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Background: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases., Methodology: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion., Significance: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.

Huntington's disease (HD) is a neurodegenerative disease that affects four to seven individuals per 100,000. The onset of symptoms usually begins in middle age, although approximately 5% become symptomatic as juveniles. Death occurs approximately 15 years following the onset of symptoms, which include choreic movements, cognitive decline and psychiatric changes. HD is an autosomal dominant inherited disease that is associated with an expansion of a trinucleotide (CAG) repeat located on chromosome 4. Physicians rely on a positive family history, and diagnostic and genetic tests to detect the expansion in the number of CAG trinucleotide repeats in the HD gene to confirm the diagnosis. More than 99% of HD patients have 40 or more CAG triplet repeats and, therefore, targeted mutational analysis is greater than 99% sensitive. Individuals with 26 triplet repeats or less are normal, and while those with 27-35 repeats may not demonstrate symptoms themselves, their offspring may have the disease. Individuals with 36-39 repeats may or may not exhibit symptoms. The College of American Pathology/American College of Medical Genetics Biochemical and Molecular Genetics Resource Committee has emphasized the need to standardize the methodology for the determination of the accurate number of CAG repeats. This will prevent false-positive or -negative results when conducting predictive or prenatal testing of at-risk individuals. The National Institute of Standards and Technology is developing a standard reference material to provide these positive and negative controls needed by clinical testing laboratories. The use of a HD standard reference material will provide the quality control and assurance that data from different laboratories are both comparable and accurate.

The length heteroplasmies in the hypervariable (HV) regions of mitochondrial DNA (mtDNA) from blood cells were examined in 57 healthy Korean donors. Interestingly, all the healthy Korean subjects displayed length heteroplasmies in both the HV1 and HV2 regions. Closer examination of the HV2 length heteroplasmies indicated that most of these donors (84%) exhibited a minimal 303-315 homopolymeric C (poly-C) tract frameshift of 1 bp (mixture of one major and minor mtDNA type). Sixteen percent of the donors however had poly-C tract frameshifts of 2 bp or more. The donor group with major length variants (two or more frameshifts) had about a two-fold decrease in mtDNA copy number compared with the group exhibiting only a 1 bp frameshift. This result supports the possibility that a severe frameshift in the 303-315 poly-C tract may also cause the impairment of mtDNA replication in hematopoietic tissue.

In 1966, a male (17 years old) was clinically examined at the National Institutes of Health (NIH) and diagnosed with Idiopathic Progressive External Ophthalmoplegia (IPEO). A muscle biopsy showing ragged-red fibers implicated mitochondrial involvement. Since the sequence of human mitochondrial DNA (mtDNA) was not determined until 1981, no genetic confirmation of the disease was possible at that time. In 1999, clinical reexamination and sequencing the entire mtDNA of the patient and living maternal relatives (mother and brother) indicated a progressive mitochondrial myopathy and the presence of the 4977 base pair (bp) deletion (the common deletion) in the patient.

Background: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications., Methods: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing., Results: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications., Conclusions: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors-37.9% +/- 3.6% heterogeneity in circulating CD34+ cells, 36.4% +/- 14.1% in T cells, 36.0% +/- 10.7% in B cells, and 47.7% +/- 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% +/- 5.7% in CD34+ cells, 15.2% +/- 9.0% in T cells, 15.4% +/- 6.7% in B cells, and 32.3% +/- 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

Human mitochondrial DNA (mtDNA) mutations are important for forensic identifications and mitochondrial disease diagnostics. Low-frequency mutations, heteroplasmies, or SNPs scattered throughout the DNA in the presence of a majority of mtDNA with the Cambridge Reference Sequence (CRS) are almost impossible to detect. Therefore, the National Institute of Science and Technology has developed heteroplasmic human mtDNA Standard Reference Material (SRM) 2394 to allow scientists to determine their sensitivity in detecting such differences. SRM 2394 is composed of mixtures ranging from 1/99 to 50/50 of two 285-bp PCR products from two cell lines that differ at one nucleotide position. Twelve laboratories using various mutation detection methods participated in a blind interlaboratory evaluation of a prototype of SRM 2394. Most of these procedures were unable to detect the mutation when present below 20%, an indication that, in many real-life cases, low-frequency mutations remain undetected and that more sensitive mutation detection techniques are urgently needed.

