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Boyd Haley

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Dr. Haley is a retired chemist.biochemist with an academic track record of innovative compound development that has use in the study of the biochemical abnormalities of nucleotide binding proteins in disease by the technology of photo affinity labeling. He has also developed a compound that has orphan drug designation by the EMA and FDA for the treatment of heavy metal toxicity, especially for mercury toxicity.

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Papers by Boyd Haley

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

by Gabriela Pavlinkova and Boyd Haley

Journal of Immunological Methods, 1997

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved ... more Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which Ž Ž . provide base stacking interactions to the purine ring of ATP Rajagopalan et al. 1996 Proc. Natl. Acad. Sci. USA 93, . 6019-6024 . Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Novel unconventional binding site in the variable region of immunoglobulins

by Boyd Haley and Gabriela Pavlinkova

Proceedings of the National Academy of Sciences, 1996

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the f... more The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein

The Journal of biological chemistry, Jan 10, 1984

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consist... more Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the h...

Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP

The Journal of biological chemistry, Jan 15, 1985

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit cont... more The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, labe...

The relationship of the toxic effects of mercury to exacerbation of the medical condition classified as Alzheimer's disease

Mercury(II) or Hg2+, is neurotoxic. When exposed to normal brain tissue homogenates, neurons in c... more Mercury(II) or Hg2+, is neurotoxic. When exposed to normal brain tissue homogenates, neurons in culture, Hg2+ (mercury(II) or mercuric mer- cury) is capable of causing many of the same biochemical aberrancies found in Alzheimer's diseased (AD) brain. Also, rats exposed to mercury(0), metallic mercury, vapor show some of these same abnormalities in their brain tissue. Specifically, the rapid inactivation of

Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker

Proceedings of the National Academy of Sciences, 1992

In this report, 8- and 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate were used to examine c... more In this report, 8- and 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate were used to examine cerebrospinal fluid (CSF) samples for the presence of an ATP binding protein unique to individuals with Alzheimer disease (AD). A 42-kDa ATP binding protein was found in the CSF of AD patients that is not observed in CSF from normal patients or other neurological controls. The photolabeling is saturated with 30 microM 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate. Photoinsertion can be totally prevented by the addition of 25 microM ATP. Photoinsertion of 2-azidoadenosine 5&#39;-triphosphate into the protein is only weakly protected by other nucleotides such as ADP and GTP, indicating that this is a specific ATP binding protein. A total of 83 CSF samples were examined in a blind manner. The 42-kDa protein was detected in 38 of 39 AD CSF samples and in only 1 of 44 control samples. This protein was identified as glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] based on similar nucleotide binding properties, comigration on two-dimensional gels, reaction with a polyclonal anti-GS antibody, and the presence of significant GS enzyme activity in AD CSF. In brain, GS plays a key role in elimination of free ammonia and also converts the neurotransmitter and excitotoxic amino acid glutamate to glutamine, which is not neurotoxic. The involvement of GS, if any, in the onset of AD is unknown. However, the presence of GS in the CSF of terminal AD patients suggests that this enzyme may be a useful diagnostic marker and that further study is warranted to determine any possible role for glutamate metabolism in the pathology of AD.

γ-Fluoroadenosine triphosphate. Synthesis, properties, and interaction with myosin and heavy meromyosin

Biochemistry, 1972

Malfeasance paper final pdf

by Janet Kern and Boyd Haley

Methodological issues and evidence of malfeasance in research purporting to show thimerosal in vaccines is safe

by Janet Kern, Boyd Haley, and Mark Geier

BioMed research international, 2014

There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compoun... more There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto-immune reaction; Well's syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. In contrast, the United States Centers for Disease Control and Prevention states that Thimerosal is safe and there is "no relationship between [T]himerosal[-]containing vaccines and autism rates in children." This is puzzling because, in a study conducted directly by CDC epidemiologists, a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy was found. The CDC's current stance that Thimerosal is s...

