Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies.

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.

Evaluation of tetrahydrocannabinolic acid (THCA) synthase polymorphisms for distinguishing between marijuana and hemp.

The Controlled Substances Act (CSA) classifies marijuana (Cannabis sativa) as a Schedule I illicit drug. However, the recent Agriculture Improvement Act of 2018 (U.S. Farm Bill) removed hemp from the definition of marijuana in the CSA, making it a legal crop. As a result, many hemp products are now available, including strains of hemp buds high in other cannabinoids such as cannabidiol (CBD) or cannabigerol (CBG). The genetic inheritance of chemical phenotype (chemotype) has been widely studied, with the tetrahydrocannabinolic acid (THCA) synthase gene at the forefront. Previous studies have speculated that there are two forms of the THCA gene, one that produces an active enzyme (present in marijuana) and one that cannot produce a functional enzyme (present in hemp). A DNA analysis method is desirable for determining crop type in sample types inconducive to chemical analysis, such as immature crops, trace residues, small leaf fragments, seeds, and root material. This study optimized and evaluated a previously reported single nucleotide polymorphism (SNP) assay for determining C. sativa crop type. Furthermore, the presence or absence of 15 cannabinoids, including THC and THCA, was reported in cannabis reference materials and 15 legal hemp flower samples. The SNP assay correctly identified crop type in most samples. However, several marijuana samples were classified as hemp, and several hemp seeds were classified as marijuana. Two strains of legal CBG hemp flowers were also classified as marijuana, indicating that factors other than the genetic variation of the THCA synthase gene should be considered when determining crop type.

Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing.

BACKGROUND: Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA, and the use of DNA barcodes could assist drug trafficking investigations by indicating the biogeographical origin and crop type of a sample and providing a means for linking cases. Additionally, the legality of marijuana in the USA remains complicated with some states fully legalizing marijuana for recreational use while federally marijuana remains completely illegal. Massively parallel sequencing (MPS) offers distinct advantages over capillary electrophoresis (CE), including more comprehensive coverage of target loci, analysis of hundreds of markers simultaneously, and high throughput capabilities. METHODS: This study reports on the development of a MiSeq FGx® assay targeting seven "hotspot" regions in the Cannabis sativa chloroplast genome that are highly polymorphic and informative in attempts to determine biogeographical origin and distinguishing between marijuana and hemp. Sequencing results were compared to previous studies that used CE-based genotyping methods. RESULTS: A total of 49 polymorphisms were observed, 16 of which have not been previously reported. Additionally, sequence data revealed isoalleles at one locus, which were able to differentiate two samples that had the same haplotype using CE-based methods. This study reports preliminary results from sequencing 14 hemp and marijuana samples from different countries using the developed MPS assay. CONCLUSION: Future studies should genotype a more comprehensive sample set from around the world to build a haplotype database, which could be used to provide investigative leads for law enforcement agencies investigating marijuana trafficking.

Use of Eucalyptus DNA profiling in a case of illegal logging.

Eucalyptus is grown world-wide for paper pulp, solid wood, and other industries. Theft or illegal cutting of the trees causes hardship to owners of plantations and countries whose economies rely on the sale and export of eucalyptus products. Unfortunately, many of these crimes go unpunished due to lack of forensic evidence. Over 1200 short tandem repeat (STR) markers have been identified in the genomes of genus Eucalyptus and related species. However, their importance and utility in aiding forensic investigations of wood theft have not been explored. This study evaluated nine STRs for diversity and applied them to a case involving suspected wood theft. As expected, three dinucleotide STR markers showed greater variability but resulted in harder to interpret profiles. Four STR tetranucleotide markers evaluated in this study were found to contain additional repeat structures (dinucleotide or trinucleotide) that enhanced their variability but resulted in profiles with peaks at multiple stutter positions and heterozygote peak imbalance. The most promising STR markers were EGM37 and EMBRA 1374. Though less variable, they yielded robust and reproducible DNA profiles. All nine STR markers were applied to a case involving suspected wood theft. Samples were collected from seized wood and from remaining stumps in a plantation. No DNA match was found, thus eliminating the evidence samples as having originated from the forest. Dendrochronology analysis also resulted in an exclusion. This case study represents the first report using STR markers in any eucalyptus species to provide DNA evidence in a case of suspected wood theft.