We have reported marked mitochondrial DNA (mtDNA) sequence heterogeneity among individual CD34 clones from adult bone marrow (BM) and the age-dependent accumulation of mtDNA mutations in this mitotically active tissue. Here, we show direct evidence of clonal expansion of cells containing mtDNA mutations and that the mtDNA sequence may be easily determined by using peripheral blood (PB) as a CD34 cell source. Analysis of 594 circulating CD34 clones showed that 150 (25%) had mtDNA sequences different from the same donor's corresponding aggregate sequence. Examination of single granulocytes indicated that 103 (29%) from the same 6 individuals showed mtDNA heterogeneity, with sequences distinct from the corresponding aggregate tissue sequence and from the sequences of other single granulocytes. Circulating and BM CD34 cells showed virtually identical patterns of mtDNA heterogeneity, and the same changes were seen in progeny granulocytes as in their progenitors, indicating that blood sampling could be used in studies to determine whether mtDNA reflects an individual's cumulative or recent exposure to mutagens; as a marker of individual hematopoietic progenitors, stem cells, and their expansion; and for the detection of minimal residual disease in hematologic malignancies of CD34 cell origin.

Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in postmitotic tissues but have been postulated to be diluted and lost in continually proliferating tissues such as bone marrow (BM). Having observed marked sequence variation among healthy adult individuals' total BM cell mtDNA, we undertook analysis of the mtDNA control region in a total of 611 individual CD34+ clones from 6 adult BM donors and comparison of these results with the sequences from 580 CD34+ clones from 5 umbilical cord blood (CB) samples. On average, 25% (range, 11% to 50%) of individual CD34+ clones from adult BM showed mtDNA heterogeneity, or sequence differences from the aggregate mtDNA sequence of total BM cells of the same individual. In contrast, only 1.6% of single CD34+ clones from CB showed mtDNA sequence variation from the aggregate pattern. Thus, age-dependent accumulation of mtDNA mutations appears relatively common in a mitotically active human tissue and may provide a method to approximate the mutation rate in mammalian cells, to assess the contribution of reactive oxygen species to genomic instability, and for natural "marking" of hematopoietic stem cells; our data also have important implications for the aging process, forensic identifications, and anthropologic conclusions dependent on the mtDNA sequence.

Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA) genome of HL-60, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology (NIST) SRM 2392-I. NIST human mtDNA SRM 2392 will continue to be available and includes the DNA from two apparently healthy individuals. Both SRM 2392 and 2392-I contain all the information (e.g. the sequences of 58 unique primer sets) needed to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392, the HL-60 mtDNA in SRM 2392-I has two tRNA differences and more polymorphisms resulting in amino acid changes. Four of these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM10742A) from an individual with LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European descent or may mean that haplogroup J is more prone to LHON. Both these mtDNA SRMs will provide enhanced quality control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.

The transmission of a C16,291C/T heteroplasmy in the HV1 region of human mitochondrial DNA (mtDNA) was examined in buccal cells from 13 maternally-related individuals across three generations and in additional tissues (hair, blood, or finger nails) from three members of this family. The ratio of C:T at nucleotide position (np) 16,291 showed wide intra- and intergenerational variation as well as tissue variation within individuals. Our results demonstrate that one or two sequence differences between samples in the mtDNA does not warrant an exclusion. To avoid false exclusions especially when comparing mtDNA from hair samples, we recommend the analysis of as many samples as possible in order to minimize the possibility that the detection of a rare polymorphism in a single sample would be considered an exclusion when it is really a match. The observation that the transmission of a mtDNA heteroplasmy from one individual to her offspring is likely to differ among the first-generation offspring and between that generation and subsequent generations lends further credence to the bottleneck theory of inheritance of human mtDNA.

