Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene.

BACKGROUND: Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities. OBJECTIVES: To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII. ANIMALS: Affected kitten and 80 healthy cats. METHODS: Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the ß-glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted. RESULTS: A 3-month-old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations. CONCLUSIONS AND CLINIC IMPORTANCE: The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.

Low-level constitutional mosaicism of a de novoBRCA1 gene mutation.

BACKGROUND: Pathogenic BRCA1 mutations are usually inherited. Constitutional low-level BRCA1 mosaicism has never been reported. METHODS: Next-generation sequencing (NGS) of cancer gene panel of germline and tumour DNA in a patient with early onset, triple-negative breast cancer. RESULTS: Constitutional de novo mosaicism (5%) for a pathogenic (c.1953dupG; p.Lys652Glufs*21) BRCA1mutation was detected in leukocytes, buccal tissue and normal breast tissue DNA, with ∼50% mutation in tumorous breast tissue. CONCLUSION: This is the first reported case of low-level, multiple tissue, constitutional mosaicism in BRCA1, and highlights the need to consider deep sequencing in affected individuals clinically suspected of having cancer predisposition whose tumours display a BRCA mutation.

Assessment of a decision aid to assist genetic testing research participants in the informed consent process.

Limited attention has been given to applying decision-making theories from psychology to the content and process of informed consent in genetic testing research. Data are presented from a study that developed and assessed a psychological theory-based decision aid as part of the informed consent process. This innovative approach assisted at-risk women in assessing the consequences of participating in a research project that offered them free hemophilia A genetic carrier testing. Results suggest: (1) the decision aid can be incorporated into the consent process with few problems; (2) women of varying educational backgrounds can complete the decision aid; (3) while women consider many consequences of genetic testing, their primary focus is on the implications for their family; and (4) this is in marked contrast to the typical benefit-harm statements prepared by researchers for genetic testing.

[Crystallochemistry of copper (II) and zinc (II) chelates by nonsteroidal antiinflammatory drugs]. / Cristallochimie des complexes à visée thérapeutique du cuivre (II) et du zinc (II) par des anti-inflammatoires non-stéroïdiens.

Copper(II) and zinc(II) chelates by some non-steroidal antiinflammatory drugs NSAIDs (niflumic acid, indomethacin) and 3,5-diisopropylsalicylic acid (DIPS) were characterized by single X-ray diffraction methods. Copper(II) complexes by these two types of chelates are binuclear compounds, with Cu(2)(DIPS)(4)L(2) or Cu(2)(AINS)(4)L(2) formula (L=axial non-NSAID ligand such as diethylether, dimethylsulfoxide DMSO). In zinc(II) complex by DIPS, the metal ion is tetrahedrally coordinated and the corresponding compound is mononuclear with Zn(DIPS)(2)(DMSO)(2) formula. These copper(II) and zinc(II) complexes were found to be more active than their parent drugs from the antiinflammatory and anticonvulsant properties. It was pointed out that the Cu(2)(DIPS)(4)L(2) complexes (L=diethylether, N,N-dimethylformamide) exhibited no rotorod toxicity when examined for anticonvulsant activity using the seizure produced by maximal electroshock, following oral administration to rats.

Prion diseases: copper deficiency states associated with impaired nitrogen monoxide or carbon monoxide transduction and translocation.

