Cell cycle and apoptotic proteins in relation to ovarian epithelial morphology.

OBJECTIVE: To assess the relationship between the expression of cell cycle and apoptotic proteins and the morphological appearance of the surface epithelium in non-neoplastic ovaries. METHODS: The subjects for this study were 79 women who had undergone oophorectomy for benign conditions at the North Middlesex Hospital, London, and Royal Free Hospital, London, and whose ovaries had been reported on routine histology as entirely normal or containing physiological cysts or endometriosis. The epithelial morphology was reassessed on haematoxylin and eosin-stained paraffin wax sections using nine cytological and architectural parameters associated with premalignant intraepithelial changes. A 'score' was obtained for each ovary. Expression of p53, Ki67, cyclin D1 and Bcl-2 in the surface, cystic and endometriotic epithelium was assessed in corresponding sections using standard immunohistochemistry. RESULTS: The median score for the morphological changes was significantly higher in the sections, which expressed p53 compared to those which did not. This difference remained significant in a subanalysis of the sections, which did not contain endometriosis. No relationship was identified between the morphological score and the expression of Ki67, Bcl-2 and cyclin D1. CONCLUSION: Increased intraepithelial abnormality as assessed by an epithelial morphological score of ovarian sections is associated with expression of the p53 cell cycle protein. This lends credence to the hypothesis that the ovarian surface or cystic epithelium goes through an identifiable precursor or "premalignant" phase before the development of invasive disease. Further work is required to characterise the changes that take place before the development of malignancy in ovarian epithelium.

Diaphragm disease of the small bowel--a case report and literature review.

A 75-year-old man who had an emergency laparotomy for small bowel obstruction was found at operation to have multiple mid-ileal strictures. Histology of the resected specimen confirmed diaphragm disease of the bowel. The pathogenesis of this disease remains unclear but it is associated with long-term use of NSAID. Diagnosis is often difficult as many clinicians are unaware of this condition. The relevant literature has been reviewed.

Ovarian epithelial dysplasia in relation to ovulation induction and nulliparity.

OBJECTIVE: The goal of this study was to assess the relationship between ovulation induction, nulliparity, and ovarian epithelial dysplasia. METHODS: This retrospective cohort study was performed in one teaching and one district general hospital in London. The subjects, 83 women who had undergone hysterectomy and bilateral oophorectomy and whose ovaries were reported as "normal," were divided into three groups: ovulation induction (13), nulliparity (20), and fertile controls (50). These ovaries were independently reviewed by two pathologists who assigned a score of 0, 1, or 2 to nine epithelial cytological and architectural features. The main outcome measure was the total dysplasia score, which was used to quantify the degree of ovarian epithelial abnormality in the three groups. RESULTS: The mean dysplasia score was significantly higher in the women who had undergone ovulation induction than in the fertile controls (7.92 vs 5.70, P = 0.012). The magnitude of the difference between the ovulation induction group and controls remained similar after adjusting for age, parity, and duration of oral contraceptive use (2.17, 95% CI: -0.11-4.44). However, the statistical significance of this difference was reduced (P = 0.062). We did not find any evidence of a difference in dysplasia score between nulliparous women and controls, neither before (P = 0.85) nor after adjusting for age and duration of oral contraceptive use (P = 0.87). CONCLUSIONS: These results suggest a possible association between ovarian epithelial dysplasia and ovulation induction therapy, in accord with previous reports of increased risk of ovarian cancer in women with a history of fertility treatment. The higher dysplasia score could be attributable to the drugs used to induce ovulation or to a genetic susceptibility to ovarian cancer.

Determination of sulfadoxine in human blood plasma using packed-column supercritical fluid chromatography.

A sensitive, rapid, selective and reproducible method has been developed to measure plasma levels of sulfadoxine, 4-Amino-N-(5, 6-dimethoxy-4-pyrimidinyl) benzensulfonamide; in healthy, human volunteers using packed-column super-critical fluid chromatography. Omeprazole, 5-methoxy-2-[[(4-methoxy-3, 5-di-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole; was used as the internal standard (i.s.) at 15.0 microg/ml. The drug and the i.s. were extracted from plasma using dichloromethane. Separation of sulfadoxine and i.s. was done on a Nucleosil (250x4.6 mm) 10 microm, RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml/min) as the mobile phase. The column temperature was 40 degrees C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 265 nm. The limit of quantification was 0.50 microg/ml using 1 ml plasma specimen. The mean extraction recovery of the drug from plasma was found to be 94.9%. The SFC method was directly compared to a published HPLC/UV method. With respect to speed and use of organic solvents SFC was found to be superior; while in all other aspects the results were similar to the published technique. The method has been successfully used to estimate the sulfadoxine levels in healthy human volunteers from 0 to 240 h following an oral dose of 500 mg of sulfadoxine in combination with 25 mg of pyrimethamine.

