[Effects of colpotomic approaches on prognosis and recurrence sites of stage â a2-â ¡a2 cervical cancer after laparoscopic radical hysterectomy].

Objective: To investigate the factors affecting the prognosis of stage Ⅰa2-Ⅱa2 cervical cancer after laparoscopic radical hysterectomy (LRH), and to compare the prognosis and recurrence sites of patients with different colpotomy paths. Methods: The clinical data of 965 patients with stage Ⅰa2-Ⅱa2 cervical cancer who underwent LRH in the First Affiliated Hospital of Army Medical University from January 2015 to December 2018 were collected. The median age was 47.0 years of all patients with a median follow-up of 62 months (48-74 months). Cox regression was used to perform the univariate and multivariate analysis of the clinicopathological factors associated with the prognosis that included disease-free survival (DFS) and overall survival (OS). Patients were categorized into LRH through vaginal colpotomy (VC group, n=475) and LRH through intracorporeal colpotomy (IC group, n=490) according to the colpotomic approaches. The prognosis and recurrence sites of patients in each group were compared. Results: (1) During the follow-up period, 137 cases recurred (14.2%, 137/965) and 98 cases died (10.2%, 98/965). The 5-year DFS and OS were 85.8% and 89.9%, respectively. In univariate analysis, positive vaginal margin (PVM) was significantly affected the 5-year OS of patients with cervical cancer (P=0.023), while clinical stage, maximum diameter of tumor, degree of pathological differentiation, lymph node metastasis (LNM), depth of cervical stromal invasion, parametrium involvement, and uterine corpus invasion (UCI) were significantly associated with 5-year DFS and OS in patients with cervical cancer (all P<0.05). In multivariate analysis, clinical stage (HR=1.882, 95%CI: 1.305-2.716), LNM (HR=2.178, 95%CI: 1.483-3.200) and UCI (HR=3.650, 95%CI: 1.906-6.988) were independent risk factors of 5-year DFS (all P<0.001). Clinical stage (HR=2.500, 95%CI: 1.580-3.956), LNM (HR=2.053, 95%CI: 1.309-3.218), UCI (HR=3.984, 95%CI: 1.917-8.280), PVM (HR=3.235, 95%CI: 1.021-10.244) were independent risk factors of 5-year OS (all P<0.05). (2) Different colpotomy paths did not significantly affect the 5-year DFS and OS of patients with stage Ⅰa2-Ⅱa2 cervical cancer. The 5-year DFS in VC group and IC group were 85.9% and 85.6% (P=0.794), and the 5-year OS were 90.8% and 89.3% (P=0.966), respectively. Recurrence patterns consisted of intraperitoneal recurrence, pelvic recurrence, vaginal stump recurrence, and lymph node and distant metastasis. The intraperitoneal recurrence rate of VC group was significantly lower than that of IC group [0.6%(3/468) vs 2.3% (11/485), P=0.037], while the rates of pelvic recurrence, vaginal stump recurrence, lymph node and distant metastasis and overall recurrence were not significantly different between two groups (all P>0.05). Subgroup analysis of patients with different clinical stages, LNM and UCI showed that statistical differences of the intraperitoneal recurrence rates between two groups were only in patients without LNM (0.5% vs 2.3%, P=0.030) or without UCI (0.7% vs 2.3%, P=0.037). Conclusions: Clinical stage, LNM, PVM and UCI are independent risk factors for the prognosis of patients with stage Ⅰa2-Ⅱa2 cervical cancer. For patients without LNM or UCI, LRH through VC could reduce the intraperitoneal recurrence rate, while it is not enough to improve 5-year DFS and OS of patients. Low proportion of intraperitoneal recurrence, intra-operative tumor cells spillage to vagina stump and pelvic cavity might be the explanation.

Expression of breast cancer anti-estrogen resistance 1 in relation to vascular endothelial growth factor, p53, and prognosis in esophageal squamous cell cancer.