Literature concerning prion diseases and Cu metabolism was examined to determine merits of various suggestions concerning the relationship between these diseases and altered Cu metabolism. There are a number of recent suggestions that the normal non-pathogenic form of the prion protein (PrP(C)) contains Cu while the abnormal pathogenic form of this protein, PrP(SC), lacks Cu. Results of experiments showing oxidant sensitivity in the presence of ionically bonded Cu and millimolar concentrations of hydrogen peroxide were found to lack relevance. Demonstrating superoxide disproportionation and a correlation with cellular Cu2Zn2SOD activity is relevant and consistent with a role for PrP(C) in Cu endocytosis. There are also a number of recent suggestions that PrP(C) has a role in nerve transmission. Serum from mice that lack cellular PrP(C) was found to have an elevated Cu content consistent with a response to overcome an inflammatory disease. Attempts to induce a 'transmissible' form of prion disease requiring intracerebral injections of somewhat purified brain homogenates were found lacking in support for an etiology occurring as the result of oral ingestion of supposedly 'infected' tissues. It is suggested that PrP(C) is a normal Cu-dependent cuproglycoprotein of unknown function that may have a role in facilitating normal nitrogen monoxide- or carbon monoxide-mediated biochemistry.

Retinal angiomatous proliferation in age-related macular degeneration.

BACKGROUND: It is known that choroidal neovascularization (CNV) in age-related macular degeneration (ARMD) may erode through the retinal pigment epithelium, infiltrate the neurosensory retina, and communicate with the retinal circulation in what has been referred to as a retinal-choroidal anastomosis (RCA). This is extremely common in the end stage of disciform disease. In recent years, the reverse also seems to be possible, as angiomatous proliferation originates from the retina and extends posteriorly into the subretinal space, eventually communicating in some cases with choroidal new vessels. This form of neovascular ARMD, termed retinal angiomatous proliferation (RAP) in this article, can be confused with CNV. PURPOSE: The purpose of this article is 1) to review the clinical and angiographic characteristics of a series of patients with RAP and 2) to propose a theoretical sequence of events that accounts for the neovascularized process. METHODS: In this retrospective clinical and angiographic analysis, 143 eyes with RAP (108 patients) were reviewed and classified based on their vasogenic nature and course. Clinical biomicroscopic examination, fluorescein angiography, and indocyanine green angiography were used to evaluate patients. RESULTS: The results of this series suggest that angiomatous proliferation within the retina is the first manifestation of the vasogenic process in this form of neovascular ARMD. Dilated retinal vessels and pre-, intra-, and subretinal hemorrhages and exudate evolve, surrounding the angiomatous proliferation as the process extends into the deep retina and subretinal space. One or more dilated compensatory retinal vessels perfuse and drain the neovascularization, sometimes forming a retinal-retinal anastomosis. Fluorescein angiography in these patients usually revealed indistinct staining simulating occult CNV. Indocyanine green angiography was useful to make an accurate diagnosis in most cases. It revealed a focal area of intense hyperfluorescence corresponding to the neovascularization ("hot spot") and other characteristic findings. Based on understanding of the nature and progression of the neovascularized process, patients with RAP were classified into three vasogenic stages. Stage I involved proliferation of intraretinal capillaries originating from the deep retinal complex (intraretinal neovascularization [IRN]). Stage II was determined by growth of the retinal vessels into the subretinal space (subretinal neovascularization [SRN]). Stage III occurred when CNV could clearly be determined clinically or angiographically. A vascularized pigment epithelial detachment and RCA were inconsistent features of this stage. CONCLUSIONS: Retinal angiomatous proliferation appears to be a distinct subgroup of neovascular ARMD. It may present in one of three vasogenic stages: IRN, SRN, or CNV. Whereas ICG angiography is helpful in diagnosing RAP and in documenting the stage of the neovascularized process, it is frequently difficult to determine the precise nature and location of the new vessel formation. It is important for clinicians to recognize the vasogenic potential and the associated manifestations of this peculiar form of neovascular ARMD so that a proper diagnosis can be made, and when possible, an appropriate management administered.

Multiple tactile maps in the human cerebellum.

Functional imaging studies of the cerebellum have mostly investigated motor performance or have been limited to the anterior lobe and therefore the somatosensory representations in the human cerebellum have not been fully demonstrated. We used fMRI of the entire cerebellum during tactile stimulation of the hand and foot in six normal subjects. Our results demonstrate that the tactile projections to the cerebellum in humans are represented in both the anterior and posterior lobes. in agreement with previous functional imaging studies, our results show a large-scale, between-limb somatotopy comparable to that shown in early animal studies.

