Enzyme dehydration using Microglassification™ preserves the protein's structure and function.

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to ß-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function.

In vitro studies of DNA damage caused by tricyclic antidepressants: a role of peroxidase in the side effects of the drugs.

Studies show that tricyclic antidepressants prescribed for migraines, anxiety, and child enuresis have numerous adverse effects in living cells. One of the undesired outcomes observed under treatment with these drugs is DNA damage. However, the mechanisms underlying damage have yet to be elucidated. We performed in vitro studies of the DNA damage caused by four tricyclic antidepressants: imipramine, amitriptyline, opipramol, and protriptyline. We focused particularly on the DNA damage aided by peroxidases. As a model of a peroxidase, we used horseradish peroxidase (HRP). At pH 7, reactions of HRP with excess hydrogen peroxide and imipramine yielded an intense purple color and a broad absorption spectrum with the maximum intensity at 522 nm. Reactions performed between DNA and imipramine in the presence of H(2)O(2) and HRP resulted in the disappearance of the DNA band. In the case of the other three drugs, this effect was not observed. Extraction of the DNA from the reaction mixture indicated that DNA is degraded in the reaction between imipramine and H(2)O(2) catalyzed by HRP. The final product of imipramine oxidation was identified as iminodibenzyl. We hypothesize that the damage to DNA was caused by an imipramine reactive intermediate.

Uterine peroxidase-catalyzed formation of diquinone methides from the selective estrogen receptor modulators raloxifene and desmethylated arzoxifene.

Long-term usage of the selective estrogen receptor modulator (SERM) tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen-induced carcinogenesis involves metabolism to reactive intermediates, such as an o-quinone, quinone methide, and carbocations. We have previously shown that the benzothiophene SERMs, raloxifene and desmethylated arzoxifene (DMA), can also be bioactivated to electrophilic quinoids by rat/human liver microsomes and rat hepatocytes [(2006) Chem. Res. Toxicol. 19, 1125-1137]. Because the uterus is a major target tissue of estrogens and antiestrogens, it was of interest to determine if quinoids could be formed from SERMs in uterine tissue potentially producing cytotoxic effects. Incubations with rat uterine microsomes showed that both raloxifene and DMA could be oxidized to electrophilic diquinone methides that were trapped as the corresponding GSH conjugates. A new raloxifene GSH-dependent conjugate was identified as raloxifene Cys-Gly that was formed from the hydrolysis of 7-glutathinyl raloxifene by gamma-glutamyl transpeptidase. Interestingly, the metabolism of raloxifene and DMA in rat uterine microsomes was not NADPH-dependent and could be inhibited by cyanide and NADPH or enhanced by H2O2. In addition, coincubations with the peroxidase substrates guaiacol or o-phenlyenediamine inhibited diquinone methide GSH conjugate formation from both SERMs. Incubations of raloxifene and DMA with horseradish peroxidase (HRP) were studied as models of the interaction between benzothiophene SERMs and peroxidase. The results showed that HRP could directly oxidize raloxifene and DMA to the corresponding dimers via the formation of phenoxyl radicals in the absence of exogenous hydrogen peroxide. In addition, GSH appears to be involved in multiple peroxidase-catalyzed oxidative metabolic pathways of benzothiophene SERMs. Finally, COATag (covert oxidatively activated tag) methodology, which involves the utilization of biotin-conjugated raloxifene and DMA, was used to identify target proteins by affinity chromatography. Incubations of raloxifene and DMA COATags with rat uterine microsomes showed several modified proteins by Western blot analysis. The protein modification could be enhanced by the addition of H2O2 and decreased by the addition of NADPH, suggesting that unlike liver metabolism the formation of quinoids in the uterus could be mediated by uterine peroxidases.

Doxorubicin degradation in cardiomyocytes.

Antitumor therapy with the anthracycline doxorubicin is limited by a severe cardiotoxicity, which seems to correlate with the cardiac levels of doxorubicin and its metabolization to reactive oxygen species. Previous biochemical studies showed that hydrogen peroxide-activated myoglobin caused an oxidative degradation of doxorubicin; however, a pharmacological evaluation of this metabolic pathway was precluded by the lack of safe and specific inhibitors of doxorubicin degradation. We found that tert-butoxycarbonyl-alanine inhibited doxorubicin degradation induced in vitro by hydrogen peroxide-activated oxyferrous myoglobin. When assessed in H9c2 cardiomyocytes, tert-butoxycarbonyl-alanine neither stimulated the cellular uptake of doxorubicin nor diminished its efflux; moreover, tert-butoxycarbonyl-alanine did not cause toxicity per se nor did it augment the toxicity induced by hydrogen peroxide or chemical agents that increased the cellular levels of reactive oxygen species. Nonetheless, tert-butoxycarbonyl-alanine increased the cellular levels of doxorubicin, its conversion to reactive oxygen species, and its concentration-related toxicity. tert-Butoxycarbonyl-alanine also aggravated the toxicity of a degradation-prone anthracycline analog, daunorubicin, but it caused a minor effect on the toxicity of a degradation-resistant analog, aclarubicin. These results suggested that tert-butoxycarbonyl-alanine increased the cellular levels and toxicity of doxorubicin by inhibiting its oxidative degradation to harmless products. Accordingly, doxorubicin samples that had been oxidized in vitro with hydrogen peroxide and oxyferrous myoglobin lacked toxicity to cardiomyocytes. The effects of tert-butoxycarbonyl-alanine were most evident at 0.1 to 1 microM doxorubicin, which may be relevant to clinical conditions. These studies identify an oxidative degradation of doxorubicin as a possible salvage mechanism for diminishing its levels and toxicity in cardiomyocytes.

