IR spectra of cytochrome c denatured with deuterated guanidine hydrochloride show increase in beta sheet

Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra are obtained for horse heart ferricytochrome c in solutions of 0-7M guanidine hydrochloride and deuterated guanidine hydrochloride. Substitutions of deuterium for hydrogen in both the denaturant and protein provide resolvable amide I spectra over a wide range of denaturant concentrations. Deuteration enhances the ability to measure the true protein IR spectrum in the amide I region in which the secondary structure can be deduced, because spectra in D(2)O are less prone to spectral distortion upon background denaturant subtraction than spectra in H(2)O. Other investigators studying equilibrium unfolded cytochrome c were limited to guanidine concentrations below 3.0M because of detector saturation. Detector saturation is avoided with the use of ATR-FTIR spectroscopy, allowing one to obtain protein spectra at high denaturant concentrations. Second derivative spectra of samples show reductions in alpha helix and increases in beta sheet at high denaturant concentrations, contrary to expectations of finding primarily a random coil secondary structure. Using this new technique, the protein was estimated to consist of 51% beta sheet and only 15% random coil in the presence of 6.6M deuterated guanidine hydrochloride.     

Synthetic procedures for the preparation of deuterium-labeled analogs of naturally occurring steroids

The object of this article is to review the procedures that have been published concerning the preparation of deuterium-labeled analogs of naturally occurring steroid hormones. In combination with mass spectrometric methods, these stable isotope-labeled compounds should be applicable for human metabolism studies or as internal standards. Deuteration techniques for the elucidation of stereochemical problems, procedures for the preparation of monodeuterated steroids, and synthesis of deuterated analogs of nonbiologic steroids have therefore not been included in this review.     

Oxalobacter formigenes and its role in oxalate metabolism in the human gut

Oxalate is ingested in a wide range of animal feeds and human foods and beverages and is formed endogenously as a waste product of metabolism. Bacterial, rather than host, enzymes are required for the intestinal degradation of oxalate in man and mammals. The bacterium primarily responsible is the strict anaerobe Oxalobacter formigenes. In humans, this organism is found in the colon. O. formigenes has an obligate requirement for oxalate as a source of energy and cell carbon. In O. formigenes, the proton motive force for energy conservation is generated by the electrogenic antiport of oxalate(2-) and formate(1-) by the oxalate-formate exchanger, OxlT. The coupling of oxalate-formate exchange to the reductive decarboxylation of oxalyl CoA forms an 'indirect' proton pump. Oxalate is voided in the urine and the loss of O. formigenes may be accompanied by elevated concentrations of urinary oxalate, increasing the risk of recurrent calcium oxalate kidney stone formation. Links between the occurrence of nephrolithiasis and the presence of Oxalobacter have led to the suggestion that antibiotic therapy may contribute to the loss of this organism from the colonic microbiota. Studies in animals and human volunteers have indicated that, when administered therapeutically, O. formigenes can establish in the gut and reduce the urinary oxalate concentration following an oxalate load, hence reducing the likely incidence of calcium oxalate kidney stone formation. The findings to date suggest that anaerobic, colonic bacteria such as O. formigenes, that are able to degrade toxic compounds in the gut, may, in future, find application for therapeutic use, with substantial benefit for human health and well-being.     

Whole body distribution of deuterated linoleic and alpha-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.     

Comparison of Drosophila cytochrome P450 metabolism of natural and model substrates

Metabolism of some insecticides and toxic natural plant compounds is known to involve cytochrome P450 enzymes. Correlations between insecticide resistance and deethylation of the model substrate, 7-ethoxycoumarin, have prompted its use in screens for potentially resistant insect populations. The applicability of this model substrate as an indicator of the enzyme activities and inductive responsiveness of cytochrome P450 isoforms involved in the metabolism of carnegine was investigated. This toxic isoquinoline alkaloid is found in the host-plants of some species of cactophilic Drosophila. The results show that the ethoxycoumarin (ECOD) assay does not accurately predict carnegine metabolism either quantitatively or with respect to the overall pattern of activity. Therefore, the ECOD assay may be as isozyme-specific in insects as has already been demonstrated in mammals and its use as an indicator of general P450 activity is questionable.     