Tamoxifen retinopathy in a male patient.

PURPOSE: To report a case of tamoxifen retinopathy in a male patient. METHODS: Case report. A 68-year-old man, who had received a cumulative tamoxifen dose of 60 g over 33 months for unresectable hepatocellular carcinoma, was evaluated. RESULTS: A peculiar, bilateral, symmetric, inner retinal crystalline deposition associated with mild macular edema was discovered. No other ocular toxicity of tamoxifen was observed. CONCLUSION: To our knowledge, this is the first report of tamoxifen retinopathy in a male.

When the perpetrator gets killed: effects of observing the death of a handgun user in a televised public service announcement.

This study evaluates the cognitive effects of an anti-handgun violence public service announcement (PSA) on sixth-, seventh-, and eighth-grade students (N = 294). Participants were randomly assigned to a treatment group, which viewed a PSA depicting the death of an aggressive handgun user, or a comparison group, which viewed identical content except that the PSA showed no negative consequence for the handgun user. Logistic regression analysis, adjusting for race and gender, revealed that the treatment group was more likely to report negative expected outcomes for aggressively using a handgun and lower behavioral intentions to aggressively use a handgun compared with the comparison group. These findings suggest that observing handgun violence on television that depicts death as a negative physical consequence for the perpetrator may produce lower handgun-encouraging beliefs compared with observing no consequence for the perpetrator--the norm for most televised violence today.

Endoscopic vitreoretinal surgery for complicated proliferative diabetic retinopathy.

PURPOSE: To evaluate the indication for endoscopic vitreoretinal surgery in proliferative diabetic retinopathy (PDR). METHODS: Chart review of consecutive cases of vitreoretinal surgery for PDR performed by one of the authors (Y.L.F.) over a 2-year period. RESULTS: Endoscopic vitreoretinal surgery was performed in 8 of 41 (19.5%) eyes. The surgical indications were small pupil (3), hyphema (3), pseudophakia with fibrotic posterior capsule (1), and pars plana neovascularization with anterior tractional retinal detachment (6). CONCLUSION: Endoscopic vitreoretinal surgery, by enhancing the visualization of the retroirideal space, is a useful technique in PDR with opaque ocular media and/or neovascularization of the pars plana and ciliary body.

Essential metalloelement chelates facilitate repair of radiation injury.

Treatment with essential metalloelement (Cu, Fe, Mn, and Zn) chelates or combinations of them before and/or after radiation injury is a useful approach to overcoming radiation injury. No other agents are known to increase survival when they are used to treat after irradiation, in a radiorecovery treatment paradigm. These chelates may be useful in facilitating de novo syntheses of essential metalloelement-dependent enzymes required to repair radiation injury. Reports of radioprotection, which involves treatment before irradiation, with calcium-channel blockers, acyl Melatonin homologs, and substituted anilines, which may serve as chelating agents after biochemical modification in vivo, as well as Curcumin, which is a chelating agent, have been included in this review. These inclusions are intended to suggest additional approaches to combination treatments that may be useful in facilitating radiation recovery. These approaches to radioprotection and radiorecovery offer promise in facilitating recovery from radiation-induced injury experienced by patients undergoing radiotherapy for neoplastic disease and by individuals who experience environmental, occupational, or accidental exposure to ultraviolet, x-ray, or gamma-ray radiation. Since there are no existing treatments of radiation-injury intended to facilitate tissue repair, studies of essential metalloelement chelates and combinations of them, as well as combinations of them with existing organic radioprotectants, seem worthwhile.

Opportunities for public health genetics trainees: results of an employer/workplace survey.