Thimerosal as discrimination: vaccine disparity in the UN Minamata Convention on mercury

by Janet Kern, Boyd Haley, Carmen Chaigneau, and Mark Geier

Indian journal of medical ethics

When addressing toxins, one unmistakable parallel exists between biology and politics: developing... more When addressing toxins, one unmistakable parallel exists between biology and politics: developing children and developing nations are those most vulnerable to toxic exposures. This disturbing parallel is the subject of this critical review, which examines the use and distribution of the mercury (Hg)-based compound, thimerosal, in vaccines. Developed in 1927, thimerosal is 49.55% Hg by weight and breaks down in the body into ethyl-Hg chloride, ethyl-Hg hydroxide and sodium thiosalicylate. Since the early 1930s, there has been evidence indicating that thimerosal poses a hazard to the health of human beings and is ineffective as an antimicrobial agent. While children in the developed and predominantly western nations receive doses of mostly no-thimerosal and reduced-thimerosal vaccines, children in the developing nations receive many doses of several unreduced thimerosal-containing vaccines (TCVs). Thus, thimerosal has continued to be a part of the global vaccine supply and its accepta...

Evidence supporting a link between dental amalgams and chronic illness, fatigue, depression, anxiety, and suicide

by Kristin Homme, Boyd Haley, and Geir Bjørklund

Neuro endocrinology letters, Jan 24, 2014

The purpose of this review is to examine the evidence for a relationship between mercury (Hg) exp... more The purpose of this review is to examine the evidence for a relationship between mercury (Hg) exposure from dental amalgams and certain idiopathic chronic illnesses - chronic fatigue syndrome (CFS), fibromyalgia (FM), depression, anxiety, and suicide. Dental amalgam is a commonly used dental restorative material that contains approximately 50% elemental mercury (Hg0) by weight and releases Hg0 vapor. Studies have shown that chronic Hg exposure from various sources including dental amalgams is associated with numerous health complaints, including fatigue, anxiety, and depression - and these are among the main symptoms that are associated with CFS and FM. In addition, several studies have shown that the removal of amalgams is associated with improvement in these symptoms. Although the issue of amalgam safety is still under debate, the preponderance of evidence suggests that Hg exposure from dental amalgams may cause or contribute to many chronic conditions. Thus, consideration of Hg t...

Mercury and autism: accelerating evidence?

Neuro endocrinology letters, 2005

The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk... more The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury exposure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a correlation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymorphism. In vitro, mercury and thimerosal in levels found several days after vaccination inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain developmen...

Response to Dr. Ari Brown and the Immunization Action Coalition

Medical …, 2009

Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates

by Boyd Haley and J. Meyer

Molecular Carcinogenesis, 1989

The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product req... more The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

by G. Pavlinkova and Boyd Haley

Journal of Immunological Methods, 1997

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved ... more Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which Ž Ž . provide base stacking interactions to the purine ring of ATP Rajagopalan et al. 1996 Proc. Natl. Acad. Sci. USA 93, . 6019-6024 . Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3?,5?-cyclic monophoshate

by Boyd Haley and Patrick Schoff

Journal of Cellular Biochemistry, 1982

Baby Hair, Mercury Toxicity and Autism

International Journal of Toxicology, 2004

Concerns continue to be raised regarding the potential role of Q1 mercury exposure in the etiolog... more

Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N'-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aorti...

by Boyd Haley and Sainath Kotha MD

International Journal of Toxicology, 2011

Here, we investigated thiol-redox-mediated phospholipase D (PLD) signaling as a mechanism of merc... more Here, we investigated thiol-redox-mediated phospholipase D (PLD) signaling as a mechanism of mercury cytotoxicity in mouse aortic endothelial cell (MAEC) in vitro model utilizing the novel lipid-soluble thiol-redox antioxidant and heavy metal chelator, N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bis(2-mercaptoethyl)isophthalamide (NBMI) and the novel PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). Our results demonstrated (i) mercury in the form of mercury(II) chloride, methylmercury, and thimerosal induced PLD activation in a dose- and time-dependent manner; (ii) NBMI and FIPI completely attenuated mercury- and oxidant-induced PLD activation; (iii) mercury induced upstream phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) leading to downstream threonine phosphorylation of PLD(1) which was attenuated by NBMI; (iv) mercury caused loss of intracellular glutathione which was restored by NBMI; and (v) NBMI and FIPI attenuated mercury- and oxidant-induced cytotoxicity in MAECs. For the first time, this study demonstrated that redox-dependent and PLD-mediated bioactive lipid signaling was involved in mercury-induced vascular EC cytotoxicity which was protected by NBMI and FIPI.

