Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and β-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-tyrosine) and lipid peroxidation (malondialdehyde) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.

Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy

Isotretinoin (13-cis retinoic acid) is frequently prescribed for severe acne [Peck, G. L., Olsen, T. G., Yoder, F. W., Strauss, J. S., Downing, D. T., Pandya, M., Butkus, D. & Arnaud-Battandier, J. (1979) N. Engl. J. Med. 300, 329–333] but can impair night vision [Fraunfelder, F. T., LaBraico, J. M. & Meyer, S. M. (1985) Am. J. Ophthalmol. 100, 534–537] shortly after the beginning of therapy [Shulman, S. R. (1989) Am. J. Public Health 79, 1565–1568]. As rod photoreceptors are responsible for night vision, we administered isotretinoin to rats to learn whether night blindness resulted from rod cell death or from rod functional impairment. High-dose isotretinoin was given daily for 2 months and produced systemic toxicity, but this caused no histological loss of rod photoreceptors, and rod-driven electroretinogram amplitudes were normal after prolonged dark adaptation. Additional studies showed, however, that even a single dose of isotretinoin slowed the recovery of rod signaling after exposure to an intense bleaching light, and that rhodopsin regeneration was markedly slowed. When only a single dose was given, rod function recovered to normal within several days. Rods and cones both showed slow recovery from bleach after isotretinoin in rats and in mice. HPLC analysis of ocular retinoids after isotretinoin and an intense bleach showed decreased levels of rhodopsin chromophore, 11-cis retinal, and the accumulation of the biosynthetic intermediates, 11-cis and all-trans retinyl esters. Isotretinoin was also found to protect rat photoreceptors from light-induced damage, suggesting that strategies of altering retinoid cycling may have therapeutic implications for some forms of retinal and macular degeneration.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Altered stability of pulmonary surfactant in SP-C-deficient mice

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (−/−) mice were produced. The SP-C (−/−) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (−/−) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (−/−) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (−/−) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (−/−) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (−/−) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Chimeric anti-angiogenin antibody cAb 26–2F inhibits the formation of human breast cancer xenografts in athymic mice

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

A single in vivo administration of bombesin antagonist RC-3095 reduces the levels and mRNA expression of epidermal growth factor receptors in MXT mouse mammary cancers

Epidermal growth factor (EGF) and its receptors (EGFR) play important roles in tumorigenesis. In various experimental cancers, treatment with antagonists of bombesin/gastrin-releasing peptide (BN/GRP) produces a reduction in EGFRs, concomitant to inhibition of tumor growth. To investigate the mechanisms involved, we monitored concentrations of BN/GRP antagonist RC-3095 in serum of mice, rats, and hamsters given a single subcutaneous or intravenous injection of this analog. In parallel studies, we measured levels and mRNA expression of EGFRs in estrogen-dependent and independent MXT mouse mammary cancers, following a single subcutaneous administration of RC-3095 to tumor-bearing mice. Peak values of RC-3095 in serum were detected 2 min after intravenous or 15 min after subcutaneous injection. The levels of RC-3095 declined rapidly and became undetectable after 3–5 hr. In the estrogen-dependent MXT tumors, the concentration of EGF receptors was reduced by about 60% 6 hr following injection and returned to original level after 24 hr. Levels of mRNA for EGFR fell parallel with the receptor number and were nearly normal after 24 hr. In the hormone-independent MXT cancers, the number of EGFRs decreased progressively, becoming undetectable 6 hr after injection of RC-3095, and returned to normal values at 24 hr, but EGFR mRNA levels remained lower for 48 hr. Thus, in spite of rapid elimination from serum, BN/GRP antagonist RC-3095 can induce a prolonged decrease in levels and mRNA expression of EGFRs. These findings may explain how single daily injections of BN/GRP antagonists can maintain tumor growth inhibition.

ZK200775: A phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma

Stroke and head trauma are worldwide public health problems and leading causes of death and disability in humans, yet, no adequate neuroprotective treatment is available for therapy. Glutamate antagonists are considered major drug candidates for neuroprotection in stroke and trauma. However, N-methyl-d-aspartate antagonists failed clinical trials because of unacceptable side effects and short therapeutic time window. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonists derived from the quinoxalinedione scaffold cannot be used in humans because of their insolubility and resulting renal toxicity. Therefore, achieving water solubility of quinoxalinediones without loss of selectivity and potency profiles becomes a major challenge for medicinal chemistry. One of the major tenets in the chemistry of glutamate antagonists is that the incorporation of phosphonate into the glutamate framework results in preferential N-methyl-d-aspartate antagonism. Therefore, synthesis of phosphonate derivatives of quinoxalinediones was not pursued because of a predicted loss of their selectivity toward AMPA. Here, we report that introduction of a methylphosphonate group into the quinoxalinedione skeleton leaves potency as AMPA antagonists and selectivity for the AMPA receptor unchanged and dramatically improves solubility. One such novel phosphonate quinoxalinedione derivative and competitive AMPA antagonist ZK200775 exhibited a surprisingly long therapeutic time window of >4 h after permanent occlusion of the middle cerebral artery in rats and was devoid of renal toxicity. Furthermore, delayed treatment with ZK200775 commencing 2 h after onset of reperfusion in transient middle cerebral artery occlusion resulted in a dramatic reduction of the infarct size. ZK200775 alleviated also both cortical and hippocampal damage induced by head trauma in the rat. These observations suggest that phosphonate quinoxalinedione-based AMPA antagonists may offer new prospects for treatment of stroke and trauma in humans.