OBJECTIVE: To conduct the first employer/workplace survey identifying employment opportunities for graduates of programs with training in public health genetics in the USA, and to determine whether employment opportunities will increase in coming years. METHODS: Six public health genetics training competencies were developed. A survey about workplace and employment opportunities was then conducted with mailings to (1) departments in schools of public health and departments of preventive medicine, (2) local and regional public health officials, (3) insurance companies and health management organizations (HMOs), and (4) biotechnology and pharmaceutical companies. RESULTS: A total of 196 surveys were returned among 1,464 that were mailed. Response rates varied from 5.8 to 46.5% among the target groups. The percent of responding organizations currently employing individuals with skills in genetics ranged from 20 to 62%. The percent currently employing individuals with skills in public health ranged from 39 to 96%. Training opportunities such as internships or practicum experiences are reported for one-third of respondents. For all of the competencies, approximately half of survey respondents who rated the competency important or very important already employ individuals with public health genetics skills. Similarly, at least a quarter of survey respondents who rated the competency important or very important plan to hire individuals with that skill in the next 5 years. Overall, approximately 40% of those surveyed are planning to hire individuals with competencies in public health genetics in the next 5 years. CONCLUSION: Employment opportunities already exist and new positions are becoming available in schools of public health and departments of preventive medicine, departments of public health, insurance companies and HMOs for professionals with public health genetics training. Based on our survey findings, skills and training in public health genetics are important in the workplace.

Matching simulation and experiment: a new simplified model for simulating protein folding.

Simulations of simplified protein folding models have provided much insight into solving the protein folding problem. We propose here a new off-lattice bead model, capable of simulating several different fold classes of small proteins. We present the sequence for an alpha/beta protein resembling the IgG-binding proteins L and G. The thermodynamics of the folding process for this model are characterized using the multiple multihistogram method combined with constant-temperature Langevin simulations. The folding is shown to be highly cooperative, with chain collapse nearly accompanying folding. Two parallel folding pathways are shown to exist on the folding free energy landscape. One pathway contains an intermediate--similar to experiments on protein G, and one pathway contains no intermediates-similar to experiments on protein L. The folding kinetics are characterized by tabulating mean-first passage times, and we show that the onset of glasslike kinetics occurs at much lower temperatures than the folding temperature. This model is expected to be useful in many future contexts: investigating questions of the role of local versus nonlocal interactions in various fold classes, addressing the effect of sequence mutations affecting secondary structure propensities, and providing a computationally feasible model for studying the role of solvation forces in protein folding.

Compensation of susceptibility-induced signal loss in echo-planar imaging for functional applications.

Functional magnetic resonance imaging favors the use of multi-slice gradient-recalled echo-planar imaging due to its short image acquisition times, whole brain coverage and sensitivity to BOLD contrast. However, despite its advantages, gradient-recalled echo-planar imaging also is sensitive to static magnetic field gradients arising primarily from air-tissue interfaces. This can lead to image artifacts such as voxel shifts and complete signal loss. A method to recover signal loss by adjusting the refocusing gradient amplitude in the slice-select direction, preferably axially, is proposed. This method is implemented as an automated computer algorithm that partitions echo-planar images into regions of recoverable signal intensities using a histogram analysis and determines each region's proper refocusing gradient amplitude. As an example, different refocusing gradient amplitudes are interleaved in a fMRI acquisition to maximize the signal to noise ratio and obtain functional activation in normal and dropout regions. The effectiveness of this method is demonstrated by recovering signal voids in the orbitofrontal cortex, parahippocampal/amygdala region, and inferior visual association cortex near the cerebellum.

Low-temperature crystal structures of tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodico pper(II) and tetrakis-mu-3,5-diisopropylsalicylatobis-diethyletheratodicopp er(II) and their role in modulating polymorphonuclear leukocyte activity in overcoming seizures.

