Toxicology study for magnetic injection of prednisolone into the rat cochlea.

This paper investigates the safety of a novel 'magnetic injection' method of delivering therapy to the cochlea, in a rodent model. In this method of administration, a magnetic field is employed to actively transport drug-eluting superparamagnetic iron-oxide core nanoparticles into the cochlea, where they then release their drug payload (we delivered the steroid prednisolone). Our study design and selection of control groups was based on published regulatory guidance for safety studies that involve local drug delivery. We tested for both single and multiple delivery doses to the cochlea, and found that magnetic delivery did not harm hearing. There was no statistical difference in hearing between magnetically treated ears versus ears that received intra-tympanic steroid (a mimic of a standard-of-care for sudden sensorineural hearing loss), both 2 and 30 days after treatment. Since our treatment is local to the ear, the levels of steroid and iron circulating systemically after our treatment were low, below mass-spectrometry detection limits for the steroid and no different from normal for iron. No adverse findings were observed in ear tissue histopathology or in animal gross behavior. At 2 and 30 days after treatment, inflammatory changes examined in the ear were limited to the middle ear, were very mild in severity, and by day 90 there was ongoing and almost complete reversibility of these changes. There were no ear tissue scarring or hemorrhage trends associated with magnetic delivery. In summary, after conducting a pre-clinical safety study, no adverse safety issues were observed.

Dynamic inversion enables external magnets to concentrate ferromagnetic rods to a central target.

The ability to use magnets external to the body to focus therapy to deep tissue targets has remained an elusive goal in magnetic drug targeting. Researchers have hitherto been able to manipulate magnetic nanotherapeutics in vivo with nearby magnets but have remained unable to focus these therapies to targets deep within the body using magnets external to the body. One of the factors that has made focusing of therapy to central targets between magnets challenging is Samuel Earnshaw's theorem as applied to Maxwell's equations. These mathematical formulations imply that external static magnets cannot create a stable potential energy well between them. We posited that fast magnetic pulses could act on ferromagnetic rods before they could realign with the magnetic field. Mathematically, this is equivalent to reversing the sign of the potential energy term in Earnshaw's theorem, thus enabling a quasi-static stable trap between magnets. With in vitro experiments, we demonstrated that quick, shaped magnetic pulses can be successfully used to create inward pointing magnetic forces that, on average, enable external magnets to concentrate ferromagnetic rods to a central location.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus.

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.

Blood distribution to the anterior spinal artery from each segment of intercostal and lumbar arteries.

AIM: Prevention of paraplegia, a serious complication of surgery for thoracoabdominal aortic aneurysm, has been well documented. However no assured prophylaxis against this complication has yet been found. Spinal ischemia is believed to be the major cause of paraplegia. We conducted an experimental study to define the development of paraplegia with regard to the blood supply to the spinal cord. METHODS: A porcine model was used to evaluate blood distribution to the anterior spinal artery. Colored silastic agent was selectively injected into the intercostal and lumbar arteries, and distribution to the anterior spinal artery was evaluated on 50 animals. The intercostal and lumbar arteries were ligated in the segments where the blood supply to the anterior spinal artery would be interrupted. Whether or not paraplegia developed was checked 2 days later. RESULTS: Colored silastic agent arrived at the anterior spinal artery from all segments of the 8th intercostal to 4th lumbar arteries. Two of 9 pigs (22.2%) that underwent ligation of the segments from the 9th intercostal to 2(nd) lumbar artery suffered paraplegia. In 3 non-paraplegic pigs, colored silastic agent injected into the preserved arteries was found to have covered a wider range. CONCLUSION: All the intercostal and lumbar arteries supplied blood to the anterior spinal artery. When large segments of intercostal and lumbar arteries were ligated, the blood flow from the preserved segments acquired increased dominance. The possibility exists that any intercostal and lumbar artery can supply blood to the spinal cord and become collateral circulation to the anterior spinal artery.

