Susceptibility of complete bacteria and spheroplasts of Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi to rolliniastatin-2.

Rolliniastatin-2 is an acetogenin with elevated cytotoxic activity whose effect on bacteria has not yet been analysed and was isolated by CC from the hexanic extract of Annona diversifolia Saff (Annonaceae) seeds. In this study, we evaluated the susceptibility of Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi as well as spheroplasts of these bacteria to the compound, determining percent inhibition by turbidimetry. The complete bacteria were susceptible to rolliniastatin-2 (28-40% at 2 mg mL(-1)), while spheroplasts showed an increased susceptibility (55-58%, at 1 ng mL(-1)). The most susceptible bacterium was P. aeruginosa, while the spheroplasts of E. coli and S. typhi were most sensitive to the compound's effects.

Molecular pathology of prostate cancer.

This chapter includes discussion of the molecular pathology of tissue, blood, urine, and expressed prostatic secretions. Because we are unable to reliably image the disease in vivo, a 12 core method that oversamples the peripheral zone is widely used. This generates large numbers of cores that need to be carefully processed and sampled. In spite of the large number of tissue cores, the amount of tumor available for study is often quite limited. This is a particular challenge for research, as new biomarker assays will need to preserve tissue architecture intact for histopathology. Methods of processing and reporting pathology are discussed. With the exception of ductal variants, recognized subtypes of prostate cancer are largely confined to research applications, and most prostate cancers are acinar. Biomarker discovery in urine and expressed prostatic secretions would be useful since these are readily obtained and are proximate fluids. The well-known challenges of biomarker discovery in blood and urine are referenced and discussed. Mediators of carcinogenesis can serve as biomarkers as exemplified by mutations in PTEN and TMPRSS2:ERG fusion. The use of proteomics in biomarker discovery with an emphasis on imaging mass spectroscopy of tissues is discussed. Small RNAs are of great interest, however, their usefulness as biomarkers in clinical decision making remains the subject of ongoing research. The chapter concludes with an overview of blood biomarkers such as circulating nucleic acids and tumor cells and bound/free isoforms of prostate specific antigen (PSA).

Discrete serum protein signatures discriminate between human retrovirus-associated hematologic and neurologic disease.

The human T-cell leukemia virus type I (HTLV-I) is the causative agent for adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Approximately 5% of infected individuals will develop either disease and currently there are no diagnostic tools for early detection or accurate assessment of disease state. We have employed high-throughput expression profiling of serum proteins using mass spectrometry to identify protein expression patterns that can discern between disease states of HTLV-I-infected individuals. Our study group consisted of 42 ATL, 50 HAM/TSP, and 38 normal controls. Spectral peaks corresponding to peptide ions were generated from MS-TOF data. We applied Classification and Regression Tree analysis to build a decision algorithm, which achieved 77% correct classification rate across the three groups. A second cohort of 10 ATL, 10 HAM and 10 control samples was used to validate this result. Linear discriminate analysis was performed to verify and visualize class separation. Affinity and sizing chromatography coupled with tandem mass spectrometry was used to identify three peaks specifically overexpressed in ATL: an 11.7 kDa fragment of alpha trypsin inhibitor, and two contiguous fragments (19.9 and 11.9 kDa) of haproglobin-2. To the best of our knowledge, this is the first application of protein profiling to distinguish between two disease states resulting from a single infectious agent.

Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease.

The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility.

Overexpression of p53 in prostate carcinoma is associated with improved overall survival but not predictive of response to radiotherapy.

p53 gene mutations are among the most common specific genetic alterations in human cancer. Inactivation of p53 and subsequent protein accumulation has been implicated in a variety of human malignancies and associated with prostate cancer progression. In this study, we assessed p53 protein overexpression and gene mutations in prostate carcinoma and investigated associations between p53 alterations and clinicopathological parameters, survival, and response to radiotherapy. We evaluated 58 archival formalin-fixed, paraffin-embedded prostate carcinomas to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. p53 mutational status of tumor DNA was evaluated using polymerase chain reaction-single-strand conformation polymorphism analysis of exons 5-9 and confirmed by direct DNA sequencing. Univariate and multivariate statistical analysis was used to determine the association of p53 status with clinical characteristics and response to radiotherapy. Overexpression of p53 was detected in 42 (72%) of 58 primary prostate carcinomas, but was undetectable in 7 samples of benign prostatic hyperplasias or 5 samples of normal prostate tissue. p53 exon 5-9 mutations were detected in 8 (14%) of 58 patient specimens. p53 mutational status, but not overexpression, was associated with higher Gleason scores (p=0.0145). Neither p53 overexpression nor mutation was associated with clinical stage, biochemical disease-free probability, or predictive of response to radiotherapy. p53 protein accumulation was inversely associated with improved overall survival (p=0.0108). Our studies demonstrate that p53 protein accumulation is a frequent alteration in prostate cancer. The disparity between p53 protein overexpression and p53 exon 5-9 mutations suggests the possibility of mutations outside this region or stabilization of wild-type p53 by alternative mechanisms. In our patient population, p53 protein overexpression or mutational status was not predictive of outcome in patients treated with radiation therapy. Additional studies are needed to further evaluate the association between p53 protein overexpression and improved overall survival.