Two binuclear copper(II) complexes of 3,5-diisopropylsalicylic acid were characterized by single crystal X-ray diffraction methods and examined for anti-inflammatory activity using activated polymorphonuclear leukocytes and for anticonvulsant activities using electroshock and metrazol models of seizures. These complexes were crystallized from dimethylformamide (DMF) or diethylether. Tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodicop per(II) [Cu(II)2(3,5-DIPS)4(DMF)2] I is in space group P 1; a = 10.393 (2), b = 11.258 (2), c = 12.734 (2) A, alpha = 96.64 (2), beta = 92.95 (2), gamma = 94.90 (2) degrees; V = 1471.7 (4) A3; Z = 1. Tetrakis-mu-3,5-diisopropylsalicylatobis-etheratodicopper(II ) [Cu(II)2(3,5-DIPS)4(ether)2] II is in space group P 1; a = 10.409 (3), b = 11.901 (4), c = 12.687 (6) A, alpha = 91.12 (5), beta = 90.84 (5), gamma = 100.90 (4) degrees; V = 1542 (1) A3; Z = 1. The structure of I was determined at 140 K from 4361 unique reflections (I > 2sigma(1)) and refined on F2 to R1 = 0.04 and wR2 = 0.09. The structure of II was determined at 180 K from 4605 unique reflections (I > 2sigma(I)) and refined on F2 to R1 = 0.05 and wR2 = 0.13. Each compound is a crystallographically centrosymmetric binuclear complex with Cu atoms bridged by four 3,5-diisopropylsalicylate ligands related by a symmetry center [Cu-Cu(i): 2.6139 (9) A in I and 2.613 (1) in II]. The four nearest O atoms around each Cu atom form a nearly rectangular planar arrangement with the square pyramidal coordination completed by the dimethylformamide (or diethylether) oxygen atom occupying an apical position, at a distance of 2.129 (2) A in I and 2.230 (3) A in II. Each Cu atom is displaced towards the DMF (or diethylether) ligand, by 0.189 A in I and 0.184 A in II, from the plane of the four O atoms. The crystal structures of I and II are essentially similar to each other, except for the DMF or diethylether accommodation. Many disorder phenomena were found in the crystal structure of I. Copper(II)2(3,5-DIPS)4(DMF)2 inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism in vitro. This effect was concentration related and significant for concentrations higher than 10 microg or 0.68 nmol/ml. Copper(II)2(3,5-DIPS)4(DMF)2 was more active than the parent ligand, 3,5-DIPS, as has been demonstrated with copper complexes of other non-steroidal anti-inflammatory drugs. The DMF and diethylether ternary complexes of Cu(II)2(3,5-DIPS)4 were found to have anticonvulsant activity in the maximal electroshock model of grand mal epilepsy in doses ranging from 26 to 258 micromol/kg of body mass following intraperitoneal, subcutaneous, or oral treatment. The DMF ternary complex was also found to be effective in the subcutaneous injection of metrazol model of petit mal epilepsy. We conclude that both ternary copper complexes are lipophilic and bioavailable, capable of facilitating the inflammatory response to brain injury and causing the subsidence of this response in bringing about remission of these disease states.

Retinal choroidal anastomoses and occult choroidal neovascularization in age-related macular degeneration.

