Treatment with PB125® Increases Femoral Long Bone Strength in 15-Month-Old Female Hartley Guinea Pigs.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a transcription factor that serves as a master regulator of anti-inflammatory agents, phase I xenobiotic, and phase II antioxidant enzymes, all of which provide a cytoprotective role during disease progression. We hypothesized that oral administration of a purported phytochemical Nrf2-activator, PB125®, would increase long bone strength in aging Hartley guinea pigs, a model prone to musculoskeletal decline. Male (N = 56) and female (N = 56) guinea pigs were randomly assigned to receive daily oral treatment with either PB125® or vehicle control. Animals were treated for a consecutive 3-months (starting at 2-months of age) or 10-months (starting at 5-months of age) and sacrificed at 5-months or 15-months of age, respectively. Outcome measures included: (1) ANY-maze™ enclosure monitoring, (2) quantitative microcomputed tomography, and (3) biomechanical testing. Treatment with PB125® for 10 months resulted in increased long bone strength as determined by ultimate bending stress in female Hartley guinea pigs. In control groups, increasing age resulted in significant effects on geometric and structural properties of long bones, as well as a trending increase in ultimate bending stress. Furthermore, both age and sex had a significant effect on the geometric properties of both cortical and trabecular bone. Collectively, this work suggests that this nutraceutical may serve as a promising target and preventive measure in managing the decline in bone mass and quality documented in aging patients. Auxiliary to this main goal, this work also capitalized upon 5 and 15-month-old male and female animals in the control group to characterize age- and sex-specific differences on long bone geometric, structural, and material properties in this animal model.

Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay.

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Purification, crystallization and preliminary X-ray analysis of NtdA, a putative pyridoxal phosphate-dependent aminotransferase from Bacillus subtilis.

NtdA is a putative sugar aminotransferase that is required for the synthesis of 3,3'-neotrehalosadiamine. The enzyme was purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized using the microbatch method. X-ray diffraction data were collected from a single crystal to 2.3 A resolution at the Canadian Light Source (CLS). The crystals belonged to space group P2(1), with unit-cell parameters a = 50.3, b = 106.7, c = 96.7 A, beta = 96.2 degrees, and contained two molecules per asymmetric unit.

Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) from Bacillus subtilis.

Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD(+)-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 A resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 A resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.

Synthesis and antifungal properties of compounds which target the alpha-aminoadipate pathway.

Fungi synthesize lysine via the alpha-aminoadipate pathway, which is not found in plants or animals. This pathway has been proposed as a target for antifungal agents, but until now no reports have appeared to test this proposal. Hampering studies on the susceptibility of filamentous fungi such as those of the clinically important genus Aspergillus is the fact that growth quantitation is notoriously difficult. We have used the recently-reported XTT-based method of biomass quantitation to measure the susceptibility of Aspergillus nidulans strain A28 to growth suppression by novel compounds designed to target early steps in the alpha-aminoadipate lysine biosynthesis pathway, specifically those steps involving (R)-homocitrate and (2R,3S)-homoisocitrate. Three compounds show moderate inhibition of fungal growth, which can be partially restored by the presence of lysine in the growth medium.

Phase I trial of two recombinant vaccines containing the 19kd carboxy terminal fragment of Plasmodium falciparum merozoite surface protein 1 (msp-1(19)) and T helper epitopes of tetanus toxoid.

The safety and immunogenicity of 2 yeast-derived, blood-stage malaria vaccines were evaluated in a phase l trial. Healthy adults were given 2 or 3 doses of alum-adsorbed vaccine containing the 19 kDa carboxy-terminal fragment of the merozoite surface protein-1 (MSP-1(19)) derived from the 3D7 or the FVO strain of Plasmodium falciparum fused to tetanus toxoid T-helper epitopes P30 and P2. The first 2 doses of MSP-1(19) were well tolerated. Hypersensitivity reactions occurred in 3 subjects after the third dose of MSP-1(19), including bilateral injection site reactions in 2 (one with generalized skin rash), and probable histamine-associated hypotension in 1. Serum antibody responses to MSP-1(19) occurred in 5/16, 9/16 and 0/8 subjects given 20 microg of MSP-1(19), 200 microg of MSP-1(19), and control vaccines (hepatitis B or Td), respectively. Both MSP-1(19) vaccines were immunogenic in humans, but changes in formulation will be necessary to improve safety and immunogenicity profiles.

Unexpected divergence of enzyme function and sequence: "N-acylamino acid racemase" is o-succinylbenzoate synthase.

