of cardiopulmonary bypass and global ischemia on human and canine left ventricular mass: evidence for interspecies differences.

Previous studies in dogs suggest that global ischemia with cardiopulmonary bypass causes increased left ventricular (LV) mass and water content. To investigate effects in humans, we developed a simplified method for mass determination by intraoperative two-dimensional echocardiography. LV mass was measured as echocardiographic short-axis myocardial area. This was validated by linear regression versus postmortem LV mass in 10 dogs (r = 0.89) and versus single-plane angiography in 18 patients (r = 0.73). According to this method, there was no change in LV mass (209 gm versus 208 gm; NS) at constant preload in 20 patients during routine operations (eight coronary revascularizations, 10 aortic valve replacements, and two mitral valve replacements). The same method used in 10 dogs after 2 hours of bypass, 60 minutes of normothermic global ischemia, and reperfusion revealed an LV mass increase from 113 +/- 13 gm (SE) to 150 +/- 16 gm (p less than 0.01) at matched preload. In addition, in 14 dogs after 2 hours of bypass alone, LV mass was unchanged (98 +/- 5 gm versus 101 +/- 5 gm; NS) at matched preload. Data recently derived from a separate study in our laboratory revealed a statistically significant increase in canine LV mass when conditions of human cardiopulmonary bypass and cardioplegic arrest were reproduced. We conclude that uncomplicated cardiac operations in humans do not alter LV mass. This supports the safety of crystalloid cardioplegia in humans. While present evidence is not conclusive, it appears that the threshold for edema formation after ischemic injury may be higher in humans than it is in dogs. The clinical relevance of studies of cardioplegia in edematous dog hearts thus deserves careful scrutiny.

Brain alpha erythroid spectrin: identification, compartmentalization, and beta spectrin associations.

Using isoform and subunit specific antibodies we have determined the presence, localization, and beta spectrin associations of alpha erythroid spectrin, alpha SpI sigma*, as well as alpha non-erythroid spectrin, alpha SpII sigma 1, in mouse brain. Peptide specific antibodies against unique sequences within the beta SpII sigma 1, non-erythroid beta spectrin isoform, and within beta SpI sigma 1, erythrocyte beta spectrin isoform were used to compare the immunolocalization of beta spectrin subunit isoforms with that of alpha spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific alpha and beta subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the alpha and beta spectrin isoforms revealed that antibodies specific for the alpha subunit of erythrocyte spectrin (alpha SpI sigma 1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI sigma 2). In contrast antibodies specific for the non-erythroid alpha subunit (alpha SpII sigma 1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpII sigma 1). The distinct localization of antigens by antisera which recognize either the alpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. Immunoprecipitation experiments of cerebellar extracts using beta spectrin peptide specific antibodies followed by immunoblotting analysis confirmed the association of an erythroid alpha subunit isoform with a beta erythroid subunit isoform, as well as the association of non-erythroid alpha and beta subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two alpha spectrin subunits, one erythroid alpha SpI sigma* and one non-erythroid alpha SpII sigma 1; these associate with an erythroid beta SpI sigma 1, and a non-erythroid beta SpII sigma 1 in the cerebellum of mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Opiate-enhanced toxicity and noradrenergic sprouting in rats treated with 6-hydroxydopa.

Because endorphin receptor activation alters the function of the central noradrenergic system, opiates may change the regenerative sprouting of neurons in response to adrenergic neurotoxins. To test this hypothesis, newborn rats were treated with several opioids and 6-hydroxydopa (6-OHDOPA) and the development of the noradrenergic system was evaluated. In combination with 6-OHDOPA morphine and naloxone potentiated the development of norepinephrine (NE) levels in the pons-medulla and cerebellum by four weeks of age, beta-Endorphin, Leu- and Met-enkephalin and d-Ala2-enkephalinamide produced a similar effect in the pons-medulla. The effect of morphine was partially attenuated by naloxone. Increased cerebellar noradrenergic histofluorescent staining was observed with the morphine + 6-OHDOPA and naloxone + 6-OHDOPA treatments. Both naloxone and morphine decreased NE levels in the pons-medulla of adult rats treated with 6-OHDOPA. These results suggest that opiates and endorphins may enhance sprouting of noradrenergic neurons following neonatal treatment with 6-OHDOPA, by increasing the toxicity of this neurotoxin.

