Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.

Iron is a crucial transition metal for virtually all life. Two major destinations of iron within mammalian cells are the cytosolic iron-storage protein, ferritin, and mitochondria. In mitochondria, iron is utilized in critical anabolic pathways, including: iron-storage in mitochondrial ferritin, heme synthesis, and iron-sulfur cluster (ISC) biogenesis. Although the pathways involved in ISC synthesis in the mitochondria and cytosol have begun to be characterized, many crucial details remain unknown. In this review, we discuss major aspects of the journey of iron from its initial cellular uptake, its modes of trafficking within cells, to an overview of its downstream utilization in the cytoplasm and within mitochondria. The understanding of mitochondrial iron processing and its communication with other organelles/subcellular locations, such as the cytosol, has been elucidated by the analysis of certain diseases e.g., Friedreich's ataxia. Increased knowledge of the molecules and their mechanisms of action in iron processing pathways (e.g., ISC biogenesis) will shape the investigation of iron metabolism in human health and disease.

Glutathione S-transferase and MRP1 form an integrated system involved in the storage and transport of dinitrosyl-dithiolato iron complexes in cells.

Nitrogen monoxide (NO) is vital for many essential biological processes as a messenger and effector molecule. The physiological importance of NO is the result of its high affinity for iron in the active sites of proteins such as guanylate cyclase. Indeed, NO possesses a rich coordination chemistry with iron and the formation of dinitrosyl-dithiolato iron complexes (DNICs) is well documented. In mammals, NO generated by cytotoxic activated macrophages has been reported to play a role as a cytotoxic effector against tumor cells by binding and releasing intracellular iron. Studies from our laboratory have shown that two proteins traditionally involved in drug resistance, namely multidrug-resistance protein 1 and glutathione S-transferase, play critical roles in intracellular NO transport and storage through their interaction with DNICs (R.N. Watts et al., Proc. Natl. Acad. Sci. USA 103:7670-7675, 2006; H. Lok et al., J. Biol. Chem. 287:607-618, 2012). Notably, DNICs are present at high concentrations in cells and are biologically available. These complexes have a markedly longer half-life than free NO, making them an ideal "common currency" for this messenger molecule. Considering the many critical roles NO plays in health and disease, a better understanding of its intracellular trafficking mechanisms will be vital for the development of new therapeutics.

Deferasirox (ICL670A) effectively inhibits oesophageal cancer growth in vitro and in vivo.

BACKGROUND AND PURPOSE: Growing evidence implicates iron in the aetiology of gastrointestinal cancer. Furthermore, studies demonstrate that iron chelators possess potent anti-tumour activity, although whether iron chelators show activity against oesophageal cancer is not known. EXPERIMENTAL APPROACH: The effect of the iron chelators, deferoxamine (DFO) and deferasirox, on cellular iron metabolism, viability and proliferation was assessed in two oesophageal adenocarcinoma cell lines, OE33 and OE19, and the squamous oesophageal cell line, OE21. A murine xenograft model was employed to assess the effect of deferasirox on oesophageal tumour burden. The ability of chelators to overcome chemoresistance and to enhance the efficacy of standard chemotherapeutic agents (cisplatin, fluorouracil and epirubicin) was also assessed. KEY RESULTS: Deferasirox and DFO effectively inhibited cellular iron acquisition and promoted intracellular iron mobilization. The resulting reduction in cellular iron levels was reflected by increased transferrin receptor 1 expression and reduced cellular viability and proliferation. Treating oesophageal tumour cell lines with an iron chelator in addition to a standard chemotherapeutic agent resulted in a reduction in cellular viability and proliferation compared with the chemotherapeutic agent alone. Both DFO and deferasirox were able to overcome cisplatin resistance. Furthermore, in human xenograft models, deferasirox was able to significantly suppress tumour growth, which was associated with decreased tumour iron levels. CONCLUSIONS AND IMPLICATIONS: The clinically established iron chelators, DFO and deferasirox, effectively deplete iron from oesophageal tumour cells, resulting in growth suppression. These data provide a platform for assessing the utility of these chelators in the treatment of oesophageal cancer patients.