Receptors and enzymes for medical sensing of L-glutamate.

Medical sensing systems using isolated or intact glutamate receptor (GluR) ion channels and glutamate oxidase (GluOx) are discussed for L-glutamate, one of the principal neurotransmitter in the central nervous systems of mammalian brain, and related agonists. The GluR-based sensing system used for the evaluation of signal transduction ability of GluR channels demonstrate that the agonist selectivity based on the signal transduction ability is not parallel to that of the binding assay. On the other hand, the appropriate design of the enzyme system, namely glutamate oxidase (GluOx), in combination with horseradish peroxidase (HRP), enables to real-time monitoring of L-glutamate in vivo and in vitro and also to visualize its release in submerged, acute mouse hippocampal slices.

PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis.

The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P2-producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyveWT or PIKfyveK1831E expression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyveK1831E but not PIKfyveWT expression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyveK1831E showed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyveK1831E-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PIKfyveK1831E-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.

Increased retinal endothelial cell monolayer permeability induced by the diabetic milieu: role of advanced non-enzymatic glycation and polyol pathway activation.

BACKGROUND: Increased vascular permeability could be involved in the pathogenesis of diabetic retinopathy. The present study was aimed at assessing whether high glucose concentrations can impair retinal endothelial cell barrier function directly, irrespective of changes in other determinants of permeability, and the role of non-enzymatic glycation and polyol pathway activation in these alterations. METHODS: Bovine retinal endothelial cells (BREC) were exposed for various periods to high glucose vs iso-osmolar mannitol and normal glucose containing media+/-agents mimicking or inhibiting advanced glycation end product (AGE) formation and polyol pathway activation. Monolayer permeability was assessed by measuring the transendothelial passage of (125)I-labeled proteins. RESULTS: Permeability increased significantly (up to +70%) in BREC exposed to high glucose, but not to mannitol, for 1-30 days, vs normal glucose control cells. Exposure to AGE-modified bovine serum albumin (BSA) (> or = 90%) and, to a lesser extent, sorbitol (+28%) mimicked the high glucose effect. The AGE formation and nitric oxide synthase (NOS) inhibitor aminoguanidine significantly reduced (by 60%) changes induced by 30-day exposure to high glucose, whereas methylguanidine, which inhibits only NOS activity, did not affect permeability. Aldose reductase or sorbitol dehydrogenase inhibitors decreased (by approximately 40%) the enhanced leakage produced by 1-day, but not 30-day, incubation in high glucose. CONCLUSIONS: The present results indicate that high glucose is capable of impairing retinal endothelial cell barrier function directly and that non-enzymatic glycation and polyol pathway activation may mediate these changes, with AGEs participating in the long-term alterations and increased flux through the sorbitol pathway in the short-term effect.

Peroxidase-catalyzed in vitro formation of polychlorinated dibenzo-p-dioxins and dibenzofurans from chlorophenols.

Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by a peroxidase-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra- and pentachlorophenol with plant horseradish peroxidase and with myeloperoxidase recovered from human leukocytes, each in the presence of hydrogen peroxide. The yield, the reaction and the PCDD/F-pattern found are dependent on the CP. The amounts of PCDD/F formed within 4 or 24 h are in the micromol/mol-range for all substrates and both peroxidases. The experiments suggest that biochemical formation of PCDD/F from precursors such as CPs can take place in the human body and that this metabolic pathway may lead to a higher inner exposure to PCDD/F than up to now assumed based on intake data for PCDD/F.

Brainstem neurons projecting to different levels of the spinal cord of the dogfish Scyliorhinus canicula.