This paper discusses results of a supercritical fluid extraction-gas chromatography/mass spectrometry (SFE-GC/MS) study of small samples ( 100 microg to 1 mg) of human scalp hair. The method offers a number of benefits including greater sensitivity than liquid extraction methods because the entire extractable mass is transferred to the analytical system, compared with only a few percent from a conventional liquid extraction/injection. The project's goals were to determine if SFE-GC/MS analyses of the surface-extractable components of an individual's hair yield consistent chemical profiles and to investigate if the profiles are sufficiently different to distinguish them from those of other individuals. In addition, the mtDNA sequences from ten of the same individuals used in the SFE-GC/MS study from four family units were determined, and, while the families were distinguishable, the maternal relations yielded identical sequences. In tandem, SFE-GC/MS and mtDNA techniques may provide valuable complementary data from forensic hair samples.

We undertook to systematically analyze the entire mitochondrial genome by gene amplification and direct sequencing in 10 patients with myelodysplasia; results were compared with concomitantly studied 8 healthy volunteers as well as mtDNA sequences in a standard database. Nucleotide changes that were present in our healthy controls as well as those in published databases were counted as polymorphisms. Overall, there was no increase in the number of mtDNA genes harboring polymorphisms or "new" mutations between our patients and healthy controls, although there were a few more mtDNA changes resulting in amino acid changes in myelodysplasia (9 in 8 controls versus 16 in 10 patients). Thirty new mutations, all nucleotide substitutions, were found among the 10 patients, distributed throughout the mitochondrial genome; 5 mutations resulted in amino acid changes. None of the mutations in controls produced amino acid changes. We were not able to confirm previously described mutations in sideroblastic anemia or "hot spots" in the cytochrome c oxidase I and II genes. Our data do not support a major role for mitochondrial genomic instability in myelodysplasia, and they fail to reproduce previous reports of significant or widespread mitochondrial mutations in this disease. Modest changes in mutation numbers and mitochondrial microsatellites may be evidence of increased mutagenesis in mtDNA, or, more likely, a reflection of limited clonality among hematopoietic stem cells in this bone marrow failure syndrome.

Background: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood., Methods: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing., Results: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence., Conclusion: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.

MitoAnalyzer provides information about the effects of single nucleotide polymorphisms (SNPs) and mutations in human mitochondrial DNA (mtDNA). This program determines if a single base pair (bp) change is in the non-coding or coding region, in the first, second or third bp of the codon, in a rRNA, tRNA or a protein, causes an amino acid (aa) change, the nature of that change, the position of the aa change in the protein, and the new aa sequence of the changed protein. Mutations associated with published mitochondrial diseases are noted. This program, thus, facilitates rapid analysis and evaluation of SNPs and mutations in human mtDNA.

The Standard Reference Materials Program at the US National Institute of Standards and Technology (NIST) has three human DNA standard reference materials (SRM 2390, SRM 2391a, and SRM 2392) currently available [1, 2]. Both the DNA profiling SRM 2390 and the polymerase chain reaction (PCR)-based DNA profiling SRM 2391a are intended for use in forensic and paternity identifications, for instructional law enforcement, or for non-clinical research purposes and are not intended for clinical diagnostics. The mitochondrial DNA (mtDNA) SRM 2392 is to provide standardization and quality control when performing PCR and sequencing any segment or the entire 16,569 base pairs that comprise human mitochondrial DNA. SRM 2392 is designed for use by the forensic, medical, and toxicological communities for human identification, disease diagnosis or mutation detection.

For Mohs surgical wounds that show exposed bone (ie, bone denuded of periosteum), healing by secondary intention may be preferable to surgical reconstruction. To determine the appropriateness of secondary intention healing, we reviewed surgical outcome in 205 patients with Mohs wounds of the scalp and forehead that had healed by secondary intention. Of these patients, 38 had Mohs wounds showing exposed bone. The mean area of exposed bone was 1074 mm(2); the mean area of exposed soft tissue was 1575 mm(2). The mean time for wounds with intact periosteum to epithelialize was 7 weeks; the mean time for bare bone to epithelialize was 13 weeks. All wounds healed without infection or tissue breakdown. We conclude that secondary intention healing of scalp and forehead wounds showing exposed bone is a safe and effective method of wound management after Mohs surgery.