Investigation of chloroplast regions rps16 and clpP for determination of Cannabis sativa crop type and biogeographical origin.

Cannabis sativa can be classified as either hemp (a legal crop containing less than 0.3% delta-9-tetrahydrocannabinol, THC) or marijuana (an illegal drug containing more than 0.3% THC). Despite its legalization in 33 states for medicinal or recreational use, marijuana remains the most commonly used illicit drug in the USA, and it is heavily trafficked into and within the country. Discriminating between marijuana and hemp is critical to the legal process. Genetic analysis provides a means of analyzing samples unsuitable for chemical analysis, and in addition to discriminating between crop types, DNA may be able to determine the biogeographical origin of samples. In addition, the sharing of rare haplotypes between different seizures may be useful for linking cases and providing investigative leads to law enforcement. This study evaluates the potential of two highly polymorphic regions of the chloroplast genome of C. sativa, rps16 and clpP, to be used for determination of crop type and biogeographical origin. Custom fragment analysis and SNaPshot™ assays were developed to genotype nine polymorphic loci in hemp samples from the USA and Canada, marijuana samples from USA-Mexico and Chile, and medical marijuana samples from Chile. Haplotype analysis revealed eight haplotypes. Only Canadian hemp could be completely differentiated from the other sample groups by haplotype. Phylogenetic analysis and principal component analysis suggested a closer relationship among USA-Mexico marijuana, Chilean marijuana and medical marijuana, and USA hemp. Genotyping additional polymorphisms in future studies is expected to reveal further differences between these sample groups.

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum.

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration's (DEA) Heroin Signature Program, which seeks to combat rising opioid use. The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.

Efficacy of "touch" DNA recovery and room-temperature storage from assault rifle magazines.

Crimes committed with assault rifles are becoming increasingly prevalent in the United States. In the absence of other evidence, DNA analysis can often provide informative leads. Unfortunately, any DNA transferred to rifle components left behind at a crime scene is likely to be low in quantity and/or quality. Furthermore, collected evidence is unlikely to be processed immediately and may require storage. Long-term storage can subject DNA to damage and degradation, which ultimately affects DNA profile interpretation and may prevent the identification of potential suspects. This study assessed the ability of a new swab storage device, the SwabSaver®, to preserve "touch" DNA from AR-15 magazine rifles using three different collection devices. Three volunteers loaded bullet cartridges into plastic polymer and aluminum AR-15 magazines. DNA was collected with traditional cotton swabs, layered cotton paper swabs, or nylon-flocked swabs. Collection devices were then stored at room-temperature for up to two months in either the SwabSaver® device or an empty centrifuge tube. The results suggest that substrate and swab type had less of an effect on profile completeness than storage type. Furthermore, SwabSaver® storage yielded DNA quantities comparable to "touch" DNA extracted after 24 h.

Thioredoxin overexpression in mitochondria showed minimum effects on aging and age-related diseases in male C57BL/6 mice.

