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Biomedical subjects

J C Williams

Publications and source records attributed to J C Williams.

At least 19 recordsLinked to original sources
PubMed2006

Iron as a bound secondary electron donor in modified bacterial reaction centers.

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.

Bicarbonates↗
PubMed2006

Dose determination of the persistent activity of moxidectin long-acting injectable formulations against various nematode species in cattle.

The effectiveness, safety and production-enhancing benefit (improved weight gains) of moxidectin long-acting injection given subcutaneously in the ear at the rates of 0.75, 1.0 and 1.5mg/kg bw were evaluated in three studies under common protocol. The only adverse reaction to treatment was a mild (<2 tablespoons in volume), and for the most part transient (<28 days for the treatment rate of 1.0mg/kg bw) injection site swelling as noted in a minority of the animals (12.2% of the animals treated at the rate of 1.0mg/kg bw). Regardless of study site, post-treatment interval or dose rate, average daily gains were improved over control cattle by approximately 33%. Reductions in strongyle EPG counts relative to controls were > or = 90% for all dose rates of moxidectin for a post-treatment period of 42 days (Wisconsin), 84 days (Arkansas) and 140 days (Louisiana). In Arkansas and Louisiana, the majority (>80%) of post-treatment strongyle eggs, as determined by coproculture, were Cooperia spp. As determined by sequential necropsies, periods of continuous, post-treatment protection (> or = 90% efficacy in at least two out of three studies) for moxidectin long-acting injection given at the rate of 1.0 mg/kg bw were 90 days (adult Haemonchus spp.), 120 days (Dictyocaulus viviparus and adult Ostertagia and Oesophagostomum) and 150 days (Ostertagia spp. EL4).

Animals↗
PubMed2006

Microbial community structure of activated sludge during aerobic granulation in an annular gap bioreactor.

A novel annular gap reactor was designed to create a controlled shear environment in which aerobic granular sludge could be developed. The bacterial and eukaryal community structures during two aerobic granular sludge experiments were tracked using denaturing gradient gel electrophoresis (DGGE). The first granule cultivation experiment, using an organic loading rate of 1.6 kg/m3d COD, resulted in biomass that was dominated by filamentous bacteria and Zoogloea ramigera colonies. A second experiment with a higher organic loading rate of 6 kg/m3d COD developed a granule-like morphology but was ultimately dominated by filamentous fungi. Species identification via DGGE band purification and DNA sequencing closely matched the observed sludge morphology and behavior.

Aerobiosis↗
PubMed2005

Proton release due to manganese binding and oxidation in modified bacterial reaction centers.

The pH dependence of binding and oxidation of Mn2+ in highly oxidizing reaction centers with designed metal-binding sites was characterized by light-minus-dark optical difference spectroscopy and direct measurements of proton uptake/release. These mutants bind a Mn2+ ion that can efficiently transfer an electron to the oxidized bacteriochlorophyll dimer, as described earlier [Thielges et al. (2005) Biochemistry 44, 7389-7394]. The dissociation constant, KD, significantly increased with decreasing pH. The pH dependence of KD between pH 7 and pH 8 was consistent with the binding of Mn2+ being stabilized by the electrostatic release of two protons. The strong pH dependence of proton release upon Mn2+ binding, with a maximal release of 1.4 H+ per reaction center, was interpreted as being a result of a shift in the pKa values of the coordinating residues and possibly other nearby residues. A small amount of proton release associated with Mn2+ oxidation was observed upon illumination. These results show that functional metal-binding sites can be incorporated into proteins upon consideration of both the metal coordination and protonation states of the ligands.

Binding Sites↗
PubMed2005

New tetragonal form of reaction centers from Rhodobacter sphaeroides and the involvement of a manganese ion at a crystal contact point.

