Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

1. The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.

2. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.

3. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.

4. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K_m value was relatively comparable to that for marmoset livers.

5. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

     

Topically injected adrenocorticotropic hormone induces mechanical hypersensitivity on a full‐thickness cutaneous wound model in rats

Cutaneous wound pain causes physical and psychological stress for patients with wounds. Previous studies reported that stress induces hyperalgesia and deteriorates wound healing. However, the effect of the stress response such as in hypothalamic‐pituitary‐adrenal (HPA) axis on local wound area is unclear. We aimed to investigate the effects of a stress response on the mechanical withdrawal threshold in the local wound area and describe the identification of a wound pain exacerbation. We topically injected adrenocorticotropic hormone (ACTH) into the granulation tissue of full‐thickness cutaneous wound model rats on the fifth day postwounding and measured the mechanical withdrawal thresholds, cytochrome P450 2Bs levels and concentration of 5,6‐epoxyeicosatrienoic acid in wound exudate. We found that ACTH induced mechanical hypersensitivity at 4 and 6 hours after injection (P = .004 and .021, respectively), and increased gene expression of cytochrome P450 2B12 expression (P = .046). Concentration of 5,6‐EET in the wound exudate was moderately correlated with the mechanical withdrawal threshold (r = ?.630). Finally, the mechanical withdrawal threshold in the 5,6‐EET group was significantly lower than that in the control group at 2 hours after the injection (P = .015). We propose that 5,6‐EET is one of the most promising contributors to the wound pain exacerbation. These findings could guide clinical wound and pain management.     

Impact of mediastinal lymph node dissection on octogenarians with non-small cell lung cancer

Objective: Impacts of mediastinal lymph node dissection on a patient’s course after pulmonary resection is unclear in octogenarians with non-small cell lung cancer. Methods: Retrospectively identified subjects included 39 octogenarians and 1 nonagenarian, with grades according to the Charlson Comorbidity Index ranging from only 0 to 2. We performed mediastinal lymph node dissection in 19 patients (D group), and just lymph node sampling biopsy in the other 21 (S group). We compared clinicopathologic features and outcome after surgery between both groups. Results: Deterioration of performance status at the time of discharge, evident in 17 patients overall, was significantly more frequent in the D group. Postoperative complications occurred in 27 patients overall and there was no significant difference between the two groups. Survival rates in younger patients at 1, 3, and 5 years were 86, 59, and 49%, respectively; in octogenarians these were 83, 58, and 42% (no significant difference). Nor did survival differ significantly by surgical management of mediastinal lymph nodes; 1-, 3-, and 5-year survival rates were 94, 63, and 40%, respectively in the D group and 78, 66, and 43%, respectively in the S group. Conclusion: Octogenarians with non-small cell lung cancer should be treated by urgent pulmonary resection whenever possible. Since mediastinal lymph node dissection has little effect on long-term survival or the carried risk of worsening performance status at discharge, pulmonary resection without complete mediastinal lymph node dissection should be considered.     

Cerebral perfusion scintigraphy in patients with cerebrovascular disease by using 99mTc-ECD: comparative study with 123I-IMP SPECT

99mTc-ECD SPECT was performed in 19 patients with cerebrovascular disease, and location, extent, and severity of the lesions on 99mTc-ECD SPECT were compared with those on 123I-IMP SPECT. The initial brain uptake was 5.5 +/- 0.7% of the injected dose at 10 minutes after injection, 5.3 +/- 1.3% at 90 minutes, and clearance from the brain is slow. The distribution in the brain was changed, especially washout from the thalamus was slower than that from other regions. The count ratio of perfusion defect to normal area (D/N) on 99mTc-ECD SPECT was unchanged over the time, and had no significant differences from that on 123I-IMP SPECT. 99mTc-ECD SPECT was superior in detection of the lesion in the basal ganglia, and showed the images with superior spatial resolution due to physical characteristics of 99mTc. However, mild ischemic lesion and peri-infarct area was not clearly visualized, while 123I-IMP SPECT could demonstrate these lesions with better contrast.     

Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells

Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity.     

Characterization of estrogen receptor in human gastric cancer

Estrogen receptors (ER) were examined in cytosol, nuclear potassium chloride (KCl) extractable fraction, and nuclear KCl unextractable fraction by the dextran-coated charcoal adsorption method in various gastric cancer tissue. The overall ER-positive rate in the cytosol and nuclear fraction was 19.2%. The maximum binding site (Bmax) was 36.0 to 175.0 fmol/mg of protein, and the dissociation constant (Kd) was 0.6 to 1.6 X 10(-9) in cytosol fraction. In the nuclear fraction, Bmax was 7.5 fmol/mg of DNA and Kd was 2.3 X 10(-9). Estrogen receptors were characterized in cytosol protein. In cytosol, the estrogen (E2)-ER complex was sedimented at approximately the 5S and 8S regions by 5% to 20% linear sucrose gradient centrifugation. A steroid specificity study of ER showed the presence of an binder in gastric cancer tissue. In conclusion, these results that gastric cancer tissue has E2 binding sites with the same biochemical characteristics as in breast cancer and endometrial cancer strongly suggest the hormonal dependency of gastric cancer.     