The purpose of this study was to clarify the role of breast cancer anti-estrogen resistance 1 (BCAR1) expression in relation to vascular endothelial growth factor (VEGF), p53, and proliferation in esophageal squamous cell cancer (ESCC). Expression of BCAR1, VEGF, p53, and the ki-67 proliferative index were examined by tissue microarray and immunohistochemistry in 106 specimens with ESCC and matched adjacent normal tissues. Among them, 40 cases were simultaneously examined by Western blot. Both Western blot and immunohistochemistry showed that BCAR1 expression was substantially higher in ESCC than in adjacent normal tissues (P < 0.001). BCAR1 expression was significantly connected with degree of tumor differentiation, with poorly differentiated tumors showing higher BCAR1 expression (P < 0.001). BCAR1 expression was significantly and positively correlated with VEGF and p53 expression levels (r= 0.541, P < 0.001; r= 0.374; P < 0.001) but not proliferative index (r= 0.44; P= 0.066). Additionally, a significant relationship was also observed between VEGF and p53 (r= 0.321; P= 0.001). Kaplan-Meier survival analysis revealed that patients with high BCAR1 expression had significantly shorter survival times than those with low BCAR1 expression levels (median survival 40 months vs. 27 months, P= 0.09). Multivariate analysis also revealed that levels of BCAR1 expression (hazard ratio 2.250, P= 0.015) was a significant and independent prognostic indicator. High expression of BCAR1 is associated with elevated VEGF and p53 expression levels, as well as poor prognosis in ESCC. Therefore, BCAR1 may be a potential candidate for predicting prognosis and a new therapy target for ESCC.

Reconstruction of hypopharyngeal and cervical esophageal defect after resection of hypopharyngeal carcinoma: a new technique based on the use of bilateral platysma myocutaneous flaps.

Commonly used procedures for reconstructing hypopharyngeal and cervical esophageal defects resulting from total laryngopharyngectomy (TL) are the gastric conduit or colon transposition as well as microvascularized free flaps. Herein we designed an alternative procedure utilizing bilateral platysma myocutaneous flaps (PMCFs) for the reconstruction of hypopharyngeal and cervical esophageal defects. This report summarizes the technical description of this procedure. TL and cervical esophagectomy were performed and bilateral PMCFs were harvested for reconstruction of hypopharyngeal and cervical esophageal defects in 25 patients aged between 46 and 73 years (mean 58.7 ± 16.2 years). All these patients had advanced-stage (IV) cancer with involvement of the cervical esophagus. Operative time ranged from 176 to 382 minutes (average 243 ± 91 minutes) and the mean intraoperative blood loss was 294 ± 119mL. There were six cases of anastomotic leak (24.0%) and two of them (8.0%) developed anastomotic stricture. Neither flap necrosis nor postoperative death was observed. The majority of our patients (68.0%) were restored to a normal unrestricted oral diet after surgery. The 3-year and 5-year actuarial survival rates were approximately 54.7% and 26.1%, respectively. We conclude that reconstruction of the cervical esophagus with bilateral PMCFs is a valuable method for treating advanced hypopharyngeal carcinoma.

Management of delayed intrathoracic esophageal perforation with modified intraluminal esophageal stent.

In this article, we reviewed our experience of treatment of the delayed intrathoracic nonmalignant esophageal perforation employing modified intraluminal esophageal stent. Between February 1990 and August 2006, eight patients were included in this study. Five patients experienced sepsis. The interval time between perforation and stent placement ranged from 36 h to 27 days (average, 8.6 days). Esophageal stenting and throracotomy for foreign body removal were performed in four patients. The remaining four patients underwent stent placement and thoracostomy. Nutrition was initiated through gastrostomy after 7 to 10 days after the stenting. The stent was removed after the patients resumed oral intake of food and the esophagogram showed that perforation was closed. There was no death in this group. Signs of sepsis remitted 1 week after stent placement. Complications included stress ulcer, stimulative cough, and pneumonia each. Stent removal ranged 32 to 120 days (average 66.7) after its placement. The stent was kept in place for 4 months to prevent formation of esophageal stricture in one patient with caustic esophageal burns. The follow-up was completed in all the patients. The mean follow-up period was 59 months (range 12-180). One patient with caustic esophageal burn underwent cicatricial esophagectomy and gastric transposition 3 years later due to the esophageal stricture. Barium swallow demonstrated that there was a diverticulum-like outpouching in one patient and slight esophageal stricture at T2 and T3 level in another. One patient developed reflux esophagitis 5 years after stent removal. All the patients finally had a normal intake of food. Modified esophageal stenting is an effective method to manage the delayed intrathoracic esophageal perforation. Prevention of stent migration and its convenient adjustment might be the major advantages of this method.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

ZNRD1 might mediate UV irradiation related DNA damage and repair in human esophageal cancer cells by regulation of ERCC1.

