Ivax Pharmaceuticals
Ivax Pharmaceuticals launched its glyburide/metformin hvdrochloride tablets in 100 mg, 300 mg and 400 mg strengths under 180 days of marketing exclusivity afforded under its first-to-file status. The drug is the generic version of Glucovance, which Bristol-Myers Squibb markets for the treatment of diabetes. Glucovance had 2003 U.S. sales of nearly $500 million.
Ivax also received tentative approval for gabapentin tablets in 600 mg and 800 mg dosage strengths, as well as first-to-file status for gabapentin tablets in 100 mg, 300 mg and 400 mg strengths. The drug is the generic version of Pfizer’s epilepsy treatment Neurontin, with U.S. sales of approximately $2.4 billion in 2003.
Pfizer manufactures Neurontin in five dosage strengths: 100 mg, 300 mg and 400 mg capsules and 600 mg and 800 mg tablets. Tablets in the 100 mg, 300 mg and 400 mg strengths are not marketed currently.
Ivax, however is in litigation over whether its gabapentin products infringe a Pfizer patent. A trial in the U.S. District Court of New Jersey has yet to be scheduled.
Teva Pharmaceutical Industries and Alpharma also plan to co-market various dosage strengths of gabapentin.
Mylan Laboratories to Acquire King Pharmaceuticals for 2.65 Times Revenue
The Deal: Mylan Laboratories Inc. has agreed to purchase King Pharmaceuticals in a deal valued at approximately $4 billion. Under the terms of the agreement, each share of King Pharmaceuticals will be exchanged for 0.9 common shares of Mylan Laboratories. Based on recent closing prices, the deal values King at $16.70 per share. After the transaction closes, current Mylan shareholders will own about 56 percent of the combined company’s outstanding shares while current King shareholders will own the remaining 44 percent. The deal is expected to close by the end of 2004.
King Pharmaceuticals produces both generic and brand-name drugs. The company’s leading products include Altace, a cardiovascular treatment, and Levoxyl, a thyroid disorder drug. The company markets to physicians and health care facilities through its Monarch Pharmaceuticals subsidiary.
Mylan Laboratories is the second largest generic-drug company in the world and one of the largest manufacturers of prescription generic drugs in the United States. Company products include antibiotics, antidepressants, anti-inflammatories, beta-blockers, and laxatives. The company also develops and sells branded drugs through its subsidiaries, Bertek Pharmaceuticals and Mylan Tech.
Modeling diverse range of potassium channels with Brownian dynamics
ABSTRACT Using the experimentally determined KcsA structure as a template, we propose a plausible explanation for the diversity of potassium channels seen in nature. A simplified model of KcsA is constructed from its atomic resolution structure by smoothing out the protein-water boundary and representing the atoms forming the channel protein as a homogeneous, low dielectric medium. The properties of the simplified and atomic-detail models, deduced from electrostatic calculations and Brownian dynamics simulations, are shown to be qualitatively similar. We then study how the current flowing across the simplified model channel changes as the shape of the intrapore region is modified. This is achieved by increasing the radius of the intracellular pore systematically from 1.5 to 5 A while leaving the dimensions of the selectivity filter and inner chamber unaltered. The strengths of the dipoles located near the entrances of the channel, the carbonyl groups lining the selectivity filter, and the helix macrodipoles are kept constant. The channel conductance increases steadily as the radius of the intracellular pore is increased. The rate-limiting step for both the outward and inward current is the time it takes for an ion to cross the residual energy barrier located in the intrapore region. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than ~100 mV but deviates slightly from Ohm’s law at higher applied potentials. The nonlinearity in the current-voltage curve becomes less pronounced as the radius of the intracellular pore is increased. When the strengths of the dipoles near the intracellular entrance are reduced, the channel shows a pronounced inward rectification. Finally, the conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings on the transport of ions across the potassium channels and membrane channels in general.
Determination of the crystal structure of the KcsA potassium channel and its subsequent refinement at 2.0-Angstrom resolution have provided valuable information on this biologically important class of channels, especially on the operation of the selectivity filter. There are many different types of potassium channels, which differ widely in their conductances and gating characteristics Some of these are voltage gated or activated by intracellular Ca^sup 2+^ ions, whereas the activation of the bacterial KcsA channel depends sensitively on internal pH.
