An adaptable multiple power source for mass spectrometry and other scientific instruments.

An Adaptable Multiple Power Source (AMPS) system has been designed and constructed. The AMPS system can provide up to 16 direct current (DC) (±400 V; 5 mA), 4 radio frequency (RF) (two 500 VPP sinusoidal signals each, 0.5-5 MHz) channels, 2 high voltage sources (±6 kV), and one ∼40 W, 250 °C temperature-regulated heater. The system is controlled by a microcontroller, capable of communicating with its front panel or a computer. It can assign not only pre-saved fixed DC and RF signals but also profiled DC voltages. The AMPS system is capable of driving many mass spectrometry components and ancillary devices and can be adapted to other instrumentation/engineering projects.

[Immunohistochemical and molecular analyis of mismatch repair genes in germ cell tumors]. / Análisis inmunohistoquimico y molecular de los genes de reparación en cáncer testicular.

UNLABELLED: Correction of misincorporated nucleotides during DNA replication (mismatch repair) distinguishes histologically similar cancers with distinct biological and clinical behavior. We investigated expression of two mismatch repair genes in testis cancer to determine the expression pattern in histologically distinct subtypes, correlate expression with genetic instability and correlate expression and genetic instability with clinical outcome. PATIENTS AND METHODS: 118 cases of testis cancer were analyzed. Immunohistochemical analysis of paraffin embedded specimens utilized monoclonal antibody for hMLH1 and hMSH2 mismatch repair proteins. Genetic instability was determined by comparing genomic DNA from microdissected matched normal and tumor cells. PCR amplification of 10 genetic markers assessed loss of heterozygosity and/or microsatellite instability. RESULTS: hMSH2 staining was associated with pathologic stage (p < 0.001) while hMLH1 staining was associated with cancer specific survival (p = 0.036). Genetic instability was detected in 94% of low hMLH1 and 92% of low hMSH2 staining tumors. Relapse and cancer specific death correlated with genetic instability (p = 0.01 and 0.04 respectively). Overall 9% of tumors exhibited reduced mismatch repair expression, microsatellite instability and an unfavorable clinical outcome. CONCLUSIONS: Mismatch repair expression and genetic instability define testis cancers with distinct molecular properties and clinical behavior. In conjunction with pathologic examination and serum tumor markers, mismatch repair expression may be an important determinant for clinical management of men with this malignancy.

Análisis inmunohistoquimico y molecular de los genes de reparación en cáncer testicular / Immunohistochemical and molecular analysis of mismatch repair genes in germ cell tumors

La corrección de los nucleótidos mal incorporados durante la replicación del ADN (sistema de reparación de genes) puede determinar un potencial biológico y clínico diferente en los tumores. En este trabajo investigamos la expresión inmunohistoquímica de los genes de reparación en cáncer de testículo en los distintos tipos histológicos, correlacionando el grado de expresión con la presencia de inestabilidad microsatélite y correlacionando ambas con la evolución clínica. Pacientes y método: 118 casos de tumores testiculares fueron analizados molecularmente realizando inmunohistoquímica para hMLH1 y hMSH2. El análisis de inestabilidad microsatélite y LOH se realizó comparando ADN micro disecado de tejido tumoral y normal, el que fue amplificado mediante PCR con 10 marcadores preestablecidos. Resultados: El grado de expresión de hMSH2 se correlaciona con el estadio del tumor (p<0,001) mientras que la sobreviva cáncer específica se correlaciona con el grado de expresión de hMLH1 (p=0,036). Inestabilidad microsatélite se detectó en un 94% y 92% de los cánceres con baja expresión de hMLH1 y hMSH2 respectivamente. La recaída y muerte cáncer específica se correlacionan con el grado de inestabilidad genética (p=0,01, p=0,04). Un 9% de los tumores presentan una bajo grado de expresión de los genes de reparación, inestabilidad microsatélite y un mal pronóstico. Conclusiones: El grado de expresión de los genes de reparación así como la frecuencia de inestabilidad logran definir cánceres testiculares con diferentes propiedades moleculares y diferente pronóstico. Los genes de reparación usados en conjunto con la histología, los marcadores serológicos pueden ser determinantes en el manejo de los pacientes con tumores testiculares (AU)

Correction of misincorporated nucleotides during DNA replication (mismatch repair) distinguishes histologically similar cancers with distinct biological and clinical behavior. We investigated expression of two mismatch repair genes in testis cancer to determine the expression pattern in histologically distinct subtypes, correlate expression with genetic instability and correlate expression and genetic instability with clinical outcome. Patients and methods: 118 cases of testis cancer were analyzed. Immunohistochemical analysis of paraffin embedded specimens utilized monoclonal antibody for hMLH1 and hMSH2 mismatch repair proteins. Genetic instability was determined by comparing genomic DNA from microdissected matched normal and tumor cells. PCR amplification of 10 genetic markers assessed loss of heterozygosity and/or microsatellite instability. Results: hMSH2 staining was associated with pathologic stage (p<0.001) while hMLH1 staining was associated with cancer specific survival (p=0.036). Genetic instability was detected in 94% of low hMLH1 and 92% of low hMSH2 staining tumors. Relapse and cancer specific death correlated with genetic instability (p=0.01 and 0.04 respectively). Overall 9% of tumors exhibited reduced mismatch repair expression, microsatellite instability and an unfavorable clinical outcome. Conclusions: Mismatch repair expression and genetic instability define testis cancers with distinct molecular properties and clinical behavior. In conjunction with pathologic examination and serum tumor markers, mismatch repair expression may be an important determinant for clinical management of men with this malignancy (AU)