Objective: In this study, the effects of overexpression of thioredoxin 2 (Trx2) on aging and age-related diseases were examined using Trx2 transgenic mice [Tg(TXN2]+/0]. Because our previous studies demonstrated that thioredoxin (Trx) overexpression in the cytosol (Trx1) did not extend maximum lifespan, this study was conducted to test if increased Trx2 expression in mitochondria shows beneficial effects on aging and age-related pathology. Methods: Trx2 transgenic mice were generated using a fragment of the human genome containing the TXN2 gene. Effects of Trx2 overexpression on survival, age-related pathology, oxidative stress, and redox-sensitive signaling pathways were examined in male Tg(TXN2)+/0 mice. Results: Trx2 levels were significantly higher (approximately 1.6- to 5-fold) in all of the tissues we examined in Tg(TXN2)+/0 mice compared to wild-type (WT) littermates, and the expression levels were maintained during aging (up to 22-24 months old). Trx2 overexpression did not alter the levels of Trx1, glutaredoxin, glutathione, or other major antioxidant enzymes. Overexpression of Trx2 was associated with reduced reactive oxygen species (ROS) production from mitochondria and lower isoprostane levels compared to WT mice. When we conducted the survival study, male Tg(TXN2)+/0 mice showed a slight extension (approximately 8-9%] of mean, median, and 10th percentile lifespans; however, the survival curve was not significantly different from WT mice. Cross-sectional pathological analysis (22-24 months old) showed that Tg(TXN2)+/0 mice had a slightly higher severity of lymphoma; however, tumor burden, disease burden, and severity of glomerulonephritis and inflammation were similar to WT mice. Trx2 overexpression was also associated with higher c-Jun and c-Fos levels; however, mTOR activity and levels of NFκB p65 and p50 were similar to WT littermates. Conclusions: Our findings suggest that the increased levels of Trx2 in mitochondria over the lifespan in Tg(TXN2)+/0 mice showed a slight life-extending effect, reduced ROS production from mitochondria and oxidative damage to lipids, but showed no significant effects on aging and age-related diseases.

Thioredoxin and aging: What have we learned from the survival studies?

Our laboratory has conducted the first systematic survival studies to examine the biological effects of the antioxidant protein thioredoxin (Trx) on aging and age-related pathology. Our studies with C57BL/6 mice overexpressing Trx1 [Tg(act-TRX1)+/0 and Tg(TXN)+/0) demonstrated a slight extension in early lifespan compared to wild-type (WT) mice; however, no significant effects were observed in the later part of life. Overexpression of Trx2 in male C57BL/6 mice [Tg(TXN2)+/0] demonstrated a slightly extended lifespan compared to WT mice. The pathology results from two lines of Trx1 transgenic mice showed a slightly higher incidence of age-related neoplastic diseases compared to WT mice, and a slight increase in the severity of lymphoma, a major neoplastic disease, was observed in Trx2 transgenic mice. Together these studies indicate that Trx overexpression in one compartment of the cell (cytosol or mitochondria alone) has marginal beneficial effects on lifespan. On the other hand, down-regulation of Trx in either the cytosol (Trx1KO) or mitochondria (Trx2KO) showed no significant changes in lifespan compared to WT mice, despite several changes in pathophysiology of these knockout mice. When we examined the synergetic effects of overexpressing Trx1 and Trx2, TXNTg x TXN2Tg mice showed a significantly shorter lifespan with accelerated cancer development compared to WT mice. These results suggest that synergetic effects of Trx overexpression in both the cytosol and mitochondria on aging are deleterious and the development of age-related cancer is accelerated. On the other hand, we have recently found that down-regulation of Trx in both the cytosol and mitochondria in Trx1KO x Trx2KO mice has beneficial effects on aging. The results generated from our lab along with our ongoing study using Trx1KO x Trx2KO mice could elucidate the key pathways (i.e., apoptosis and autophagy) that prevent accumulation of damaged cells and genomic instability leading to reduced cancer formation.

Characterization of new chloroplast markers to determine biogeographical origin and crop type of Cannabis sativa.

Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA. Despite its schedule I classification by the federal government, 33 states and the District of Columbia have legalized its use for medicinal or recreational purposes. This state-specific legalization has created a new problem for law enforcement: preventing and tracking the diversion of legally obtained Cannabis to states where it remains illegal. In addition, trafficking of the drug at the border with Mexico remains an issue for law enforcement agencies. C. sativa crops can be classified as marijuana (a drug containing the psychoactive chemical delta-9-tetrahydrocannabinol) or hemp (the non-drug form of the plant). Differentiation between crop types is important for forensic purposes. In addition, investigation of trafficking routes into and within the USA requires genetic association of samples from different seizures, and determining where the crop originated could provide important leads. This project seeks to exploit sequence variations in C. sativa chloroplast DNA (cpDNA) to allow genetic determination of biogeographic origin, discrimination between marijuana and hemp, and association between cases for C. sativa samples. Due to the limited discriminatory ability of common barcoding markers, the authors sought to discover more informative polymorphic regions. By comparing published whole genome cpDNA sequences, 58 polymorphisms and seven hotspot regions were identified. Hemp samples from the USA and Canada, marijuana samples from Mexico and Chile, and medical marijuana samples from Chile were evaluated using two cpDNA hotspot regions, rpl32-trnL and trnS-trnG. Principal component analysis supported some differences between the groups based on their crop type and biogeographic origin.