Iron Chelators: Development of Novel Compounds with High and Selective Anti-Tumour Activity.

Targeting essential nutrients (eg., those required for DNA synthesis) to inhibit cancer cell growth is a well established therapeutic strategy. A good example is the highly successful folate antagonist, methotrexate. However, up until recently, strategies to target iron which is also crucial for DNA synthesis have not been systematically explored to develop agents for the treatment of cancer. Over the last 15 years, our laboratory has embarked upon structure-activity studies designed to develop novel Fe chelators with anti-cancer efficacy. These studies have led to the development of the dipyridyl thiosemicarbazone chelators that show potent and selective anti-cancer activity and which overcome resistance to other cytotoxic agents. This class of compounds include the chelator, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which at optimal doses markedly inhibits tumour growth and is well tolerated. Moreover, this ligand does not induce overt Fe-depletion in vivo, probably because very low doses (0.4 mg/kg) are effective at inhibiting tumour growth. Importantly, our compounds are far more active and less toxic than the chelator, Triapine®, that is being assessed in a wide variety of international clinical trials. A vital part of the mechanism of action of these compounds is their ability to form a redox-active Fe complex that generates radicals to inhibit tumour growth. Due to their relatively high lipophilicity and low molecular weight of this class of compounds, oral activity may be expected in addition to their well known efficacy via the intravenous route.

Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents.

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.

Protection against hydrogen peroxide-mediated cytotoxicity in Friedreich's ataxia fibroblasts using novel iron chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone class.

Iron-loading diseases remain an important problem because of the toxicity of iron-catalyzed redox reactions. Iron loading occurs in the mitochondria of Friedreich's ataxia (FA) patients and may play a role in its pathogenesis. This suggests that iron chelation therapy could be useful. We developed previously the lipophilic iron chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) ligands and identified 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH) as the most promising analog. Hence, this study assessed the efficacy of PCTH and other PCIH analogs compared with various chelators, including deferiprone and desferrioxamine (DFO). Age- and sex-matched control and FA fibroblasts were preincubated with iron chelators and subsequently challenged with 50 microM H2O2 for up to 24 h. The current study demonstrates an interesting structure-activity relationship among the closely related PCIH series of ligands, with only PCTH being highly effective at preventing H2O2-induced cytotoxicity. PCTH increased FA fibroblast cell viability by up to 70%, whereas DFO rescued viability by 1 to 5% only. Hence, PCTH, which was well tolerated by cells was far more effective than DFO at preventing oxidative stress. It is noteworthy that kinetic studies demonstrated PCTH to rapidly penetrate cells to induce 59Fe efflux, whereas DFO, PCIH, 2-pyridylcarboxaldehyde benzoyl hydrazone, and 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone were far slower, indicating it is the rate of chelator permeation that is crucial for protection against H2O2. In addition, PCTH was found to be as effective as or more effective than conventional radical scavengers or the antioxidant idebenone (which has undergone clinical trials) at protecting cells against H2O2-mediated cytotoxicity. These findings further indicate the potential of PCTH for treatment of iron overload.

Investigating the antiproliferative activity of quinoline-5,8-diones and styrylquinolinecarboxylic acids on tumor cell lines.

The structure-activity relationships of new quinoline based compounds were investigated. Quinoline-5,8-dione and styrylquinoline scaffolds were used for the design of potentially active compounds. The novel analogues had comparable antiproliferative activity to cisplatin when evaluated in a bioassay against the P388 leukemia cell line. However, these compounds appeared far less efficient against SK-N-MC neuroepithelioma cells. Analogues without the 5,8-dione structure but containing the 8-carboxylic acid group were also found to induce antiproliferative activity. Hydrophobicity as measured by HPLC did not correlate with antiproliferative activity.