Utilization of juvenile animal studies to determine the human effects and risks of environmental toxicants during postnatal developmental stages

BACKGROUND: Toxicology studies utilizing animals and in vitro cellular or tissue preparations have been used to study the toxic effects and mechanism of action of drugs and chemicals and to determine the effective and safe dose of drugs in humans and the risk of toxicity from chemical exposures. Testing in animals could be improved if animal dosing using the mg/kg basis was abandoned and drugs and chemicals were administered to compare the effects of pharmacokinetically and toxicokinetically equivalent serum levels in the animal model and human. Because alert physicians or epidemiology studies, not animal studies, have discovered most human teratogens and toxicities in children, animal studies play a minor role in discovering teratogens and agents that are deleterious to infants and children. In vitro studies play even a less important role, although they are helpful in describing the cellular or tissue effects of the drugs or chemicals and their mechanism of action. One cannot determine the magnitude of human risks from in vitro studies when they are the only source of toxicology data. METHODS: Toxicology studies on adult animals is carried out by pharmaceutical companies, chemical companies, the Food and Drug Administration (FDA), many laboratories at the National Institutes of Health, and scientific investigators in laboratories throughout the world. Although there is a vast amount of animal toxicology studies carried out on pregnant animals and adult animals, there is a paucity of animal studies utilizing newborn, infant, and juvenile animals. This deficiency is compounded by the fact that there are very few toxicology studies carried out in children. That is one reason why pregnant women and children are referred to as "therapeutic orphans." RESULTS: When animal studies are carried out with newborn and developing animals, the results demonstrate that generalizations are less applicable and less predictable than the toxicology studies in pregnant animals. Although many studies show that infants and developing animals may have difficulty in metabolizing drugs and are more vulnerable to the toxic effects of environmental chemicals, there are exceptions that indicate that infants and developing animals may be less vulnerable and more resilient to some drugs and chemicals. In other words, the generalization indicating that developing animals are always more sensitive to environmental toxicants is not valid. For animal toxicology studies to be useful, animal studies have to utilize modern concepts of pharmacokinetics and toxicokinetics, as well as "mechanism of action" (MOA) studies to determine whether animal data can be utilized for determining human risk. One example is the inability to determine carcinogenic risks in humans for some drugs and chemicals that produce tumors in rodents, When the oncogenesis is the result of peroxisome proliferation, a reaction that is of diminished importance in humans. CONCLUSIONS: Scientists can utilize animal studies to study the toxicokinetic and toxicodynamic aspects of drugs and environmental toxicants. But they have to be carried out with the most modern techniques and interpreted with the highest level of scholarship and objectivity. Threshold exposures, no-adverse-effect level (NOAEL) exposures, and toxic effects can be determined in animals, but have to be interpreted with caution when applying them to the human. Adult problems in growth, endocrine dysfunction, neurobehavioral abnormalities, and oncogenesis may be related to exposures to drugs, chemicals, and physical agents during development and may be fruitful areas for investigation. Maximum permissible exposures have to be based on data, not on generalizations that are applied to all drugs and chemicals. Epidemiology studies are still the best methodology for determining the human risk and the effects of environmental toxicants. Carrying out these focused studies in developing humans will be difficult. Animal studies may be our only alternative for answering many questions with regard to specific postnatal developmental vulnerabilities.     

Biological activity of probiotic microorganisms

Adaptation of bifidobacteria and lactic acid bacteria to nutrient media with increased concentrations of bile (1%) and protein substrates of animal origin allowed the variants resistant to bile and displaying a high production of proteolytic enzymes (active within the pH range of 2.5–9.0) to be selected. Administration of the preparations involving the selected bifidobacteria and lactic acid bacteria assisted in the normalization of the intestinal microflora and activation of protein metabolism in the organism of animals. Specifically, it increased the total protein level in blood serum and redistributed protein fractions, increasing the content of globulins and decreasing albumin concentration.     

Biological activity of probiotic microorganisms

Adaptation of bifidobacteria and lactic acid bacteria to nutrient media with increased concentrations of bile (1%) and protein substrates of animal origin allowed the variants resistant to bile and displaying a high production of proteolytic enzymes (active within the pH range of 2.5-9.0) to be selected. Administration of the preparations involving the selected bifidobacteria and lactic acid bacteria assisted in the normalization of the intestinal microflora and activation of protein metabolism in the organism of animals. Specifically, it increased the total protein level in blood serum and redistributed protein fractions, increasing the content of globulins and decreasing albumin concentration.     

