Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.     

Formation of glycolate by a reconstituted spinach chloroplast preparation

A reconstituted preparation requiring fructose 6-phosphate, transketolase, triphosphopyridine nucleotide, ferredoxin, fragmented spinach chloroplasts, and light capable of forming glycolate at rates of about 10 micromoles per milligram of chlorophyll per hour has been characterized. The glycolaldehyde-transketolase addition product could be substituted for fructose 6-phosphate and transketolase. The stoichiometry of the reaction was: 1 mole of fructose 6-phosphate consumed for each mole of glycolate and of reduced triphosphopyridine nucleotide produced. Evidence was presented indicating that glycolate formation was coupled to the photosystems of the photosynthetic electron transport chain. Synthesis of glycolate is envisaged as the result of either (a) a reaction between the upper two carbon atoms derived from fructose 6-phosphate and an uncharacterized oxidant generated by photosystem 2 or (b) hydrogen peroxide produced by the reoxidation of reduced triphos-phopyridine nucleotide or reduced ferredoxin by molecular oxygen.     

Electrophysiological properties of human oviduct smooth muscle cells in dissociated cell culture.

Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was -35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 Momega and 96 msec, compared to connected cells, 26 Momega and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current pulses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5 . 20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.     

Chemosensitivity of single smooth muscle cells to acetylcholine, noradrenaline, and histamine in vitro

Electrical responses to acetylcholine, noradrenaline, and histamine were recorded from solitary smooth muscle cells. Iontophoresis of each transmitter elicited three fast responses: a hyperpolarization, a depolarization, or a biphasic hyperpolarization-depolarization. Each transmitter activated a specific receptor since responses were specifically blocked by antagonists, two transmitters elicited different responses in solitary cells, and desensitization of response to one transmitter did not cause desensitization of responses to other transmitters. Responses were due to increased ion conductances since input resistance decreased during responses and reversal potentials were measured for depolarizing responses (-5 mV) and hyperpolarizing responses (-60 mV). Regional differences in transmitter sensitivity were mapped on solitary cells. Biphasic responses were due to simultaneous activation of receptors mediating hyperpolarizing responses and receptors mediating depolarizing responses which were segregated in the cell membrane. Noradrenaline enhanced action potential amplitude by regulation of voltage-dependent ion conductances. Finally, noradrenaline and histamine elicited periodic hyperpolarizing potentials, which may be due to increased intracellular Ca++.     

Analysis of TaqMan Array Cards Data by an Assumption-Free Improvement of the maxRatio Algorithm Is More Accurate than the Cycle-Threshold Method

Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke''s Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss'' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss'' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings.     

Constitutive RelB activation in v-Src-transformed fibroblasts: requirement for IkappaB degradation

RelB, an NF-kappaB/Rel-related transacting factor, was initially identified as an immediate-early gene product in fibroblasts and subsequently shown to exhibit constitutive DNA binding activity in lymphoid cells. The data presented in this report show that RelB is also constitutively active, as monitored by electrophoretic mobility shift assay, in the v-Src-transformed fibroblast cell line, SR1. By contrast, nontransformed parental (3Y1) cells displayed inducible NF-kappaB activity; RelB activity was also observed, although to a lesser extent, in two additional v-Src-transformed fibroblast lines. RelB activation in SR1 cells did not require an increase in RelB expression or result from a decrease in the levels of IkappaB alpha or p105, proteins previously shown to bind to and inhibit the activity of the Rel proteins. Numerous studies have shown that stimulus-dependent Rel activation requires degradation of IkappaB alpha, p105 or other member of the IkappaB family, and that this process is precluded by agents that inhibit proteasome activity. We show that treatment of SR1 cells with proteasome inhibitors abolishes RelB activity and thus suggest that RelB in these cells is associated with IkappaB and that v-Src transformation activates RelB by accelerating IkappaB proteolysis. Additional data show that serum and tumor necrosis factor-alpha (TNF-alpha) increase RelB protein levels in 3Y1 cells and that this process is blocked by proteasome inhibitors.     

The national DBS brain tissue network pilot study: need for more tissue and more standardization

Over 70,000 DBS devices have been implanted worldwide; however, there remains a paucity of well-characterized post-mortem DBS brains available to researchers. We propose that the overall understanding of DBS can be improved through the establishment of a Deep Brain Stimulation-Brain Tissue Network (DBS-BTN), which will further our understanding of DBS and brain function. The objectives of the tissue bank are twofold: (a) to provide a complete (clinical, imaging and pathological) database for DBS brain tissue samples, and (b) to make available DBS tissue samples to researchers, which will help our understanding of disease and underlying brain circuitry. Standard operating procedures for processing DBS brains were developed as part of the pilot project. Complete data files were created for individual patients and included demographic information, clinical information, imaging data, pathology, and DBS lead locations/settings. 19 DBS brains were collected from 11 geographically dispersed centers from across the U.S. The average age at the time of death was 69.3 years (51?C92, with a standard deviation or SD of 10.13). The male:female ratio was almost 3:1. Average post-mortem interval from death to brain collection was 10.6 h (SD of 7.17). The DBS targets included: subthalamic nucleus, globus pallidus interna, and ventralis intermedius nucleus of the thalamus. In 16.7% of cases the clinical diagnosis failed to match the pathological diagnosis. We provide neuropathological findings from the cohort, and perilead responses to DBS. One of the most important observations made in this pilot study was the missing data, which was approximately 25% of all available data fields. Preliminary results demonstrated the feasibility and utility of creating a National DBS-BTN resource for the scientific community. We plan to improve our techniques to remedy omitted clinical/research data, and expand the Network to include a larger donor pool. We will enhance sample preparation to facilitate advanced molecular studies and progenitor cell retrieval.     

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp—Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1→6 is homologous to antistasin, a leech anticoagulant, and Cys7→12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.