Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca2+ channels.

Structurally novel compounds able to block voltage-gated Ca2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca2+ concentration ([Ca2+](i)) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K+-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K+-depolarised Ca2+ influx, with IC(50) values calculated to be 234, 548 and 209 microg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

Hysterectomy prevalence and adjusted cervical and uterine cancer rates in England and Wales.

OBJECTIVE: To present recent trends in cervical and uterine cancer adjusted for true population at risk, using accurate estimates of the prevalence of hysterectomy where the cervix has been removed or not. To describe trends and projections of hysterectomy incidence and prevalence with and without cervix removal. DESIGN: Collation of available NHS and private sector information. SETTING: England and Wales. SAMPLE: NHS operations from Hospital Inpatient Enquiry, Hospital Episode Statistics and Hospital Activity Analysis for England and Wales. Private sector data from surveys with up to 97% coverage. METHODS AND MAIN OUTCOME: Measures NHS data by 5-year age group, year and operation type were collated for 1961-1995. non-NHS operations for 1981, 1986, and 1992/3 were back-projected. Hysterectomy incidence rates, 1961-95, were back-projected to estimate prevalence rates by accumulation. True populations at risk of disease and hysterectomy were calculated by applying one minus the relevant hysterectomy prevalence rates to the population by age group and year. RESULTS: When based on the true population at risk, the age standardised cervical cancer incidence rate in 1992 was 14.4 per 100,000, compared with 12.6 when based on the all women population estimate. Incidence rates for earlier years were also affected, but there was no important effect on the rate of change over time. Absolute changes for uterine cancer are greater because the true population at risk is proportionally smaller particularly at the older ages, but there are again no major effects on the rate of change. By 1995 2.3 million women in England and Wales were without a uterus, with a peak prevalence of 21.3% in the age group 55-59. Projections based on 1995 incidence rates show hysterectomy prevalence for the screened age groups, 25-64, will now fall. Subtotal hysterectomy is 3.5% of operations and increasing. CONCLUSIONS: True populations at risk must be used to assess the impact of screening if further reductions in cervix cancer incidence rates are not to be masked. It is essential to monitor hysterectomy by type, as subtotal hysterectomy is becoming more common. Hysterectomy incidence may have peaked. Hysterectomy prevalence in England and Wales may not be as high as would be estimated from some regional studies.

Polyoxygenated dysidea sterols that inhibit the binding of [I125] IL-8 to the human recombinant IL-8 receptor type A.

The combined CH(2)Cl(2) and MeOH crude extract of a new species of the marine sponge Dysidea, collected in Northern Australia was found to inhibit the binding of [I125] interleukin-8 [IL-8] to the human recombinant IL-8 receptor type A at 500 microg/mL. Bioassay-guided fractionation led to the isolation of three new polyoxygenated sterols 3, 4, and 5. Their structures were assigned on the basis of 1D and 2D NMR experiments, and relative stereochemistries were established by ROESY correlations and analysis of coupling constants. The IC(50) values for inhibition of IL-8Ra for sterols 3, 4, and 5 were 20, 5.5, and 4.5 microM, respectively.

The induction and potentiation of c-Jun and c-Fos expression in spinal neurons mediated by NK1 and NK2 receptors.

Specific antagonists were used to determine NK1 and NK2 receptor mediation of the expression of c-Jun and c-Fos inducible transcription factors. (ITFs) in dorsal horn neurons. The induction of c-Jun by C-fiber stimulation was strongly reduced in the superficial laminae by NK1 and NK2 antagonists, but only weakly in the deep laminae by NK2 antagonism. c-Fos induction was reduced in all laminae by both NK1 and NK2 antagonism but less than with c-Jun. The potentiation of c-Jun expression, caused by a preceding stimulus, was abolished in all laminae by NK1 and NK2 antagonists, whereas that of c-Fos was reduced in all laminae but again less than that of c-Jun. Thus, the expressions of ITFs mediated by NK1 and NK2 receptors are complex, depending upon the neurons and stimulus considered, and they are not a measure of the neurons' electrophysiological responsiveness.

The incidence and prognosis of nail apparatus melanoma. A retrospective study of 105 patients in four English regions.

Our population-based study establishes epidemiological data on age-specific incidence rates, clinical presentation, Breslow microstaging, treatment and survival of nail apparatus melanoma (NAM) patients in England. Four cancer registries, covering a population of 10.6 million, recorded 105 cases of NAM during the period 1984-93. During the same decade there was a total of 7585 patients with cutaneous melanoma and NAM represents 1.4% of all cutaneous melanoma. The incidence rate of NAM in English patients is 0.1 per 100,000 of the population per annum. Amelanotic melanoma was the clinical presentation in 24 of our NAM cases. The overall prognosis is poor with an observed 5 year survival of only 51%. Patients with NAM less than 2.5 mm Breslow depth have a 5 year survival of 88% and are twice as likely to survive compared with those with tumours greater or equal to 2.5 mm in thickness (P < 0. 05). NAM patients are best managed by a multidisciplinary team approach in a few key skin cancer centres.