The TVGYGD amino acid sequence of the peptide chains lining the selectivity filter of all potassium channels is known to be highly conserved (Heginbotham et al., 1992; Schrempf et al., 1995; Doyle et al., 1998, MacKinnon et al., 1998). The only charged residue in the external pore region of KcsA that is not conserved is the glutamic acid E^sub 71^. Because this residue appears to be protonated (Ranatunga et al., 2001), its presence is not essential to channel function. Thus, it is likely that the diversity of potassium channels results from structural changes on the protein architecture near the intracellular segment of the pore, which have very different sequences. While the search for the complete tertiary structure of potassium channels continues (for example, see Hong and Miller 2000; Li-Smerin et al., 2000; Perozo, 2000; Lu et al., 2001), useful insights to the structure of the pore may be obtained from a study of the inverse problem, that is, predicting relevant aspects of the channel structure from its functional properties.
Appearance of the tertiary structure of the KcsA protein has stimulated much activity in the modeling of ion permeation in potassium channels (for reviews, see Roux et al., 2000; Kuyucak et al., 2001; Tieleman et al., 2001). Most of these studies focus on the selectivity filter and attempt to understand its operational principles by performing molecular dynamics (MD) simulations (Aqvist and Luzhkov, 2000; Guidoni et al., 2000; Shrivastava and Sansom, 2000; Allen et al., 2000; Berneche and Roux, 2001). However, MD simulations are too slow at present to determine the channel conductance, which is the most important functional property of an ion channel. To facilitate the calculation of conductance, one has to use a more coarse-grained simulation method such as Brownian dynamics (BD) or Poisson-Nernst-Planck equations. In view of the difficulties associated with the application of continuum theories of electro-diffusion to narrow pores (Corry et al., 2000; Graf et al., 2000), BD appears to be the method of choice for this purpose .
In our previous studies of the KcsA channel we investigated its permeation properties by calculating the electrostatic potential energy profiles for multiple K+ ions and performing three-dimensional BD simulations. Electrostatic calculations show that, without the stabilizing effect of backbone dipole and charged side-chains in the protein, a potassium ion attempting to cross-the channel would face an insurmountable energy barrier. When these charges are placed inside the channel wall, this large barrier turns into a deep potential well, which accommodates two K+ ions in the selectivity filter and one in the cavity in the absence of an applied potential. By simulating the trajectories of ions using BD, we deduced many of the experimentally observed properties of the channel. Among these are the channel conductance, the current-voltage relationships, the conductance-concentration curves, and the reversal potentials with asymmetrical ionic concentrations in the two sides of the channel.
Here we use BD simulations to explore whether the widely differing properties of potassium channels found in nature can be understood by small modifications of the channel geometry. For this purpose, we first construct a conducting state of the KcsA potassium channel that includes all the experimentally determined channel protein. From this detailed model, we extract a simplified KcsA model by replacing the atoms in the protein with a homogeneous, low dielectric medium, and representing the polar groups and oppositely charged pairs, deemed responsible for ion permeation, as dipoles. Also the irregular water– protein interface found in the crystal structure is smoothed out. The atomic-detail and simplified models of KcsA are compared to ascertain that they have qualitatively similar functional properties. These simplifications reduce the computational time for BD simulations, which is important for a time consuming systematic study. But more importantly, the simplified structure provides a template for modeling of the diverse range of potassium channels using BD. Using a simplified structure for this purpose (rather than the atomic structure of KcsA) is more sensible because, as noted above, there are large variations in their sequences. Here, we explore the influence of the intrapore geometry on the diversity question by changing the radius of the pore entrance on the intracellular side systematically from 1.5 to 5 A while keeping the dimensions of the selectivity filter and cavity constant. We make several predictions about the properties of the potassium channels with large and small conductances.
It is worthwhile to emphasize that such a procedure would be much harder to use with the MD method, even if the current time constraint for the calculation of conductance were not a problem. This is because MD is much more sensitive to details in channel structure, and finding the correct atomic structure that would reproduce the properties of the channel would be a very time consuming task.