Crystals have been obtained of wild-type reaction centers from Rhodobacter sphaeroides using manganese chloride as a precipitating agent. The crystals belong to the tetragonal space group P4(2)22, with unit-cell parameters a = b = 207.8, c = 107.5 A. The crystal structure has been determined to a resolution limit of 4.6 A using a previously determined structure of the reaction center as a molecular-replacement model. The calculated electron-density maps show the presence of a manganese ion at one of the crystal contact points bridging two symmetry-related histidine residues, suggesting that the metal plays a key role in facilitating the crystallization of the protein in this form.

Crystallization↗
PubMed2005

Design of a redox-linked active metal site: manganese bound to bacterial reaction centers at a site resembling that of photosystem II.

Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganese-binding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highest-affinity site had a dissociation constant of approximately 1 microM. For the highest-affinity mutant, a first-order rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 muM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex.

Amino Acid Substitution↗
PubMed2005

Exsheathment of Ostertagia ostertagi infective larvae following exposure to bovine rumen contents derived from low and high roughage diets.

The objective of this study was to characterize the exsheathment kinetics of Ostertagia ostertagi infective larvae (L3) following in vivo exposure to bovine rumen contents derived from low and high roughage diets. O. ostertagi L3 were placed in disposable dialysis bags and incubated for various time points between 0 and 360 min in the rumen of a fistulated steer maintained on a 71% grain diet or a 100% grass diet. The maximum percentage of exsheathed L3 was observed 120 min post-exposure to grass-derived rumen contents, while maximum exsheathment for L3 exposed to grain-derived rumen contents did not occur until 360 min. This work provides the first report of the in vivo exsheathment kinetics for O. ostertagi in its bovine host. Results of this study also support earlier reports that rumen pH may affect the exsheathment efficiency of abomasal trichostrongylids.

Animals↗
PubMed2005

Strategies for improved shock wave lithotripsy.

Research in lithotripsy that started with the effort to characterize acute shock wave damage to the kidney has led to advances on several fronts, including discovery of strategies that have improved clinical treatment. It is appreciated now that shock wave trauma is primarily a vascular lesion, that injury is dose dependent, and that hemorrhage can be severe and can lead to a permanent loss of functional renal mass. Studies of the renal functional response to lithotripsy have shown that shock wave treatment triggers vasoconstriction in the kidney. This finding has been turned to advantage, and it is now known that when treatment is begun using low amplitude pulses, subsequent high amplitude shock waves are far less damaging. Thus, when shock waves are delivered judiciously, treatment can have a protective effect. The finding that cavitation is a key mechanism in vessel rupture has led to the development of novel experimental methods of shock wave delivery that can suppress bubble expansion and minimize tissue damage. Progress has also been made in understanding the physical mechanisms involved in stone comminution, and it is seen that the forces generated by cavitation, shear stress and circumferential squeezing act synergistically to fragment stones. Recent work suggests that a broad focal zone may be an advantage, allowing stones to be broken with lower amplitude pulses. Cavitation has been shown to play a critical role in reducing stone fragments to a size that can be voided. Cavitation is also the factor that limits the rate at which treatment can be performed, as stones break significantly better at slow rate than at fast ratean observation from basic research that is now appreciated in clinical practice. The current environment in lithotripsy research is encouraging. There is great interest in developing new technology, and in finding ways to improve how lithotripsy is performed.

Animals↗
PubMed2004

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides.

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.

Amino Acid Substitution↗
PubMed2004

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference.

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.

Darkness↗
PubMed2003

Larvicidal effects of several chemicals on Strongyloides infective larvae.

The larvicidal effects of 11 anthelmintics, 7 pesticides and 4 disinfectants were evaluated with infective larvae of Strongyloides papillosus (SPL) and Strongyloides venezuelensis (SVZ). The lethal concentrations against SPL and SVZ were found to be similar. Three chemicals (dichlorvos, levamisole and trichlorfon) showed highest larvicidal effects. The 50% lethal concentration (LC(50)) values for the three compounds against SPL larvae were 0.08, 0.24, and 0.59 ppm, respectively.