Loss of nerve cell bodies in caudal locus coeruleus following treatment of neonates with 6-hydroxydopa.

The locus coeruleus is a well defined nucleus in cresylechtviolet preparations and the perikarya are easily distinguished. The coeruleus neurons are thought to be noradrenergic and during development can be selectively affected by the neurotoxin, 6-hydroxydopa (6-OHDOPA). In 6-month-old rats that were treated on day of birth with 6-OHDOPA (60 mg/kg, i.p.) there was a 32% loss of nerve cell bodies in the locus coeruleus. While it was apparent that loss of cell bodies occurred throughout the entire nucleus, the greatest loss of perikarya was from the caudal extent of the nucleus. It is known that sprouting of noradrenergic terminals occurs in the cerebellum of rats following treatment of newborns with 6-OHDOPA. That there are fewer cell bodies to contribute additional terminals further dramatizes this sprouting phenomenon.

Effect of sympathetic input on ontogeny of beta-adrenergic receptors in rat pineal gland.

The ontogeny of beta-adrenergic receptors was investigated in the pineal glands of rats 1-64 days of age. The density of beta-receptors increased about 3-fold between 1 and 16 days of age and decreased slightly by 64 days, correlating temporally with the development of the sensitivity of adenylate cyclase to norepinephrine in pineal gland. Preventing adrenergic innervation of the pineal gland by neonatal ganglionectomy or decentralization failed to prevent the development of beta-adrenergic receptors in pineal gland. Bilateral adrenal demedullation alone or in combination with ganglionectomy also failed to prevent the normal developmental increase of beta-receptors in the gland. These results, showing that the ontogeny of pineal beta-receptors correlated temporally with that of the responsiveness of adenylate cyclase to adrenergic neurohormones, support the hypothesis that the responsiveness of tissues ot beta-agonists is dependent on the development of the beta-receptor. In addition, these experiments show that the beta-adrenergic receptor can develop even in the absence of sympathetic innervation or circulating catecholamines produced by the adrenal medullae.

Brain spectrin: of mice and men.

This article reviews our current knowledge of the structure of alpha spectrins and beta spectrins in the brain, as well as their location and expression within neural tissue. We discuss the known protein interactions of brain spectrin isoforms, and then describe results that suggest an important role for spectrin (alpha SpII sigma 1/beta SpII sigma 1) in the Ca(2+)-regulated release of neurotransmitters. Evidence that supports a role for spectrin in the docking of synaptic vesicles to the presynaptic plasma membrane and as a Ca2+ sensor protein that unclamps the fusion machinery is described, along with the Casting the Line model, which summarizes the information. We finish with a discussion of the value of spectrin and ankyrin-deficient mouse models in deciphering spectrin function in neural tissue.

A promotion ladder for teachers at Harvard Medical School: experience and challenges.

The authors describe the development, implementation, and institutionalization at Harvard Medical School of a promotion ladder that recognizes the teaching and scholarly contributions of full-time clinical faculty. They also discuss the challenges that arose during this process, for example, how to make the new track creditable and attractive to both the appointed faculty and the faculty at large. The criteria developed for promotion focus on a candidate's skills and accomplishments in teaching, scholarship, clinical work, and departmental service. The authors present the elements of these criteria for the three professional levels of the ladder and outline the appointment process, including the steps for consideration of a given promotion. The development of this teacher-clinician ladder has had a positive influence on faculty who are committed to teaching by allowing recognition of their contributions in a track held to be the equal of the other full-time tracks in a medical faculty traditionally committed to research and patient care. Data are given for the 70 faculty who were promoted over the five years from 1989 to 1994. The true success of this promotion ladder will be measured only over time through its impact on the educational enterprise within the medical school and its hospitals, and its capacity to both successfully advance the careers of qualified medical educators and further the development of the field of medical education.

Correlation between lipid-associated sialic acid and tumor burden in melanoma.

Recent studies have suggested that lipid-associated sialic acid (LSA) may be a useful tumor marker for monitoring patients with melanoma, but the relationship between LSA and tumor burden has not been previously studied. We therefore examined LSA levels in 240 patients of whom 169 had no clinical evidence of disease (NED) and 71 had metastatic disease. There was a statistically significant difference in LSA levels in patients with NED compared with metastatic disease as well as those with high tumor burden compared with low or intermediate tumor burden. There was no difference between the groups with low and intermediate tumor burden. An LSA level of 25 mg/dl provided a positive predictive value of 70% and a negative value of approximately 80%. Our data show that LSA levels correlate with tumor burden in patients with melanoma.

