Epigenetic origin of evolutionary novel centromeres.

Most evolutionary new centromeres (ENC) are composed of large arrays of satellite DNA and surrounded by segmental duplications. However, the hypothesis is that ENCs are seeded in an anonymous sequence and only over time have acquired the complexity of "normal" centromeres. Up to now evidence to test this hypothesis was lacking. We recently discovered that the well-known polymorphism of orangutan chromosome 12 was due to the presence of an ENC. We sequenced the genome of an orangutan homozygous for the ENC, and we focused our analysis on the comparison of the ENC domain with respect to its wild type counterpart. No significant variations were found. This finding is the first clear evidence that ENC seedings are epigenetic in nature. The compaction of the ENC domain was found significantly higher than the corresponding WT region and, interestingly, the expression of the only gene embedded in the region was significantly repressed.

Fluorescence In Situ Hybridization Probe Preparation.

The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A "golden path" of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800-1000 bp) at each end allowed them to be very precisely mapped on the human genome.The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56-64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.

A comprehensive molecular cytogenetic analysis of chromosome rearrangements in gibbons.

Chromosome rearrangements in small apes are up to 20 times more frequent than in most mammals. Because of their complexity, the full extent of chromosome evolution in these hominoids is not yet fully documented. However, previous work with array painting, BAC-FISH, and selective sequencing in two of the four karyomorphs has shown that high-resolution methods can precisely define chromosome breakpoints and map the complex flow of evolutionary chromosome rearrangements. Here we use these tools to precisely define the rearrangements that have occurred in the remaining two karyomorphs, genera Symphalangus (2n = 50) and Hoolock (2n = 38). This research provides the most comprehensive insight into the evolutionary origins of chromosome rearrangements involved in transforming small apes genome. Bioinformatics analyses of the human-gibbon synteny breakpoints revealed association with transposable elements and segmental duplications, providing some insight into the mechanisms that might have promoted rearrangements in small apes. In the near future, the comparison of gibbon genome sequences will provide novel insights to test hypotheses concerning the mechanisms of chromosome evolution. The precise definition of synteny block boundaries and orientation, chromosomal fusions, and centromere repositioning events presented here will facilitate genome sequence assembly for these close relatives of humans.

Expression of leading region genes on IncI1 plasmid ColIb-P9: genetic evidence for single-stranded DNA transcription.

The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.

Blood lead levels among children of lead-exposed workers: A meta-analysis.

BACKGROUND: To further assess the utility of targeted blood lead screening for children from households with members having occupational lead exposures, we conducted a meta-analysis of all available reports of take-home lead exposures. Our objective was to estimate the blood lead levels among U.S. children (ages 1-5) from households with lead-exposed workers. METHODS: Reports considered for inclusion were cited in Medline, Toxline, Excerpta Medica, and Bio-Med plus all unpublished reports available at the National Institute for Occupational Safety and Health through 1994. The a priori criteria for inclusion of U.S. reports required their having data on: (1) venous blood lead levels for children, (2) children's ages, (3) data for at least five children, (4) workers' occupations, (5) workers' blood lead levels, and (6) data collection methods. RESULTS: Based on a meta-analysis of 10 reports from 1987 through 1994, the children (n=139) of lead-exposed workers (n=222) had a geometric mean blood lead level of 9.3 microg/dL compared to a U.S. population geometric mean of 3.6 microg/dL (P=0.0006). Also in this group, 52% of the children had blood lead levels (BLLs) >/= 10 microg/dL compared to 8.9% in the U.S. (P=.0010), and 21% of the children had BLLs >/= 20 microg/dL compared to 1.1% in the U.S. (P=. 0258). CONCLUSIONS: We estimate, based on 1981-83 survey data, that there are about 48,000 families with children under six living with household members occupationally exposed to lead. If the findings from this meta-analysis (admittedly limited by small numbers) are generalizable, about half of the young children in these families may have BLLs >/= 10 microg/dL. Data were too sparse to determine if children of workers with elevated blood leads were at greater risk than children whose parents were only known to be lead exposed. Our findings support the position that children of lead-exposed workers should be targeted for blood lead screening. Am. J. Ind. Med. 36:475-481, 1999. Published 1999 Wiley-Liss, Inc.