Animals↗
PubMed2003

Correlation of proton release and electrochromic shifts of the optical spectrum due to oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides.

Reaction centers from the Y(L167) mutant of Rhodobacter sphaeroides, containing a highly oxidizing bacteriochlorophyll dimer and a tyrosine residue substituted at Phe L167, were compared to reaction centers from the Y(M) mutant, with a tyrosine at M164, and a quadruple mutant containing a highly oxidizing dimer but no nearby tyrosine residue. Distinctive features in the light-induced optical and EPR spectra showed that the oxidized bacteriochlorophyll dimer was reduced by Tyr L167 in the Y(L167) mutant, resulting in a tyrosyl radical, as has been found for Tyr M164 in the Y(M) mutant. In the Y(L167) mutant, the net proton uptake after formation of the tyrosyl radical and the reduced primary quinone ranged from +0.1 to +0.3 H(+)/reaction center between pH 6 and pH 10, with a dependence that is similar to the quadruple mutant but different than the large proton release observed in the Y(M) mutant. In the light-induced absorption spectrum in the 700-1000 nm region, the Y(L167) mutant exhibited unique changes that can be assigned as arising primarily from an approximately 30 nm blue shift of the dimer absorption band. The optical signals in the Y(L167) mutant were pH dependent, with a pK(a) value of approximately 8.7, indicating that the tyrosyl radical is stabilized at high pH. The results are modeled by assuming that the phenolic proton of Tyr L167 is trapped in the protein after oxidation of the tyrosine, resulting in electrostatic interactions with the tetrapyrroles and nearby residues.

Amino Acid Substitution↗
PubMed2003

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides.

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.

Bacterial Proteins↗
PubMed2003

Dose confirmation of moxidectin 0.5% pour-on against adults and fourth-stage larvae of various Cooperia spp. and Trichostrongylus colubriformis in Louisiana.

Thirty-five castrate or intact Holstein male calves, ranging in weight from 122 to 210kg, were used in the study. On study Days -15 and -14, all study calves were treated with fenbendazole 10% paste at 10mg/kg to remove existing nematode infections. All cattle had zero egg counts on Day -1. Experimental infections of a mixed species inoculum were administered to all calves on Day 0. The inoculum consisted of Cooperia spp. (primarily C. punctata, but also C. pectinata, C. spatulata, C. oncophora and C. surnabada-total 40,961); Ostertagia ostertagi-1550; Trichostrongylus colubriformis-4996; and Oesophagostomum radiatum-38. Necropsy results from two of three monitor calves slaughtered on Day 6 after infection indicated that Day 6 was an appropriate time to evaluate efficacy of moxidectin against fourth larval stages (L(4)). The remaining 32 calves were randomly allotted to four groups of eight based on body weights. Eight calves (Group 1) were treated with moxidectin 0.5% pour-on at 0.5mg/kg on Day 6 to evaluate efficacy against nematode larval stages; eight control calves (Group 2) were matched with these principals. Eight calves (Group 3) were also treated with moxidectin pour-on at the same dosage on Day 23 in order to determine efficacy against adult nematodes; eight control calves (Group 4) were matched with these principals. In both cases, principals and controls of the Days 6 and 23 treatments were necropsied at 14-15 days after treatment. C. punctata was the only species found in a sufficient number of controls to evaluate moxidectin efficacy against the L(4). Moxidectin pour-on was not effective (P<0.05) against C. punctata L(4) by arithmetic means, but was highly effective based on geometric means. Regardless of whether arithmetic or geometric means were used for percent efficacy calculations, moxidectin pour-on was demonstrated to be highly effective (>99%) against the adult stages of C. oncophora, C. punctata, C. spatulata, C. surnabada, Cooperia spp. adult females and T. colubriformis adults.

Administration, Topical↗
PubMed2003

Proton release upon oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides.