Thus MD studies of eukaryotic potassium channels have to wait for the solution of their crystal structures, which may take a long time. In contrast, the implicit treatment of water and averaging over the atomic structure of protein in BD renders it insensitive to such details, making it an ideal tool for exploring the structure-function relationships in a diverse range of potassium channels.
Unclogging a pipe: Potassium channel pinball
Ion channels are characterized by three main properties: their gating characteristics, their selectivities, and their conductivity patterns. The crystal structure of the KcsA potassium channel pore (Doyle et al., 1998) provided immediate qualitative answers to most questions about potassium channel selectivity. It has led to an explosion of computational papers that illustrate in great detail how the various structural features function to exclude anions, stabilize cations, select for potassium over sodium, and promote divalent block. What remains wide open is understanding their enormous diversity: they are gated in many ways and differ greatly in their conductance behavior. Selective potassium channels have five strictly conserved residues (the signature sequence) and similar inner helix sequences, motifs that form the filter and the mid-channel aqueous cavity. These features promote multi-ion stabilization, a property initially deduced by Hodgkin and Keynes (1955). Regardless of their gating mechanisms, they exhibit wide-ranging electrical properties. Maximal conductivities span a nearly 100-fold range.
The crystallographic pore structure represents the channel in a closed state, the constriction on the intracellular side (the inner pore) being too narrow to permit ion (or water) passage. As has been pointed out repeatedly, structural modification of this inner pore is required for current to flow. In this issue, Chung et al. (2002) provide a reasonable and intuitive, albeit speculative, proposal for specific changes that could account for the broad spread of limiting conductances. The finding is striking: a small adjustment of the inner pore radius drastically alters channel resistivity.
In a series of papers Chung and his coworkers have exploited the advantages of Brownian dynamics (BD) to monitor ionic movement through channels and mimic experimentally observed current-voltage-concentration (I-V-c) profiles. The great virtue of BD is that, unlike molecular dynamics, large (picosecond) time steps can be used so that, with ultra-high-speed computers, multiple simulations can be run for hundreds of nanoseconds, long enough to ensure statistical reliability and observe repeated ion passage through the pore, thus generating sets of concentration dependent IN curves. Of course, much molecular detail is suppressed with simplification. In applications to the potassium channel, the protein is replaced by a few selected electrical features probably common to all such channels (the oxygen containing dipolar moieties forming the selectivity filter, the macrodipoles oriented toward the aqueous cavity, and acidic guard groups at each mouth) embedded in a low dielectric milieu. The aqueous pore becomes a high dielectric viscous continuum, in which the filter radius is fixed, and the radius of the inner pore and both pore mouths are adjustable. In contrast to continuum treatments like Poisson– Nernst-Planck modeling (Eisenberg, 1999), ions are charged spheres of finite size, assigned their crystal radii. This aspect of the model permits quite rigorous treatment of two crucial features: ionic repulsion and dielectric variability. From the Brownian perspective ion movement is basically electrodiffusive, driven by the potential due to the fixed model charges and the applied voltage, subject to a diffusive viscous drag, to random forces mimicking thermal coupling to the surroundings, to inter-ionic repulsion and to reaction fields induced by dielectric variation.
Previous work with this model showed that BD provides a semiquantitative description of potassium conductance. The mechanism for controlling ionic currents turns out to be marvelously simple. It hinges first and foremost on the filter and mid-channel aqueous cavity being a region of high negative charge density, always multiply cation occupied. In effect the channel is blocked by its own permeant ions. Conduction entails relief of this block. Outward conductance requires an ion to enter the inner pore, penetrate the filter and drive out one of the resident ions. For inward conductance an ion must exit the filter region and traverse the inner pore. In all cases, glutamates at the inner pore entrance form an ion-binding site. The inner pore is ionophobic. As an ion moves outward from the glutamates it must surmount a substantial energy barrier until it is attracted by the field of the macrodipoles surrounding the mid-channel cavity; it then accelerates rapidly, enters the filter and effects conduction by knock-off. Inward movement begins by overpopulating the filter; the extra ion then surmounts the pore’s internal barrier, resides near the inner mouth glutamates, and ultimately escapes. Increasing the inner pore radius reduces its ionophobic barrier height, thus increasing current flow in either direction. A change from 2 to 2.5 A would drop the barrier by almost 4 kT, leading to a nearly 25-fold increase in current. Small alterations in inner pore radius can account for the great diversity in potassium channel conductances.