OBJECTIVE: This study was designed to identify the incidence of retinal choroidal anastomoses in patients with occult choroidal neovascularization (CNV) and focal hot spots on indocyanine green (ICG) angiography, to identify the clinical and angiographic features that would assist in their identification, and to determine if the presence of these anastomotic lesions affect the outcome of laser therapy. DESIGN: Combined prospective and retrospective cross-sectional study. PARTICIPANTS: One hundred fifty consecutive patients with newly diagnosed occult CNV secondary to exudative age-related macular degeneration and focal hot spots on ICG angiography were evaluated prospectively. In addition, a retrospective review was performed on 79 eyes previously reported to have undergone laser photocoagulation treatment with ICG guidance. METHODS AND TESTING: In all cases, stereo color and red-free photographs, and stereo fluorescein and digital ICG angiograms were obtained for evaluation. MAIN OUTCOME MEASURES: Images obtained by all four techniques were evaluated for the presence of a retinal choroidal anastomosis. Associated clinical and angiographic findings were noted. In the retrospective review, the success rate of laser treatment was correlated with the presence or absence of a retinal choroidal anastomosis. RESULTS: Of the 150 eyes evaluated prospectively, 31 (21%) were found to have a retinal choroidal anastomosis. Retinal choroidal anastomoses were found in 27% of patients with associated serous pigment epithelial detachment (PED), whereas 13% were found in those without an associated elevation of the retinal pigment epithelium. Seventy-one percent of eyes had multiple anastomotic connections. Ninety percent of eyes had at least one retinal vein involved in the anastomotic connection. Clinical evidence of preretinal and intraretinal hemorrhage and cystic edema coupled with angiographic evidence of intraretinal dye leakage were key features of retinal choroidal anastomoses. In the retrospective review, seven patients were found to have retinal choroidal anastomoses with associated serous PED and demonstrated a very low (14%) success rate for laser treatment. CONCLUSIONS: Retinal choroidal anastomoses can present as a primary manifestation of the exudative process in age-related macular degeneration. They may be seen in eyes with and without detachment of the retinal pigment epithelium. Specific clinical and angiographic features have been identified that can aid in the diagnosis of these vascular anomalies. Their presence represents a poor prognostic sign for successful ICG-guided laser treatment.

Polypoidal choroidal vasculopathy masquerading as central serous chorioretinopathy.

OBJECTIVE: To differentiate polypoidal choroidal vasculopathy (PCV) from central serous chorioretinopathy (CSC). DESIGN: A retrospective, observational case series. PARTICIPANTS: Thirteen patients originally diagnosed with CSC proved to have PCV after more extensive evaluation and follow-up. METHODS: A clinical and angiographic review of patients with manifestations of CSC, including macular detachment. MAIN OUTCOME MEASURES: Demographic data, funduscopic examination, and fluorescein and indocyanine green (ICG) angiographic findings. RESULTS: Thirteen patients initially suspected of having CSC were ultimately diagnosed as having PCV. These eyes had exudative macular detachments secondary to a small caliber, polypoidal choroidal vascular abnormality or so-called polypoidal choroidal neovascularization. The clinical manifestations in the fundus varied. They included multiple, variably sized serous pigment epithelial detachments, neurosensory retinal detachment, lipid deposition, patchy atrophy of the pigment epithelium and indistinct staining from decompensation of the posterior blood-retinal barrier on fluorescein angiography. In reality, the suspected PEDs proved to be polypoidal lesions of PCV when imaged with ICG angiography. CONCLUSIONS: The clinical diagnosis of CSC or PCV generally poses little challenge to the experienced retinal specialist. However, in CSC with persistent and/or recurrent exudation, a myriad of retinal pigment epithelial changes may evolve that make it difficult to differentiate these two entities. In such patients, ICG angiography is useful in differentiating CSC from PCV. An accurate clinical diagnosis is important since each of these entities, CSC and PCV, may differ in terms of their risk factors, natural course, and visual prognosis.

Calcium(II)(3) (3,5-Diisopropylsalicylate)(6)(H(2)O)(6) Activates Nitric Oxide Synthase: An Accounting for its Action in Decreasing Platelet Aggregation.