Markedly different light-induced protonational changes were measured in two reaction center mutants of Rhodobacter sphaeroides. A quadruple mutant containing alterations, at residues L131, M160, M197, and M210, that elevate the midpoint potential of the bacteriochlorophyll dimer was compared to the Y(M) mutant, which contains these alterations plus a tyrosine at M164 serving as a secondary electron donor [Kálmán et al., Nature 402 (1999) 696]. In the quadruple mutant, a proton uptake of 0.1-0.3 H(+)/reaction center between pH 6 and 10 resulted from formation of the oxidized bacteriochlorophyll donor and reduced primary quinone. In the Y(M) mutant, a maximal proton release of -0.5 H(+)/reaction center at pH 8 was attributed to formation of the tyrosyl radical and modeled using electrostatic and direct proton-releasing mechanisms.

Amino Acid Substitution↗
PubMed2003

Current and future strategies for the treatment of malignant brain tumors.

Glioblastoma (GB) is the most common subtype of primary brain tumor in adults. These tumors are highly invasive, very aggressive, and often infiltrate critical neurological areas within the brain. The mean survival time after diagnosis of GB has remained unchanged during the last few decades, in spite of advances in surgical techniques, radiotherapy, and also chemotherapy; patients' survival ranges from 9 to 12 months after initial diagnosis. In the same time frame, with our increasing understanding and knowledge of the physiopathology of several cancers, meaningful advances have been made in the treatment and control of several cancers, such as breast, prostate, and hematopoietic malignancies. Although a number of the genetic lesions present in GB have been elucidated and our understanding of the progressions of this cancer has increased dramatically over the last few years, it has not yet been possible to harness this information towards developing effective cures. In this review, we will focus on the classical ways in which GB is currently being treated, and will introduce a novel therapeutic modality, i.e., gene therapy, which we believe will be used in combination with classical treatment strategies to prolong the life-span of patients and to ultimately be able to control and/or cure these brain tumors. We will discuss the use of several vector systems that are needed to introduce the therapeutic genes within either the tumor mass, if these are not resectable, or the tumor bed, after successful tumor resection. We also discuss different therapeutic modalities that could be exploited using gene therapy, i.e., conditional cytotoxic approach, direct cytotoxicity, immunotherapy, inhibition of angiogenesis, and the use of pro-apoptotic genes. The advantages and disadvantages of each of the current vector systems available to transfer genes into the CNS are also discussed. With the advances in molecular techniques, both towards the elucidation of the physiopathology of GB and the development of novel, more efficient and less toxic vectors to deliver putative therapeutic genes into the CNS, it should be possible to develop new rationale and effective therapeutic approaches to treat this devastating cancer.

Animals↗
PubMed2002

Influence of the protein environment on the properties of a tyrosyl radical in reaction centers from Rhodobacter sphaeroides.

The influence of the local environment on the formation of a tyrosyl radical was investigated in modified photosynthetic reaction centers from Rhodobacter sphaeroides. The reaction centers contain a tyrosine residue placed approximately 10 A from a highly oxidizing bacteriochlorophyll dimer. Measurements by both optical and electron paramagnetic resonance spectroscopy revealed spectral features that are assigned as arising primarily from an oxidized bacteriochlorophyll dimer at low pH values and from a tyrosyl radical at high pH values, with a well-defined transition that occurred with a pK(a) of 6.9. A model based on the wild-type structure indicated that the Tyr at M164 is likely to form a hydrogen bond with His M193 and to interact weakly with Glu M173. Substitution of Tyr or Glu for His at M193 increased the pK(a) for the transition from 6.9 to 8.9, while substitution of Gln for His M193 resulted in a higher pK(a) value. Substitution of Glu M173 with Gln resulted in loss of the partial formation of the tyrosyl that occurs in the other mutants at low pH values. The results are interpreted in terms of the ability of the residues to act as proton acceptors for the oxidized tyrosine, with the pK(a) values reflecting those of either the putative proton acceptor or the tyrosine, in accord with general models of amino acid radicals.

Arginine↗