The paper makes predictions about rectification (unidirectional ion passage). According to Chung et al. (2002), were the inner mouth glutamates fully protonated there would be essentially no outward current; presumably without charged sites to attract ions they wouldn’t enter the pore from the intracellular side. Inward current would still flow because escape from the central cavity and overcoming the pore’s internal barrier is ratelimiting; the charge state of inner mouth guard groups matters little here. What about modifying the aspartates at the outer mouth? Complete protonation would make the channel slightly outwardly rectifying, but the effect would be much less dramatic. Unlike at the inner mouth, where guard groups are the sole force promoting ion entry, entrance at the outer mouth also reflects the influence of the filter field.
High Potassium Diets Reduce Blood Pressure Of Black Adolescents
Black individuals are at an increased risk of developing essential hypertension (EH) when compared to other ethnic groups. Therefore, it is important to prevent the onset of this chronic medical problem early on in life. In order to do so, precursors or markers for essential hypertension in children and adolescents need to be determined. Previous research has shown modifiable risk factors for hypertension, including nondipping status of ambulatory blood pressure (ABP; defined as [is less than] 10% decrease in blood pressure from awake to asleep), and cardiovascular reactivity (CVR; defined as an increase in BP in response to stress). One possible intervention for decreasing the risk of developing EH in black children is to increase potassium (K+) intake. There has been consistent evidence that increasing dietary K+ can significantly lower BP in salt-sensitive (SS) individuals. SS individuals experience a rise in BP in response to a high sodium (Na) intake.
A recent study attempted to examine the effects of increased dietary potassium on the nondipping status of ABP and CVR in salt-sensitive and salt-resistant black youths. Fifty-eight normotensive black teenagers comprised the sample. The subjects first participated in a five-day low-sodium diet, which was immediately followed by a 10-day high sodium diet to determine their salt sensitivity status. Individuals were classified as salt-sensitive if their mean blood pressure increase was [is greater than] 5 mm Hg from the low to the high Na diet. Following this protocol, sixteen subjects were identified to be salt sensitive while the remaining 42 were considered to be salt resistant. The subjects were then randomly instructed on either a three-week high-potassium diet, including 80 [micro]mol of K+ per day, or were instructed to follow their regular dietary habits. Compliance was determined utilizing results of urinary K+ excretion analysis.
At the initiation of the intervention, a significantly greater amount of salt-sensitive subjects were nondippers in ABP when compared to the salt-resistant individuals. Following the dietary intervention, all of the salt-sensitive adolescents in the high K+ group achieved dipper status due to a drop in their nighttime blood pressures. There were no significant differences in the CVR between the two groups.
Based upon these results, it can be concluded that high-potassium diets improve cardiovascular risk factors among black adolescents, particularly among salt-sensitive adolescents. Potassium can be found in high concentrations in fresh fruits and vegetables. It appears that encouraging increased consumption of these foods, typically minimally consumed in the adolescent population, may help to reduce their risk of developing hypertension later in life.
Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines
Recombinant, immunodominant antigens derived from Mycobacterium tuberculosis can be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cation ic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates ThI T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of ThI responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, against M. tuberculosis infection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.-Authors’ Abstract.
perchlorate pollutant masculinizes fish
Known largely as a component of rocket fuel, perchlorate is a pollutant that often turns up in soil and water. In dozens of studies, it has perturbed thyroid-hormone concentrations, which can affect growth and neurological development. Data from fish now indicate that perchlorate can also disrupt sexual development.
Some of the changes were so dramatic that scientists initially mistook female fish for males. Several females displayed male-courtship behavior and produced sperm.
Richard R. Bernhardt of the University of Alaska in Anchorage and his colleagues focused on threespine sticklebacks (Gasterosteus aculeatus), a tiny marine species. For 3 weeks, the researchers incubated wild-captured adults in clean water or in water treated with 30, 60, or 100 parts per million (ppm) perchlorate. The adults spawned during that period.