Purposes of these studies were first; to determine whether or not Calcium(II)(3) (3,5- diisopropylsalicylate)(6)(H(2)O)(6) [Ca(II)(3)(3,5-DIPS)(6)], a lipophilic calcium complex, could decrease activated-platelet aggregation, and second; to determine whether or not it is plausible that Ca(II)(3)(3,5-DIPS)(6) decreases activated-platelet aggregation by facilitating the synthesis of Nitric Oxide (NO) by Nitric Oxide Synthase (NOS). The influence of Ca(II)(3)(3,5-DIPS)(6) on the initial rate of activated-platelet aggregation was determined by measuring the decrease in rate of increase in transmission at 550 nm for a suspension of Thrombin-CaCl(2) activated platelets following the addition of 0, 50, 100, 250, or 500 muM Ca(II)(3)(3,5-DIPS)(6). To establish that the Ca(lI)(3)(3,5- DIPS)(6)-mediated decrease in aggregation was due to activation of NOS, the effect of L-NMMA, an inhibitor of NOS, on the inhibition of platelet aggregation by Ca(II)(3)(3,5-DIPS)(6) was determined using a suspension of activated platelets contaimng 0 or 250 muM Ca(II)(3)(3,5-DIPS)(6) without or with 1 mM L-NMMA. An in vitro Bovine Brain NOS reaction mixture, containing CaCl(2) for the activation of Phosphodiesterase-3' ,5'-Cyclic Nucleotide Activator required for the activation of NOS, was used to determine whether or not Ca(II)(3)(3,5-DIPS)(6) could be used as a substitute for the addition of Ca. The decrease in absorbance at 340 nm, lambda maximum for NADPH, was measured to determine NOS activity following the addition of NOS to the complete reaction mixture containing either CaCl(2), Ca(II)(3)(3,5-DIPS)(6), or neither Ca compound. Increasing the concentration of Ca(II)(3)(3,5-DIPS)(6) caused a concentration related decrease in activated platelet aggregation. The addition of L-NMMA to activated platelets, in the absence of Ca(II)(3)(3,5-DIPS)(6), caused a 129% increase in initial rate of platelet aggregation. The initial rate of platelet aggregation decreased 74% with the addition of 250 muM Ca(II)(3)(3,5-DIPS)(6) and the addition of L-NMMA plus 250 muM Ca(II)(3)(3,5-DIPS)(6) caused a 197% decrease in initial rate of aggregation compared to the initial rate observed width the presence of 1 mM L-NMMA alone. There was only a small, 27%, increase in initial rate of 0.4 mM NADPH oxidation when 0.9 mM CaCl(2) was added to the NOS reaction mixture in comparison to the initial rate of NADPH oxidation with no addition of CaCI(2). Addition of an equivalent amount of Ca in the form of Ca(II)(3)(3,5-DIPS)(6), 333 muM, caused a 37% increase in initial rate of NADPH oxidation compared to the addition of 0.9 mM CaCl(2). Addition of increasing concentrations of L-NMMA plus 0.9 mM CaCl(2) or 333 muM Ca(II)(3)(3,5-DIPS)(6) to the NOS reaction mixture caused a concentration related increase in initial rate of NADPH oxidation. Addition of L-NMMA while expected to decrease NADPH oxidation actually increased the rate of NADPH oxidation. Additions of 133 muM or 267 muM Ca(II)(3)(3,5- DIPS)(6) also caused concentration related increases in initial rate of NADPH oxidation in the presence of 113 muM L-NMMA. However, the addition of 533 muM Ca(II)(3)(3,5-DIPS)(6) caused a dramatic decrease in initial rate of NADPH oxidation by NOS. It is concluded that: 1) Ca(II)(3)(3,5- DIPS)(6) activates platelet NOS in preventing platelet aggregation, 2) in vitro NOS activity can be observed spectrophotometrically by following the consumption of NADPH as a decrease in absorbance at 340 nm, 3) Ca(II)(3)(3,5-DIPS)(6) plays a role in enhancing Bovine Brain NOS activity resulting in an increased rate of NADPH oxidation by NOS, 4) Ca(II)(3)(3,5-DIPS)(6) is a useful form of Ca in activating NOS and superior to CaCl(2) with regard to the facilitation of a NADPH oxidation, and 5) L-NMMA stimulates Bovine Brain NOS activity rather than causing an inhibition of this enzyme and must serve as a reducible substrate for Bovine Brain NOS.