The downregulation of zinc ribbon domain-containing 1 (ZNRD1) protein was recently found to partially reverse the resistance of human leukemia cells toward chemical therapeutic drugs. Therefore, the ZNRD1 protein might be involved in the process of DNA damage and repair. To explore the possible protective effects of ZNRD1 on DNA damage induced by ultraviolet (UV)-C irradiation in human esophageal squamous cancer cell line EC109, we designed and transfected a expression vector into EC109 cells, and established an overexpression cell line. The single-cell gel electrophoresis (comet assay) was used to investigate the DNA damage and repair in UV-C-irradiated control and transfected cells. It was found that the ZNRD1-expressing cells exhibited a significant enhanced DNA repair capacity. Moreover, the overexpression of ZNRD1 could upregulate the expression of excision repair cross-complementing 1 (ERCC1) gene. Collectively, these findings suggested that ZNRD1 might play an important role in the process of DNA damage and repair by regulating the expression of ERCC1.

Prevention and management of complications after colon interposition for corrosive esophageal burns.

We present our experience in the management of complications after a colon interposition for corrosive esophageal burns. From April 1976 to December 2006, 85 patients with caustic esophageal burns were included in this study. The superior belly median incision with an anterior border incision of the left sternocleidomastoid was used. Anastomosis between the colon and the cervical esophagus was performed in 68 and between the colon and pharyngeal portion in 14 patients. An esophageal scar part resection and gastric-esophageal anastomosis was performed in one patient who had been given an unsuccessful colon and jejunum interposition at another institute. An anastomotic modeling operation was performed in one patient with anastomotic stricture who had been managed with colon interposition at another institute. Exploratory thoracotomy and gastrostomy was performed in one patient who had an unsuccessful colon interposition at another institute. Seven of 14 patients (8.5% of 17.1%) died with serious complications such as aspirated pneumonia, interposition colon necrosis, abdominal wound dehiscence and degradation of swallowing and concordance function. However, others with such serious complications survived and were discharged for rehabilitation after corresponding treatment. The 25 patients (30.1%) with other mild complications were discharged for rehabilitation and corresponding management. Two patients from other institutes were discharged for rehabilitation and one was lost to follow-up. The most dangerous complication of this procedure is colon necrosis, and the stomach is the best organ for re-operation. Otherwise, aspiration in infants due to hypoplasia and degradation of swallowing co-ordination needs attention. Peri-operative management is very important, including the control of mediastinal and pulmonary infection and systemic nutritional support to avoid abdominal wound dehiscence. The platysma flap is an excellent method for the treatment of anastomotic stricture.

Antimitotic and antitubulin activity of the tumor inhibitor steganacin.

Steganacin, a newly isolated tumor inhibitor, completely inhibits cleavage in sea urchin eggs at 3 X 10(-7) M by preventing the formation of the mitotic apparatus. Steganacin inhibits the polymerization of tubulin in vitro and also causes a slow depolymerization of preformed microtubules. Optical ultracentrifuge studies of steganacin-treated tubulin show a small reduction in 20 S and 30 S peaks at 0 degree. In electron microscope studies the ring structure of tubulin is seen at 0 degree but disappears if the temperature of tubulin incubated with steganacin is raised to 37 degrees. Steganacin inhibits the binding of colchicine to tubulin and thus resembles podophyllotoxin, which also competitively inhibits colchicine binding. Steganacin had a trimethoxybenzene ring and probably interacts with that portion of the colchicine-binding site that recognizes the trimethoxybenzene ring of colchicine.

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1-1: microconformational changes induced by S-hexyl glutathione.