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase C isoforms in young and aged rats

Relationships were examined between spatial learning and hippocampal concentrations of the α, β2, and γ isoforms of protein kinase C (PKC), an enzyme implicated in neuronal plasticity and memory formation. Concentrations of PKC were determined for individual 6-month-old (n = 13) and 24-month-old (n = 27) male Long–Evans rats trained in the water maze on a standard place-learning task and a transfer task designed for rapid acquisition. The results showed significant relationships between spatial learning and the amount of PKC among individual subjects, and those relationships differed according to age, isoform, and subcellular fraction. Among 6-month-old rats, those with the best spatial memory were those with the highest concentrations of PKCγ in the particulate fraction and of PKCβ2 in the soluble fraction. Aged rats had increased hippocampal PKCγ concentrations in both subcellular fractions in comparison with young rats, and memory impairment was correlated with higher PKCγ concentrations in the soluble fraction. No age difference or correlations with behavior were found for concentrations of PKCγ in a comparison structure, the neostriatum, or for PKCα in the hippocampus. Relationships between spatial learning and hippocampal concentrations of calcium-dependent PKC are isoform-specific. Moreover, age-related spatial memory impairment is associated with altered subcellular concentrations of PKCγ and may be indicative of deficient signal transduction and neuronal plasticity in the hippocampal formation.

Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function

Alterations in serotonin (5-hydroxytriptamine, 5-HT), norepinephrine, and γ-aminobutyric acid have been linked to the pathophysiology of anxiety and depression, and medications that modulate these neurotransmitters are widely used to treat mood disorders. Recently, the neuropeptide substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), have been proposed as possible targets for new antidepressant and anxiolytic therapies. However, animal and human studies have so far failed to provide a clear consensus on the role of SP in the modulation of emotional states. Here we show that both genetic disruption and acute pharmacological blockade of the NK1R in mice result in a marked reduction of anxiety and stress-related responses. These behavioral changes are paralleled by an increase in the firing rate of 5-HT neurons in the dorsal raphe nucleus, a major source of serotonergic input to the forebrain. NK1R disruption also results in a selective desensitization of 5-HT1A inhibitory autoreceptors, which resembles the effect of sustained antidepressant treatment. Together these results indicate that the SP system powerfully modulates anxiety and suggest that this effect is at least in part mediated by changes in the 5-HT system.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Theory for normal and impaired experience-dependent plasticity in neocortex of adult rats

We model experience-dependent plasticity in the cortical representation of whiskers (the barrel cortex) in normal adult rats, and in adult rats that were prenatally exposed to alcohol. Prenatal exposure to alcohol (PAE) caused marked deficits in experience-dependent plasticity in a cortical barrel-column. Cortical plasticity was induced by trimming all whiskers on one side of the face except two. This manipulation produces high activity from the intact whiskers that contrasts with low activity from the cut whiskers while avoiding any nerve damage. By a computational model, we show that the evolution of neuronal responses in a single barrel-column after this sensory bias is consistent with the synaptic modifications that follow the rules of the Bienenstock, Cooper, and Munro (BCM) theory. The BCM theory postulates that a neuron possesses a moving synaptic modification threshold, θM, that dictates whether the neuron's activity at any given instant will lead to strengthening or weakening of its input synapses. The current value of θM changes proportionally to the square of the neuron's activity averaged over some recent past. In the model of alcohol impaired cortex, the effective θM has been set to a level unattainable by the depressed levels of cortical activity leading to “impaired” synaptic plasticity that is consistent with experimental findings. Based on experimental and computational results, we discuss how elevated θM may be related to (i) reduced levels of neurotransmitters modulating plasticity, (ii) abnormally low expression of N-methyl-d-aspartate receptors (NMDARs), and (iii) the membrane translocation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in adult rat cortex subjected to prenatal alcohol exposure.

The second STE12 homologue of Cryptococcus neoformans is MATa-specific and plays an important role in virulence

Cryptococcus neoformans STE12α, a homologue of Saccharomyces cerevisiae STE12, exists only in MATα strains. We identified another STE12 homologue, STE12a, which is MATa specific. As in the case with Δste12α, the mating efficiency for Δste12a was reduced significantly. The Δste12a strains surprisingly still mated with Δste12α strains. In MATα strains, STE12a functionally complemented STE12α for mating efficacy, haploid fruiting, and regulation of capsule size in the mouse brain. Furthermore, when STE12a was replaced with two copies of STE12α, the resulting MATa strain produced hyphae on filament agar. STE12a regulates mRNA levels of several genes that are important for virulence including CNLAC1 and CAP genes. STE12a also modulates enzyme activities of phospholipase and superoxide dismutase. Importantly, deletion of STE12a markedly reduced the virulence in mice, as is the case with STE12α. Brain smears of mice infected with the Δste12a strain showed yeast cells with a considerable reduction in capsule size compared with those infected with STE12a strains. When the disrupted locus of ste12a was replaced with a wild-type STE12a gene, both in vivo and in vitro mutant phenotypes were reversed. These results suggest that STE12a and STE12α have similar functions, and that the mating type of the cells influences the alleles to exert their biological effects. C. neoformans, thus, is the first fungal species that contains a mating-type-specific STE12 homologue in each mating type. Our results demonstrate that mating-type-specific genes are not only important for saprobic reproduction but also play an important role for survival of the organism in host tissue.