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM 2392 includes DNA from two lymphoblast cell cultures (CHR and 9947A) and cloned DNA from the CHR HV1 region, which contains a C stretch and is difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area or the entire mtDNA (16,569 bp) to be amplified and sequenced. While none of the differences in these three templates correspond to published mutations associated with specific diseases, some of these differences did result in animo acid changes compared with that published by S. Anderson et al. (1981, Nature 290: 457-465). An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified and sequenced correctly., (Copyright 1999 Academic Press.)

Background: Facial defects after Mohs surgery may be closed at the time of surgery, shortly thereafter, or allowed to heal spontaneously. Selection of appropriate management options requires accurate prediction of the final cosmetic result., Objective: To determine the relative merits of treatment options for facial defects resulting from Mohs surgery., Methods: We prospectively and retrospectively analyzed outcomes for 132 wounds of the cheek resulting from Mohs surgery and allowed to heal spontaneously. The cheek was delineated into topographic areas and the wound location, size, and depth were recorded. Patients were observed intermittently, and a final evaluation of cosmesis was made after > or = 6 months., Results: Most wounds in the nasolabial fold and preauricular areas healed with excellent results and half the wounds in the medial area of the cheek healed with good or excellent results. Central and mandibular areas of the cheek healed unpredictably, and results were unsatisfactory for most of these wounds., Conclusion: Final cosmetic results of Mohs surgery in the cheek area can be predicted on the basis of location, size, and depth of the wound, enabling physicians to knowledgeably select the most suitable treatment option.

Accurate prediction of the course of wound healing allows patients with Mohs surgical wounds on the face to be objectively selected either for surgical repair or for spontaneous healing. Parameters affecting wound healing--location, depth and size of wound--were documented at intervals for 135 patients who had full-thickness defects in the forehead after Mohs surgery, which were left to heal by secondary intention. Quality of cosmesis was determined at least four months after surgery. Final cosmetic result was predictable on the basis of wound location, size and depth. Allowing selected Mohs surgical wounds to heal spontaneously offers optimal cancer surveillance, simplified wound care and elimination of reconstructive procedures along with their associated costs and potential complications.

Human mitochondrial DNA has become a useful tool in forensic investigations. Its polymorphic nature and maternal inheritance are characteristics that have, combined with its sequence information, enabled investigators to identify missing persons, war casualties and individuals involved in mass disasters and criminal cases. Various screening procedures have been developed to reduce the need to sequence samples that do not match, but DNA-sequence information is still necessary to verify a match. Even though several challenges remain before mitochondrial-DNA-sequence information can be used unambiguously, comparative mitochondrial-DNA-sequence analysis appears to be a reliable and powerful means for human identification.

Two new research approaches in combustion toxicology are: 1. the prediction of smoke toxicity from mathematical equations, which are empirically derived from, experiments on the toxicological interactions of complex fire gas mixtures and 2. the use of toxicant suppressants in materials or products to prevent the formation of toxic combustion products. The predictive approach consists of burning materials using a bench-scale method that simulates realistic fire conditions, measuring the concentrations of the primary fire gases--CO, CO2, low O2, HCN, HCl, HBr, and NO2--and predicting the toxicity of the smoke using either the 6- or 7-gas N-Gas Model. These models are based on the results of toxicological studies of these primary gases as individual gases and as complex mixtures. The predicted toxic potency is checked with a small number of animal (Fischer 344 male rats) tests to assure that an unanticipated toxic gas is not generated or an unexpected synergistic or antagonistic effect has not occurred. The results indicate if the smoke from a material or product is extremely toxic (based on mass consumed at the predicted toxic level) or unusually toxic (based on the gases deemed responsible). The predictions based on bench-scale laboratory tests have been validated with full-scale room burns of a limited number of materials of widely differing characteristics chosen to challenge the system. The advantages of this new approach are 1. the number of test animals is minimized by predicting the toxic potency from the chemical analysis of the smoke, 2. smoke may be produced under conditions that simulate the fire scenario of concern, 3. fewer tests are needed, thereby reducing the overall cost of the testing and 4, information is obtained on both the toxic potency of the smoke and the responsible gases. The N-Gas Models have been developed into the N-Gas Method (described in this paper) and these results have been used in computations of fire hazard. The 6-Gas Model is now part of the international standard ISO 13344 approved by 16 member countries of the International Standards Organization (ISO) and is also included in the U.S. national standard ASTM E1678 approved by the American Society for Testing and Materials (ASTM). In addition, the 6-Gas Model is used in the American National Standard--NFPA 269--approved by the National Fire Protection Association (Quincy, MA). The second new research approach, toxicant suppressants, examines the potential of chemical compounds, which when added to a material, to inhibit or reduce the concentration of a specific toxic gas normally generated during the material's thermal decomposition. The effectiveness of this approach was demonstrated at the National Institute of Standards and Technology (NIST) when HCN generation was reduced by 90% and the resultant toxicity of the combustion products was lowered by 50% when a flexible polyurethane foam (FPU) was treated with 0.1% (by weight) cuprous oxide (Cu2O). Copper and cupric oxide (CuO) also reduced the HCN generation but were not as efficient as Cu2O. Although melamine-treated FPU foams are being promoted as more fire safe than standard foams, a melamine-treated foam generated 10 times more HCN than a foam without melamine. The addition of Cu2O to this melamine foam also reduced the HCN generation by 90%.