The glutathione S-transferase (GST) isoenzyme A1-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

Economic reforms and the acute inpatient care of patients with schizophrenia: the Chinese experience.

OBJECTIVE: This study compared insured and uninsured schizophrenic inpatients in China and examined changes in the acute inpatient care of schizophrenic patients during China's economic reform era. METHOD: Detailed chart reviews of 50 randomly selected inpatients discharged from a hospital in central China each year from 1984 through 1993 identified 321 patients with schizophrenia. Demographic, insurance, treatment, and cost data of these patients were collected from the charts. RESULTS: With logistic regression models to control for confounding variables, the analyses showed that the 129 insured patients were significantly more likely than the 192 uninsured patients to be urban residents, to be older, to have had 7 or more years of schooling, and to have had more psychiatric hospitalizations; moreover, their index admissions were longer and were more likely to include use of traditional Chinese medications. The estimated 19% of schizophrenic individuals in the community with health insurance receive inpatient treatment 2.8 times more frequently than the 81% without insurance. Compared to admissions in 1984-1988, admissions in 1989-1993 were significantly shorter and involved longer periods of polypharmacy with multiple antipsychotic medications but included lower mean chlorpromazine-equivalent doses of medication. The relative cost of inpatient care for an acute episode of schizophrenia increased 3.5-fold over the 10-year period, from 11% of mean annual household income in 1984 to 37% in 1993. CONCLUSIONS: Changes in the incentive system for care providers and rapid increases in the cost of care during the economic reform era have resulted in increasingly restricted availability of services for the many schizophrenic patients without health insurance.

Structural requirements for antileukemic activity among the naturally occurring and semisynthetic maytansinoids.

In an effort to determine the structural requirements for the significant antileukemic, cytotoxic, antitubulin, and antimitotic activity exhibited by the novel ansa macrolide, maytansine (1), four new C-3 ester and six new C-9 ether homologues were synthesized. The biological activities of these compounds were assayed and compared to the activities of previously reported, naturally occurring maytansinoids. From the data, it is apparent that presence of the C-3 ester is necessary for significant activity, and variations in the ester group are not accompanied by marked changes in activity. However, elimination of the ester group, as in maytansinol (7), maysine (8), normaysine (9), and maysenine (10), results in a significant decrease in biological activity. Blockage of the C-9 carbinolamide via etherification markedly reduces antileukemic and cytotoxic activity and slightly reduces antitubulin activity but has relatively little effect on antimitotic activity against sea urchin eggs. Thus, a free carbinolamide at C-9 is advantageous for optimal activity.

Effects of the immunosuppressant FK-506 and its analog FK-520 on hepatic and renal cytochrome P450 mixed-function oxidase.

The novel immunosuppressant FK-506 and its analog FK-520 were found to inhibit the hepatic microsomal mixed-function oxidase system in male Sprague-Dawley rats. At 5 and 10 mg/kg/day, s.c., for 6 days they caused 30-80% decreases in cytochrome P450 levels, NADPH-cytochrome P450 reductase, and benzphetamine N-demethylase activities. The metabolism of FK-506 itself was inhibited by 50%. FK-506 and FK-520 had a minimal effect on the renal cytochrome P450 levels unlike cyclosporin A which produced a 67% increase after six daily 25 mg/kg doses. A single dose of FK-506 (25 mg/kg, s.c.) had a minimal effect on the hepatic or renal metabolizing enzyme system. In vitro, addition of FK-506 and FK-520 to human and control rat liver microsomes resulted in a concentration-dependent inhibition of benzphetamine N-demethylation (10-20% at 50 microM, 60-75% at 250 microM). We suggest that in view of its potential to inhibit hepatic cytochrome P450-dependent mixed-function oxidase, resulting in the inhibition of its own metabolism, FK-506 should be administered with caution to transplant patients.

Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes.

The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3''-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3''-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).

[Stimulative effect of electro-blunt-tip needle on the points of human skin and its analgesia in rats].

