Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2.

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Non-templated hydrothermal growth of anisotropic magnetite nanostructures using hexamine as the directing agent.

Anisotropic growth of magnetite (Fe3O4) nanoparticles is achieved in a hydrothermal growth process using hexamine to play a dual role of oxide forming and directing agent. The samples are characterized by X-ray diffraction, scanning electron microscopy, high resolution transmission electron microscopy, squid magnetometry, ferromagnetic resonance technique, diffuse reflectance spectroscopy and Mössbauer spectroscopy, which collectively establish the formation of Fe3O4 phase. Anisotropic structures such as nanorods and nanotubules are revealed and these are shown to exhibit good humidity sensing properties.

RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Invasive cervical resorption: a case report.

Invasive cervical resorption (ICR) is a relatively uncommon form of external resorption, which may occur in any tooth in the permanent dentition. Characterized by its cervical location and invasive nature, this resorptive process leads to progressive and usually destructive loss of the tooth structure, the clinical features of which often resemble internal resorption ("pink tooth"). This article describes a case report of ICR and its management. The salient features were a large resorptive defect and localized fibrous in-growth located almost wholly on the cervicolabial aspect of the maxillary incisor crown involving the enamel and dentin.

Clinical evaluation of postoperative sensitivity in composite resin restorations using various liners.

Light-cured resin composites are a viable option for restoring Class I and Class II cavities; however, several researchers have reported postoperative sensitivity in such restorations. In this in vivo study, 80 teeth with Class I and Class II cavities were selected. They were divided into four groups of 20 teeth each, to be restored with composite resin using different treatment protocols. Postoperative sensitivity to cold water at various temperatures was assessed for up to three months. The data was interpreted using Chi Square Test for homogeneity among the groups, and Z values of significance for pair-wise evaluation were determined. It was found that use of self-etching primers as bonding agents underneath the composite resin restorations were associated with significantly decreased incidence of postoperative sensitivity to cold stimuli.

Incidence of Postoperative Pain after Single Visit and Two Visit Root Canal Therapy: A Randomized Controlled Trial.

INTRODUCTION: Root Canal Treatment (RCT) has become a mainstream procedure in dentistry. A successful RCT is presented by absence of clinical signs and symptoms in teeth without any radiographic evidence of periodontal involvement. Completing this procedure in one visit or multiple visits has long been a topic of discussion. AIM: To evaluate the incidence of postoperative pain after root canal therapy performed in single visit and two visits. MATERIAL AND METHODS: An unblinded/ open label randomized controlled trial was carried out in the endodontic department of the Dental Institute, where 78 patients were recruited from the regular pool of patients. A total of 66 maxillary central incisors requiring root canal therapy fulfilled the inclusion and exclusion criteria. Using simple randomization by biased coin randomization method, the selected patients were assigned into two groups: group A (n=33) and group B (n=33). Single visit root canal treatment was performed for group A and two visit root canal treatment for group B. Independent sample t-test was used for statistical analysis. RESULTS: Thirty three patients were allotted to group A where endodontic treatment was completed in single visit while 33 patients were allotted to group B where endodontic treatment was completed in two visits. One patient dropped-out from Group A. Hence in Group A, 32 patients were analysed while in Group B, 33 patients were analysed. After 6 hours, 12 hours and 24 hours of obturation, pain was significantly higher in Group B as compared to Group A. However, there was no significant difference in the pain experienced by the patients 48 hours after treatment in both the groups. CONCLUSION: Incidence of pain after endodontic treatment being performed in one-visit or two-visits is not significantly different.

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple forms of cytochrome P450 and associated monooxygenase activities in human brain mitochondria.

We have investigated cytochrome P450 (P450) and associated monooxygenase activities in human brain mitochondria isolated from eight regions of four human brain samples obtained at autopsy. P450-associated monooxygenase activities including aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD), p-nitrophenol hydroxylase (PNPH), and N-nitrosodimethylamine N-demethylase (ND-MAD) were detectable in the mitochondria from human brain regions. Immunoblot experiments using antisera to purified rat liver microsomal P450, namely P4502B1/2, P4501A1/2, and P4502E1, revealed immunoreactive bands in isolated mitochondria from different regions of the human brain. The antibody to P4502B1/2 and P4501A1/2 inhibited the human brain mitochondrial APD and ECD activities, respectively. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450-associated monooxygenase activities, although it completely inhibited the corresponding activities in brain microsomes. Overall, the present study demonstrates, in human brain mitochondria, the presence of multiple forms of P450 belonging to the 1A, 2B, and 2E subfamilies that are involved in xenobiotic metabolism.

Further characterization of rat brain flavin-containing monooxygenase. Metabolism of imipramine to its N-oxide.