Surveillance of work-related asthma in selected U.S. states using surveillance guidelines for state health departments--California, Massachusetts, Michigan, and New Jersey, 1993-1995.

PROBLEM/CONDITION: Cases of work-related asthma (WRA) are sentinel health events that indicate the need for preventive intervention. WRA includes new-onset asthma caused by workplace exposure to sensitizers or irritants and preexisting asthma exacerbated by workplace exposures. REPORTING PERIOD: This report reviews cases of WRA identified by state health departments from January 1, 1993, through December 31, 1995, as well as follow-up investigations of cases and associated workplaces conducted through June 30, 1998. DESCRIPTION OF THE SYSTEMS: State-based surveillance and intervention programs for WRA are conducted in California, Massachusetts, Michigan, and New Jersey as part of the Sentinel Event Notification Systems for Occupational Risks (SENSOR) cooperative agreement program, initiated by CDC's National Institute for Occupational Safety and Health (NIOSH). RESULTS: From 1993 through 1995, a total of 1,101 cases of WRA were identified by SENSOR surveillance staff members in California, Massachusetts, Michigan, and New Jersey. Of these 1,101 cases, 19.1% were classified as work-aggravated asthma, and 80.9% were classified as new-onset asthma. Objective evidence substantiating asthma work-relatedness was documented in the medical records of 3.4% of WRA cases identified in the two states (Michigan and New Jersey) where medical records are routinely reviewed for this information. Indoor air pollutants, dusts, cleaning materials, lubricants (e.g., metalworking fluids), and diisocyanates were among the most frequently reported causes of WRA. In addition, a well-recognized cause of occupational asthma - natural rubber latex - was identified in a new setting, the healthcare industry. The most common industries associated with WRA cases included transportation equipment manufacturing (19.3%), health services (14.2%), and educational services (8.7%). Air sampling for agents known to induce occupational asthma was performed in Michigan for comparison with established federal time-weighted average exposure limits. Sixteen (13.4%) of 119 workplaces tested had airborne concentrations exceeding NIOSH recommended exposure limits (RELs); 11 (9.1%) of 121 workplaces had concentrations exceeding permissible exposure limits (PELs) of the Michigan Occupational Safety and Health Act (MIOSHA) program. INTERPRETATION: The surveillance data findings confirm well-recognized causes of asthma and have identified new putative causes (e.g., cleaning materials and metalworking fluids). Because the surveillance program depends on physicians' recognizing asthma work-relatedness and reporting diagnosed cases, the data are considered an underestimate of the magnitude of the WRA problem. The data also indicate that physicians are not commonly performing objective physiologic tests to substantiate a WRA diagnosis. Workplace findings suggest a need to evaluate existing exposure standards for specific agents known to induce occupational asthma (e.g., diisocyanates). Case-based surveillance can help improve the recognition, control, and prevention of WRA. The SENSOR model also provides a mechanism for workers and physicians to request workplace investigations aimed at primary prevention for other workers. PUBLIC HEALTH ACTION: NIOSH and state health department representatives are working to establish a long-term agenda for state-based surveillance of work-related conditions and hazards. The results from the SENSOR WRA programs described in this report support inclusion of WRA as a priority condition warranting surveillance at the state level.

Occupational noise-induced hearing loss surveillance in Michigan.

