Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model.

The analysis of rapid changes of protein expression in living systems in response to insults requires rigorous methods of complexity reduction. To control dynamic pattern of hundreds or even thousands of protein isoforms, we applied a novel method of differential molecular analysis to a cellular model which is suited to study ischemia. Neural derivatives of murine embryonic stem cells were exposed to chemical ischemia. The model was used to obtain starting material for a quantitative differential proteomics analysis. Fractionation of phosphoproteins from these samples and subsequent identification by mass spectrometry of differential proteins provide proof of principle of how novel molecular analytical tools provide new insight into the network of neuroprotective molecular events during specific situations of neuronal stress and related pharmaceutical intervention. Our results indicate a particular role of an isoform of the acidic calcium-independent phospholipase A2 in this type of insult.

An epidemiological approach to study fatigue in the working population: the Maastricht Cohort Study.

In 1998, a large scale prospective cohort study of prolonged fatigue in the working population was started in the Netherlands. The ultimate goal of this Maastricht Cohort Study was to identify risk factors involved in the aetiology and natural course of prolonged fatigue in the working population and to develop preventive measures and treatments that can be used in occupational health settings. In this paper, a conceptual model for epidemiological research on prolonged fatigue is presented. This model is the basis for the Maastricht Cohort Study. Alongside the model and design, the characteristics of the study population, the prevalence and one year cumulative incidence of prolonged fatigue, as well as its relation with secondary health outcomes (psychological distress, need for recovery, and burnout) are presented. Furthermore, model, design, and the presented results are discussed.

Analysis of the co-amplified STR loci D1S1656, D12S391 and D18S51: population data and validation study for a highly discriminating triplex-PCR.

A multiplex-PCR composed of the three highly variable STR loci D1S1656, D12S391 and D18S51 has been established. The non-overlapping fragment sizes allow allele detection using a monochrome automated laser fluorescent sequencer (A.L.F. express, Pharmacia Biotech). The typing results of the triplex-PCR showed no difference to those of singleplex-PCR. Allele frequencies were determined in a Western German population of 228 individuals from Cologne. The heterozygosities and exclusion chances (D1S1656, 0.982; D12S391, 0.979; D18S51, 0.97) are very high compared to other short tandem repeats used for forensic applications. No deviations from the Hardy-Weinberg equilibrium were found. Successful typing of DNA amounts down to 50-100 pg is possible. Mixtures of up to 1:10 can be identified. In conclusion, the high combined exclusion chance due to the well-balanced allelic distribution and its high sensitivity make this triplex-PCR a valuable tool for forensic casework.

Properties of an electron-density map derived from a limited number of experimentally determined triplet phases.

In a previous communication [Weckert et al. (1999). Acta Cryst. D55, 1320-1328], the feasibility of the measurement of a large set of triplet phases by three-beam interference was demonstrated. This paper reports the methodology for the calculation of an electron-density map from this limited amount of experimental phase information and the map's properties with respect to model building and refinement. The tetragonal form of hen egg-white lysozyme (HEWL) was chosen as a test structure for the development of this method. The quality of the electron-density map obtained from all measured triplet phases allows a straightforward and nearly complete interpretation. The starting model was refined to a final R value of 17.4%. In a second step, the minimum number of phased reflections needed for the interpretation of an electron-density map was investigated, applying criteria based on |F| and resolution.

Feasibility study for the measurement of a large set of triplet phases from a small protein.

The feasibility of measuring a set of triplet phases large enough to solve the structure of a small protein has been evaluated. A total of about 850 triplet phases have been measured from the tetragonal form of hen egg-white lysozyme. From these triplet phases, about 750 single phases can be derived. The experimental details of these measurements as well as the results, the values of the measured triplet phases, are reported. Additional experimental data from other small proteins are also presented.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C(1) has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-I and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Hölzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C(1) crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C1 has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-1 and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Holzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C1 crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Adenomatoid tumors of the pleura.

We report two cases of small pleural nodules showing the distinctive histologic appearance of adenomatoid tumor. Both lesions were discovered incidentally during surgery in patients undergoing lung resection for unrelated intrapulmonary masses: lung carcinoma in one case and histoplasmosis in the other. The tumors were composed of a focal proliferation of epithelioid cells forming vacuoles and tubular spaces in a fibrous stroma, as seen in adenomatoid tumors from other sites. The differential diagnosis in both cases included metastatic signet ring cell carcinoma. The mesothelial nature of the lesions was supported by immunohistochemical and ultrastructural evidence. The tumor cells in both cases were positive for cytokeratin but negative for carcinoembryonic antigen and LeuM1. One case was also negative for BER-EP4, B72.3, CD34, and Factor VIII. Electron microscopy in this case demonstrated well-developed basal laminae, desmosomes, and numerous slender microvilli along the luminal surfaces of the tumor cells. Adenomatoid tumors are regarded as a benign variant of mesothelioma. Despite the abundance of mesothelial cells in the pleura, adenomatoid tumors are apparently extremely rare in this location. Separation from malignant lesions such as adenocarcinoma and epithelioid hemangioendothelioma is important.