Cardiovascular health risks related to overweight.

Cross-sectional surveys of the civilian noninstitutionalized population of the United States, including in-home interviews and clinical examinations, were employed to examine trends in consumption of energy and fat, prevalence of overweight in the population, the association of overweight with levels of blood pressure and blood cholesterol, and the prevalence of high blood pressure and high blood cholesterol among the overweight compared with the nonoverweight. Data from participants 20 years of age and older are reported. Study results suggest that total mean energy intake, although generally accepted to be underreported in dietary surveys, may have increased. Total fat and saturated fat intake as a percent of energy decreased, but remained above recommended levels. Overweight has increased in the population, despite decreases in the prevalence of high blood pressure and high blood cholesterol levels. Increased levels of overweight, reported as body mass index, are associated with increased cardiovascular risk factors of high blood pressure and high blood cholesterol. These data suggest the need for health care practitioners to emphasize the requirement for energy balance (or weight loss if overweight, ie, not at a "healthy weight"). A focus on fat intake alone without emphasis on energy balance is inadequate for achieving and maintaining recommended weight.

Influence of contextual features on the activation of ambiguous word meanings.

Three studies examined whether initial meaning activation is sensitive to context. Experiment 1 demonstrated that contextually appropriate targets were activated more than inappropriate targets. Experiment 2 evaluated activation across intervals of 0, 300, and 600 ms. Constraining sentences activated contextually appropriate meanings over inappropriate meanings. This was maintained across the intervals for highly salient targets. Less-salient targets, although initially activated, were no longer activated 300 ms following the homograph. Experiment 3 converged on context-sensitive activation following a 50-ms exposure of the sentence-final homograph. Conclusions are (a) initial meaning activation can be sensitive to context, (b) when a homograph is instantiated, it is congruent with a broad scope of targets, and (c) less-salient targets receive less activation over the time course.

Neuron-specific enolase as a tumor marker in metastatic melanoma.

Neuron-specific enolase (NSE) has been shown by some investigators to be a useful tumor marker for melanoma, but the relationship between NSE and tumor burden has not been extensively studied. We therefore examined NSE levels in 240 patients of whom 169 had no clinical evidence of disease (NED) and 71 had metastatic disease. There was no statistically significant difference in NSE levels in patients with NED compared with metastatic disease as well as those with high tumor burden compared with low or intermediate tumor burden. In addition, the mean absolute values of NSE, despite a slight elevation with tumor burden, were within the normal range (< 20 ng/ml). Our data suggest that NSE levels measured by the method used in our study are of no benefit in melanoma.

Effect of changing the light/dark schedule, the time of onset of the light or dark period, or the daylength, on rhythms of epidermal cell proliferation.

Rhythms of labeling and mitotic indices were studied in the hindlimb epidermis of the anuran tadpole Rana pipiens under different light/dark (LD) cycles and daylengths in order to examine the role of the various parameters of the lighting regimen in setting the periods of the rhythms and the timing of the cell proliferation peaks. Altering the time of, or inverting, the 12 h light period on a 24 h day resulted in phase shifting of basically bimodal circadian rhythms with peaks in the light and dark. Thus the cell proliferation rhythms were entrained to the LD cycle. These rhythms also entrained to noncircadian schedules since they lengthened on a 15L:15D cycle and shortened on a 9L:9D cycle, although the bimodal characteristic of a peak in the light and a peak in the dark remained. Studies of 18L:6D and 6L:18D cycles in which either the time of onset of light or dark was changed relative to the 12L:12D control indicated that the onset of dark may regulate the timing of the labeling index peaks while the onset of light may determine the time of occurrence of mitotic index peaks. Control of the timing of labeling and mitotic index peaks by different parameters of the LD cycle suggests a mechanism for cell cycle regulation by the environmental lighting schedule. Analysis of the rhythms on all the cycles studied suggested that labeling index rhythms equal the length of, or twice the length of, the dark period. Mitotic index rhythms equal the daylength or a multiple of the length of the dark period.