Assay of tolnaftate and related impurities by isocratic supercritical fluid chromatography.

Tolnaftate, an antifungal drug (TF) and related impurities arising from synthesis, viz., N-methyl-m-toluidine (NMmT) and beta-naphthol-1-chlorothio carbamate (beta-NCTC) can be determined by supercritical fluid chromatography. Even though it was possible to elute TF completely with neat SCF CO2, the peaks of the impurities were found to merge. The chromatographic figures of merit of the three analytes such as retention time (tR), capacity factor (k), selectivity factor (alpha), no. of theoretical plates (N), were optimized. The three compounds can be resolved in 5 min on a Hypersil (250 x 4.0 mm) 5 mu, C18 column with supercritical carbon dioxide, modified with 1.96% methanol as the mobile phase at 9.81 MPa and at 40 degrees C. Detection was carried out at 220 nm. The data as evaluated by the linear regression least squares fit method gave linearity ranges from 0.2 to 10.0 microg/mL for TF and NMmT and 0.3 to 10.0 microg/mL for beta-NCTC with correlation coefficients > 0.99. The method was successfully employed to estimate levels of 0.01% for NMmT and 0.02% for beta-NCTC with respect to TF.

Application of packed column supercritical fluid chromatography to the simultaneous determination of seven anticonvulsant drugs.

Studies of speed, resolution, and selectivity have shown that packed column supercritical fluid chromatography (PCSFC) is a viable technique for the isocratic, isothermal and isobaric separation of seven anticonvulsants, viz., phenobarbitone, phenytoin sodium, phethenylate sodium, nitrazepam, clonazepam, carbamazepine, and primidone, and their simultaneous estimation. The drugs were eluted from a JASCO, RP-C(18) (250 x 4.6 mm) 10 micro packed column with a binary mobile phase of carbon dioxide and methanol, using ibuprofen as the internal standard. The effect of pressure, temperature, modifier concentration, and the rate of flow of CO(2) on retention and selectivity of all the analytes were studied and the parameters optimised. Without methanol in the mobile phase none of the solutes eluted. Changing modifier concentration was the most effective physical parameter for changing retention and selectivity. The analytes were detected using a UV detector at 215 nm. An arbitrary mixture of eight components was baseline resolved in approximately 7 min. The study includes a successful attempt at quantification of the drugs. Chromatographic and analytical figures of merit have been listed. The present work holds promise for a possible replacement of HPLC with SFC for the separation and assay of drugs of different families.

Separation and estimation of seven vasodilators using packed column supercritical fluid chromatography.

This paper reports a method for an isocratic separation and simultaneous estimation of seven vasodilators: isosorbide mononitrate (ISMN), isosorbide dinitrate (ISDN), cyclandelate, nimodipine, amlodipine, pentifylline and pentoxifylline using packed column supercritical fluid chromatography (SFC). An arbitrary choice of vasodilatory compounds with respect to their chemical structures was made to examine the viability of this technique for analysis of drugs and pharmaceuticals. Elution was performed on a RP-C18 column. SFC offers several degrees of freedom: temperature, pressure and modifier concentration to attain optimum resolution and sensitivity. The effects of these parameters on retention time have been studied using methanol modified carbon dioxide. The analytes were identified and measured by UV-detection. The chromatographic points of merit have been listed. Detection limits appear to be similar to those found in liquid chromatography. Modifier concentration does generally make major changes in retention and selectivity. A full scale validation for the seven vasodilators has been attempted and the statistical quality evaluated.

Isocratic, simultaneous reversed-phase high-performance liquid chromatographic estimation of six drugs for combined hypertension therapy.