Modifiers of lung cancer risk in uranium miners from the Colorado Plateau.

Given the scientific consensus that exposure to radon decay products causes lung cancer, most recent studies have focused on the nature of the exposure-response relationship. Since residential radon exposure is now a primary public health issue, a better understanding of the effects of low levels of radon as well as factors modifying risk estimates has become very important. Several factors are shown to affect risk estimates in the latest update of the vital status follow-up (through 1990) and smoking history for the cohort of underground uranium miners in the Colorado Plateau. This analysis confirms earlier results indicating a strong dependence of relative risk estimates upon attained age. Quantitative estimates of relative risk as a function of cumulative exposure to radon decay products (WLM) are provided for three age strata. The non-linearity often reported in the Colorado Plateau data is shown to be at least partially due to an inverse exposure-rate effect, i.e., low exposure rates for long periods are more hazardous than equivalent cumulative exposure received at higher rates for shorter periods of time. However, this effect is shown to diminish at lower exposure rates and cumulative exposures. In addition, use of the new smoking data indicates that the radon/smoking interaction is submultiplicative and may depend upon attained age.

An update of mortality from all causes among white uranium miners from the Colorado Plateau Study Group.

To place previously recognized mortality risks into the context of the total mortality from all causes, an updated retrospective cohort mortality study was conducted on 3,238 white males from the US Public Health Service cohort of Colorado Plateau uranium miners. Vital status was followed from 1960 through 1990. Life-table analyses used combined New Mexico, Arizona, Utah, and Colorado mortality rates for external comparison and mortality risks within the lowest radon-exposure or duration-employed category for internal comparison. Significantly elevated SMRs were found for pneumoconioses (SMR = 24.1, 95% CI 16.0-33.7), lung cancer (SMR = 5.8, 95% CI 5.2-6.4), tuberculosis (SMR = 3.7, 95% CI 1.9-6.2), chronic obstructive respiratory diseases (SMR = 2.8, 95% CI 2.2-3.5), emphysema (SMR = 2.5, 95% CI 1.9-3.2), benign and unspecified tumors (SMR = 2.4, 95% CI 1.0-4.6), and diseases of the blood and blood-forming organs (SMR = 2.4, 95% CI 1.0-5.0). No significantly lowered SMRs were found for any disease. For lung cancer and pneumoconioses standardized rate ratios increased with increasing exposure to radon progeny or duration of employment. Most findings from this update are consistent with previous studies. Not observed were previously elevated SMRs for chronic nephritis and for acute alcoholism. New findings observed were elevated SMRs for benign and unspecified tumors and for diseases of the blood and blood-forming organs. The most important long-term mortality risks for the white uranium-miners continue to be lung cancer and pneumoconioses, for which SMRs remain significantly elevated after a mean period of 22.4 years since last uranium mining.

Pooled reanalysis of cancer mortality among five cohorts of workers in wood-related industries.

OBJECTIVES: To provide more information regarding the risk of cancer associated with wood dust, a pooled reanalysis of data from five cohort studies was performed. METHODS: The combined cohort consisted of 28,704 persons from five studies: British furniture workers, members of the union representing furniture workers in the United States, two cohorts of plywood workers, and one of wood model makers, among whom 7665 deaths occurred. Pooled analyses were carried out for all of the cohorts combined, the two furniture worker cohorts combined, and the two plywood workers cohorts combined. RESULTS: Significant excesses of nasal [observed 11, standardized mortality ratio (SMR) 3.1, 95% confidence interval (95% CI) 1.6-5.6] and nasopharyngeal (observed 9, SMR 2.4, 95% CI 1.1-4.5) cancer were observed. That for nasal cancer appeared to be associated with exposure to wood dust but was based solely on cases from the British furniture worker cohort, while that of nasopharyngeal cancer was observed for furniture and plywood workers and was associated with both high and low probability of wood dust exposure. Some support for an excess risk of multiple myeloma was also observed but was less clearly associated with wood dust exposure. No excesses of lung, larynx, stomach, or colon cancer were found to be associated with any surrogate indicators of wood dust exposure. CONCLUSIONS: Workers exposed to wood dust may have an excess risk of nasopharyngeal cancer and multiple myeloma in addition to sinonasal cancer. The limitations of this study would tend to obscure relationships, rather than create false positive findings.