[Impact of multi-segmental aortic clamping and distal aortic perfusion on the prevention of postoperative paraplegia during thoracoabdominal aortic graft replacement]. / Multisegmentale Aortenabklemmung und distale Aortenperfusion zur Verhinderung der postoperativen Paraplegie bei thorakoabdomineller Aortenprothesenimplantation.

OBJECTIVE: We present the impact of multisegmental aortic clamping under distal aortic perfusion and segmental artery reimplantation on the prevention of postoperative paraplegia during thoracoabdominal aortic graft replacement. PATIENTS: During the last 14 years in 47 patients (age range: 22 to 82 years; average: 57,9 +/- 13,2 years; 16 females and 31 males) with thoracoabdominal aortic aneurysm a graft replacement was performed with adjuncts of normothermic partial bypass and multisegmental aortic clamping. As many patent segmental arteries as possible were reimplanted. RESULTS: Five patients died during hospitalization, for an in-hospital mortality rate of 10,6 %. In the elective patients (n = 40), the hospital mortality rate was 7,5 %. The average number of segmental aortic clampings per patient was 2,83 +/- 1,19 times. In 39 patients (82,9 %), 117 segmental arteries were reimplanted or preserved by beveled anastomosis. Eighty-three out of 117 segmental arteries (70,9 %) were located between TH9 and L2. Postoperative paraplegia/paraparesis did not occur in any patient. CONCLUSION: In view of our results reimplantation of as many segmental arteries as possible under multisegmental aortic clamping with adequate distal aortic perfusion can be recommended for effective prevention of spinal cord ischemia in TAAA surgery.

Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN-BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter.

GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine-containing neuroblastoma cell line SK-N-BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br-cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br-cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5'-flanking sequence. Cis-acting elements required for maximal basal and cAMP-dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK-N-BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1-like site and the other a combined CRE-Sp1-CCAAT-box element. EMSA and supershift assays demonstrated that the combined CRE-Sp1-CCAAT-box element recruits ATF-2 and NF-Y but not Sp1-4 or Egr-1-3. NF-Y binding was confirmed using pure recombinant human NF-Y protein. Transcription of the human GTPCH gene in human SK-N-BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF-Y and ATF-2.

One-stage operation for descending thoracic aortic aneurysm and left lung cancer: a case report.

We performed concomitant graft replacement for descending thoracic aortic aneurysm and pulmonary resection for squamous cell carcinoma of the left upper lobe in a 79-year-old man. The tumor reached the parietal pleura. No distance metastasis was found, and the tumor was diagnosed preoperatively as a stage IIB (N0, M0, T3) tumor. The descending thoracic aortic aneurysm was saccular, with greatest diameter being 55 mm, and extending from TH5 to TH8. A left upper lobectomy was performed, and after irrigation with a large volume of saline diluted with povidone iodine, graft replacement for the aortic aneurysm was performed under femoro-femoral partial bypass. To prevent postoperative graft infection, the greater omentum was dissected and placed over the resected pulmonary hilum and the graft. The patient's postoperative course was uneventful. There was no sign of infection, and the patient was discharged 1 month after surgery. Artificial graft wrapping with the greater omentum was useful for the prevention of the postoperative graft infection in this case of surgical treatment of lung cancer and descending thoracic aortic aneurysm.

Behavior, appetite, and urinary cortisol responses by adult female pigtailed macaques to cage size, cage level, room change, and ketamine sedation.