Identification of a superimmunoglobulin gene family member overexpressed in benign prostatic hyperplasia .

BACKGROUND: Benign prostate hyperplasia (BPH), a nonmalignant disease with an increasing rate of occurrence associated with advancing age, requires auxiliary markers to help identify its presence and distinguish its progression from prostate cancer. METHODS: Hybridoma technology was used to generate an antibody against a BPH antigen, which was subsequently characterized by Western blot analysis, sequence homology, and RT-PCR. RESULTS: A BPH-associated protein, designated P25/26, was identified that showed a strong sequence similarity with superimmunoglobulin family members, overexpressed in BPH, with lower expression observed in both normal and prostate cancer tissues. CONCLUSIONS: Further studies appear warranted to assess the role that this and other superimmunoglobulin family members may have in the pathogenesis of BPH, and to determine if these glycoproteins have any clinical utility in the differential diagnosis or therapeutic monitoring of BPH.

Cloning and characterization of UROC28, a novel gene overexpressed in prostate, breast, and bladder cancers.

A novel gene, designated UROC28, was identified by an agarose gel-based differential display technique, and it was found to be up-regulated in prostate, breast, and bladder cancer. Expression of UROC28 was also up-regulated in prostate cancer cells in the presence of androgens as demonstrated by relative quantitative reverse transcription-PCR. The elevated expression of this gene was observed to increase in surgically removed tissues concomitantly with rising Gleason grade and was most elevated in metastatic tissue. UROC28 protein was detected in serum by Western slot blot analyses, and a significant higher UROC28 protein level was found in sera of prostate cancer individuals compared with normal individuals and individuals with nonmalignant prostatic hyperplasia. Northern analyses in normal tissues showed that the UROC28 cDNA hybridizes to two mRNAs at about 2.1 and 2.5 kb. Nucleic acid sequence analyses indicated that these two alternatively spliced mRNA variants differ only at the 3' untranslated region. These two mRNAs encode the same protein with 135 amino acids. Bioinformation analyses suggest that there is a possible transmembrane domain from amino acid aa34 to aa50, three protein kinase-C phosphorylation sites at aa62 (SQK), aa89 (TMK), and aa94 (SMK), and one myristylation site at aa118 (GLECCL). Genomic Southern hybridization and chromosomal mapping demonstrated that UROC28 is encoded by a single copy of gene at chromosome 6q23-24. In situ hybridization and immunohistochemistry experiments further confirmed up-regulation of this gene in prostate and breast cancers with the expression localizing to the glandular epithelium. This gene did not demonstrate increased expression in lung and colon cancer tissues.

Prostate-specific membrane antigen levels in sera from healthy men and patients with benign prostate hyperplasia or prostate cancer.

Prostate-specific membrane antigen (PSMA) serum levels have been proposed to be of prognostic significance in patients with advanced prostate disease. The objective of the present study was to confirm PSMA serum expression by Western blot techniques, to determine whether such data could assist in the differentiation of benign from malignant prostatic disease, and to determine the suitability of serum PSMA measurements in predicting recurrent or progressive prostate malignancies. We measured PSMA, a transmembrane glycoprotein identified in prostate epithelial cells, in the sera of 236 normal individuals and cancer patients by Western blot analysis. Within the normal male population, PSMA levels increase with age and were found to be significantly elevated in subjects more than 50 years of age when compared to those of younger men. We did not confirm previous reports that serum PSMA measurements could distinguish late-stage prostate carcinoma from early-stage prostate carcinoma, nor did we find PSMA to be more effective than prostate-specific antigen in monitoring prostate cancer patient prognosis. Furthermore, we found elevated serum PSMA in healthy females, and, similar to the healthy male population, the levels increased with age, with the highest levels found in the sera from breast cancer patients. These latter observations further support that PSMA is not a specific biomarker for prostate cancer and that a variety of normal and diseased tissue may contribute to the serum levels of PSMA.