Enhancement by sulpiride of the inhibitory effects of cysteamine on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of sulpiride on cysteamine inhibition of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on the BUdR labelling index of gastric mucosa were investigated in inbred Wistar rats. After 25 weeks of oral treatment with MNNG, rats received one of the following alternate-day injections: cysteamine (2 doses), cysteamine (2 doses) plus sulpiride or sulpiride. At week 52, prolonged administration of cysteamine significantly reduced the incidence of adenocarcinomas of the glandular stomach. Cysteamine at low dose had no effect on the incidence of gastric cancers, but a combination of low-dose cysteamine and sulpiride caused a significantly greater reduction in the incidence of gastric cancers. Administration of sulpiride alone had no influence on gastric carcinogenesis. The labelling index of the antral mucosa was significantly lower in rats treated with high but not low doses of cysteamine. However, a combination of low-dose cysteamine and sulpiride significantly decreased the labelling index of the antral mucosa. Our findings indicate that cysteamine suppressed gastric carcinogenesis and that sulpiride enhanced this inhibition. Because sulpiride is a dopamine antagonist, these findings also indicate that dopamine may play an important role in cysteamine inhibition of gastric carcinogenesis.     

Lectin-cytochemical study on epithelial mucus glycoprotein of conjunctiva and pterygium

The epithelium of pterygium and conjunctiva was studied with reference to cytochemical reactivity to six fluorescein-labeled lectins that recognize a certain carbohydrate residue(s) of cellular membrane-bound or secretory glycoprotein: Ulex europaeus agglutinin-1 (UEA-1, specific for fucose); Dolichos biflorus agglutinin (DBA, specific for N-acetylgalactosamine); peanut agglutinin (PNA, specific for galactose-beta 1-3N-acetylgalactosamine): wheat germ agglutinin (WGA, specific for N-acetylglucosamine and N-acetylneuraminic acid); Concanavalia ensiformis (Con A, specific for mannose); Ricinus communis agglutinin-1 (RCA-1, specific for galactose). Non-goblet epithelial cells of pterygium were labeled with UEA-1, DBA and PNA, while those of conjunctiva were not. Distribution density of goblet cells was larger in pterygium than in conjunctiva, but there was no distinct difference in lectin reactivity between the two tissues, with marked label with WGA, PNA and RCA-1. Con A did not bind to either pterygium or conjunctiva. The observations suggest the presence of anomalous mucus glycoproteins secreted from pterygium.     

The effects of oral adsorbent (AST-120) in the experimental model of chronic renal failure--pathophysiological study on renal function, glomerular hypertrophy, mesangial function and glomerular histology

Oral adsorbent (AST-120) reduces blood levels of urea and creatinine in experimental studies. It has also been shown to retard the progression of chronic renal failure in clinical studies. In the present study, the effect of AST-120 was examined in the rat model of subtotal nephrectomy (sNPX). This experimental model of chronic renal failure is characterized by glomerular hyperfunction, glomerular hypertrophy, increased mesangial trapment of macromolecules and subsequent glomerular sclerosis. We report the effect of AST-120 on glomerular hyperfunction, glomerular hypertrophy and mesangial trapment of macromolecules in the early stage and glomerular function and histology in the late stage of the rat model of sNPX. From 2 days after sNPX, rats were fed regular rat chow with (AST group: AST) or without (control) AST-120. At 2 weeks, iron dextran (ID) was injected intravenously. Three days after the injection, mesangial trapment of ID was largely ameliorated in AST when compared with control (p less than 0.02). The value of mean planar area of glomerulus (PAmean) in AST was significantly lower than that in control (p less than 0.05). At 2 and 9 weeks, the values of GFR and RPF in AST were all statistically higher than those in control. At 9 weeks, whereas average glomerular sclerosis index (SI: 0-4 scale) was 1.07 in control, significantly lower SI (0.57) was noted in AST (p less than 0.05). Thus, AST-120 has effects on glomerular hypertrophy, increased mesangial trapment of macromoleculus and finally the progression of chronic renal failure in the rat model of sNPX. The effects are not through reducing glomerular hyperfunction.(ABSTRACT TRUNCATED AT 250 WORDS)