Pigtailed macaques (Macaca nemestrina) and longtailed macaques (M. fascicularis) show behavioral, ecological, and possible temperament differences, and their responses to the laboratory environment might therefore be quite different. We tested pigtailed macaques under the same conditions that were investigated in a previous study with longtailed macaques, using the same comprehensive set of physiological and behavioral measures of stress. First, eight adult females' adaptation to a new room in regulation-size cages was monitored, and in the third week their responses to ketamine sedation were measured. Then they spent two weeks singly housed in each of four cage sizes (USDA regulation size, one size larger, one size smaller, and a very small cage). Half of the subjects were in upper-level cages and the remainder in lower-level cages for the entire study. Cage size, ranging from 20% to 148% of USDA regulation floor area, was not significantly related to abnormal behavior, self-grooming, manipulating the environment, eating/drinking, activity cycle, cortisol excretion, or biscuit consumption. Locomotion and frequency of behavior change were significantly reduced in the smallest cage, but did not differ in cage sizes ranging from 77% to 148% of regulation size. The only manipulation to produce an unequivocal stress response, as measured by cortisol elevation and appetite suppression, was ketamine sedation. Room change and cage changes were associated with minimal cortisol elevation and appetite suppression. Wild-born females showed more appetite suppression after room change than captive-born females. No differences were related to cage level. Pigtailed macaques strongly resembled longtailed macaques except they showed weaker responses to the new room and cage change, probably because the pigtails had spent more time in captivity. These findings support the conclusion that increasing cage size to the next regulation size category would not have measurable positive effects on the psychological well-being of two species of laboratory macaques.

Antioxidant and hepatoprotective actions of the medicinal herb Artemisia campestris from the Okinawa Islands.

The antioxidant action of Artemisia campestris was examined in vitro and in vivo. A water extract of A. campestris showed a strong scavenging action of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Depression of the elevation of serum enzyme levels after CCl4-treatment was also observed by oral administration of the extract. In that case, CCl4-derived lipid peroxidation in the liver was decreased by the extract treatment. These results suggest that the extract of A. campestris scavenges radicals formed by CCl4 treatment resulting in protection against CCl4-induced liver toxicity.

Successful surgical treatment of impending rupture of thoracoabdominal aortic aneurysm in an elderly patient with severe pulmonary emphysema.

In a case of successful surgery for impending thoracoabdominal aortic aneurysmic rupture, an 83-year-old man with severe pulmonary emphysema was transferred to our hospital diagnosed with impending aneurysmic rupture. The aneurysm had been pointed out 2.5 years ago but surgical repair was not undertaken due to the patient's severe pulmonary emphysema. After admission, computed tomography showed an enlarging saccular thoracoabdominal aortic aneurysm. Emergency surgery was conducted because of severe pain below the left costal margin. We resected the wall of the saccular aortic aneurysm and reconstructed the aorta with an on-lay patch under femoro-femoral bypass and selective visceral organ perfusion. Tracheostomy provided respiratory care on the day following surgery. The patient was weaned from respiratory support 6 days after surgery. Postoperative aortography showed that the reconstructed thoracoabdominal aorta functioned satisfactorily. The patient remains in good health 18 months after surgery.

Two-stage operation for multiple aneurysms of the thoracic aorta, abdominal aorta, and left common iliac artery in an octogenarian.

Multiple aortic aneurysms are well described in the surgical literature. However, there are many problems related to surgical treatment of elderly patients with such aneurysms. This report presents the case, an octogenarian with multiple aortic aneurysms that were successfully treated by graft replacement. An 82-year-old man with a descending aortic aneurysm was referred to our institution for surgery. In addition to the previously diagnosed aneurysm, computed tomography and aortography showed an abdominal aortic aneurysm and a left common iliac aneurysm. Since the patient was an elderly man with chronic obstructive pulmonary disease, a two-stage operation was performed. The abdominal aortic aneurysm and left common iliac aneurysm were resected first due to the risk of thromboembolism from the abdominal aortic aneurysm during surgery involving replacement of the descending aorta under femoro-femoral (F-F) bypass. Fifty-two days after the first operation, a second operation was performed to repair the descending aortic aneurysm. The postoperative course was uneventful. Angiography after the operation showed satisfactory replacement of the multiple aortic aneurysms. The patient was discharged 25 days after the second operation.

Surgical treatment of traumatic aneurysm of the ascending aorta.