Occupational noise-induced hearing loss (NIHL) is an important yet often overlooked illness that can affect an individual's safety and performance at work. This article describes a state-based surveillance system for occupational NIHL. The Michigan surveillance system enables us to describe the magnitude of occupational NIHL among Michigan workers and direct public health interventions in the form of enforcement workplace inspections. The data presented are based on interviews of individuals with occupational NIHL reported to the Michigan Department of Consumer and Industry Services (MDCIS) by Michigan's audiologists and otolaryngologists from 1992-1997. From 1992-1997, 1378 individuals with occupational NIHL were reported to the MDCIS and interviewed about their exposures to noise at work. Over 70% of the workplace noise exposure were in manufacturing. At the most recent company where these individuals were exposed to noise, approximately 46% were not provided regular hearing testing. Regular hearing testing was more likely to occur in the larger companies and in industries covered by regulations requiring such testing to be performed. There were improvements over time in the percentages of companies providing regular hearing testing and hearing protection. Construction workers are employees among a group of industries that are not adequately protected from excessive noise exposures by occupational regulations. Regular hearing testing was not provided for over 90% of construction jobs, although hearing protection such as earplugs or earmuffs was provided for approximately half of these jobs. Forty-three state enforcement inspections were conducted at the companies reported by the patients interviewed, because these companies were reported to provide no regular hearing testing or no hearing protection despite exposures to excessive levels of noise. During the 43 inspections, 23 companies had noise levels above dBA, and 17 of those had either no hearing conservation program (HCP) or had one that was cited as being incomplete. The inspections potentially protected 758 similarly exposed workers in the companies with the high noise levels that lacked an HCP or that had a deficient HCP. The number of patients with occupational NIHL is likely a gross underestimate of the true magnitude of the disease. However, the surveillance system has identified workplaces with hazardous levels of noise and no HCP, thereby protecting similarly exposed coworkers of the index patients from further exposures to noise and hearing loss.

Work-related asthma and respiratory symptoms among workers exposed to metal-working fluids.

The objective of this work was to determine whether the prevalence of respiratory symptoms differed among workers exposed to different types of metal-working fluids. As part of a mandatory surveillance system for occupational illness, from 1988-1994, the Michigan Department of Public Health received, 86 occupational disease reports of work-related asthma secondary to exposure to metal-working fluids. As part of a public health program, follow-up industrial hygiene inspections, including medical interviews of the workforce, were performed at companies where the reported cases had become ill. Metal-working fluids were the second most common cause of work-related asthma reported in the state. Most of the reports were from the automobile industry. Follow-up inspections were conducted at 37 facilities where the individuals with work-related asthma had worked. Seven hundred and fifty-five workers at these facilities were interviewed. Only one facility was above the allowable oil mist standard. Despite the exposure levels being within the legal limits, approximately 20% of the fellow workers of the reported cases had daily or weekly respiratory symptoms suggestive of work-related asthma. Workers exposed to emulsified, semisynthetic, or synthetic machining coolants were more likely to have chronic bronchitis; to have visited a doctor for shortness of breath; to have visited a doctor for a sinus problem; to be bothered at work by nasal stuffiness, runny nose, or sore throat; and to have an increased prevalence of respiratory symptoms consistent with work-related asthma, compared to workers exposed to mineral oil metal-working fluids. These findings were found in individuals who currently smoked, had never smoked or were ex-cigarette smokers. Further research to determine the chemical components or microbial contaminants responsible for these findings is needed.

A state-based surveillance system for work-related asthma.

The current national surveillance system for occupational illnesses underestimates the incidence of work-related asthma. This article describes a state-based surveillance system for work-related asthma. The Michigan surveillance system enables us to estimate the incidence of work-related asthma, describe the characteristics of affected individuals, and facilitate public health interventions in the form of workplace inspections. The data presented are based on interviews with a case-series of individuals with work-related asthma reported to the Michigan Department of Public Health (MDPH) from 1988 to 1994. We also present cross-sectional data on coworkers of the index cases, who were interviewed during the workplace investigations, and exposure measurements from those investigations. Potential cases were reported by physicians, hospitals, or the Michigan Department of Labor. Case eligibility was based on the criteria for work-related asthma developed by the National Institute for Occupational Safety and Health (NIOSH). Between 1988 and 1994, 725 people who met the NIOSH criteria for work-related asthma were reported to the MDPH. Seventy-six percent of the reports were from physicians, 17.1% were from hospitals, 7.3% were from workers' compensation records, and 3.5% were from other health professionals. Eighty-three percent of the reports were for individuals with the onset of newly diagnosed asthma after a period of symptomless exposure, 7.3% were for aggravation of preexisting asthma, and 9.5% were for reactive airway dysfunction syndrome (RADS). The overall annual average incidence rate of work-related asthma in Michigan was 2.9 cases per 100,000 workers. Rates were 0.8/100,000 in the service industry and 8.5/100,000 in manufacturing. Isocyanates and machining coolants were the two most common causes of asthma among workers reported to the surveillance system. Demographics of the individuals reported are described. During workplace follow-up investigations, 861 fellow workers were identified as having possible work-related asthma. Another 151 coworkers were identified from the company-maintained injury and illness logs as having possible work-related asthma. In addition, the investigations identified two new causes of work-related asthma. The primary limitations of the surveillance system include a lack of objective testing to confirm the diagnosis of work-related asthma and underreporting of cases. Despite these limitations, this state-based surveillance system has proven successful in identifying new cause of asthma and identifying workplaces with a high prevalence of workers with respiratory symptoms who may benefit from public health interventions.