Cytodiagnosis of metastatic amelanotic melanomas by fine-needle aspiration biopsy: adjunctival value of immunocytochemistry and electron microscopy.

BACKGROUND: The cytologic diagnosis (CD) of metastatic amelanotic melanomas (MAM) is challenging because the tumor cells may mimic those of a carcinoma or a sarcoma in cytologic materials obtained by fine-needle aspiration biopsy (FNAB). METHODS: Thirty-five well documented cases of MAM at different anatomic sites with cytologic evaluation by FNAB were reviewed. RESULTS: In 31 cases a correct CD was made based on the cytologic and immunocytochemical (IM) findings. The MAMs were characterized by abundant dyshesive pleomorphic malignant cells containing prominent nucleoli and occasional intranuclear cytoplasmic inclusions. The tumor cells stained positively with S-100 and/ or HMB-45 antibodies. There were four cases with unusual cytologic manifestations and equivocal IM characteristics requiring electron microscopic examination (EME) of the aspirated tumor cells to identify intracytoplasmic melanosomes, pre-melanosomes, or microtubules within cisternae of rough endoplasmic reticulum (MCRER) to establish a diagnosis of MAM. CONCLUSIONS: A correct CD of MAMs may be made in a large number of cases by routine cytologic findings and IM staining with S-100 protein and HMB-45 antibodies. However, EME is necessary to demonstrate melanosomes, premelanosomes, or MCRER in cases with unusual cytologic and equivocal IM manifestations.

Improving survival of melanoma patients in Europe since 1978. EUROCARE Working Group.

Within the EUROCARE study 45 cancer registries have contributed survival data on 3.5 million cancer sufferers within 17 countries in Europe. This paper reports on survival in 16,113 cutaneous malignant melanoma cases diagnosed between 1985 and 1989. Relative survival rates were calculated according to the Hakulinen computer program with data stratified according to country, age group and period of diagnosis. Relative 5-year survival was higher in women (81%) than in men (69%) but there was wide variation in relative survival across Europe at both 1 and 5 years, ranging at 5 years between 54 and 89%. At both 1 and 5 years, survival was lowest in the Eastern countries. Between 1978 and 1989 there was an improvement in survival rates probably attributable to the detection of increasing proportions of better prognosis, thinner tumours. In the younger age groups, this may be related to public awareness campaigns. The discrepancy in survival between men and women also narrowed. The single most important prognostic factor is the depth of invasion at diagnosis and there is some evidence that underlying variation in stage at presentation as well as histological type accounts for much of the observed variation in survival.

Accelerated breakdown and enhanced expression of c-Fos in the rat brain after noxious stimulation.

c-Fos expression was examined in rat brains at increasing times after a single noxious stimulus to one hindpaw. In some nuclei the expression peaked at 1 h and was gone by 6 h; in others it was biphasic with a larger peak appearing 6 h after the first. In other rats a second, contralateral stimulus was given at increasing times after the first, and c-Fos examined after a further 1.5 h. In some nuclei the first stimulus potentiated c-Fos expression caused by the second stimulus; in others the second stimulus erased any c-Fos still present from the first. Thus two similar stimuli can interact in very different ways in effecting c-Fos expression in different central nervous system nuclei, and rapid down-regulation might represent a novel type of interaction.

Aspiration biopsy of granulosa-cell tumor of the ovary: cytologic findings and differential diagnosis.

Fine-needle aspirates from four histologically confirmed adult granulosa-cell tumors of the ovary revealed irregular sheets, loose aggregates or tight clusters of small tumor cells with round or oval nuclei and scant cytoplasm. Nuclear grooves and indentations were noted in a small number of cells in two cases. Tumor cells arranged in follicular pattern mimicking Call-Exner bodies were not identified in all cases. A cytologic differential diagnosis of "small-cell" tumors of the ovary is briefly discussed.

Aspiration biopsy cytology of papillary carcinoma of the breast.

The fine-needle aspirates of two cases of noninfiltrating papillary carcinoma (PC) and three examples of early invasive PC of the breast were examined. In three cases in which the tumors displayed cuboidal or polygonal cells the aspirates showed papilla-like clusters of tumor cells with relatively "strong" cellular cohesiveness. Single and small aggregates of tumor cells as well as hemosiderin-laden or foamy macrophages were also present. Aspirates from the two PCs predominantly consisting of tall columnar epithelial cells revealed only monolayered and multilayered epithelial fragments with folding in one case. In the other case large epithelial fragments and small tight clusters of polygonal tumor cells were present. No bipolar nuclei of myoepithelial cells were identified in all cases. No specific cellular features permitting the differentiation between noninfiltrating and early invasive breast PCs were identified in this small series. Staining for carcinoembryonic antigen using the peroxidase-antiperoxidase technique was performed on aspiration smears of three cases. It revealed a positive cytoplasmic reaction in two cases.