Parotid involvement by desmoplastic melanoma.

Desmoplastic malignant melanoma often arises in sun damaged skin of the head and neck and shows frequent neurotropism. Although metastatic melanoma frequently involve the parotid, direct spread to the parotid has been rarely reported. We evaluated five cases of desmoplastic malignant melanoma involving the parotid gland with clinical and pathological evidence of precursor cutaneous lesions in four of the five cases. The parotid involvement in four cases was tumoural, and three of these were not clinically suspected to be melanoma. The histological appearance in all five cases was that of a sarcomatoid tumour. Immunohistochemistry and electronmicroscopy performed on three of the cases showed only evidence of schwannian differentiation: the tumour cells were positive for S-100 protein and vimentin, and negative for cytokeratin and HMB-45. Electronmicroscopy showed no evidence of melanogenesis. All five tumours showed histological evidence of prominent neurotropism with one case demonstrating extension from overlying skin along cutaneous nerves to the superficial parotid. Thus, desmoplastic malignant melanoma may involve the parotid by neurotropic spread and can be pathologically indistinguishable from malignant schwannoma, a diagnosis which may be made erroneously in the absence of clinical information.

ras and p53 genes in male breast cancer.

We have studied the roles of Ki-ras oncogene and p53 tumor suppressor gene in a series of 20 cases of male breast cancer and one papilloma of the male breast. Ki-ras was detected in 50-microns sections after digestion with proteinase K and SDS. DNA was amplified by polymerase chain reaction, dot blotted, and mutations were screened with labeled ras mutation-specific oligonucleotides. Wild-type and mutant p53 protein were detected with antibodies CM1 and DO7, using the avidin-biotin-peroxidase method. Two of 17 carcinomas showed Ki-ras mutations, both in codon 12 (gly --> lys and gly --> arg). Five of 20 male breast cancers (25%), including one large intraductal carcinoma, expressed mutant p53 protein. Although the incidence of mutant p53 expression in male breast cancer is similar to that in women, Ki-ras mutations are not significantly increased.

Improvements in lysozyme protein crystal perfection through microgravity growth.

Microgravity offers an environment for protein crystallization where there is an absence of convection and sedimentation. We have investigated the effect of microgravity conditions on the perfection of protein crystals. The quality of crystals for X-ray diffraction studies is characterized by a number of factors, namely size, mosaicity and the resolution limit. By using tetragonal lysozyme crystals as a test case we show, with crystal growth in two separate Space Shuttle missions, that the mosaicity is improved by a factor of three to four over earth-grown ground control values. These microgravity-grown protein crystals are then essentially perfect diffraction gratings. As a result the peak to background of individual X-ray diffraction reflections is enhanced by a similar factor to the reduction in the mosaicity. This then offers a particularly important opportunity for improving the measurement of weak reflections such as occur at high diffraction resolution. These microgravity results set a benchmark for all future microgravity and earth-based protein crystallography procedures.

Immunohistochemical assessment of tumor vascularity in recurrent Clark II melanomas using antibody to type IV collagen.

Thin melanomas, measuring 0.76 mm or less, are generally associated with an excellent prognosis. However, a certain subset of these seemingly innocuous lesions have been reported to develop recurrences. Therefore, the predictive values of currently accepted prognostic indicators have been questioned in thin melanomas. Several studies concerning tumor vascularity in melanoma and certain non-melanocytic malignancies suggest that the degree of vascularization correlates with growth rate and biologic aggressiveness. In the present study, we determined the vascularity of a small group of Clark level II melanomas that resulted in recurrence, and compared these results to an equal number of nonrecurrent lesions with similar prognostic indicators. Blood vessels were labeled by immunoperoxidase staining techniques for Type IV collagen, and quantified by image analysis. No statistical difference was found between the two groups when mean blood vessel counts and percent vascular area were measured. The recurrent tumors had a mean PVA of 4.68 compared to 4.34 for the nonrecurrent group (p = 0.677). The mean blood vessel count beneath the recurrent group was 29.6 per 400 x field, and the corresponding value for the nonrecurrent group was 31.8 (p = 0.681). Our data is preliminary within this limited group of tumors, yet it suggests that tumor vascularity is not a distinctive prognostic indicator by which eventual outcome can be predicted in thin Clark level II malignant melanomas.

The orderly progression of melanoma nodal metastases.