Silicosis in the 1990s.

OBJECTIVE: To describe state-based surveillance for silicosis that estimates prevalence of this condition, describes characteristics of affected individuals, and targets public health interventions. DESIGN: The data presented are a case series of patients with silicosis reported to a state health department. Patients were interviewed using a standardized questionnaire, chest radiographs interpreted by a "B-reader," pulmonary function tests obtained from medical records, and follow-back investigations conducted at the worksites where the cases had been exposed to silica. SETTING: All individuals with silicosis in the state of Michigan reported to the Michigan Department of Public Health (MDPH). SUBJECTS: Individuals included in this article were reported from 1987 through 1995. Cases were reported by hospitals, physicians, the state workers' compensation bureau, or from death certificates. Only data on individuals who met the criteria for silicosis developed by the National Institute for Occupational Safety and Health (NIOSH) are included. RESULTS: Between 1987 and 1995, 577 people were reported to MDPH who met the NIOSH criteria for silicosis. About 60% of the reports came from hospitals. The disease is occurring mainly among men born before 1940 who began working in a Michigan ferrous foundry in the 1930s or 1940s and worked there > 20 years. Over 40% of the patients are black. The overall annual average incidence rate of silicosis among black men (14.3 cases per 100,000) is seven times higher than among white men (2.1 cases per 100,000). The individuals identified with silicosis generally have severe disease. Almost 30% have progressive massive fibrosis and another 31.7% have advanced simple silicosis. Only about a third of all patients have normal results of breathing tests. Thirteen percent had been told they had tuberculosis (includes both clinical disease and a positive skin test). They have an increase of over 300% in the likelihood of dying of nonmalignant respiratory disease, both restrictive and obstructive, and an 80% increase in the likelihood of dying of lung cancer. Despite the severity of disease, over 45% of the individuals had not applied for workers' compensation. Although silicosis typically occurs after a long duration of exposure to silica, some individuals developed silicosis after a relatively short time. Three people developed silicosis who began working with silica in the 1980s, 18 in the 1970s, and 66 in the 1960s. Initial industrial hygiene follow-up inspections where the individuals had worked showed ongoing exposure above recommended and/or legal levels. Repeated inspections to these same facilities have subsequently shown reductions in silica exposure. CONCLUSION: This state-based surveillance system has proved useful in characterizing individuals with silicosis, estimating its prevalence, increasing the medical community's awareness of the condition, and targeting effective public health interventions.

Surveillance for silicosis, 1993--Illinois, Michigan, New Jersey, North Carolina, Ohio, Texas, and Wisconsin.