We report an isocratic, HPLC procedure for assay of the orally administered hypertension drugs [atenolol, amlodipine, nifedipine, nitrendipine, nimodipine and felodipine given in retention order] of which atenolol, an aryloxypropanolamine beta-blocker is administered with anyone of the other five dihydropyridine calcium channel blockers in combined hypertension therapy. The drugs were dissolved in methanol and 20 microliters of a mix of the drugs was injected onto a reverse phase JASCO-metaphase ODS (250 x 4.0 mm) 5 mu column. Any one of the six drugs could be used as the internal standard. The drugs were resolved by elution with a pH 4.5 equivolume mobile phase of acetonitrile-0.01 M KH2PO4, with pH adjustments done with H3PO4 (flow-rate 1.5 ml min-1). The column effluent was monitored at 250 nm. The detector response (peak height ratio) was linear in the dynamic range of 25-3200 ng ml-1 of these drugs, with the detection limits at approximately 15 ng ml-1. Full statistical evaluation of the data including linear regression (least-square fit) analysis was performed. The suggested procedure has the advantage that all the five dihydropyridine derivatives can be quantified alone or in formulation with atenolol.

Packed column supercritical fluid chromatographic separation and estimation of acetaminophen, diclofenac sodium and methocarbamol in pharmaceutical dosage forms.

A reproducible and fast method has been developed for the assay of acetaminophen, methocarbamol, and diclofenac sodium in bulk and drug forms using packed column supercritical fluid chromatography employing internal standard method. The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 11.1% (v/v) methanol on a Shendon-Phenyl (250x4.6 mm) 5 mum column with detection monitored spectrophotometrically at 225 nm. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on chromatograhic figures like retention time (t(R), min), retention factor (k(')) etc. Modifier concentration proved to be the most effective means for changing both retention and selectivity. Calibration data and recovery of the drug from spiked concentrations were determined to assess the viability of the method. The supercritical fluid chromatography (SFC) method was directly compared to an HPLC assay, developed in the laboratory, of the same analytes. With respect to speed and use of organic solvents SFC was found to be superior, while in all other aspects the results were similar to HPLC. The method has been successfully used for the assay of two formulations containing a combination of (A) acetaminophen and methocarbamol and (B) acetaminophen and diclofenac sodium. There was no interference from excipients. The present work validates the recent proposition that supercritical fluid chromatography using CO(2) and modifiers is a viable, faster alternative to reverse phase HPLC.

A comparative study using HPLC and packed column supercritical fluid chromatography for the assay of three anti-psychotic dosage forms.

A reproducible and selective method was developed for the analysis of three anti-pschycotics, i.e. haloperidol, trifluoperazine and trihexyphenidyl in bulk and dosage forms using packed column supercritical fluid chromatography (SFC). The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 16.67% (v/v) methanol containing 0.8% isopropylamine. Parallel studies were performed by HPLC using ion pairing reagent and a comparison is discussed. The method was successfully used for the assay of three formulations containing a combination of: (1) haloperidol-trihexyphenidyl; (2) haloperidol-trifluoperazine; (3) trifluoperazine-trihexyphenidyl.

Genome wide search for genetic damage in p53 transgenic mouse lung tumours reveals consistent loss of chromosome 4.

The tumour which develops most frequently in mice carrying a p53 Val135 transgene is adenocarcinoma of the lung. We established 10 cell lines from these tumours and investigated their karyotypes by detailed cytogenetic analysis using a complete set of mouse chromosome-specific paints. Consistent loss of chromosome 4 material was noted in 9 out of 10 cell lines; this loss was detected in tetraploid but not diploid cells of the same cell line, suggesting that mouse chromosome 4 plays a critical role in the progression of lung adenocarcinomas. Other frequently observed chromosome aberrations involved chromosomes 7, 5 and 8. Atypical bronchial epithelium was observed together with lung tumours and in tumour-free, apparently normal lungs indicating that mouse lung tumours induced due to the presence of a mutant p53 transgene may develop via pre-invasive lesions and thus may be effective models for the study of lung tumour progression.

Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

A simultaneous packed column supercritical fluid chromatographic method for ibuprofen, chlorzoxazone and acetaminophen in bulk and dosage forms.