Each group’s offspring were then raised to sexual maturity in similarly treated or untreated water. At spawning age, 10 apparent males per treatment group were each given their own aquariums. Once a day, each male received a 10-minute visit from an egg-swollen female in the same treatment group.
The first sign of something amiss: Among perchlorate-exposed fish, many would-be dads lacked the electric-blue and red coloration that normally signals readiness to spawn. Most of these fish didn’t exhibit typical reproductive behaviors, such as nest building, and many ignored prospective mates. Among cleanwater males, 80 percent spawned. As the perchlorate concentration climbed from 30 to 100 ppm, successful spawning fell from 50 percent to zero.
Eventually, the bellies of three apparent males began swelling with eggs. They proved to be hermaphroditic females, bearing both fertile eggs and fertile sperm.
The perchlorate-exposed true males developed unusually long testes.
Last January, the Environmental Protection Agency suggested limiting perchlorate contamination in natural bodies of water to 24.5 parts per billion. The concentrations used in the new study were at least 1,000 times that limit.
However, these doses are still environmentally relevant, argue aquatic toxicologist Bernhardt and his colleagues in the August Environmental Toxicology and Chemistry. They say that the test concentrations are similar to or less than those at several contaminated U.S. sites.
The “big surprise” was that perchlorate could produce hermaphroditic females and males with superlarge testes, says ecotoxicologist Gerald T. Ankley of EPA’s Mid-Continent Ecology Division in Duluth, Minn. Clearly, that’s “not something you would have anticipated [from] the way we think perchlorate works,” he adds.
All the changes observed suggest that perchlorate “is acting like an androgen,” or male-sex hormone, notes fish physiologist Ann Cheek of the University of Texas Health Science Center at Houston. Confirming this would require cellular analyses of testes and thyroid tissue.
Christopher W. Theodorakis of Southern Illinois University in Edwardsville argues that the “intriguing” masculinization may instead point to a new role for thyroid hormones–preservation of reproductive function.
“This paper may be telling us there’s more to perchlorate–and its effects on the thyroid–than we’d realized,” agrees R. Thomas Zoeller, a thyroid endocrinologist at the University of Massachusetts in Amherst. That “could be pretty profound,” he says.
Effects of ammonium perchlorate on thyroid function in developing fathead minnows, pimephales promelas
Perchlorate is a known environmental contaminant, largely due to widespread military use as a propellant. Perchlorate acts pharmacologically as a competitive inhibitor of thyroidal iodide uptake in mammals, but the impacts of perchlorate contamination in aquatic ecosystems and, in particular, the effects on fish are unclear. Our studies aimed to investigate the effects of concentrations of ammonium perchlorate that can occur in the environment (1, 10, and 100 mg/L) on the development of fathead minnows, Pimephales promelas. For these studies, exposures started with embryos of < 24-hr postfertilization and were terminated after 28 days. Serial sectioning of thyroid follicles showed thyroid hyperplasia with increased follicular epithelial cell height and reduced colloid in all groups of fish that had been exposed to perchlorate for 28 days, compared with control fish. Whole-body thyroxine ([T.sub.4]) content (a measure of total circulating [T.sub.4]) in fish exposed to 100 mg/L perchlorate was elevated compared with the [T.sub.4] content of control fish, but 3,5,3′-triiodothyronine ([T.sub.3]) content was not significantly affected in any exposure group. Despite the apparent regulation of [T.sub.3], after 28 days of exposure to ammonium perchlorate, fish exposed to the two higher levels (10 and 100 mg/L) were developmentally retarded, with a lack of scales and poor pigmentation, and significantly lower wet weight and standard length than were control fish. Our study indicates that environmental levels of ammonium perchlorate affect thyroid function in fish and that in the early life stages these effects may be associated with developmental retardation. Key words: development, endocrine disruption, fathead minnow, perchlorate, thyroid, thyroxine, triiodothyronine.