Continuous overexpression of thioredoxin 1 enhances cancer development and does not extend maximum lifespan in male C57BL/6 mice.

We examined the effects of continuous overexpression of thioredoxin (Trx) 1 on aging in Trx1 transgenic mice [Tg(TXN)+/0]. This study was conducted to test whether increased thioredoxin expression over the lifespan in mice would alter aging and age-related pathology because our previous study demonstrated that Tg(act-TXN)+/0 mice had no significant maximum life extension, possibly due to the use of actin as a promoter, which may have resulted in loss of Trx1 overexpression during aging. To test this hypothesis, we generated new Trx1 transgenic mice using a fragment of the human genome containing the TXN gene with an endogenous promoter to ensure continuous overexpression of Trx1 throughout the lifespan. Universal overexpression of Trx1 was observed, and Trx1 overexpression was maintained during aging (up to 22-24 months old) in the Tg(TXN)+/0 mice. The levels of Trx1 are significantly higher (approximately 4 to 31 fold) in all of the tissues examined in the Tg(TXN)+/0 mice compared to the wild-type (WT) littermates. The overexpression of Trx1 did not cause any changes in the levels of Trx2, glutaredoxin, glutathione, or other major antioxidant enzymes. The survival study demonstrated that male Tg(TXN)+/0 mice slightly extended the earlier part of the lifespan compared to WT littermates, but no significant life extension was observed over the lifespan. The cross-sectional pathological analysis (22-25 months old) showed that Tg(TXN)+/0 mice had a significantly higher severity of lymphoma and more tumor burden than WT mice, which was associated with the suppression of the apoptosis signal-regulating kinase 1 (ASK1) pathway. Our findings suggest that the increased levels of Trx1 over the lifespan in Tg(TXN)+/0 mice showed some beneficial effects (slight extension of lifespan) in the earlier part of life but had no significant effects on median or maximum lifespans, and increased Trx1 levels enhanced tumor development in old mice.

Thioredoxin overexpression in both the cytosol and mitochondria accelerates age-related disease and shortens lifespan in male C57BL/6 mice.

To investigate the role of increased levels of thioredoxin (Trx) in both the cytosol (Trx1) and mitochondria (Trx2) on aging, we have conducted a study to examine survival and age-related diseases using male mice overexpressing Trx1 and Trx2 (TXNTg × TXN2Tg). Our study demonstrated that the upregulation of Trx in both the cytosol and mitochondria in male TXNTg × TXN2Tg C57BL/6 mice resulted in a significantly shorter lifespan compared to wild-type (WT) mice. Cross-sectional pathology data showed a slightly higher incidence of neoplastic diseases in TXNTg × TXN2Tg mice than WT mice. The incidence of lymphoma, a major neoplastic disease in C57BL/6 mice, was slightly higher in TXNTg × TXN2Tg mice than in WT mice, and more importantly, the severity of lymphoma was significantly higher in TXNTg × TXN2Tg mice compared to WT mice. Furthermore, the total number of histopathological changes in the whole body (disease burden) was significantly higher in TXNTg × TXN2Tg mice compared to WT mice. Therefore, our study suggests that overexpression of Trx in both the cytosol and mitochondria resulted in deleterious effects on aging and accelerated the development of age-related diseases, especially cancer, in male C57BL/6 mice.

In-field collection and preservation of decomposing human tissues to facilitate rapid purification and STR typing.