Proteinchip(R) surface enhanced laser desorption/ionization (SELDI) mass spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures.

Improving early detection, diagnosis, treatment monitoring and prognosis of cancer will require rapid and high throughput detection, identification, and measurement of multiple biomarkers. In this study, we demonstrate the versatility of the innovative SELDI ProteinChip(R) MS technology for the rapid, reproducible and simultaneous identification of four well-characterized prostate cancer-associated (PCA) biomarkers, prostate specific antigen (free and complexed forms), prostate specific peptide, prostate acid phophatase and prostate specific membrane antigen in cell lysates, serum and seminal plasma. Proteins corresponding to the mass of these biomarkers could readily be captured and detected using either chemically defined or antibody coated ProteinChip(R) arrays. Several (yet to be identified) proteins were found upregulated in cell lysates of pure populations of PCA cells procured by laser capture microdissection (LCM) when compared with mass spectra of normal cell lysates. Coupling LCM with SELDI provides tremendous opportunities to discover and identify the signature proteins associated with each stage of tumor development. Collectively, these observations demonstrate the potential of SELDI for the discovery and simultaneous detection of and clinical assay development for PCA biomarkers in complex biological mixtures.

Accessing the embryo interior without microinjection.

For decades it has been assumed that in order to insert macromolecules into the embryo blastocoel for numerous experimental purposes, microinjection was required. Microinjection, however, can be only performed on a few embryos at a time, thus precluding many studies that could involve large populations of embryos. Laser scanning confocal microscopy, with its optical sectioning advantage, showed that fluorochrome-labeled macromolecular lectins and bovine albumin enter the blastocoel of living, swimming sea urchin embryos following a period of incubation without microinjection. A procedure is also described that shows macromolecular entry is substantially accelerated in low calcium seawater. The information gained from this study should greatly facilitate experiments on entire populations of millions of embryos at a time that require access of macromolecules to the embryo interior.

[Fracture of the maxillary tuberosity in the hemophiliac patient]. / Fractura de la tuberosidad maxilar en el paciente hemofílico.

Fracture of the left maxillary tuberosity during surgical extraction of multiple teeth, involving the maxillary sinus and pterigomaxillary fossa in a male patient with classic hemophilia due to factor VIII deficiency, is reported. The subject was treated by a private dentist and afterwards admitted at the Oral and Maxillofacial Surgery unit of the "20 de Noviembre" Hospital, ISSSTE, where extraction was completed and the fracture detected at surgery. The patient was then referred to Hematology and substitute therapy of factor VIII for two weeks was prescribed but, eventually, persistent bleeding led to suturing the external carotid.

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.

Correlation of nonspecific macromolecular labeling with environmental parameters during [(3)H]Thymidine incorporation in the waters of southwest florida.

During routine [(3)H]thymidine incorporation measurements of environmental samples, significant amounts of radioactivity are often incorporated into macromolecules other than DNA. Although the percentage of nonspecific labeling varies both temporally and spatially, the cause(s) of these variations remain unknown. Correlations between the percent incorporated radioactivity in DNA and a variety of experimental and environmental parameters measured in the Alfia River, Crystal River, Medard Reservoir, and Bayboro Harbor were examined. The amount of radioactivity incorporated into DNA ranged from 6 to 95% ([Formula: see text]; n=121). Nonspecific labeling began immediately upon the addition of [(3)H]thymidine and was linear over time. Labeling patterns were independent of both the amount of thymidine added and cell-size fraction. A two year study of Bayboro Harbor indicated no conclusive relationship between nonspecific labeling and seasonality. The amount of radioactivity incorporated into DNA was inversely correlated with total rates of thymidine incorporation and a strong diurnal pattern was observed in the Crystal River. No consistent relationship was observed between labeling patterns and primary productivity, chlorophylla, particulate DNA, dissolved DNA, bacterial cell numbers, temperature, salinity, and dissolved organic carbon. The only relationship with dissolved inorganic nutrients (N and P) occurred in the Crystal River. In this phosphate limited river, the percent of radioactivity incorporated into DNA was positively correlated with phosphate concentrations. These results indicate that nonspecific labeling is not dependent on any one parameter but may be a function of many interacting environmental factors or a function of the specific ambient bacterial population.

Turnover of extracellular DNA in eutrophic and oligotrophic freshwater environments of southwest Florida.

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 x 10 cells per liter and 28.6 mug of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 +/- 3.6 h in the Crystal River and 10.5 +/- 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.