Signal pathway profiling of prostate cancer using reverse phase protein arrays

Reverse phase protein arrays represent a new proteomics microarray technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM) and analyzing the relative states of several key phosphorylation points within the cellular circuitry. We have applied reverse phase protein array technology to analyze the status of key points in cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to invasive prostate cancer. Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Focused analysis of phospho-specific endpoints revealed changes in cellular signaling events through disease progression and between patients. We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma directly from human tissue specimens. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring of the signaling events within actual human biopsies will be critical for patient-tailored therapy.     

Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes

Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human alpha2-macroglobulin (alpha2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.     

Transit peptide cleavage sites of integral thylakoid membrane proteins

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.     

The pollination mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): sexual mimicry and trap-flowers

The pollination process in Trigonidium obtusum Lindl. (Epidendroideae: Maxillariinae) is documented. The flowers are pollinated by sexually excited drones of Plebeia droryana (Meliponinae). When attempting to copulate either with sepals or petals, these bees slip on the waxy perianth surface and become trapped in the funnel-like flower tube. Bees trying to escape from the flowers may instead access the space between the column and lip, fixing the pollinarium on their scutellum. Pollinarium-bearing bees may pollinate the flowers when repeating the above-mentioned steps, leaving pollinia on the concave stigmatic surface, thus effecting pollination. Recently removed pollinaria are too broad to enter the stigma but they begin to dehydrate and within 40 min of removal are small enough to fit the stigmatic cavity. This mechanism prevents insect-mediated self-pollination and promotes cross-pollination. Preliminary evidence based on experiments with cultivated plants suggests that they are self-compatible but that fruit set is pollinator-dependent. The data obtained are discussed in a phylogenetic context. It is suggested that the pseudocopulatory syndrome in Trigonidium could have evolved from rewardless (food advertising) ancestors. Pseudocopulation in the context of the long flowering period of this orchid species (about 7 months) is understandable since the eusocial Plebeia bees produce fertile individuals several times a year.     

Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins

Thyroid hormone (T3) plays a crucial role in several steps of cerebellar ontogenesis. By using a neuron-astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-astrocytes presented a 40-60% increase on the total neurite length and an increment in the number of neurites. Treatment of astrocytes with epidermal growth factor (EGF) yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte neurite permissiveness induced by these treatments is mostly due to modulation of extracellular matrix (ECM) components. Such increase in ECM protein expression as well as astrocyte permissiveness to neurite outgrowth was reversed by the specific EGF receptor tyrosine kinase inhibitor, tyrphostin. Moreover, studies using selective inhibitors of several transduction-signaling cascades indicated that modulation of ECM proteins by EGF is mainly through a synergistic activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this work, we provide evidence of a novel role of EGF as an intermediary factor of T3 action on cerebellar ontogenesis. By modulating the content of ECM proteins, EGF increases neurite outgrowth. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of EGF and mitogen-activated protein kinase/phosphatidylinositol 3-kinase pathways on this process.     

Physiology and pathophysiology of nitric oxide in the nervous system,with special mention of the islands of Calleja and the circunventricular organs

Nitric oxide (NO) has been recognized as a key regulatory factor in many physiological processes, including central nervous system function, development, and phatophysiology. NO is produced by a class of enzymes known as NO synthases (NOS) and in normal adult animals only the neuronal isoform (nNOS) is detectable. During cortical development, nNOS was found at E14 in neuroblasts of the marginal zone and its expression raised to a zenith by P5, decreasing afterwards until reaching a steady level by P10. At that time, nNOS was found mainly in pyramidal neurons. Interestingly, the inducible isoform of the enzyme (iNOS) was also active from P3 to P7, but it disappeared almost completely by P20. The neurodegeneration observed during normal aging and following hypoxic accidents seems to be the result of cumulative free radical damage, and excessive production of NO may be at the basis of the cascade. After ischemic events we observed an elevation in the number of neurons expressing nNOS coincident with an elevation in Ca2+-dependent NOS activity for up to 120 min. After this period, nNOS activity began to decrease but it was substituted by a rapid increase in Ca2+-independent activity coincident with the histological appearance of previously undetectable iNOS-immunoreactive neurons. These increases in NO production were accompanied by specific patterns of protein nitration, a process that seems to result in loss of protein function. In particular, we observed a correlation between exposure to ischemia-reperfusion and nitration of cytochrome c. This process was coincident with the exit of the cytochrome from the mitochondria to the surrounding cytoplasm, an early event in neuronal apoptosis. Interestingly, most of the morphological and molecular changes associated with ischemic damage were prevented by treatment with inhibitors of NO production, indicating a clear path in the search for efficacious drugs in the battle against cerebrovascular accidents.     