In recent years there has been increasing concern about the presence of perchlorate in ground and surface waters and the percolation of perchlorate into drinking waters [Urbansky 1998; U.S. Environmental Protection Agency The major source of ground and surface water contamination is ammonium perchlorate, the primary ingredient of the solid propellant in rockets and missiles (Logan 2001; U.S. EPA 2002). Perchlorate salts are also used in smaller amounts as components of air bag inflators, road flares, and fireworks; in electroplating and in tanning and finishing leathers; and as mordants for fabrics and in producing paints and enamels (Logan 2001; U.S. EPA 2002). Discharge from rocket fuel manufacturing plants, demilitarization of weapons, and the washing out and refueling of rockets are responsible for most of the ammonium perchlorate released into the environment (Urbansky 1998; U.S. EPA 2002). Indeed, at the Longhorn Army Ammunition Plant in Texas (USA), perchlorate has been measured at 30-31 mg/L in a water treatment holding pond (Smith et al. 2001).
Perchlorate has several chemical properties that make environmental contamination difficult to resolve and decontamination difficult to achieve (Logan 2001). The perchlorate anion is persistent because of its tetrahedral structure (Wolff 1998). Perchlorate salts completely ionize in solution, and the perchlorate anion is highly mobile (Logan 2001). As a result of these properties, groundwater contamination inevitably presents a risk to drinking water quality, and perchlorate has been detected in many drinking water supplies. In Nevada, 4-24 [micro]g/L was detected in drinking water (Xiao et al. 2001), and in California a number of drinking water wells showed peaks of 4-820 [micro]g/L (California Department of Health Services 2004). As a result, the U.S. EPA has estimated that perchlorate affects the quality of drinking water for 15 million people in the United States (Logan 2001).
Based on U.S. EPA guidance, and assessment of toxicity data, several U.S. states have set advisory levels for perchlorate in drinking water that vary between 1 and 18 [micro]g/L. The most recent reappraisal in California set a public heath goal for drinking water (maximum contaminant level) of 6 [micro]g/L (Office of Environmental Health Hazard Assessment 2004).
There is a long history of clinical use of perchlorate as a pharmacologic inhibitor of thyroid hormone synthesis (Hobson 1961; Wolff 1998). Thyroid gland follicles trap iodide required for the iodination of tyrosine molecules. The resulting iodothyronines are then reversibly combined with the storage protein, thyroglobulin, within the lumen of each of the thyroid follicles (Leatherland 1988, 1993). Perchlorate competitively inhibits iodide uptake by the sodium/iodide symporter at the basolateral membrane of the follicles (Capen 1997; Wolff 1998) and induces iodide efflux from the follicles by an as yet unexplained mechanism (Wolff 1998). These pharmacologic actions might be predicted to reduce circulating levels of thyroid hormones, and several studies in mammals given drinking water containing perchlorate at target doses of 0.01-100 mg/kg/day .
Histology. Whole fish (n = 5) fixed in formalin were decalcified for 14 days in 5% formic acid in 5% formaldehyde. Fish were wax embedded and serially sectioned (6/am) through all the thyroid follicles. Each follicle in each fish (5-13 follicles/fish) was traced through its entirety, and epithelial cell height was measured at the largest point.
Thyroid hormone extractions. Thyroid hormones were extracted from fathead minnow larvae based on the technique described by Greenblatt et al. (1989). Larvae were placed in Teflon tubes on ice, and 2 mL 95% ethanol containing 1 mM 6-N-propyl-2-thiouracil (PTU) was added. Samples were homogenized (Ultra Turax T25; Janke and Kunkel, Staufen, Germany) and sonicated for 20 sec (Vibra-Cell, 50% output; Sonics and Materials, Meryin/Satigny, Switzerland). A further 2 mL of 95% ethanol with 1 mM PTU was added, and samples were vortexed. Samples were centrifuged for 10 min (10,000g; 4[degrees]C), the supernatant was decanted into clean Teflon tubes, and 2 mL 95% ethanol containing PTU was added to the pellets. Tubes were vortexed vigorously and recentrifuged for 10 min (10,000g, 4[degrees]C). Supematants were pooled and evaporated to dryness under nitrogen, and desiccated samples were resuspended in 0.25 mL barbital buffer containing 2.5 mg/mL anilino naphthalene sulfonic acid (to disrupt the coupling between thyroid hormones and serum proteins, including lipoproteins), 0.25 mL ethanol, and 1 mL chloroform. Tubes were vigorously vortexed and then centrifuged for 10 min (1,500g; 4[degrees]C), producing two phases. The top ethanolic layer was removed using a glass pipette for radioimmunoassay (RIA) of thyroid hormones. The recovery of thyroid hormones was determined by addition of radioiodinated [T.sub.4] or [T.sub.3] after homogenization of whole larvae (n = 5). The recovery of 59.5 [+ or -] 3.25% [T.sub.4] and 63.9 [+ or -] 3.27% [T.sub.3] was comparable with those recoveries reported for larvae of other fish species.