Mortality among Navajo uranium miners.

OBJECTIVES: To update mortality risks for Navajo uranium miners, a retrospective cohort mortality study was conducted of 757 Navajos from the cohort of Colorado Plateau uranium miners. METHODS: Vital status was followed from 1960 to 1990. Standardized mortality ratios were estimated, with combined New Mexico and Arizona non-White mortality rates used for comparison. Cox regression models were used to evaluate exposure-response relationships. RESULTS: Elevated standardized mortality ratios were found for lung cancer (3.3), tuberculosis (2.6), and pneumoconioses and other respiratory diseases (2.6). Lowered ratios were found for heart disease (0.6), circulatory disease (0.4), and liver cirrhosis (0.5). The estimated relative risk for a 5-year duration of exposure vs none was 3.7 for lung cancer, 2.1 for pneumoconioses and other respiratory diseases, and 2.0 for tuberculosis. The relative risk for lung cancer was 6.9 for the midrange of cumulative exposure to radon progeny compared with the least exposed. CONCLUSIONS: Findings were consistent with those from previous studies. Twenty-three years after their last exposure to radon progeny, these light-smoking Navajo miners continue to face excess mortality risks from lung cancer and pneumoconioses and other respiratory diseases.

Mortality patterns among construction workers in the United States.

Thirteen authors from the National Institute for Occupational Safety and Health contribute to this summary of recent and ongoing national occupational mortality surveillance studies of construction workers, including studies conducted under NIOSH's Fatality Assessment and Control Evaluation project, Sentinel Health Events project, National Occupational Mortality Surveillance System, and other projects.

Modifiers of exposure-response estimates for lung cancer among miners exposed to radon progeny.

The association between lung cancer and exposure to radon decay products has been well established. Despite agreement on this point, there is still some degree of uncertainty regarding characteristics of the exposure-response relationship. The use of studies of underground miners to estimate lung cancer risks due to residential radon exposure depends upon a better understanding of factors potentially modifying the exposure-response relationship. Given the diversity in study populations regarding smoking status, mining conditions, risk analysis methodology, and referent populations, the risk estimates across studies are quite similar. However, several factors partially contributing to differences in risk estimates are modified by attained age, time since last exposure, exposure rate, and cigarette smoking patterns. There is growing agreement across studies that relative risk decreases with attained age and time since last exposure. Several studies have also found an inverse exposure-rate effect, i.e., low exposure rates for protracted duration of exposure are more hazardous than equivalent cumulative exposures received at higher rates for shorter periods of time. Additionally, the interaction between radon exposure and cigarette smoking appears to be intermediate between additive and multiplicative in a growing number of studies. Quantitative estimates of these modifying factors are given using a new analysis of data from the latest update of the Colorado Plateau uranium miners cohort.

A unique population of CD34+ cells in cord blood.