Three research areas -- a 7-gas N-Gas Model, toxicant suppressants, and genetic toxicology -- are presented as new research approaches in toxicology. The current 6-gas N-Gas Model predicts the toxic potency of the combustion products of materials based on the toxicological interactions of the fire gases carbon monoxide (CO), carbon dioxide (CO2), low oxygen (O2) concentrations, hydrogen cyanide (HCN), hydrogen chloride, and hydrogen bromide. The present research includes nitrogen dioxide (NO2) in a new 7-gas model which incorporates the synergistic effects of NO2 and CO2, the antagonistic effects of NO2 and HCN, and the additive effects of NO2 with CO and low O2. The area of toxicant suppressants concerns chemicals, which when added to a material, will inhibit or reduce the concentration of a specific toxic gas normally generated during thermal decomposition of that material. The effectiveness of this approach was demonstrated at the US National Institute of Standards and Technology when HCN generation was reduced by 90% and the resultant toxicity of the combustion products was lowered by 50% when a flexible polyurethane (FPU) foam was treated with 0.1% (by weight) cuprous oxide (Cu2O). Although melamine-treated FPU foams are being promoted as more fire safe than standard foams, a melamine-treated foam generated 10 times more HCN than a foam without melamine. The addition of Cu2O to this melamine foam also reduced the HCN generation by 90%. The genetic toxicology research entails the examination of DNA damage that results from the exposure of human cells to various environmental toxicants and gases.

Wounds of the lip and chin resulting from microscopically controlled (Mohs) surgery are often repaired immediately. However, wounds allowed to heal by secondary intention have the advantage of optimal cancer surveillance, simplified wound care, and avoidance of the costs and potential complications associated with reconstructive procedures. Accurate prediction of the course of wound healing would allow a rational approach to selection of surgery or healing by secondary intention. The authors evaluated 105 patients with defects of the lip and chin after Mohs excision for cancer who healed by secondary intention. Forty-six patients treated surgically were followed for comparison. The lip and chin were divided into subunits and the wound location, size, and depth were recorded. Patients were followed at intervals and a final determination regarding cosmesis was made after 6 or more months. It is concluded that the final cosmetic result can be confidently predicted on the basis of location by subunit, size, and depth of the wound.

Uvulopalatopharyngoplasty (UPPP) has become a widely practiced procedure in the management of snoring. In a number of studies, all based on short-term follow-up, snoring was reduced or eliminated in 75% to 95% of patients. Because a decline in the initial success rate was casually observed, a formal analysis of the initial and long-term results of UPPP for snoring was made in the present study. From 1985 to 1989, 69 patients with severe habitual snoring were retrospectively analyzed. A scoring system was devised for snoring, and each patient was interviewed regarding the effects of UPPP on his or her snoring 16 to 75 (mean 44) months after surgery. In 60 (87%) of 69 patients, snoring was initially significantly reduced or totally eliminated. After 13 months the success rate dropped to 46% (32 of 69 patients). Most failures occurred between 6 and 12 months of surgery, after which time the success rate remained relatively stable. The authors' conclusion that the long-term success rate of UPPP for snoring declines significantly with time has obvious implications regarding presurgical patient counseling.