Neurons in the brain that project to different levels of the spinal cord in the dogfish Scyliorhinus canicula have been identified by retrograde labelling with horseradish peroxidase. Injections have been made at four different levels, namely, cervical (segments 3-6), pectoral (segments 16-18), pelvic (segments 34-36) and caudal (segments 60-80). Labelled neurons were located in the diencephalon, mesencephalon and rhombencephalon. Twenty nuclei contained labelled cells following cervical injections. Fourteen of these contained labelled cells following pectoral injections as did nine following pelvic injections and only seven after caudal injections. Of the three diencephalic nuclei projecting to the cord only nucleus thalamus ventralis pars medialis projected further than cervical regions. From the mesencephalon, the tectospinal projection, which arises from tectal and tectotegmental regions, reaches only as far as the cervical cord. The reticulospinal system, arising from cell groups in both the mesencephalon and rhombencephalon, provides the major descending pathway and reaches the most caudal levels. From the octaval region a vestibulospinal pathway arises from nucleus octavus magnocellularis, to reach all levels of the cord, and from nucleus octavus descendens, to project only as far as the pectoral cord. Other rhombencephalic cell groups--the trigeminal nuclei and nuclei B, F and G--project at least as far as the pectoral cord. We conclude that few brainstem nuclei can directly affect the whole spinal cord, whereas many nuclei may have an impact on its most rostral regions; here we have found labelled spinal interneurons with long descending axons that reach the most caudal cord levels.

Change in the capillary permeability of the stria vascularis by different methods of death and fixation.

The permeability of the stria vascularis to horseradish peroxidase (HRP) was examined. Ten minutes after an intravenous injection of HRP, 15 guinea pigs were killed and fixed by four different methods: cochleas fixed 3 minutes after decapitation, administration of an overdose of pentobarbital, cochleas fixed for 5 minutes before decapitation, and cochleas fixed immediately after decapitation. In the animals in group 1, HRP leakage was observed in almost all strial vessels (86%), and blood sludging and amorphous HRP reaction product in 98% of strial capillaries. In the animals in group 2, strial capillaries were observed both with noticeable HRP leakage and with no evidence of leakage. Blood sludging was associated with amorphous HRP reaction product, while no blood sludging was associated with granular HRP reaction product. In group 3 animals, only faint or no HRP reaction product was observed in the perivascular spaces. Only 3% of the capillaries had blood sludging. In the animals in group 4, blood sludging but no HRP leakage was observed in almost all capillaries (82%). It is speculated that HRP does not normally leak from the strial capillaries, although many researchers have reported to the contrary. It appears that HRP leaks from the strial capillaries only when acute anoxia or hypotension continues for a few minutes. These conditions induce blood sludging and are followed by the appearance of many marginal folds in these capillaries.

Edematous necrosis in thiamine-deficient encephalopathy of the mouse.

Acute encephalopathy was produced in the adult male Swiss mouse by pyrithiamine injection in conjunction with a thiamine-deficient diet. The condition of some mice was reversed within 24 hours by a treatment of a high dose of thiamine. The lesions occurred selectively in the thalamus, pontine tegmentum, and mammillary body and were manifested by hemorrhage and edematous necrosis consisting of severe edema of astrocytes, myelin sheaths, and neuronal dendrites. Before thiamine treatment, these degenerative changes were not associated with any mesenchymal reaction. At 48 and 96 hours after thiamine treatment, these edematous changes persisted. Fat-laden macrophages appeared in the lesion. Some axons showed Wallerian-type degeneration. After three weeks of thiamine treatment, macrophages became thin and rod-shaped. Wallerian-type degeneration and myelin edema persisted. The oligodendrocytes and astrocytes were hypertrophic. These lesions of thiamine-treated encephalopathy of the mouse closely resembled the non-hemorrhagic lesions of human Wernicke encephalopathy. Mice which were concomitantly-induced with hyperglycemia and encephalopathy showed no significant differences in clinical and morphologic manifestations from the encephalopathic mice with normal blood sugar levels. Vascular permeability to horseradish peroxidase was increased only slightly at the initial stage, but was reversed in the mice which clinically responded quickly to thiamine treatment. Occasionally, persistent increase of permeability was seen in 21-day-old lesions. These findings suggested that, in thiamine-deficient encephalopathy, both nervous and vascular components in the brain were involved and that the morphologic manifestations of the nervous component were far more extensive than those of the blood vessels.

The effect of nitrogen dioxide on tracheal uptake and transport of horseradish peroxidase in the guinea pig.

The effect of 5 and 15 ppm of nitrogen dioxide (NO2) on airway transepithelial permeability to horseradish peroxidase (HRP) (molecular weight, approximately 40,000 daltons) was studied in the guinea pig. Age- and sex-matched guinea pigs (n = 34) were exposed to NO2 for 2 and for 14 days. In the control guinea pigs, 0.05% of the intratracheally instilled dosage HRP was present in the plasma within 10 min, as detected by radioimmunoassay, and increased thereafter at amean rate of 0.0016%/min. After exposure to NO2, there was a significant increase in the rate of plasma HRP accumulation (p < 0.05) with the maximal increase detected in the group exposed to 15 ppm of NO2 for 2 days. The morphologic correlates of increased permeability to HRP appear to be (a) an increase in pinocytotic activity within the hyperplastic globlet cells, and (b) transjunctional transport by leaky tight junctions that was noted only in animals exposed to 15 ppm for 14 days (p < 0.01). Our data suggests that pinocytotic vesicular transport of proteins in the secretory cells of the airway may be an important mechanism for the movement of exogenous nonlipid soluble macromolecules across the epithelial barrier.