Extracted samples or standard solutions (30 [micro]L) were incubated at 4[degrees]C overnight (in triplicate) with 100 [micro]L antiserum and 100 [micro]L radioiodinated solution, with additional “total counts” and “blank” tubes. The next morning, free and bound hormones were separated by addition of 100 [micro]L Sac-Cel (Immunodiagnostic Systems Limited, Tyne and Wear, UK) and a solution of cellulose-coupled antibodies (anti-sheep/goat); tubes were centrifuged, and the pellet of bound radiolabeled hormone was counted (Cobra gamma counter; Packard, Boston, MA, USA).
Ammonium nitrate a possible source - Carcinogens
Farmers use ammonium nitrate as a crop fertilizer and plants produce it to use as an energy source in the absence of light. The problem is that nitrate could be responsible for starting a chain of events in the body that leads to the formation of cancer-causing agents, according to Richard Loeppky, professor of chemistry, University of Missouri-Columbia. When nitrate is ingested, either through eating or drinking contaminated water, it is transferred into the bloodstream through normal digestive processes. Some of the nitrate then is transported to the salivary glands where bacteria transform it into nitrite. The nitrite is swallowed into the stomach again where it bonds with amino acids that are not combined with any particular protein. At this point, nitrolic acid is formed. This newly identified compound has properties that could lead to the formation of cancer in various areas of the body.
“The stomach is an ideal reactor for the kinds of chemistry that we are talking about,” Loeppky explains. “In addition, other researchers have suspected that amino acids played a role in forming these cancerous compounds, but until we made this discovery, no one had much of an idea how this happened.”
Nitrate also might combine with other compounds in the stomach to form cancer-causing agents, many of which must be metabolized before they are able to induce any damage. Because of this, Loeppky recommends that producers and consumers limit their use of nitrates as much as possible. “While we don’t know much about this newly discovered compound, the evidence suggests that it can alter or damage DNA. What is completely unknown is the pathway on how this is accomplished”.
On the Equivalence Point for Ammonium (De)protonation during Its Transport through the AmtB Channel
Structural characterization of the bacterial channel, AmtB, provides a glimpse of how members of its family might control the protonated state of permeant ammonium to allow for its selective passage across the membrane. In a recent study, we employed a combination of simulation techniques that suggested ammonium is deprotonated and reprotonated near dehydrative phenylalanine landmarks (F107 and F31, respectively) during its passage from the periplasm to the cytoplasm. At these landmarks, ammonium is forced to maintain a critical number (~3) of hydrogen bonds, suggesting that the channel controls ammonium (de)protonation by controlling its coordination/hydration. In the work presented here, a free energy-based analysis of ammonium hydration in dilute aqueous solution indicates, explicitly, that at biological pH, the transition from ammonium (NH^sup +^^sub 4^) to ammonia ? (NH^sub 3^) occurs when these species are constrained to donate three hydrogen bonds or less. This result demonstrates the viability of the proposal that AmtB indirectly controls ammonium (de)protonation by directly controlling its hydration.
AmtB exists in the membrane as a homotrimer. Each monomer of this protein forms a channel that passively transports ammonium (NH^sup +^^sub 4^) in the form of its “gas” ammonia (NHa) intermediate across the membranes of bacteria; for conciseness we will henceforth refer to both NH^sup +^^sub 4^ and NH^sub 3^ species, together, as Am. Structural models of AmtB resulting from x-ray diffraction (1,2) have provided initial configurations for a plethora of computational (3-10,13) studies aimed at understanding this channel’s mechanistic aspects and implications for homologous human counterparts.