Traumatic aneurysm of the ascending aorta is a rare event. This case describes a patient with such an aneurysm, resulting from injuries received in a motorcycle accident. The patient was admitted to the emergency room of a local hospital complaining of chest pain, and was subsequently referred to our institution. On admission, a chest x-ray showed mediastinal widening. Computed tomography and aortography revealed an ascending aortic aneurysm and contusion of the upper lobe of the right lung. Due to concerns about bleeding from the lung contusion, surgery was delayed for one week. During surgery, intimal tears were detected at two sites in the ascending aorta. The wall of the ascending aorta was subsequently resected and a prosthetic graft inserted. The postoperative period was uneventful and a postoperative aortogram showed that the graft had molded well.

GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L-phenylalanine-induced increase in tetrahydrobiopterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by L-phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity.

GTP cyclohydrolase I gene expression in the brains of male and female hph-1 mice.

The hph-1 mouse is characterized by low levels of GTP cyclohydrolase I (GTPCH) and tetrahydrobiopterin. A quantitative double-label in situ hybridization technique was used to examine CNS GTPCH mRNA expression within serotonin, dopamine, and norepinephrine neurons of male and female wild-type and hph-1 mice. In wild-type male and female animals the highest levels of GTPCH mRNA expression were observed within serotonin neurons, followed by norepinephrine and then dopamine neurons. Wild-type female animals were found to express lower levels of GTPCH mRNA in each cell type when compared with levels seen in wild-type males. GTPCH mRNA abundance in all three cell types was lower in hph-1 male than in wild-type male mice, with the greatest reduction in serotonin neurons. GTPCH mRNA levels were also lower in hph-1 female than in wild-type female mice, again with the greatest reduction occurring in serotonin neurons. Comparison of hph-1 male and hph-1 female mice revealed that the sex-linked difference in GTPCH mRNA expression observed in wild-type neurons was only present within female dopamine neurons. Overall, these results indicate that not only are basal levels of GTPCH mRNA expression heterogeneous across wild-type murine monoamine cell types but that gene expression is also modified in a sex-linked and cell-specific fashion by the hph-1 gene locus. The hph-1 mutation does not lie within the GTPCH mRNA coding region. The 5' flanking region of the GTPCH gene was cloned and sequenced and shown to be identical for both wild-type and hph-1 genomic DNA. Transient transfection assays performed in PC12 cells demonstrated that this 5' flanking region was sufficient to initiate transcription of a luciferase reporter gene. Although the hph-1 mutation does not lie within the 5' flanking region of the GTPCH gene, this region of the gene can function as a core promoter and is thus crucial to the control of GTPCH gene expression.

Successful treatment of bilateral deep femoral aneurysms and multiple iliac aneurysms associated with severe aortic valve disease: report of a case.

We report herein the case of a patient in whom aneurysms of the bilateral deep femoral arteries (DFA) and multiple iliac aneurysms associated with severe aortic valve disease were successfully treated by a two-staged operation. The patient was a 74-year-old man who had dense calcification of the ascending aorta and aortic arch. Prior to aortic valve replacement (AVR), the aneurysms of the DFA and internal iliac arteries were resected. The terminal end of the abdominal aorta and bilateral common iliac arteries were then reconstructed with a Y graft to be used as a possible alternative arterial input route in place of the ascending aorta for extracorporeal circulation during the AVR. The inferior mesenteric artery (IMA) was well developed, and the external iliac arteries and their branches were preserved at aneurysmectomy. Postoperatively, there was no ischemia of the pelvic organs or the hip muscles. The AVR was subsequently performed 5 weeks after the first operation, and the patient was discharged after an uneventful postoperative course.

A ruptured syphilitic descending thoracic aortic aneurysm. The characteristic findings on computed tomography for the etiological diagnosis of aneurysm.

We report the case of a 72-year-old man with a ruptured syphilitic descending thoracic aneurysm who underwent an emergency operation and successful graft replacement. Preoperative physical examination showed a pulsative mass on the left back. Preoperative computed tomography showed bone destruction in the TH6 to TH10 thoracic vertebrae and ribs and penetration (or rupture) of the aneurysm into the subcutaneous tissue. During the period of preoperative evaluations, free wall rupture of the aneurysm occurred and emergency operation for graft replacement was performed. The microscopical examination of the aneurysmal wall revealed the syphilitic changes. In literature, the vertebral destruction by atherosclerotic aneurysm is usually located at the TH12 to L3 of vertebral bodies. From the findings of this patient and a study of existing literature, we concluded that the finding of vertebral bone beyond TH12 to L3 region on CT examination of the aneurysm could be a etiological characteristic finding for syphilitic aortic aneurysm.