A comprehensive methodology has been developed for obtaining and using smoke toxicity data for fire hazard analysis. This bench-scale method can simulate diverse fire conditions and identify extremely toxic smoke under both pre- and post-flashover conditions. However, incidence data show that most of the fire deaths in the U.S. occur outside the room of fire origin from smoke and toxic gases that are generated from a fire under post-flashover conditions. Therefore, the most relevant real-scale combustion conditions to simulate in the bench-scale apparatus would be the post-flashover conditions which are achieved by using radiant heat, a high heat flux, and correcting the bench-scale carbon monoxide (CO) results to agree with CO yields observed in real-scale post-flashover fires. The number of test animals (Fischer 344 male rats) is minimized by using the N-Gas Model to estimate the LC50 value from the chemical analysis of the smoke. The current N-Gas Model predicts the toxicity of complex fire gas mixtures based on a large data base of experimental results of individual and mixed gases that include CO, CO2, reduced O2, HCN, HCl, HBr, and NOx. The prediction is checked with a small number of animal tests and an approximate LC50 value is determined. The bench-scale results have been validated with full-scale room wall burns of a limited number of materials of widely differing characteristics chosen to challenge the system. The toxic potency values are assessed to determine if the smoke from a material or product is unusually or extremely toxic and can then be used in computations of fire hazard.

A standard reference material (SRM 1049) has been developed for the University of Pittsburgh smoke toxicity method. SRM 1049 is a nylon 6/6 and has the molecular structure of [-NH(CH 2 ) 6 NHCO(CH 2 ) 4 CO-] n . This SRM is for calibrating the apparatus and providing confidence that the method is being conducted in a correct manner and that the equipment is functioning properly. The certified figure of merit is a LC 50 value plus its 95% prediction interval which were calculated and found to be 4.4 + 3.4 g. The 95% prediction interval indicates the range in which the next determined LC 50 value would be expected to fall. Thus, if an investigator were to test this SRM under their laboratory conditions according to the specifications of the University of Pittsburgh test procedure and found the LC 50 value fell within the certified 95% prediction interval, the probability is good that the test is being conducted correctly.

A standard reference material (SRM 1048) has been developed for use with the cup furnace smoke toxicity method. This SRM is to be used to calibrate the apparatus and to enable the user to have confidence that the method is being conducted in a correct manner and that the equipment is functioning properly. The toxicological results from this SRM should not be used to compare with those of other materials (i.e., to determine if the combustion products of a test material are more or less toxic than those from this SRM). SRM 1048 is an acrylonitrile-butadiene-styrene (ABS) and is the same as SRM 1007B which is used for calibrating the flaming mode of the Smoke Density Chamber test method (ASTM E-662 and NFPA 258). For the purposes of calibrating the cup furnace smoke toxicity method, LC 50 and N-Gas values plus their respective 95% confidence limits have been determined and certified for two combustion modes (flaming and nonflaming) and two observation periods (for the 30 min exposure only and for the 30 min exposure plus a 14 d post-exposure period). The certified LC 50 values plus 95% confidence intervals (in g/m 3 ) are 27 ± 3 (30 min, flaming); 25 ± 3 (30 min+ 14 d, flaming); 58 ± 15 (30 min, nonflaming); and 53 + 12 (30 min+ 14 d, nonflaming). The certified N-Gas values plus 95% confidence intervals are 1.4 ± 0.2 (30 min, flaming); 1.5 ± 0.2 (30 min+ 14 d, flaming); 1.2 ± 0.2 (30 min, nonflaming); and 1.4 ± 0.2 (30 min+ 14 d, nonflaming). It is recommended that this SRM be used with the N-Gas approach to calibrate the cup furnace smoke toxicity method rather than to determine the complete LC 50 values. The N-Gas approach has the advantage of providing information on the gases responsible for the lethalities as well as the toxic potency of the smoke. In addition, the N-Gas approach reduces the number of experimental animals, the time necessary to complete the calibration, and the expense.