The stimulative effect of electro-blunt-tip needle (a kind of electroacupuncture stimulating on the surface of skin point) on the 120 points of human skin was observed in 12 healthy volunteers. It was shown that the substitute typical electro-blunt-tip needle stimulator and its special pen-like electrode by a general electroacupuncture stimulator and a tape-like adjustable electrode and similar therapeutic effects. It was found in animal experiment that the pain threshold determined with rat tail flick analgesia method was raised by electroacupuncture and electro-blunt-tip needle, the optimal stimulating time being 10-20 minutes for both, the percentages of the maximal changing pain threshold being 206.6% and 175.4%, and the corresponding voltage being 2v and 7v respectively.

Management of achalasia with transabdominal esophagocardiomyotomy and partial posterior fundoplication.

In this article we present our experience in the management of achalasia. From May 1988 through August 2005, 71 patients with achalasia underwent transabdominal esophagocardiomyotomy and partial posterior fundoplication. Barium swallow, manometry, and 24-h pH studies were performed in all patients preoperatively. Manometry and 24-h pH monitoring were only carried out in 58 patients at the third post-operative week and in 43 patients during follow-up, even though 52 patients were included in the follow-up. There were no operative deaths or complications. All the 71 patients were able to eat semifluid or solid food without dysphagia and heartburn at discharge. Esophageal barium studies showed that the maximum esophageal diameter decreased 2.2 cm and the minimum gastroesophageal junction diameter increased 8.4 mm after operation. Manometry examination in 58 patients revealed that the lower esophageal sphincter resting pressure decreased 15.0 mmHg in the wake of the procedure. Twenty-four hour pH monitoring demonstrated that reflux events were within the normal post-operative range. Fifty-five of the 58 patients had normal DeMeester scores. Among the patients with a mean 90-month follow-up, 49 patients had normal intake of food without reflux, the remaining three had mild dysphagia without requiring treatment. All the patients resumed their preoperative work and social activities. The manometry and 24-h pH studies in the 43 patients showed there were no significant changes between the third post-operative week and during follow-up. Transabdominal esophagocardiomyotomy and posterior partial fundoplication are able to relieve the functional outflow obstruction of the lower esophageal sphincter, obviate the rehealing of the myotomy edge and prevent gastroesophageal reflux in patients who have undergone myotomy alone.

Visualization and elution of unstained proteins from polyacrylamide gels.

Proteins fractionated by electrophoresis on 18% polyacrylamide gels with low crosslinking can be directly visualized by ultraviolet light-induced fluorescence and can be recovered by electroelution.

Inhibitory anti-peptide antibody against human CYP3A4.

An inhibitory anti-peptide antibody was raised against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ) corresponding to residues 253-273 of human cytochrome P450 3A4. High titer antibodies were produced by rabbits immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA. Anti-peptide antibody recognized a single protein band in microsomes prepared from cells expressing recombinant human CYP3A4 in immunoblotting analysis. No immunodetectable proteins were found in microsomes containing other cytochrome P450 isoforms. In addition, the antibody did not recognize CYP3A5, a closely related isoform in the CYP3A family. In human liver microsomes, only one protein band which comigrated with human CYP3A4 was recognized by this antibody and the relative blotting intensity of this protein band correlated significantly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase activities (r = 0.96). More importantly, this antibody exhibited greater than 90-95% inhibition of testosterone 6 beta-hydroxylation, while other cytochrome P450-mediated reactions in human liver microsomes were not inhibited. Because of its specificity and inhibitory potency, this anti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.

Instrumental noise effect in an optical heterodyne profiler.

An optical heterodyne profiler has been developed for measuring surface roughness at Brookhaven National Laboratory. The height measurement sensitivity and lateral resolution are 1.1 Å and 4 µm, respectively, when a 40× objective is used. A Zeeman-split He-Ne laser is the light source. A noncontact measurement system is designed as an optical common-path interferometer. Optical and electronic common-mode rejection techniques are employed to minimize the effects of environmental conditions. The effect of the system noise is analyzed in detail. The effect of varying the number of samples at each sampling point is shown. The comparisons of the system noises with different objectives, 5×, 10×, 20×, and 40×, are presented.

Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli.

A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259, 5182-5188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using MaeI endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tac promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Bal31 to remove extra nucleotides from the 5' noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cytosol is apparently blocked.