OBJECTIVE: The aim of this study was to determine the order of melanoma nodal metastases. SUMMARY BACKGROUND DATA: Most solid tumors are thought to demonstrate a random nodal metastatic pattern. The incidence of skip nodal metastases precluded the use of sampling procedures of first station nodal basins to achieve adequate pathological staging. Malignant melanoma may be different from other malignancies in that the cutaneous lymphatic flow is better defined and can be mapped accurately. The concept of an orderly progression of nodal metastases is radically different than what is thought to occur in the natural history of metastases from most other solid malignancies. METHODS: The investigators performed preoperative and intraoperative mapping of the cutaneous lymphatics from the primary melanoma in an attempt to identify the "sentinel" lymph node in the regional basin. All patients had primary melanomas with tumor thicknesses > 0.76 mm and were considered candidates for elective lymph node dissection. The sentinel lymph node was harvested and submitted separately to pathology, followed by a complete node dissection. The null hypothesis tested was whether nodal metastases from malignant melanoma occurred in equal proportions among sentinel and nonsentinel nodes. RESULTS: Forty-two patients met the criteria of the protocol based on prognostic factors of their primary melanoma. Thirty-four patients had histologically negative sentinel nodes, with the rest of the nodes in the basin also being negative. Thus, there were no skip metastases documented. Eight patients had positive sentinel nodes, with seven of the eight having the sentinel node as the only site of disease. In these seven patients, the frequency of sentinel nodal metastases was 92%, whereas none of the higher nodes had documented metastatic disease. Nodal involvement was compared between the sentinel and nonsentinel nodal groups, based on the binomial distribution. Under the null hypothesis of equality in distribution of nodal metastases, the probability that all seven unpaired observations would demonstrate that involvement of the sentinel node is 0.008. CONCLUSIONS: The data presented demonstrate that nodal metastases from cutaneous melanoma are not random events. The sentinel lymph nodes in the lymphatic basins can be mapped and identified individually, and they have been shown to contain the first evidence of melanoma metastases. This information can be used to revolutionize melanoma care so that only those patients with evidence of nodal metastatic disease are subjected to the morbidity and expense of a complete node dissection. Because sentinel node histology accurately reflects the histology of the remainder of the lymphatic basin, information gained from the sentinel node biopsy can be used as a prognostic factor for melanoma. These findings demonstrate effective pathologic staging, no decrease in standards of care, and a reduction of morbidity with a less aggressive, rational surgical approach.

Age as a prognostic factor in the malignant melanoma population.

BACKGROUND: The incidence of malignant melanoma is increasing faster than any other cancer, and the state of Florida has one of the highest incidence of melanoma in the United States. This increased incidence is thought to be due to the intense sunlight exposure and ultraviolet radiation exposure in the elderly population. With the increased emphasis on issues of aging, it is appropriate to study the role of age as a prognostic factor for malignant melanoma in the Florida population. METHODS: A retrospective, computer-aided search identified 442 consecutively registered patients with malignant melanoma at the Cutaneous Oncology Program. All patients had stage 1 or 2 disease (cutaneous disease only) at diagnosis. Prognostic variables analyzed included the most powerful factors for stage 1 and 2 melanoma, tumor thickness, ulceration, and Clark level of invasion. Other prognostic variables included in the analysis were the clinical variables of sex and primary site (axial vs. extremity). The population was divided into patients < or = 65 and > 65 years of age. RESULTS: Significant disease-free survival differences were encountered in the older population, with only 55% of the elderly population being disease free at 5 years compared with 65% for the younger population (p = 0.0073). However, a greater percentage of patients with melanoma who were > 65 years of age had ulcerated lesions (17.5% vs. 12.9%) and a greater percentage of thick lesions at diagnosis (67.2% vs. 62.7%). Both of these prognostic factors would bias the older population with a poorer survival. A stepwise regression analysis of the entire population was performed, treating age as a continuous variable. Surprisingly, increasing age along with tumor thickness were the only significant predictors for disease-free survival. After inclusion of these two prognostic variables, none of the other prognostic factors, including Clark level, ulceration, sex, and primary site, added to the prognostic model. CONCLUSIONS: From this analysis, it is apparent that geriatric patients with melanoma have a worse prognosis than a younger control population, even after the correction for the more commonly cited prognostic factors. This information should be used in mathematical modeling to identify high-risk populations who are candidates for perhaps more aggressive primary or adjuvant therapies.

Histopathologic study of recurrent Clark level II melanomas.