Flavin-containing monooxygenase (FMO) activity was compared in rat liver and brain microsomes by estimating the actual amount of imipramine N-oxide relative to the corresponding activity, measured using substrate-stimulated rates of NADPH oxidation. The activities measured as NADPH oxidation rates were significantly higher than those estimated from the N-oxide formed. The brain FMO activity was detectable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The antibody to rabbit pulmonary FMO selectively inhibited imipramine N-oxidation. The antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect on imipramine N-oxidation, indicating the noninvolvement of cytochrome P-450 in the above metabolic pathway. A flavin-containing monooxygenase was partially purified from the rat brain microsomes using sequential chromatography on n-octylamino-Sepharose 4B, DEAE-Sephacel and 2',5'-ADP agarose. The purified FMO was resolved by SDS-PAGE into two bands (approximately 57 and 61 KDa, respectively) both of which cross-reacted with antibody to rabbit pulmonary FMO. The purified enzyme metabolized imipramine and the model substrate methimazole to their respective N-oxide and S-oxides.

Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain.

We have investigated the in vivo effects of the tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on antioxidant defense systems in the mitochondrial, microsomal, and cytosolic compartments of rat brain, lung, and liver. Nicotine induced maximum oxidative stress in brain mitochondria, as seen from a 1.9-fold (P < 0.001) increase in thiobarbituric acid-reactive substance (TBARS) and a 2-fold (P < 0.001) increase in glutathione S-transferase (GST) A4-4 (also referred to as rGST 8-8) activities. These changes were accompanied by a 25-40% increase in reactive oxygen species and a 20-30% decrease in alcohol dehydrogenase activities. The 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-induced oxidative damage was apparent in the microsomal fraction of brain, lung, and liver, and it also increased 4-hydroxynonenal specific GST A4-4 activity in the brain and lung mitochondrial matrix fraction. The levels of microsomal thiobarbituric acid reactive substance, cytochrome P4502E1 activity, and reactive oxygen species were also increased significantly (P < 0.001) in all tissues. Both of these toxins induced the level of GST A4-4 mRNA in the brain, while they caused a marked reduction in the liver GST A4-4 mRNA pool. Additionally, the brain mitochondrial matrix showed a markedly higher level of 4-hydroxynonenal specific GST activity and mGST A4-4 antibody-reactive protein than did the cytosolic fraction. In conclusion, the present study provides evidence for the occurrence of GST A4-4 enzyme activity in mammalian mitochondria, in addition to demonstrating that both mitochondria and microsomes are intracellular targets for nicotine- and NNK-induced organ toxicity.

Differential response of cytosolic, microsomal, and mitochondrial glutathione S-transferases to xenobiotic inducers.

Subcellular levels of different isoenzymes of glutathione S-transferases (GSTs) and their catalytic activities in rat liver, lung and brain tissues were compared following treatment with phenobarbital (PB), -naphthoflavone (BNF) and dexamethasone (DEX). The constitutive expression of á and mu classes of GSTs, but not the GST , was maximum in the liver cytosol as compared to other tissues. Cytosolic GST activity using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate was 2-4 fold higher than that in the microsomal and mitochondrial fractions. Glutathione peroxidase activity with cumene hydroperoxide as a substrate was also highest in the rat liver cytosol. PB and BNF treatments markedly induced the amount of GST proteins in all the tissues studied with the maximum induction in the cytosol after 4 days of PB and 10 days of BNF treatments, respectively. The longer duration of treatments had a suppressive effect on the GST activity, particularly in the mitochondrial and microsomal fractions. DEX treatment, on the other hand, only marginally induced the cytosolic GST, while the mitochondrial GST and the membrane bound microsomal GST activities were mostly decreased. Northern blot analysis also showed an increase in the GST-á mRNA level indicating a possible upregulation of the GST gene expression by the xenobiotic agent. Differences between the subcellular GSTs were studied by the in vitro addition of N-ethylmaleimide (NEM), a selective activator of the microsomal GST. The cytosolic GST activity, both in livers of uninduced and PB-treated, was inhibited to about 50% of the control levels by NEM. The mitochondrial activity, on the other hand, was significantly activated by the addition of NEM, similar to that reported for the microsomal GST. These results suggest selectivity in the effects of different xenobiotics on the expression and catalytic activity of GST isoenzymes from different subcellular compartments of tissues. More importantly, these observations are also relevant in studies on xenobiotic induced organ-specific toxicity and carcinogenicity.

Flavin-containing monooxygenase mediated metabolism of psychoactive drugs by human brain microsomes.