The mismatch repair gene hMSH2 is mutated in the prostate cancer cell line LNCaP.

PURPOSE: Mismatch repair genes are responsible for the coordinated correction of misincorporated nucleotides formed during DNA replication. Inactivating and inherited mutations in the prototypic mismatch repair gene hMSH2 have been described in a cancer predisposition syndrome known as hereditary nonpolyposis colon cancer. Patients with hereditary nonpolyposis colon cancer are at increased risk for colon cancer and extracolonic cancers such as upper tract transitional cell carcinoma but not prostate cancer. We investigated expression of hMSH2 in prostate cancer cell lines using genetic and molecular analysis. MATERIALS AND METHODS: We used the 3 well described prostate cancer cell lines, DU145, LNCaP and PC3. Western blot analysis with monoclonal antibody to hMSH2 was used to assess expression. Southern blot and polymerase chain reaction of genomic DNA were used to identify genetic alterations in the hMSH2 gene. Single cell cloning, dinucleotide repeats and BAT-26 were used to assess the cell lines for microsatellite instability. RESULTS: The prostate cancer cell line LNCaP did not express hMSH2 and was found to have a homozygous deletion of hMSH2 exons 9 to 16, resulting in truncation of the protein. While microsatellite analysis did not reveal alterations at the BAT-26 locus, single cell cloning produced several LNCaP subclones with alteration at 1 dinucleotide repeat. CONCLUSIONS: The well described prostate cancer cell line LNCaP has a mutation in the hMSH2 gene, resulting in loss of expression and possible evidence of microsatellite instability. To our knowledge our finding is the first demonstration of a genetic alteration in hMSH2 in a prostate cancer cell line.

Rare presentation of actinomycosis as an abdominal mass: report of a case.

PURPOSE: The purpose of this article was to report an unusual presentation of abdominal actinomycosis masquerading as a tumor. METHODS: The patient was a 54-year-old male who presented with vague abdominal discomfort and a palpable left lower quadrant mass defined on CT scan. Multiple intraoperative core biopsies were nondiagnostic, and he underwent en bloc resection of the mass and adjacent organs for a presumed tumor. RESULTS: Examination of tissue from deep within the excised specimen revealed sulfur granules diagnostic for actinomycosis. CONCLUSION: Abdominal actinomycosis is an extremely rare infection that can mimic multiple disease processes and requires accurate diagnosis for successful therapy. This novel presentation and a review of the literature are reported.

Expression of the human mismatch repair gene hMSH2: a potential marker for urothelial malignancy.

BACKGROUND: The human mismatch repair (MMR) gene hMSH2 (human mutS homolog-2) is a DNA repair gene that has been reported to be mutated in 40% of hereditary nonpolyposis colon cancer (HNPCC) kindreds and a small percentage of sporadic tumors. HNPCC is a cancer predisposition syndrome with an increased risk of carcinoma of the colon, endometrium, stomach, small intestine, ovary, ureter, and renal pelvis. Immunohistochemical analysis demonstrated increased hMSH2 expression in sporadic colon carcinoma and in the replicative compartment of normal epithelium. A recent immunohistochemical analysis of hMSH2 in bladder tumors correlated reduced hMSH2 expression with recurrence and higher tumor grade. In the current study, we examined hMSH2 expression in urothelial malignancy using immunohistochemical analysis and developed a molecular assay for the detection of hMSH2 expression in bladder washes. METHODS: Immunohistochemical analysis of 17 tumors from the genitourinary tract and reverse transcription coupled with polymerase chain reaction (RT-PCR) of 40 bladder washes were used to investigate hMSH2 expression in noninvasive and invasive urothelial malignancies. RESULTS: Increased expression of hMSH2 was detected in all tumors examined using immunohistochemical analysis independent of grade or stage. Reverse transcription-PCR of hMSH2 mRNA from bladder washes detected 17 of 21 patients with primary or recurrent urothelial neoplasms or tumors involving the urothelial system. Four patients with urothelial malignancies without detectable hMSH2 expression from their bladder washes had high grade lesions. Ten of 13 patients without pathologic or cystoscopic evidence of bladder tumors were negative for hMSH2 expression in bladder washes. Two patients with bladder tumors and bladder washes that were positive for hMSH2 subsequently were found to be negative for hMSH2 after treatment of their tumors and at last follow-up had remained recurrence free for at least 1 year. CONCLUSIONS: The results of the current study suggest that hMSH2 expression is increased in low and high grade urothelial neoplasms, similar to the expression pattern in sporadic colon carcinoma. However, a fraction of high grade lesions may not express hMSH2 as detected by RT-PCR from bladder washes. The ability to detect hMSH2 expression in bladder washes may allow the use of hMSH2 expression as a marker for urothelial malignancy. In addition, the ability to define hMSH2 deficient tumors using bladder washes may have prognostic significance in the treatment of patients with urothelial carcinoma.