A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp. is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1). Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome. Both proteins efficiently catalyze the o-succinylbenzoate synthase reaction in menaquinone biosynthesis (kcat/Km = 2.5 x 10(5) and 7.5 x 10(5) M-1 s-1, respectively), suggesting that this is their "correct" metabolic function. Their membership in the mechanistically diverse enolase superfamily provides an explanation for the catalytic promiscuity of the protein from Amycolaptosis. The adventitious promiscuity may provide an example of a protein poised for evolution of a new enzymatic function in the enolase superfamily. This study demonstrates that the correct assignment of function to new proteins in functional and structural genomics may require an understanding of the metabolism of the organism.

Evolution of enzymatic activities in the enolase superfamily: partitioning of reactive intermediates by (D)-glucarate dehydratase from Pseudomonas putida.

Glucarate dehydratase (GlucD) from Pseudomonas putida catalyzes the dehydration of both (D)-glucarate and (L)-idarate to 3-deoxy-(L)-threo-2-hexulosarate as well as their epimerization. (D)-[6-13C]Glucarate and (L)-[6-13C]idarate have been synthesized for use in continuous assay of the reactions catalyzed by GlucD by both 13C and 1H NMR spectroscopies, thereby allowing the simultaneous measure of both the dehydration and epimerization reactions. Substrate and solvent isotope effects for the dehydration reactions have been quantitated. The mechanism of the GlucD-catalyzed reaction is discussed in the context of that previously established for the homologous mandelate racemase from P. putida, also a member of the enolase superfamily whose members catalyze reactions initiated by abstraction of a proton alpha to a carboxylate group.

Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.

Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate catabolic pathway in Escherichia coli.

The genes encoding the enzymes in the (D)-glucarate/galactarate catabolic pathway have been identified in the Escherichia coli genome. These encode, in three transcriptional units, (D)-glucarate dehydratase (GlucD), galactarate dehydratase, 5-keto-4-deoxy-(D)-glucarate aldolase, tartronate semialdehyde reductase, a glycerate kinase that generates 2-phosphoglycerate as product, and two hexaric acid transporters. We also have identified a gene proximal to that encoding GlucD that encodes a protein that is 72% identical in primary sequence to GlucD (GlucD-related protein or GlucDRP). However, whereas GlucD catalyzes the efficient dehydration of both (D)-glucarate and (L)-idarate as well as their epimerization, GlucDRP is significantly impaired in both reactions. Perhaps GlucDRP is an example of gene duplication and evolution in progress in the E. coli chromosome.

Variation in the sensitivity of HBsAg screening kits.

Fifteen HBsAg kits from 14 manufacturers were assessed. Their sensitivity was evaluated by testing 150 HBsAg-positive sera, sera from four donors who were low-level HBsAg carriers, and sequential specimens from 22 seroconverting individuals together with dilutions of six of these specimens. The British HBsAg Working Standard (0.5 IU mL-1) and the NIBSC/UKBTS HBsAg Monitor Sample (0.125 IU mL-1) were also tested. Five assays failed to detect one of the 150 routine HBsAg-positive sera. Four assays (Auszyme Monoclonal; Monolisa Ag HBs 2nd generation; Murex HBsAg; Ortho HBsAg Test Systems 3) were able to detect HBsAg in all but one of the six sera from low-level carriers, whereas one assay (MicroTrak II HBsAg) detected only one of the six. The most sensitive kit (Monolisa Ag HBs 2nd generation) detected HBsAg in 79 specimens from the seroconversion panels; four other kits detected HBsAg in at least 70 specimens, seven in 60-69, two in 50-59 and the least sensitive in 31. Further analysis of the findings on seroconverters indicated a median reduction in the duration of HBsAg detection of 5 days or more for four assays when compared with the most sensitive assay. One kit (Auszyme Monoclonal) detected HBsAg in 15 of the 18 dilutions prepared from the seroconversion specimens, whereas three kits detected HBsAg in fewer than 10 dilutions. Two kits gave negative reactions with the British HBsAg Working Standard on all of five occasions and six were consistently unreactive with the NIBSC/UKBTS HBsAg Monitor Sample; only three kits (Bioelisa, Enzygnost, Murex) were always reactive. There is therefore substantial variation in sensitivity among the HBsAg kits currently available.

The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.

We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.

IgG4 responses to antigens of adult Necator americanus: potential for use in large-scale epidemiological studies.