Immediate reconstruction of full-thickness skin defects after cancer surgery is a commonly accepted surgical principle used to preserve function and minimize cosmetic deformity. Healing by secondary intention, however, offers the advantages of optimal cancer surveillance, simplified wound management, and avoidance of reconstructive procedures with their associated costs and potential complications. Accurate prediction of the course of wound healing, and thereby the final functional and cosmetic result, would allow a rational approach to selection of patients for surgical or nonsurgical repair. We observed 282 patients with full-thickness perinasal (glabella, medial canthus, dorsum, sidewall, tip, ala, philtrum, alar base, and nasolabial fold) skin defects after Mohs' surgery and documented a variety of parameters affecting wound healing, including location, depth, and size of the wound. Patients were examined at intervals, and a final determination regarding cosmesis and function was made at 6 months or later. We conclude that the most important considerations in predicting the final functional and cosmetic result include location by subunit, followed by size and depth of the wound.

This retrospective study compared elective neck dissection with elective neck radiotherapy for the control of subclinical nodal metastases. Four hundred ninety-eight patients with head and neck primary cancers and no clinically apparent neck metastases on initial presentation comprised the study population. Each patient was followed up for at least 5 years to detect failure to control neck metastases and control of the primary tumor at the time of neck recurrence. Analysis of neck recurrences occurring in patients with control of the primary tumor showed that there was no statistically significant difference between elective radiation therapy to the neck and elective neck dissection for oral cavity, oropharyngeal, and laryngeal cancers. The only statistically significant difference was noted for hypopharyngeal cancers, with radiation therapy being more effective than surgery.

The human temporal bone is an extremely complex structure. Direct axial and coronal CT sections are quite satisfactory for imaging the anatomy of the temporal bone; however, many relationships of the normal and pathologic anatomic detail of the temporal bone are better seen with direct sagittal CT sections. The sagittal projection is of interest to surgeons, as it has the advantage of following the plane of surgical approach. This article describes the advantages of using direct sagittal sections for studying various diseases of the temporal bone. The CT sections were obtained with the aid of a new head holder added to our GE CT 9800 scanner. The direct sagittal projection was found to be extremely useful for evaluating diseases involving the vertical segment of the facial nerve canal, vestibular aqueduct, tegmen tympani, sigmoid sinus plate, sinodural angle, carotid canal, jugular fossa, external auditory canal, middle ear cavity, infra- and supralabyrinthine air cells, and temporomandibular joint.

The toxicity of single and multiple fire gases is studied to determine whether the toxic effects of the combustion products from materials can be explained by the toxicological interactions (as indicated by lethality) of the primary fire gases or if minor, more obscure gases need to be considered. LC50 values for Fischer-344 rats have been calculated for the individual gases, carbon monoxide (CO), hydrogen cyanide (HCN), or decreased oxygen (O2), for 30-min exposures plus relevant postexposure periods using the NBS Toxicity Test Method. Combination experiments with CO and HCN indicate that they act in an additive manner. Synergistic effects have been found when the animals are exposed to certain combinations of CO and carbon dioxide (CO2). Five percent CO2 raised the threshold for deaths due to hypoxia and decreased the LC50 of HCN. Decreasing the O2 concentration in the presence of various mixtures of the other major fire gases increased the toxicity even further. A comparison of the concentrations of the major combustion products generated from a number of polymeric materials at their LC50 (30-min exposure plus 14-day postexposure) values with the combined pure gas results indicates that, in most cases, the observed toxicity may be explained by the toxicological interactions of the examined primary toxic fire gases. These results provide necessary information for the computer model currently being developed at the Center for Fire Research to predict the toxic hazard that people will experience under various fire scenarios.

This article reviews the authors' results using CT in the diagnosis of 18 selected patients who were clinically suspected of having otogenic intracranial complications, demonstrating with illustrative examples the important role of CT in diagnosing various stages of acute coalescent mastoiditis and its associated complications.