Thimerosal Exposure and the Role of Sulfation Chemistry and Thiol Availability in Autism

by Boyd Haley, Janet Kern, and Mark Geier

International Journal of Environmental Research and Public Health, 2013

Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the ch... more Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the children experience a developmental regression characterized by a loss of previously acquired skills and abilities. Typically reported are losses of verbal, nonverbal, and social abilities. Several recent studies suggest that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH) reserve capacity, resulting in a compromised oxidation/reduction (redox) and detoxification capacity. Research indicates that the availability of thiols, particularly GSH, can influence the effects of thimerosal (TM) and other mercury (Hg) compounds. TM is an organomercurial compound (49.55% Hg by weight) that has been, and continues to be, used as a preservative in many childhood vaccines, particularly in developing countries. Thiol-modulating mechanisms affecting the cytotoxicity of TM have been identified. Importantly, the emergence of ASD symptoms post-6 months of age temporally follows the administration of many childhood vaccines. The purpose of the present critical review is provide mechanistic insight regarding how limited thiol availability, abnormal sulfation chemistry, and decreased GSH reserve capacity in children OPEN ACCESS Int. J. Environ. Res. Public Health 2013, 10 3772

Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

Bioscience Reports, 1990

Photoactivatable (beta-32P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determ... more Photoactivatable (beta-32P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the 32P-labeled proteins were found to be 62- and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the 32P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250 nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphoryldolichol synthase.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

by Gabriela Pavlinkova and Boyd Haley

Journal of Immunological Methods, 1997

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved ... more Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which Ž Ž . provide base stacking interactions to the purine ring of ATP Rajagopalan et al. 1996 Proc. Natl. Acad. Sci. USA 93, . 6019-6024 . Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Novel unconventional binding site in the variable region of immunoglobulins

by Boyd Haley and Gabriela Pavlinkova

Proceedings of the National Academy of Sciences, 1996

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the f... more The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein

The Journal of biological chemistry, Jan 10, 1984

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consist... more Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the h...

Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP

The Journal of biological chemistry, Jan 15, 1985

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit cont... more The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, labe...

The relationship of the toxic effects of mercury to exacerbation of the medical condition classified as Alzheimer's disease

Mercury(II) or Hg2+, is neurotoxic. When exposed to normal brain tissue homogenates, neurons in c... more Mercury(II) or Hg2+, is neurotoxic. When exposed to normal brain tissue homogenates, neurons in culture, Hg2+ (mercury(II) or mercuric mer- cury) is capable of causing many of the same biochemical aberrancies found in Alzheimer's diseased (AD) brain. Also, rats exposed to mercury(0), metallic mercury, vapor show some of these same abnormalities in their brain tissue. Specifically, the rapid inactivation of

Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker

Proceedings of the National Academy of Sciences, 1992

In this report, 8- and 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate were used to examine c... more In this report, 8- and 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate were used to examine cerebrospinal fluid (CSF) samples for the presence of an ATP binding protein unique to individuals with Alzheimer disease (AD). A 42-kDa ATP binding protein was found in the CSF of AD patients that is not observed in CSF from normal patients or other neurological controls. The photolabeling is saturated with 30 microM 2-azidoadenosine 5&#39;-[gamma-32P]triphosphate. Photoinsertion can be totally prevented by the addition of 25 microM ATP. Photoinsertion of 2-azidoadenosine 5&#39;-triphosphate into the protein is only weakly protected by other nucleotides such as ADP and GTP, indicating that this is a specific ATP binding protein. A total of 83 CSF samples were examined in a blind manner. The 42-kDa protein was detected in 38 of 39 AD CSF samples and in only 1 of 44 control samples. This protein was identified as glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] based on similar nucleotide binding properties, comigration on two-dimensional gels, reaction with a polyclonal anti-GS antibody, and the presence of significant GS enzyme activity in AD CSF. In brain, GS plays a key role in elimination of free ammonia and also converts the neurotransmitter and excitotoxic amino acid glutamate to glutamine, which is not neurotoxic. The involvement of GS, if any, in the onset of AD is unknown. However, the presence of GS in the CSF of terminal AD patients suggests that this enzyme may be a useful diagnostic marker and that further study is warranted to determine any possible role for glutamate metabolism in the pathology of AD.

γ-Fluoroadenosine triphosphate. Synthesis, properties, and interaction with myosin and heavy meromyosin

Biochemistry, 1972

Malfeasance paper final pdf

by Janet Kern and Boyd Haley

Methodological issues and evidence of malfeasance in research purporting to show thimerosal in vaccines is safe

by Janet Kern, Boyd Haley, and Mark Geier

BioMed research international, 2014

There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compoun... more There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto-immune reaction; Well's syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. In contrast, the United States Centers for Disease Control and Prevention states that Thimerosal is safe and there is "no relationship between [T]himerosal[-]containing vaccines and autism rates in children." This is puzzling because, in a study conducted directly by CDC epidemiologists, a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy was found. The CDC's current stance that Thimerosal is s...

Thimerosal as discrimination: vaccine disparity in the UN Minamata Convention on mercury

by Janet Kern, Boyd Haley, Carmen Chaigneau, and Mark Geier

Indian journal of medical ethics

When addressing toxins, one unmistakable parallel exists between biology and politics: developing... more When addressing toxins, one unmistakable parallel exists between biology and politics: developing children and developing nations are those most vulnerable to toxic exposures. This disturbing parallel is the subject of this critical review, which examines the use and distribution of the mercury (Hg)-based compound, thimerosal, in vaccines. Developed in 1927, thimerosal is 49.55% Hg by weight and breaks down in the body into ethyl-Hg chloride, ethyl-Hg hydroxide and sodium thiosalicylate. Since the early 1930s, there has been evidence indicating that thimerosal poses a hazard to the health of human beings and is ineffective as an antimicrobial agent. While children in the developed and predominantly western nations receive doses of mostly no-thimerosal and reduced-thimerosal vaccines, children in the developing nations receive many doses of several unreduced thimerosal-containing vaccines (TCVs). Thus, thimerosal has continued to be a part of the global vaccine supply and its accepta...

Evidence supporting a link between dental amalgams and chronic illness, fatigue, depression, anxiety, and suicide

by Kristin Homme, Boyd Haley, and Geir Bjørklund

Neuro endocrinology letters, Jan 24, 2014

The purpose of this review is to examine the evidence for a relationship between mercury (Hg) exp... more The purpose of this review is to examine the evidence for a relationship between mercury (Hg) exposure from dental amalgams and certain idiopathic chronic illnesses - chronic fatigue syndrome (CFS), fibromyalgia (FM), depression, anxiety, and suicide. Dental amalgam is a commonly used dental restorative material that contains approximately 50% elemental mercury (Hg0) by weight and releases Hg0 vapor. Studies have shown that chronic Hg exposure from various sources including dental amalgams is associated with numerous health complaints, including fatigue, anxiety, and depression - and these are among the main symptoms that are associated with CFS and FM. In addition, several studies have shown that the removal of amalgams is associated with improvement in these symptoms. Although the issue of amalgam safety is still under debate, the preponderance of evidence suggests that Hg exposure from dental amalgams may cause or contribute to many chronic conditions. Thus, consideration of Hg t...

Mercury and autism: accelerating evidence?

Neuro endocrinology letters, 2005

The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk... more The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury exposure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a correlation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymorphism. In vitro, mercury and thimerosal in levels found several days after vaccination inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain developmen...