Gender, education and training: an international perspective.

PIP: This article examines the causes and consequences of sex discrimination in education in developing countries and considers whether the available educational structures improve gender equity or reinforce the status quo. After an introduction that distinguishes between "education" and "schooling" and identifies schooling as a means of patriarchal control, the article sketches the growing awareness of gender disparities in education. The next section describes how gender inequality in education leads to low participation of women in the labor market and limits women's access to information and services, to mobility, and to decision-making. The article then reviews the international agenda on promoting female education that has resulted in donor-driven initiatives arising from such events as the 1990 World Conference on Education For All. A look at the benefits of educating women then focuses on the "family welfare" perspective and the acknowledgement of women's full socioeconomic role. After pointing to the slow progress towards gender equity in education, the article discusses barriers to this goal posed by poverty, social conventions, early marriage, violence in schools, and curricular gender stereotyping. The article then considers problems encountered by efforts to provide informal education and training and the fact that educational initiatives are donor-driven and fail to address the causes of the gender gap. It is concluded that governments and donors must transform schools as part of a larger program of socioeconomic reforms designed to improve women's status.^ieng

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica.

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5' noncoding region is 61 bp long and the 3' noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Laparoscopic creation of a catheterizable cutaneous ureterovesicostomy.

Nephrectomy and creation of a cutaneous ureterovesicostomy for intermittent catheterization of the bladder traditionally requires two surgical procedures performed through separate incisions. Herein we report completion of these procedures using a transperitoneal laparoscopic approach, with the ureterovesicostomy stoma created at one of the laparoscopic working ports. The clinical course was remarkable for a shortened postoperative hospitalization (48 hours) with minimal incisional pain, and an excellent long-term result with complete bladder emptying and resolution of urinary infections. Laparoscopic application of the Mitrofanoff principle for creation of a catheterizable cutaneous ureterovesicostomy combines the advantages of both, allowing optimal preservation of ureteral vascularity, minimal morbidity, and efficient bladder evacuation.

Evaluation of the FHIT gene in colorectal cancers.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.

Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues.

Hereditary nonpolyposis colorectal cancer is caused by inherited mutations of mismatch repair genes. We developed monoclonal antibodies to the prototype human mismatch repair gene hMSH2 and used them to detect an immunoreactive protein of M(r) 100,000 in mismatch-proficient cell lines. In addition, a M(r) 150,000 protein coimmunoprecipitated with the hMSH2 gene product in cell lines expressing hMSH2. Immunohistochemistry demonstrated that the hMSH2 protein was exclusively nuclear. Whereas the hMSH2 protein was expressed in a variety of tissues, the most striking pattern was observed in esophageal and intestinal epithelia, where expression was limited to the replicating compartment. Neoplastic cells within benign and malignant mismatch repair-proficient tumors expressed the protein, but no hMSH2 immunoreactivity was observed in the colorectal tumors of patients with germline hMSH2 mutation. These results have implications for tumorigenic mechanisms and, potentially, for diagnosis.

Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.

Endometrial carcinoma is the second most common tumor type in women with hereditary nonpolyposis colorectal carcinoma. Microsatellite instability (MI) has been observed in the inherited (hereditary nonpolyposis colorectal carcinoma-associated) form of endometrial carcinoma as well as in approximately 20% of presumably sporadic cases. Recent studies suggest that MI in many cell lines or xenografts derived from sporadic colorectal carcinomas is not attributable to mutations in four known human DNA mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, and hPMS2). Mutational analyses of these four MMR genes in endometrial carcinomas have not been previously reported. We analyzed nine sporadic MI-positive primary endometrial carcinomas for mutations in the above four MMR genes. Mutations were detected in two tumors (in hMSH2), and both of the mutations were acquired somatically. Immunohistochemical staining revealed a lack of expression of hMSH2 protein in the two tumors containing hMSH2 mutations. Our data suggest that mutations in these four known DNA MMR genes are not responsible for MI in the majority of sporadic endometrial carcinomas displaying this phenotype.

Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity?

Light production by firefly luciferase is limited by product release resulting in flash kinetics. Several compounds (CoA, PPi, and nucleotides) transform the flash-form of light production into continuous light production. The sulfhydryl group of CoA is required; however, since nucleotides are also active, at least two mechanisms (sites) must exist.

Monoallelic mutation analysis (MAMA) for identifying germline mutations.

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).