Short tandem repeats (STR) are currently the gold standard in human identification for forensic casework purposes, and successful STR typing is dependent on sufficient quantity and quality DNA. In the aftermath of a mass disaster and some forensic cases, human remains are recovered for identification in various stages of decomposition, and ideally these remains are transported to a refrigerated facility in order to halt the decomposition process and preserve the integrity of DNA within the tissue. However, in situations where refrigeration is not available (e.g., after a mass disaster or in rural forensic casework), remains continue to be exposed to environmental insults after collection, causing further DNA damage and degradation. Therefore, successful STR typing is dependent on the time of collection and preservation of the DNA sample. This study aims to test two simple in-field collection and preservation methods for decomposing human tissues that are subsequently stored at room temperature for up to six months either in a tissue preservative solution (modified TENT buffer) or on an FTA® Elute Card. In addition, these collection and preservation methods were tested for their ability to facilitate more direct and faster processing of DNA from preserved tissues or DNA leached into the surrounding TENT preservative solution for STR typing. Pre-PCR methods tested in this study include a quick lysis of FTA® Elute Cards, silica-based purification (QIAquick®), enzyme-based extractions (PDQeX), and simple dilution of liquid preservative. The traditional DNA analysis pipeline, which includes DNA extraction and quantification, will be compared to an alternate direct PCR method, thereby allowing the elimination of these two time-consuming and costly steps. The results indicate that modified TENT preservative and FTA® Elute Cards both preserved DNA from relatively fresh tissue for up to six months at room temperature. However, mostly partial profiles were produced from decomposed tissues (day 6 - day 14 in this study) when stored for up to six months compared to when tissues were processed immediately following collection. Overall, the modified TENT preservative produced higher DNA concentrations and more successful STR results than FTA® Elute Cards. In addition, a rapid DNA extraction platform (PDQeX) generated the most successful STR typing results from the decomposed tissues stored in TENT for up to six months at room temperature. The direct PCR method used in this study generated comparable STR results to the traditional DNA analysis approach, warranting further investigation of direct PCR methods for forensic casework type samples.

The paradoxical role of thioredoxin on oxidative stress and aging.

In spite of intensive study, there is still controversy about the free radical or oxidative stress theory of aging, particularly in mammals. Our laboratory has conducted the first detailed studies on the role of thioredoxin (Trx) in the cytosol (Trx1) and in mitochondria (Trx2) on oxidative stress and aging using unique mouse models either overexpressing or down-regulating Trx1 or Trx2. The results generated from our lab and others indicate that: (1) oxidative stress and subsequent changes in signaling pathways could have different pathophysiological impacts at different stages of life; (2) changes in redox-sensitive signaling controlled by levels of oxidative stress and redox state could play more important roles in pathophysiology than accumulation of oxidative damage; (3) changes in oxidative stress and redox state in different cellular compartments (cytosol, mitochondria, or nucleus) could play different roles in pathophysiology during aging, and their combined effects show more impact on aging than changes in either oxidative stress or redox state alone; and (4) the roles of oxidative stress and redox state could have different pathophysiological consequences in different organs/tissues/cells or pathophysiological conditions. To critically test the role of oxidative stress on aging and investigate changes in redox-sensitive signaling pathways, further study is required.

Do Ames dwarf and calorie-restricted mice share common effects on age-related pathology?

Since 1996, aging studies using several strains of long-lived mutant mice have been conducted. Among these studies, Ames dwarf mice have been extensively examined to seek clues regarding the role of the growth hormone/insulin-like growth factor-1 axis in the aging process. Interestingly, these projects demonstrate that Ames dwarf mice have physiological characteristics that are similar to those seen with calorie restriction, which has been the most effective experimental manipulation capable of extending lifespan in various species. However, this introduces the question of whether Ames dwarf and calorie-restricted (CR) mice have an extended lifespan through common or independent pathways. To answer this question, we compared the disease profiles of Ames dwarf mice to their normal siblings fed either ad libitum (AL) or a CR diet. Our findings show that the changes in age-related diseases between AL-fed Ames dwarf mice and CR wild-type siblings were similar but not identical. Moreover, the effects of CR on age-related pathology showed similarities and differences between Ames dwarf mice and their normal siblings, indicating that calorie restriction and Ames dwarf mice exhibit their anti-aging effects through both independent and common mechanisms.

Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle.

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production.

Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding.

Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca(2+)/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ~3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.