Borate Transport and Cell Growth and Proliferation: Not Only in Plants

Boron is an abundant mineral essential for the life cycle of plants and may play a role in animal development and growth. Very little is known about boron homeostasis in plant and animal cells and the physiological roles of boron in animals. The recent identification of boron transporters, BOR1 in plants and NaBC1 in mammals, and that NaBC1 functions as an electrogenic Na+-coupled borate transporter essential for cell growth and proliferation open the way to probe the roles of boron in cellular function and physiology.     

Relative responses of an X-ray-resistant hybrid cell-line and its parent line to X-irradiation, ultraviolet light, actinomycin D and cordycepin.

Using colony formation as an assay, a rat-mouse hybrid cell-line (HD1) and one of its parent lines (H4) have been studied as to their abilities to survive exposure to ionizing radiation, ultraviolet light, and the drugs actinomycin D and cordycepin. HD1 cells are more resistant than H4 to ionizing radiation, actinomycin D and cordycepin. Both cell lines respond similarly to ultraviolet light. When both cell-lines were co-treated with actinomycin D or cordycepin, the toxic effect of ionizing radiation was enhanced, whereas that of ultraviolet light (U.V.L.) was unchanged. The data suggest that RNA synthesis is more important immediately after irradiation with X-rays than with U.V.L. and that cells resistant to the toxic effect of ionizing radiation are also resistant to the toxicity induced by inhibitors of RNA synthesis.     

Protection of mice from whole-body gamma radiation by deuteration of drinking water

Drinking water made available to mice was changed from ordinary tap water to tap water containing 30 atom% D2O when the animals were 6 to 8 weeks old. Twelve days later, the deuterated mice and an approximately equal number of nondeuterated control mice were subjected to whole-body gamma radiation from a 60Co source. All mice received ordinary tap water after the irradiation. Postirradiation mortality was significantly less in deuterated than in nondeuterated animals. These results may have practical implications for radiotherapy of human malignant tumors.     

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.     

The US national antimicrobial resistance monitoring system.

The use of antimicrobial agents in food animals can select for resistant bacterial pathogens that may be transmitted to humans via the commercial meat supply. In the USA, the FDA's Center for Veterinary Medicine regulatory duties require a determination that antimicrobial drugs are safe and effective for use in food animals. In addition, a qualitative assessment of risks to human health from antimicrobial resistance requires development. This risk assessment process is supported by data generated by the FDA's National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria. NARMS data on antimicrobial susceptibility among Salmonella, Campylobacter, Escherichia coli and Enterococcus is collected. Research activities defining the genetic bases of resistance helps to understand the potential public health risks posed by the spread of antimicrobial resistance from food animal antimicrobial use. These activities help insure that antimicrobials are used judiciously to promote human and animal health.     

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica

Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments.     

The impact of secondary pests on Bacillus thuringiensis (Bt) crops

The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to insecticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered insect‐resistant crops could mitigate many of the negative side effects of insecticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro‐ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here, we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if nonsusceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible long‐term ecological trophic interactions of employing this technology.     

Minimizing the release of proinflammatory and toxic bacterial products within the host: a promising approach to improve outcome in life-threatening infections

Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.     

Discovery of phytopathogenic bacteria 100 years ago: transatlantic controversies and polemics

The demonstration of a bacterial cause of some plant diseases has been claimed few years after it was commonly recognized that bacteria were able to cause diseases of human and animal. Nevertheless, some sharp controversies took place, between German and American specialists (1897-1901), before the existence of bacterial diseases of plants was accepted by all phytopathologists. Nowadays, about 350 bacteria are described, which infect plants: they are pathovars, or subspecies, belonging to 21 genera. Bacterial diseases of plants can be classified into three major categories according to the type of symptoms shown by the infected plant: necrosis and wilt, soft-rot, tumour. The interaction between bacteria and plant cells is usually established from the apoplast, although some bacteria are xylem or phloem limited. This interaction involves an original protein secretion system (which is also described in bacteria pathogenic for animals), hydrolytic enzymes (pectinases, cellulases), toxins and/or phytohormones. Bacteria of one group (Agrobacterium) modify the plant metabolism after gene transfer from a plasmid. On the economic and social point of view, these diseases may be limiting factors of some key-productions (rice, cassava). In addition, they play a role in reducing the quality of agricultural products (reduced growth, spots on leaves and fruits). Control of bacterial diseases is limited. It relies usually on a combination of prophylaxy, chemical applications, and use of resistant genotypes.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.