Described is an epidemiological investigation of hookworm infections in a rural community in Zimbabwe, where Necator americanus is the only human helminth species present. Among a cohort of 120 individuals the overall prevalence of infection was 78%. Intensity of infection was quantified both as egg counts (range: 0-2563 eggs per g of stool) and worm burden (range: 0-100 worms). Although both these measures provide useful quantitative data, they are tedious to determine in large-scale epidemiological studies and may present social and logistic difficulties. As an alternative screening method, we therefore investigated isotype-specific responses to adult worm antigens of N. americanus. The results show that specific IgG4 responses correlate positively and significantly with both measures of intensity and may be a useful marker of hookworm infection.

Antibody isotype responses to antigens of Ascaris lumbricoides in a case-control study of persistently heavily infected Bangladeshi children.

Antibody responses to Ascaris lumbricoides worm antigens were examined by ELISA in a case-control study of 2 groups of Bangladeshi children, one of which had been shown over a period of 12 months to be consistently lightly infected (controls) and the other consistently heavily infected (cases). The children showed a wide range in intensity of infection; children identified as cases were on average 4 times more heavily infected than the controls. There were no significant differences in weight, height, mid-upper arm circumference and skinfold thickness between the case or control subjects at the time blood samples for analyses by ELISA were collected. Children with repeatedly heavy infections with A. lumbricoides had higher concentrations of antibody isotypes to the antigens of A. lumbricoides than children who are repeatedly lightly infected. IgG1, IgG4 and IgE to worm antigens occurred in significantly higher concentrations in heavily infected subjects. This suggests that these antibody responses simply reflect the intensity of infection and may not play a significant role in protecting against heavy infections.

Epidemiology of human hookworm and Ascaris lumbricoides infestations in rural Gambia.

In a cross-sectional study of helminth infections in a rural village in The Gambia, West Africa, hookworm, probably Necator americanus and Ascaris lumbricoides were found to be the most prevalent helminths present at prevalence levels of 30% and 25% respectively. Other parasites present were Trichuris trichiura (2.4%) and Schistosoma mansoni (1.5%). The mean egg counts of N. americanus and A. lumbricoides in all age groups and in the total population were low. Egg counts between age and sex groups were not statistically significant. The frequency distributions of both N. americanus and A. lumbricoides were overdispersed with the majority of the sample population producing none or few eggs, and a small minority producing relatively large amounts of eggs. Large variance: mean ratios within age groups and the total study population suggested a high degree of aggregation of the parasites in this community.

Severe bilateral fibrosing pleuritis associated with chronic chylothorax in five cats and two dogs.

Severe bilateral fibrosing pleuritis was diagnosed in 5 cats and 2 dogs with chronic chylothorax. All animals were dyspneic on initial examination and remained moderately to severely dyspneic after thoracentesis. Radiographic evidence of fibrosing pleuritis included rounded lung lobes and failure of the lungs to reexpand following effective pleural drainage. Fibrosing pleuritis was also suggested in several animals with radiographic evidence of pleural fluid, in which pleural fluid could not be retrieved. Macroscopically, the lung lobes of all animals were compressed and atelectatic to various degrees, and the pleura appeared to be diffusely thickened and roughened. In several animals, fibrous adhesions were found between the parietal and visceral pleura of some lobes. Lung lobes were markedly constricted and appeared as small, smooth, rounded hilar masses in 4 animals. Mild to moderate pulmonary edema was evident in 3 animals at necropsy. Six of the 7 animals died (2) or were euthanatized (4) within 72 hours after the diagnosis of fibrosing pleuritis. The fibrosing pleuritis developed in 1 animal with lymphoblastic lymphosarcoma and chylothorax after treatment via passive pleuroperitoneal drainage; this animal was euthanatized because of underlying neoplasia. One cat, in which decortication was performed and resulted in marked reexpansion of the lung lobes, died 4 hours after surgery with signs compatible with pulmonary edema. On the basis of our findings, we suggest that animals with chronic chylothorax are at risk to develop fibrosing pleuritis. Furthermore, animals with severe bilateral fibrosing pleuritis should be given extremely guarded prognoses.

Principles of bone healing and biomechanics of external skeletal fixation.

External skeletal fixation is being used to treat an increasing number of orthopedic conditions in veterinary medicine. Study of the variables affecting the biomechanics of external fixation and bone healing is vital if patient morbidity is to be minimized. These are reviewed and incorporated into strategies that can be applied to decision making using external fixation in the clinical setting.