Human umbilical cord blood (CB) is a rich source of hematopoietic stem cells for both research and stem cell transplantation. In clinical studies, it appears that recovery from myeloablative therapy using CB requires significantly fewer cells than a typical allogeneic marrow transplant. This suggests that CB may be enriched for early hematopoietic progenitors. The present studies were undertaken to determine the presence of CD34+ cells in CB with the phenotypic characteristics of multipotential stem cells. In 22 CB harvests, the average percentage of CD34+ cells was 1.33 +/- 0.21% (SE), a value similar to that in adult normal bone marrows (BM). However, the distribution of CD34+ cells was distinctly different from either BM or granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell harvests. CB contained a defined population of brightly staining CD34+ cells with low side scatter. These CD34 (bright) cells comprised a mean of 14.5 +/- 2.5% of the CB CD34+ cells, whereas < 1% of BM CD34+ cells has been shown to be CD34- bright. Eighty-five to ninety percent were negative for three antigens expressed at an early stage of stem cell maturation: CD38, HLA-DR and LFA-1. Fifty-five percent of these CD34 (bright) cells did not express the CD45RA isoform, an additional marker of immaturity. The antigen-bright cells also lacked lineage-specific antigens including CD33, CD56, CD19, CD10 and CD7 as well as CD71. Approximately 46% were Thy-1+, and 40% expressed c-kit receptors. These data suggest that, by phenotypic criteria, CB may be a particularly enriched source of primitive hematopoietic precursors.

Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy.

The CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC-labeled anti-CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE-labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < 6 microns in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG-1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+lin- cells. Duplicate samples varied by < 10%. Initial studies indicate that this procedure can be reliably used to measure CD34+lin- cells in blood, pheresis products, and bone marrow harvests. This CD34 enumeration procedure should result in increased consistency in enumerating this stem cell population.

Colon and stomach cancer mortality among automotive wood model makers.

Automotive wood model makers have been reported to be at excess risk for colon and other cancers in recent epidemiologic studies. To further explore these risks, we conducted a retrospective cohort mortality study, with follow-up from 1940 through 1984, of 2294 white male wood model makers employed at any time until 1980 by three US auto makers. Using US mortality rates for comparison, we found elevated standardized mortality ratios of 1.2 (95% CI, 0.8-1.9) for colon cancer and 1.6 (95% CI, 0.9-2.6) for stomach cancer. We also conducted nested case-control studies for 20 colon and 17 stomach cancer cases and 543 age-matched controls. We found no trend of increased risk for colon or stomach cancer mortality with increased exposure to wood dust or to duration employed in wood model making.

Productivity: a context for excellence.

Inductively created measures of the sort described above enable the ambulatory care manager to make a crucial decision: Are the outcomes worth the cost involved? As Collins (1988, p. 235) notes, "Our nursing product is quality care based on principles and standards of practice... and substantiated and enhanced through quantifying our practice." By using nursing resources wisely, we can "moderate the march on the market...for caring and access of what we are here for" (Cunningham, 1983, p. 90). In relaying her feelings during treatment for a malignant breast tumor, Kaufman (1989) supports the contention that caring and access are essential parts of a therapeutic regime. Curtin (1987, p. 7) warns that "You cannot provide health care by taking the care out of it." This project allowed nurses to create their own measurement system that identified and quantified the professional nursing tasks involved in each workload indicator. Nonprofessional activities were also identified, quantified, and delegated to appropriate levels of staff, thus increasing the control of the nurses over their own practice environment. In the words of Curtin (1987, p. 7), "Losers count time to make heads roll. Winners use their heads to make time count. This country cannot afford to place its health in the hands of losers."

New developments in the Life Table Analysis System of the National Institute for Occupational Safety and Health.

In the 1970s, the National Institute for Occupational Safety and Health developed a Life Table Analysis System to analyze occupational cohort studies. We have updated the original system by adding two new features: direct standardization with a test for linear trend, and analyses by lagged exposure (either duration of exposure or cumulative exposure). We have also updated US reference rates through 1989. The updated systems and documentation (version F) are available upon request. In collaboration with the National Cancer Institute, we have also developed multiple cause-of-death rate files, which consider contributory as well as underlying cause. These files (also available upon request) will enable investigators to derive the expected prevalence of diseases at death, which can then be compared with the observed prevalence in an exposed cohort. Work is currently underway to produce a personal computer version of the Life Table Analysis System.