PROBLEM/CONDITION: Silicosis is an occupational respiratory disease caused by the inhalation of respirable dust containing crystalline silica. Public health surveillance programs to identify workers at risk for silicosis and target workplace-specific and other prevention efforts are currently being field-tested in seven U.S. states. REPORTING PERIOD COVERED: Confirmed cases ascertained by state health departments during the period January 1, 1993, through December 31, 1993; the cases and associated workplaces were followed through December 1994. DESCRIPTION OF SYSTEMS: As part of the Sentinel Event Notification System for Occupational Risks (SENSOR) program initiated by CDC's National Institute for Occupational Safety and Health (NIOSH), development of state-based surveillance and intervention programs for silicosis was initiated in 1987 in Michigan, New Jersey, Ohio, and Wisconsin and in 1992 in Illinois, North Carolina, and Texas. RESULTS: From January 1, 1993, through December 2, 1994, the SENSOR silicosis programs in Illinois, Michigan, New Jersey, North Carolina, Ohio, Texas, and Wisconsin confirmed 256 cases of silicosis that were initially ascertained in 1993. Overall, 185 (72%) were initially identified through review of hospital discharge data or through hospital reports of silicosis diagnoses; 188 (73%) were associated with silica exposure in manufacturing industries (e.g., foundries; stone, clay, glass, and concrete manufacturers; and industrial and commercial machinery manufacture). Overall, 42 (16%) cases were associated with silica exposure from sandblasting operations. Among the 193 confirmed cases for which information was available about duration of employment in jobs with potential exposure to silica, 37 (19%) were employed < or = 10 years in such jobs and 156 (81%) were employed > or = 11 years. A total of 192 primary workplaces associated with potentially hazardous silica exposures were identified for the 256 confirmed silicosis cases. Of these, nine (5%) workplaces were inspected by state health department (SHD) industrial hygienists, 19 (10%) were referred to the Occupational Safety and Health Administration (OSHA) for follow-up, and seven (4%) were routinely monitored by the Mine Safety and Health Administration. Of the 157 (82%) remaining workplaces, follow-up activities determined that 82 were no longer in operation, eight were no longer using silica, 18 were assigned a lower priority for follow-up, six were associated with building trades and could not be inspected because of the transient nature of work in the construction industry, and 43 workplaces were not inspected for other reasons. Fourteen (7%) of the 192 workplaces were inspected. At 10 of the 14 workplaces, airborne levels of crystalline silica were measured; in nine, silica levels exceeded the NIOSH-recommended exposure level of 0.05 mg/m, and in six, airborne silica levels also exceeded federal permissible exposure limits. ACTIONS TAKEN: Employee-specific and other preventive interventions have been initiated in response to reported cases. In addition, special silicosis prevention projects have been initiated in Michigan, New Jersey, North Carolina, Ohio, Texas, and Wisconsin. To facilitate the implementation of silicosis surveillance by other states, efforts are ongoing to identify and standardize core data needed by surveillance programs to describe cases and the workplaces where exposure occurred. These core variables will be incorporated into a user-friendly software system that states can use for data collection and reporting.

A practical technique for disinfecting electrical stimulation apparatuses used in wound treatment.

BACKGROUND AND PURPOSE: Electrical stimulation (ES) is used in wound management. Concerns, however, have been raised about the possible role ES might play in promoting or exacerbating wound infections, especially bacterial infections. The purpose of this study was to address these concerns by evaluating the efficacy of a method for disinfecting ES electrodes used in wound treatment. METHODS: Samples were taken from each wound treated in this study prior to and after ES and from sponges used with the ES electrodes prior to treatment, after treatment, and after 20 minutes of chemical disinfection. The presence and types of bacteria recovered were determined through standard microbiological techniques. RESULTS: In this study of 25 patient samples, large numbers (ie, thousands) of bacteria were recovered from the pretreatment and posttreatment wound samples and from the posttreatment sponges. Following disinfection, however, bacteria were absent from the sponges in 23 of the 25 patient samples. In the remaining 2 samples, no more than two bacterial colonies were recovered after disinfection. CONCLUSION AND DISCUSSION: Immersion of the electrodes and sponges for 20 minutes in the disinfectant resulted in reduction of bacteria to safe, noninfective levels. Disinfection either completely eliminated all bacteria from the sponges (in 92% of the samples) or eliminated nearly all bacteria (in the remaining 8% of the samples), compared with predisinfection samples, which contained very large numbers of bacteria. These results demonstrate that the disinfection method used in this study is efficacious, and it appears to be cost-effective, practical, and safe for clinical use.