Improvement in esophageal varices and liver histology postoperatively in Budd-Chiari syndrome.

BACKGROUND: In the past 17 years, 32 patients with Budd-Chiari syndrome were treated by reconstruction of the occluded inferior vena cava and reopening of the hepatic veins under femoro-femoral normothermic extracorporeal partial bypass. The mean follow-up was 8 years (range, 1.5 to 17 years). METHODS: To evaluate the benefits of our operative procedure, we compared the preoperative, early postoperative, and late postoperative endoscopic appearance of the esophageal varices and the histologic findings of the liver tissue obtained intraoperatively and at a later date. RESULTS: The esophageal varices found preoperatively in 29 patients (90.6%) had disappeared in 7 patients by the time of discharge, and in 2 patients they disappeared 4 to 7 years after surgery. In the remaining 20 patients, the grade of the esophageal varices was reduced markedly. Histologic examination of the liver showed cirrhosis in 22 patients, fibrosis in 9 patients, and severe congestion in 1 patient. Inspection of the liver in the late postoperative period (in 10 patients) showed improvement in centrilobular congestion and no increase in interlobular fibrosis. CONCLUSIONS: Gradual and steady improvement of esophageal varices and hepatic fibrosis can be achieved after our operative procedure.

Design of a novel P450: a functional bacterial-human cytochrome P450 chimera.

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.

Evaluation of cytochrome P450 mechanism and kinetics using kinetic deuterium isotope effects.

In this paper two hypotheses are tested: (i) the active oxygen species is similar in energetics for all cytochrome P450 (CYP) enzymes and (ii) linear free-energy relationships can be used to evaluate the mechanism of the reaction of these enzymes. A series of intramolecular isotope effects were determined and compared for CYPs 1A2, 2B1, 2C9, 2E1, and P450cam. The results indicate that the isotope effects are very similar for each of these isoforms of P450 and that the first hypothesis is likely to be true. Attempts to establish a linear free-energy relationship were only moderately successful: log Vmax = 0.11sigma+p + 1.73; r2 = 0.588. It was determined, through the use of intermolecular isotope effects, that the rates of hydrogen atom abstraction are masked. Thus, the second hypothesis is found to be false. This is likely to be a general result for CYP reactions, and linear free-energy relationships can only be used to determine the mechanism under very special circumstances. In all cases, the rate-limiting step should be evaluated with isotope effect experiments before any mechanistic conclusions can be drawn. If the intermolecular isotope effects are found to be masked, no mechanistic conclusion can be drawn from the linear free-energy relationship study.

Isolation of a promoter region in mouse cytochrome P450 3A (Cyp3A16) gene and its transcriptional control.

An 11.5 kb fragment of the mouse Cyp3a16 gene containing the 5' flanking region was isolated from the lambda DASHII mouse genomic library. A part of the 5' flanking region and the first exon of Cyp3a16 gene were sequenced. S1 mapping analysis showed the presence of two transcriptional initiation sites. The first exon was completely identical to Cyp3a16 cDNA. The identity of 5' flanking sequences between Cyp3a16 and Cyp3a11 genes was about 69%. A typical TATA box and a basic transcription element (BTE) were found as seen with other CYP3A genes from various animal species Moreover, some putative transcriptional regulatory elements were also found in addition to the sequence motif seen for the formation of Z-type DNA. To examine the transcriptional activity of Cyp3a11 gene, DNA fragments in the 5'-flanking region of the gene were inserted front of the luciferase structural gene, and the constructs were transfected in primary hepatocytes. The analysis of the luciferase activity indicated that the region between -146 and -56 was necessary for the transcription of CYP3a16 gene.