Flavin-containing monooxygenases (FMO) catalyze the oxidation of certain xenobiotics and drugs which contain a nucleophilic heteroatom. Here we report the first assessment of human brain flavin-containing monooxygenase from tissues obtained at autopsy from seven traffic accident victims. Human brain microsomes catalyzed the S-oxidation or N-oxidation of model substrates methimazole and N,N-dimethylaniline, respectively. The psychoactive drugs chlorpromazine, imipramine and fluoxetine, were also metabolized by human brain FMO. 'Western' immunoblot analyses revealed immunological cross-reactivity of the human brain FMO with rabbit pulmonary FMO. Immunocytochemistry further revealed the localization of the FMO predominantly in the neuronal cell bodies in the magnocellular reticular nuclei, colliculi and substantia nigra. Human brain clearly contains an active FMO system, and it is conceivable that such enzyme(s) are significantly involved in the local metabolism and modulation of pharmacological effects of psychoactive drugs.

Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

Catalytic activity and immunohistochemical localization of flavin-containing monooxygenase in rat kidney.

The presence of flavin-containing monooxygenase activity was examined in rat kidney microsomes using N,N-dimethylaniline and methimazole as substrates. Western immunoblot analysis using antisera to porcine liver and rabbit lung flavin-containing monooxygenase indicated immunological cross-reactivity between rat kidney, porcine liver and rabbit lung flavin-containing monooxygenase. Immunohistochemical studies using antisera to rabbit lung flavin-containing monooxygenase demonstrated localization of this enzyme in the proximal and distal convoluted tubules of the renal cortex, the collecting ducts in the renal medulla, but not the glomeruli. This observation indicates the colocalization of flavin-containing monooxygenase and cytochrome P-450 in the metabolically active and absorptive compartment of the renal parenchyma.

Xenobiotic metabolism in brain.

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.

Determination of the morphological irregularities in the middle and apical 1/3rd region of the root canal system of permanent maxillary incisors.

A thorough knowledge of the root canal anatomy and an understanding of its variations from the normal are mandatory for the successful root canal therapy. The assessment and exploration of the accessory canal and apical delta is necessary to combat the persistent infection at the periapical area. The purpose of this study is to determine the morphological irregularities in the middle and apical 1/3rd region of the root canal system of maxillary incisors. A total of 100 maxillary incisors were decalcified, processed, sectioned at the middle and apical 1/3rd region and observed under an ordinary microscope. The frequency of accessory canals, apical delta and type of canal configuration were studied. Accessory canals were found in 5% of the teeth. There was absence of apical delta in all the specimens. Total specimens showed single canal extended from the pulp chamber to the apex (Type 1 canal configuration).

Rat brain cytochrome P450. Reassessment of monooxygenase activities and cytochrome P450 levels.

There have been considerable interlaboratory variations in the reported levels of rat brain microsomal cytochrome P450 and associated monooxygenase activities. To ascertain if the variability could be accountable, at least in part, to different methodologies used for microsome preparation, cytochrome P450 monooxygenase components and activities were directly compared herein using brain microsome prepared by various methods. Rat brain microsome isolated using a calcium aggregation method in the presence of dithiothreitol and glycerol contained approximately 100 pmol of cytochrome P450/mg protein. Considerably lower cytochrome P450 levels (e.g. 20-40 pmol/mg protein) were found in brain microsome prepared in a more conventional manner using Tris or phosphate buffers without glycerol and dithiothreitol. The NADPH cytochrome c reductase activity was consistently approximately 23-25 nmol of cytochrome c reduced/min/mg protein, whatever the method of preparation of the brain microsome. Cytochrome P450-associated monooxygenase activities, namely morphine N-demethylase and ethoxycoumarin O-deethylase, were dependent on the amount of protein in the incubation medium, the length of incubation, and the ratio of the concentration of the substrate to the amount of protein in the incubation mixture. The specific activity of morphine N-demethylase was constant over a range of protein concentration, if the ratio of the concentration of the substrate to the protein was kept constant.

Cancer profile in Surat and its vicinity.

A retrospective study of cancer was conducted in the region of South Gujarat particularly in Surat and its vicinity over a period of 6 years (1980-85) to determine type and frequency of occurrence of various malignancies. During this period 2358 new cancer cases were recorded. A total of 60 types of cancer have been detected and confirmed histopathologically. Oral, laryngeal and oesophageal malignancies were common in males. In females, cancer of breast formed the largest group followed by cancer of cervix. A comparative high rate of lung and testicular cancers was recorded in males. In females, cancers of ovary, stomach and lung were more common. In the present study, the incidence of leukaemias was recorded to be low as compared with other types of cancer. Lack of awareness and ignorance of the disease, addiction to tobacco and alcohol and increasing environmental pollution could be some of the factors responsible for higher incidence of cancers.