A reproducible and efficient method for the separation and estimation of ibuprofen, chlorzoxazone and acetaminophen has been developed using packed column supercritical fluid chromatography (SFC). The separations were performed on an ODS-RP JASCO column employing methanol modified supercritical fluid CO(2) as the mobile phase. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on retention times. In addition a flow programming of the mobile phase helped to obtain better resolution and a faster elution for acetaminophen. The analytes were detected using a uv detector at 254 nm. The study includes a successful attempt at quantitation of the 3 drugs. Chromatographic figures of merit, linear dynamic range, limit of quantitation (LOQ), precision and accuracy etc. were determined to assess the viability of the method. The method has been extended to commercial dosage forms containing all 3 drugs.

Regulation of reactive-oxygen-species generation in fibroblasts by Rac1.

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1 beta] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.

Reproducibility of Bayesian belief network assessment of breast fine needle aspirates.

OBJECTIVE: To assess the consistency of diagnosis of fine needle aspiration biopsies of breast lesions by three experienced and five less experienced pathologists using conventional means and applying a Bayesian belief network (BBN) to 10 diagnostic features to support diagnostic decision making. STUDY DESIGN: Forty fine needle aspiration biopsies, previously assessed by one of the experienced pathologists both conventionally and using a BBN, were assessed by two further experienced pathologists and five less experienced pathologists. RESULTS: Using the BBN, the experienced pathologists arrived at diagnoses in agreement with an established consensus at a slightly lower rate than by conventional means. The less experienced pathologists arrived at the correct diagnoses no more frequently with the help of the BBN than conventionally. CONCLUSION: As used in this study, the BBN did not help less experienced pathologists to interpret their observations but did not enable less experienced pathologists to identify how their observations differed and affected their diagnoses. The prototype system used in this study has since been upgraded by providing computer graphic displays of the features to be observed so that a more uniform mental image can be held by the participating pathologists. This will be tested with the same study design.

Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

Neuropeptides and a neuronal marker in cutaneous innervation during human foetal development.

There is evidence that foetal body movements first occur at 6 weeks gestation, and that the reflex arc is functional at 8 weeks. This correlates with the detection of the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in spinal cord at 10 weeks gestation. However, the development of cutaneous neuropeptide-containing nerves is not well documented in humans. We have investigated the early appearance and distribution pattern of CGRP, SP, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), as well as those of the general neuronal marker protein gene product 9.5 (PGP) in various areas of foetal skin at different gestational ages. PGP-immunoreactive nerves were first seen in the subepidermal plexus at 6 weeks gestational age. Initially, the immunoreactive nerves are thick, club-shaped and distributed in the superficial dermis. Beaded adult-like fibres become more numerous only at later ages (10-12 weeks), and extend from this plexus to penetrate the epidermis. Histologically, the skin of the hand develops faster than that of other body areas and at 9 weeks, more PGP-immunoreactive nerves were seen in the palm than in the dorsum. Primitive sweat glands were first noted in axillary skin at 17 weeks, accompanied by a few PGP-immunoreactive nerves. Occasional, small CGRP-immunoreactive fibres were first noticed in the dermis at 7 weeks, but it was at 17 weeks that the presence of this neuropeptide was unequivocal in the subepidermal plexus. Sparse VIP-, SP- and NPY-immunoreactive fibres were not found until 16-17 weeks gestation, when they were seen in the dermis and around small blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

'Solitary' necrotic nodules of the liver: an aetiology reaffirmed.

Morphological features of lesions conforming to the description of solitary necrotic nodules of the liver were compared with liver haemangiomata and necrotic tumour metastases in the liver. An origin in haemangiomata is confirmed for most of the lesions studied, although the necrotic nodule may represent the end stage of a variety of lesions. These lesions are usually benign, although necrotic nodules are often mistaken for liver metastases. A reticulin stain is useful in assessing the lesions.

Immunoglobulin heavy chain patterns in reactive lymphadenopathy.

Thirty one lymph nodes taken from 24 benign reactive cases, three cases of angiolymphoid hyperplasia with eosinophilia, one case of Kimura's disease and three cases of Hodgkin's disease, were stained for immunoglobulin heavy chains IgG, IgM, IgA and IgE using the peroxidase-antiperoxidase method. Reticular staining of germinal centres and cells containing immunoglobulin in germinal centres and extrafollicular regions were features of all groups. No staining pattern was diagnostic for any of these conditions and in particular, the reticular staining pattern of IgE in the germinal centres that is frequently reported in Kimura's disease and in angiolymphoid hyperplasia with eosinophilia was non-specific.