Response to Dr. Ari Brown and the Immunization Action Coalition

Medical …, 2009

Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates

by Boyd Haley and J. Meyer

Molecular Carcinogenesis, 1989

The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product req... more The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

by G. Pavlinkova and Boyd Haley

Journal of Immunological Methods, 1997

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved ... more Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which Ž Ž . provide base stacking interactions to the purine ring of ATP Rajagopalan et al. 1996 Proc. Natl. Acad. Sci. USA 93, . 6019-6024 . Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3?,5?-cyclic monophoshate

by Boyd Haley and Patrick Schoff

Journal of Cellular Biochemistry, 1982

Baby Hair, Mercury Toxicity and Autism

International Journal of Toxicology, 2004

Concerns continue to be raised regarding the potential role of Q1 mercury exposure in the etiolog... more

Novel Lipid-Soluble Thiol-Redox Antioxidant and Heavy Metal Chelator, N,N'-bis(2-Mercaptoethyl)Isophthalamide (NBMI) and Phospholipase D-Specific Inhibitor, 5-Fluoro-2-Indolyl Des-Chlorohalopemide (FIPI) Attenuate Mercury-Induced Lipid Signaling Leading to Protection Against Cytotoxicity in Aorti...

by Boyd Haley and Sainath Kotha MD

International Journal of Toxicology, 2011

Here, we investigated thiol-redox-mediated phospholipase D (PLD) signaling as a mechanism of merc... more Here, we investigated thiol-redox-mediated phospholipase D (PLD) signaling as a mechanism of mercury cytotoxicity in mouse aortic endothelial cell (MAEC) in vitro model utilizing the novel lipid-soluble thiol-redox antioxidant and heavy metal chelator, N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bis(2-mercaptoethyl)isophthalamide (NBMI) and the novel PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). Our results demonstrated (i) mercury in the form of mercury(II) chloride, methylmercury, and thimerosal induced PLD activation in a dose- and time-dependent manner; (ii) NBMI and FIPI completely attenuated mercury- and oxidant-induced PLD activation; (iii) mercury induced upstream phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) leading to downstream threonine phosphorylation of PLD(1) which was attenuated by NBMI; (iv) mercury caused loss of intracellular glutathione which was restored by NBMI; and (v) NBMI and FIPI attenuated mercury- and oxidant-induced cytotoxicity in MAECs. For the first time, this study demonstrated that redox-dependent and PLD-mediated bioactive lipid signaling was involved in mercury-induced vascular EC cytotoxicity which was protected by NBMI and FIPI.

Thimerosal Exposure and the Role of Sulfation Chemistry and Thiol Availability in Autism

by Boyd Haley, Janet Kern, and Mark Geier

International Journal of Environmental Research and Public Health, 2013

Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the ch... more Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the children experience a developmental regression characterized by a loss of previously acquired skills and abilities. Typically reported are losses of verbal, nonverbal, and social abilities. Several recent studies suggest that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH) reserve capacity, resulting in a compromised oxidation/reduction (redox) and detoxification capacity. Research indicates that the availability of thiols, particularly GSH, can influence the effects of thimerosal (TM) and other mercury (Hg) compounds. TM is an organomercurial compound (49.55% Hg by weight) that has been, and continues to be, used as a preservative in many childhood vaccines, particularly in developing countries. Thiol-modulating mechanisms affecting the cytotoxicity of TM have been identified. Importantly, the emergence of ASD symptoms post-6 months of age temporally follows the administration of many childhood vaccines. The purpose of the present critical review is provide mechanistic insight regarding how limited thiol availability, abnormal sulfation chemistry, and decreased GSH reserve capacity in children OPEN ACCESS Int. J. Environ. Res. Public Health 2013, 10 3772

Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

Bioscience Reports, 1990

Photoactivatable (beta-32P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determ... more Photoactivatable (beta-32P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the 32P-labeled proteins were found to be 62- and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the 32P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250 nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphoryldolichol synthase.