The minimal growth-inhibitory amount of either hexachlorophene (HCP) or decanoate stopped growth, respiration, adenosine 5'-triphosphate synthesis, and amino acid transport of Bacillus subtilis in a culture containing amino acids and citrate as carbon sources. The electron transport system was not affected by this dose. Addition of 27.8 mM glucose or 10 mM malate to an inhibited culture did not reverse the binding of HCP or decanoate to the cells, but it allowed resumption of growth, respiration, and adenosine 5'-triphosphate synthesis, as the glucose or malate then supplied the needed carbon. The addition of glucose or malate did not reverse amino acid transport inhibition caused by decanoate, but it did reverse that due to HCP. However, if the dose of HCP was raised in the presence of glucose or malate, only growth and amino acid transport were affected; this indicates that both HCP and decanoate act at their minimal growth inhibitory doses by inhibiting substrate transport. As active transport of amino acids and ketoacids depends on the proton gradient and the membrane potential of the cells, we conclude that the primary effect of these lipophilic acids is the destruction of the proton-motive force.

Fischer 344 male rats were subjected to 30-min individual or combined exposures of carbon monoxide (CO) and carbon dioxide (CO2). All deaths from CO occurred during the exposures, and the LC50 values were 4600 and 5000 ppm, depending on experimental conditions. Animals exposed to CO2 concentrations ranging from 1.3 to 14.7% for 30 min were neither incapacitated nor fatally injured. The addition of nonlethal concentrations of CO2 (1.7 to 17.3%) to sublethal concentrations of CO (2500 to 4000 ppm) caused deaths of the exposed rats both during and following (up to 24 h) the 30-min exposures. The most toxic combination of these two gases (2500 ppm CO plus 5% CO2) increased the rate of carboxyhemoglobin (COHb) formation 1.5 times that found in rats exposed to 2500 ppm of CO alone. The COHb equilibrium levels were the same. Exposure to both CO and CO2 produced a greater degree of acidosis and a longer recovery time than that observed with either single gas. The results fit a mathematical model indicating a synergistic interaction. Combustion of 11 materials at their LC50 values indicated that CO was probably the primary toxicant in one case and that the combined CO plus CO2 was the cause of the deaths in three other cases. Additional fire gases need to be studied to explain deaths from the other materials.

More than 80% of the hexachlorophene added to a Bacillus subtilis culture binds to the cells. Complete growth inhibition requires 6 x 10(5) molecules bound per cell. In contrast, more than 99% decanoate remains in solution and 3.8 x 10(7) molecules bound per cell are needed to inhibit growth. Centrifugation and resuspension of cells in growth medium removes only decanoate, whereas the addition of 1% bovine serum albumin to the growth medium removes both inhibitors from their binding sites on the cells. The addition of untreated cells to a hexachlorophene-treated culture enables the hexachlorophene molecules to redistribute among all the cells with the result that the inhibited cells can resume growth.

High-resolution CT scanning accurately depicts the status of the structures of the temporal bone, allowing delineation of pathology prior to surgical exploration of ears with cholesteatoma. It provides information concerning location and extent of disease as well as possible anatomic variations and complications that may be encountered. The main advantages of CT scanning over polytomography are superior soft-tissue contrast resolution and improved spatial detail at a reduced radiation dose for the patient.

The inhibition of cell duplication by many lipophilic acids was measured in Bacillus subtilis and in the following mammalian cell lines, the human epithelial-type cell lines HeLa, strain R and strain L-132, the human fibroblast cell line VA-13, and the rat glial cell line C. The results were correlated to the partition coefficient and the distribution coefficient (= apparent partition coefficient at pH 7.2) of the compounds, using octanol/water partition coefficients and pKa values either from the literature or measured for this work. For B. subtilis, the logarithm of the inhibitory potency of most compounds increases linearly with the logarithm of the partition coefficient. Exceptional high potencies were observed for compounds that can efficiently delocalize the charge of the negative ion over the whole molecule. Most compounds inhibit tissue cultures at least as potently as they inhibit B. subtilis. But some compounds are significantly more potent in tissue cultures than would have been expected from the B. subtilis data; such compounds (analgesics/antipyretics, anti-inflammatory compounds, butyrate, norepinephrine) presumably inhibits mammalian cells by specific reactions with certain cell components. However, most compounds inhibit the different cell lines to a similar degree, indicating no cellular specificity; exceptions to this rule are chlorambucil, chlortetracycline and dexamethasone. Many of the lipophilic acids that are potent inhibitors of mammalian cell replication are also teratogenic. Exceptional compounds may not reach the embryo. We propose that a number of other lipophilic acids that are potenta inhibitors and to which humans are frequently exposed should be tested for their teratogenic effect.