Analysis of spontaneous frameshift mutations in REV1 and rev1-1 strains of Saccharomyces cerevisiae.

Frameshift mutations occur by a number of mechanisms. To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae. All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38. DNA sequence information was recovered using two approaches-the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR). Using these techniques, saturated mutation spectra for the spontaneous reversion of his4-38 were generated. The most frequently occurring mutational events in both strains were -1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed. Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of -1 frameshift mutations. In addition, while for -1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only -1 frameshifts and one +2 frameshift), and those recovered using PCR (-1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations). This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations.

Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay.

The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.

Mutation spectrum of spontaneous frameshift revertants in yeast using double-strand gap repair.

A mutation spectrum was constructed from a series of randomly isolated spontaneous His+ revertants of the frameshift mutant his4-38 in Saccharomyces cerevisiae. For each true revertant, a 438 bp region encompassing his4-38 on chromosome III was recovered into a shuttle vector by double-strand gap repair. Of the 45 independent His+ revertants sequenced, 44 were -1 base deletions and one revertant was a +2 base insertion. The -1 deletions exhibited a bimodal distribution. Of the bases encompassing the his4-38 region from +153-181, approximately 45% were not involved in a reversion event, although a -1 frameshift within this region will result in a viable His+ revertant. Approximately 49% of -1 events occurred within runs of 3 repeated bases. At these sites the strand-slippage model for frameshift mutation is supported. However, the -1 events occurring at sites of 2 repeated bases and the low frequency (2%) of +2 base insertions suggest that the transiently misaligned template model is a significant mechanism in reversion of his4-38. When the distribution of -1 events at repeated bases was discounted, a hotspot involving a -T at position +163 was resolved.

Efficient transformation of previously activated and dividing T lymphocytes by human T cell leukemia-lymphoma virus.

Modifying previously reported techniques, we attempted to increase the efficiency of human T cell leukemia-lymphoma virus (HTLV) transformation of human T lymphocytes. Lethally irradiated donor cells (DCs) were cultured with target mononuclear cells (TMCs). DCs included ten HTLV+ T cell lines with varying degrees of virus expression or seven cell lines that do not express HTLV. TMCs were prepared from 20 cord and 16 adult peripheral blood samples, including eight patients with acquired immunodeficiency syndrome (AIDS). TMCs were either added directly to the DCs or were first stimulated with phytohemagglutinin (PHA) (5 micrograms/mL) and grown in T cell growth factor (TCGF) prior to exposure to DCs. The presence of integrated HTLV proviral DNA in the transformed cells was determined by dot blot hybridization, utilizing a cloned probe to the HTLV-I genome. HTLV production by transformed TMCs was assessed for HTLV p19, reverse transcriptase, and virus particles. No transformation occurred with T cell donor lines that do not express HTLV. Low virus expressor DCs could only, with rare exception, transform preactivated TMCs. High-titer virus-producing DCs could transform activated and nonactivated cord blood cells and activated adult TMCs. Only MT-2 could routinely transform nonactivated normal adult and activated AIDS TMCs. HUT 102 B2 could transform only one activated AIDS sample, the cells of which initially expressed HTLV-like proteins and virions. Transformed cell lines contained subsets of mature T lymphocytes with variable HTLV expression. Prior activation and culture of the T lymphocytes increases the probability and rate of transformation by HTLV, allowing for biologic detection of low HTLV-producing cells and for in vitro expansion of T lymphocyte subsets from selected patients.

Percutaneous nephrostomy by pigtail catheter in children.

Percutaneous nephrostomy in children with the use of the pigtail catheter is discussed as a useful, noninvasive, rapid technic in cases of pediatric obstructive uropathy or in children in whom immediate surgical intervention would be undesirable. Three cases are presented, as well as technical notes.