Evolutionary stability of vigilance coordination among social foragers

Coordination can greatly improve the efficiency of anti-predatory vigilance scans by increasing predator detection for a constant proportion of time spent vigilant. However, it has been rarely found in nature and most studies have detected or assumed independent scanning by group members. In this study, we analysed the functional consequences of the coordinated alternation of vigilance scanning by group foragers. We introduce coordination by assuming that interscan intervals (ISIs) follow a modified gamma distribution. Depending on the parameters of the distribution, successive scans can be evenly spaced (coordinated scanning) or may present a high overlap (uncoordinated scanning). Comparing evolutionarily stable strategies for animals that do not coordinate their scanning with animals that do coordinate their anti-predator behaviour shows that coordination has a marked effect on survival probability. Moreover, the coordinating strategy is quite robust against mutants that scan independently with exponential distributions of ISIs. However, coordination breaks down when animals can continuously adjust their level of coordination by deciding the proportion of time they spend monitoring the behaviour of other group members. In this case, coordination is only evolutionarily stable if it can be very easily achieved.     

Microsatellite DNA library for Caiman latirostris

New genetic markers were characterized for the broad-snouted caiman (Caiman latirostris) by constructing libraries enriched for microsatellite DNA. Construction and characterization of these libraries are described in the present study. One microsatellite marker was developed from a (ACC-TGG)(n)enriched microsatellite DNA library, and 12 microsatellite markers were developed from a (AC-TG)(n)enriched microsatellite DNA library. These markers were tested in wild-caught animals, and these tests resulted in ten new polymorphic microsatellites for C. latirostris.     

Microgeographic variation in Caiman latirostris

In theory, geographic scale is related to genetic variation at the population level, whereas microgeographic scale may reveal intra-population structure such as social groups and families. In the present work, both levels of genetic variation in the broad-snouted caiman (Caiman latirostris) were evaluated in small wetlands associated with the Piracicaba River and some of its tributaries in the state of S?o Paulo, Brazil. Genetic variation was determined using microsatellite DNA markers originally developed for the American alligator (Alligator mississipiensis) and previously tested in pedigreed captive broad-snouted caimans. Using these markers, we were able to detect variability among individuals from different sites, even those within a small geographic distance. Genetic results suggest that the groups sampled at each site are composed predominantly of related individuals. A possible combination of high mortality and low natality rates results in a low number of successfully dispersed individuals per generation. Future studies using a recently constructed Caiman latirostris microsatellite library (Zucoloto et al., 2002) might help us to understand metapopulation processes that may be occurring within this species.     

Fat liquefaction: effect of low-level laser energy on adipose tissue

Low-level laser energy has been increasingly used in the treatment of a broad range of conditions and has improved wound healing, reduced edema, and relieved pain of various etiologies. This study examined whether 635-nm low-level lasers had an effect on adipose tissue in vivo and the procedural implementation of lipoplasty/liposuction techniques. The experiment investigated the effect of 635-nm, 10-mW diode laser radiation with exclusive energy dispersing optics. Total energy values of 1.2 J/cm(2), 2.4 J/cm(2), and 3.6 J/cm(2) were applied on human adipose tissue taken from lipectomy samples of 12 healthy women. The tissue samples were irradiated for 0, 2, 4, and 6 minutes with and without tumescent solution and were studied using the protocols of transmission electron microscopy and scanning electron microscopy. Nonirradiated tissue samples were taken for reference. More than 180 images were recorded and professionally evaluated. All microscopic results showed that without laser exposure the normal adipose tissue appeared as a grape-shaped node. After 4 minutes of laser exposure, 80 percent of the fat was released from the adipose cells; at 6 minutes of laser exposure, 99 percent of the fat was released from the adipocyte. The released fat was collected in the interstitial space. Transmission electron microscopic images of the adipose tissue taken at x60,000 showed a transitory pore and complete deflation of the adipocytes. The low-level laser energy affected the adipose cell by causing a transitory pore in the cell membrane to open, which permitted the fat content to go from inside to outside the cell. The cells in the interstitial space and the capillaries remained intact. Low-level laser-assisted lipoplasty has a significant impact on the procedural implementation of lipoplasty techniques.