Overall, the prognosis for thin lesions of melanoma (less than 0.76 mm) is excellent. However, a number of melanoma patients with seemingly innocuous lesions have been reported to develop recurrences. For this reason, we examined histologic sections taken from eight cases of Clark level II melanoma that unexpectedly recurred, and compared their histopathologic features with an equal number of nonrecurrent lesions in whom reliable clinical follow-up data were available. Prognostic variables including Breslow thickness, mitotic rate, ulceration, the presence or absence of regression, a vertical growth phase component, and an associated banal nevus were evaluated in a double blind manner. When attempts were made to predict outcome based on one or more prognostic variables, the only correlation of statistical significance was the Breslow thickness (P = 0.04). A Breslow thickness greater than 0.4 mm was associated with a significantly shorter disease free interval than a thickness below 0.4 mm. There was no significant correlation between predicted outcome based on the histologic features examined and the eventual outcome based on history of recurrence (P = 0.36). These data indicate that although prognostic models that predict outcome in melanoma are generally reliable, there is a sizable population of patients with thin melanomas that do worse than would be expected.

Computer database for melanoma: a clinical management and research tool to ensure continuous quality assessment.

The need for an efficient method to handle data and for chart documentation is more apparent today than at any time in the past. High volume clinics for screening, diagnosis, and treatment are the rule rather than the exception in modern practice. A papermill medical record department or tumor registry fails on a day-to-day basis because of inefficiency of data collection, filing, storage, and abstraction. Requirements of the Joint Commission for the Accreditation of Health Care Organizations (JCAHO) include the setting of standards and the ability to query data to ensure the standards are met. A PC-based software program has been developed with the following features: (1) ongoing timed queries of the data are possible on any variable collected in the database to monitor trends in the standards established for care in the clinic; (2) a daily update of the database is performed so that it may be used as a clinical management tool, acting as an electronic medical record or as a clinical research tool; (3) the software will summarize the chart by abstracting a predetermined list of key data elements to improve clinical efficiency; (4) chart documentation is improved for Medicare coding requirements to maximize physician reimbursement; and (5) clinic notes and referral letters are generated the same day as the clinic visit to allow patients to have a copy of their clinic notes or for same day FAXing to the referring doctors. To date, > 1,200 patients with melanoma have been registered and entered into the system.(ABSTRACT TRUNCATED AT 250 WORDS)

The effectiveness of skin cancer screening and continuing medical education programs toward increasing the survival of patients with malignant melanoma.

The 5-year survival rate for malignant melanoma has increased 40% over the last 50 years. During this same time period, the treatment for the disease has not changed significantly, and consists of wide excision of the primary tumour and perhaps regional node dissection. The purpose of this study was to investigate the possible impact of screening/education programs on melanoma survival. In 1987, a multimodality University-based Melanoma Treatment Center was established and programs were instituted for skin cancer screening and Continuing Medical Education (CME) of health care providers. During the last 5 years, 594 patients with newly diagnosed melanoma have been registered at the clinic. The number of patients with localized (stage 1 or 2, negative regional nodes) disease was 516 (85%). For all stages of disease, the 3-year actuarial survival for this screened population was 85%. From the National Cancer Database of 9879 patients registered with melanoma for 1988, 75% had localized disease and the 3-year survival was 76%. There were significant differences noted between the screened Florida population and the nationwide database using an odds ratio statistic. This involved a higher frequency of patients diagnosed with localized disease (odds ratio = 1.89 (1.49-2.40)) and a better survival (odds ratio = 1.79 (1.41-2.27)) in the screened population served by CME-educated community physicians.

Primary treatment of pelvic osteosarcoma. Report of five cases.

Five patients, ages 12 to 20 years, with nonresectable primary (Patients 2, 3, and 5) and metastatic (Patients 1 and 4) pelvic osteosarcomas were treated with intraarterial cisplatin and concurrent radiation therapy from 1983 to 1987. Long-term local tumor control was achieved in all five patients. Patients 1 and 3 are alive with no evidence of local recurrence or metastatic disease at 77 and 56 months of follow-up, respectively, since diagnosis of the pelvic tumor. Patients 2, 4, and 5 died of metastatic lung disease at 25, 39, and 12 months, respectively, after diagnosis of the pelvic tumor. Patient 4 had no clinical or radiologic evidence of local recurrence. Control of tumor growth in patients with pelvic osteosarcomas can be achieved with regional chemotherapy and concurrent radiation therapy. These patients also should receive adjuvant intensive systemic chemotherapy to increase the probability of eliminating potential subclinical metastatic disease.

Etiology and prognosis of local recurrence in malignant melanoma of the skin.

All patients with stage I and stage II malignant melanoma of the skin were analyzed for stage; time of local, regional, or systemic recurrence, or two or more of these events; presence or absence of ulceration of the primary tumor; thickness of the primary tumor; level of invasion according to Clark; and margins of resection. Local recurrence had a significant negative impact on the long-term survival of patients. Our data revealed that local recurrence had the same poor prognostic effect as regional or systemic recurrence, or both. Factors significant in predicting local recurrence included the primary tumor characteristics of ulceration of the primary tumor and thickness of the primary tumor. Margins of resection and level of invasion were not noted significant in predicting local recurrence.