Lung cancer mortality among nonsmoking uranium miners exposed to radon daughters.

Radon daughters, both in the workplace and in the household, are a continuing cause for concern because of the well-documented association between exposure to radon daughters and lung cancer. To estimate the risk of lung cancer mortality among nonsmokers exposed to varying levels of radon daughters, 516 white men who never smoked cigarettes, pipes, or cigars were selected from the US Public Health Service cohort of Colorado Plateau uranium miners and followed up from 1950 through 1984. Age-specific mortality rates for nonsmokers from a study of US veterans were used for comparison. Fourteen deaths from lung cancer were observed among the nonsmoking miners, while 1.1 deaths were expected, yielding a standardized mortality ratio of 12.7 with 95% confidence limits of 8.0 and 20.1. These results confirm that exposure to radon daughters in the absence of cigarette smoking is a potent carcinogen that should be strictly controlled.

Effects of halothane and enflurane on firing threshold of frog myelinated axons.

1. Firing thresholds and conduction latencies of single myelinated axons in frog sciatic nerves were monitored during impulse activity in vitro. Resting threshold and the activity dependence of threshold were studied as a function of the concentration of two inhalational anaesthetic agents, halothane and enflurane. 2. At concentrations comparable to those obtained during general anaesthesia both agents produced biphasic effects on the resting threshold. A step increase in the partial pressure of anaesthetic was followed first by a transient lowering of threshold, then by a slow rise to a steady-state level above the original baseline. Step decreases in anaesthetic were followed by transient rises before threshold dropped. Transients lasted 20-30 min. During these threshold transients, the average latency of impulse conduction changed monotonically. The prolongation of latency following an increase in anaesthetic was progressive, reaching steady state concurrently with threshold (20 min to greater than 1 h). 3. The anaesthetics reduced the long-lasting increased threshold ('depression') which normally follows repetitive impulse activity in axon membrane. 4. These actions of halothane at concentrations of 0.25-2.7% (0.14-1.54 mM) and enflurane at concentrations of 0.62-3.08% (0.35-1.73 mM) on resting threshold and on the activity-dependent increase in threshold increased monotonically with anaesthetic concentration. 5. The effects on excitability at steady state are consistent with block of voltage-dependent Na+ and K+ channels by these inhalational agents. Reduced depression may occur because the anaesthetics reduce the net ion transfer per impulse, slowing the substrate-driven Na+-K+-ATPase and thereby reducing electrogenic hyper-polarization. 6. The finding that general anaesthetics inhibit depression at clinically relevant concentrations supports the possibility that general anaesthesia is produced by inhibition of processes that modulate excitability of nerve membrane. We suggest that general anaesthetics produce unconsciousness and amnesia because they disrupt activity-dependent processes, which may thus remove temporal 'context' essential for interpreting nerve impulse patterns.

Industrial hygiene characterization of automotive wood model shops.

A suspicion of an excess cancer risk in automotive model shops prompted the Industrywide Studies Branch, NIOSH, to conduct a proportionate mortality study and an industrial hygiene characterization of operations in these shops. The mortality study showed a statistically significant excess proportion of deaths due to colon cancer and leukemia (for woodshops only). The materials used in the model shops include various natural woods, laminated woods, plastics, resins, varnishes, putties and paints. Personal breathing zone samples were collected for total and respirable dust, amines, various hydrocarbons (including styrene, and toluene), formaldehyde, and nitrosamines. Particle size distribution studies were conducted on the wood dust and bulk airborne samples of dusts were subjected to various mutagenicity test systems. Work practices, ventilation and general housekeeping were checked. Total wood dust samples ranged from 0.03 to 25 mg/m3 with an average around 1.0 mg/m3. The percent respirable dust ranged from 19 to 38% as measured with Andersen impactors. Solvent exposure samples ranged from non-detectable to about 10% of the OSHA Permissible Exposure Levels. Relevant recommendations for improvement of contaminant control were made.