The center of an AmtB monomer forms a narrow hydrophobic pore (lumen) connecting cytoplasmic and periplasmic vestibules, both accessible to aqueous solution. Diffraction studies revealed an NH^sup +^^sub 4^ binding site in the cytoplasmic vestibule (site Am1 (1,2)) where the cation donates hydrogen bonds to the backbone carbonyl group of A162, the side-chain hydroxyl oxygen of S219, and ~2-3 water molecules (3,5,7). Aromatic groups (F107 and F215) form a floor for site Am1, capping the hydrophobic lumen to help prevent entrance of water from the periplasm (see Fig. 1). These aromatic groups rotate at low free energy cost to allow translocation of Am (3,5,7) under the influence of an electrochemical gradient.
In the presence of AmSO^sub 4^, the x-ray structure (1) displayed three luminal binding sites (Am2, Am3, and Am4-see Fig, 1 A), where Am interacts closely with His residues (H168 and H318). Calculations of the apparent pK^sub a^ of luminal Am (3,10) indicate that these sites may only be occupied by neutral NH^sub 3^. As such, it would appear that the disallowance of permanently charged species in the lumen is the most Am-selective feature of AmtB. An aromatic group (F31) just below site Am4 helps to prevent hydration of the lumen, and provides a low free energy barrier for NH^sub 3^ passage to the cytoplasmic vestibule (Fig. 1, A and B). Just below the lumen, a fifth site (Am5) was revealed by a molecular dynamics (MD) study (3). At this site, calculations of the apparent pK^sub a^ (3) suggest Am must exist in its protonated form, where it donates hydrogen bonds to a carboxyl oxygen of D313, the hydroxyl oxygen of S263, and surrounding water (Fig. 1, A and B),
Combining knowledge of experimental and computational results (1-3,10), it appears that AmtB deprotonates NH^sup +^^sub 4^ between sites Am 1-2, and reprotonates NH^sub 3^ between sites Am4-5 to allow Am flux toward the cytoplasm. However, it is difficult to determine, experimentally, how the channel controls these (de)protonation events. Computational studies, though they should help clarify the (de)protonation mechanism, have proposed disparate explanations (3-5,7). Lin et al. (5) and Nygaard et al. (7) both proposed that a highly conserved Asp residue (D160), whose mutation is known to destroy AmtB’s transport capability (11), plays a key role in NH^sup +^^sub 4^ deprotonation. Lin et al. (5) observed that water forms a hydrogen bonded network between NH^sup +^^sub 4^ at Am1 and the carboxylate of D160. This led them to suggest that the charged carboxylate drives deprotonation at site Am1, and accepts a proton donated by NH^sup +^^sub 4^ using hydronium as an intermediate. On the other hand, Nygaard et al. (7) proposed that deprotonation occurs near site Am2, after NH^sup +^^sub 4^ moves from Am1 across the stacked (F107/F215) aromatic moieties. In this configuration, it was suggested that NH^sup +^^sub 4^ donates a proton to D160 via the backbone carbonyl group of A162 and the amide N-H of G163 using an imidic acid mechanism.
Luzhkov et al. (10) presented results that would suggest that D160 does not function as a proton acceptor. Rather, their calculations showed that the apparent pKa of D160’s carboxylate is downshifted (from its standard value of ~3.9) by 0.3-5.1 units when site Ami is unoccupied. When NH^sup +^^sub 4^ occupies Am1, the apparent pK^sub a^ of D160 shifts even further downward by 9.2 units, making its protonation effectively impossible. Our own results (3), as well as those of Luzhkov et al., showed that D160 is engaged in persistent hydrogen bonds with the protein, and that the negative charge of D160 stabilizes Am in its protonated form, shifting its apparent pK^sub a^ upward by ~4 units. Taken together, these results indicate that the importance of D160, as evidenced by mutational studies (11), is more likely due to recruitment of NH^sup +^^sub 4^ from the periplasm and stabilizing its binding at site Am1 rather than accepting a proton as suggested by Lin et al. and Nygaard et al.