Tissue microarray (TMA) applications: implications for molecular medicine

Modern expression-screening platforms such as complementary DNA (cDNA) arrays allow for high-throughput lead discovery in cancer and other diseases. For evaluation of promising candidate genes, however, in situ analysis of high numbers of clinical tissues samples--for example, by immunohistochemistry or fluorescence in situ hybridisation--is mandatory. Tissue microarray (TMA) technology greatly facilitates such analysis. Minute tissue cores (diameter 0.6 mm) are removed from up to a thousand different conventional paraffin blocks and re-assembled in a single empty paraffin block at predefined positions. Sections of the resulting TMA can be utilised for the range of research applicable to conventional tissue sections. Important advantages of the TMA technology are speed (parallel analysis of up to a thousand tissues), cost efficiency (the same amount of reagents required for a single large-section analysis is sufficient for a thousand samples), and standardisation (the same experimental conditions are applied to all samples). Because of the high numbers of samples usually included in TMAs, they are optimally suited to detect genotype-phenotype associations with high statistical power. Thus, TMA technology will markedly accelerate the transition from basic research to clinical applications.     

Improvement of pharmacokinetics of radioiodinated Tyr(3)-octreotide by conjugation with carbohydrates

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr(3)-octreotide (TOC). Glucose ([(125)I]Gluc-TOC), maltose ([(125)I]Malt-TOC), and maltotriose ([(125)I]Mtr-TOC) derivatives of [(125)I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [(125)I]Gluc-TOC and [(125)I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [(125)I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [(125)I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5-3.5. While tumor uptake of [(125)I]TOC (6.7 +/- 2.6%ID/g), [(125)I]Malt-TOC (5.3 +/- 1.9%ID/g), and [(125)I]Mtr-TOC (4.9 +/- 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [(125)I]Gluc-TOC was significantly increased (10.1 +/- 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and gamma-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.     

Androgen receptor agonist and antagonist reduce response of cytokine-induced killer cells on prostate cancer cells

Despite many advances, prostate cancer (PCa) is still the second most frequently diagnosed cancer and fifth leading cause of cancer death in men worldwide. So far, the promising field of onco-immunology has not yet provided a satisfactory treatment option for PCa. Here we show that the ex vivo expansion and activation of cytokine-induced killer (CIK) cells isolated from primary peripheral blood mononuclear cells induce immune-mediated apoptosis in both human PCa LNCaP and C4-2 cells. Interestingly, pretreating LNCaP and C4-2 cells with either androgen or the androgen receptor (AR) antagonist enzalutamide mediates resistance to this immunogenic attack. This is associated with a reduction of both total cell loss and apoptosis levels suggesting one possible mechanism blunting onco-immunological activity. The data also suggest that secreted factors from AR ligand-treated PCa cell suppress lymphocyte proliferation. Further, we analysed immune-mediated killing activity using conditioned media from LNCaP and C4-2 treated cells. The obtained data suggest that the conditioned media from PCa treated cells does not influence a measurable lymphocyte-mediated apoptosis. However, analysing clonal expansion of activated lymphocytes, the androgen-derived conditioned media suppresses lymphocyte proliferation/expansion suggesting inhibition of onco-immunological activity by pretreatment of PCa cells with AR ligands.     

Ringing induces the accumulation of vegetative storage proteins in poplar bark

Poplar branches were ringed in late spring to determine whether the interruption of the phloem flow could induce the accumulation of vegetative storage proteins (VSPs) in the bark of adult trees. Eight days after ringing, an increased deposition of starch as well as a premature rise in the soluble-protein level occurred in the bark tissues located 1 cm above the ring. Changes in the SDS-PAGE pattern of bark proteins were characterized by the accumulation of three polypeptides (32, 36 and 38 kDa), which exhibited the same molecular weight as VSPs described in poplar bark during winter, cross-reacted to antibodies raised against a poplar VSP, and bound to several lectins in the same way as poplar bark VSPs. These results indicate that during the vegetative period, ringing induces the accumulation of VSPs in the bark of poplar.     

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the -lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml^–1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml^–1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml^–1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml^–1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.     

The influence of externally applied organic substances on phloem translocation in detached maize leaves

Summary Solutions of organic substances show differing influences on the direction of phloem transport of ¹⁴C-labeled assimilates in predarkened maize leaf strips, when externally applied to one end of the strip. One group of substances pushes the assimilates away from the site of application. Examples of this group are 75 mM solutions of sucrose, trehalose, maltose, D-glucose, D-fructose, glucose-6-phosphate, raffinose and galactose. There is strong evidence that pushing substances are taken up from the apoplast and loaded into the phloem. Another group of substances attracts the assimilates, it seems to pull the assimilates in direction to the site of application. Examples of this second group are 75 mM solutions of arabinose, melibiose, myo-inositol, D-mannitol, polyethylene glycol 2000, and Na₂-EDTA (ethylene-diaminetetraacetate). The pulling substances obviously are not taken up into living cells. It is assumed that they accumulate in the apoplast and build up a water stress (water potential), which is counteracted by an increase of solute concentration in the parenchyma, thus creating a sink for assimilates. A third group of substances shows inert behaviour, having no perceptible influence on phloem transport, at least not, when applied as 75 mM solutions. At concentrations of more than 300 mM, inert substances tend to attract assimilates like those of the second group. Inert substances are xylose, sorbose, 2-deoxy-D-glucose, mannose and sorbitol.abbreviation EDTA ethylenediaminetetraacetate Supported by Deutsche Forschungsgemeinschaft     

Expression of functional kainate and AMPA receptors in developing lateral superior olive neurons of the rat

A functional analysis of AMPA and kainate receptors (AMPARs and KARs) in the lateral superior olive (LSO), a major nucleus in the auditory brainstem, has not been performed so far, to our knowledge. Here we investigated the presence and characteristics of such receptors in the rat LSO by means of whole-cell patch-clamp recordings in combination with pharmacology. Current responses evoked by 200 microM AMPA were completely blocked by the specific AMPAR antagonist GYKI 52466 (100 microM). Properties of the AMPAR-mediated currents (latency, activation time constant, and peak amplitude) remained constant between postnatal day 3 (P3) and P10. Current responses evoked by 100 microM KA were not completely blocked by 100 microM GYKI 52466, indicating that the residual component was mediated by KARs. Throughout development, two groups of KAR-mediated currents (fast I(KA) and slow I(KA)) were distinguished because they had significantly different mean activation time constants. Moreover, the mean peak amplitude of fast I(KA) was significantly higher than that of slow I(KA). The differentiation into fast I(KA) and slow I(KA) can be explained by the existence of two groups of LSO neurons displaying different KAR densities, distributions, and/or diverse types with differences in conductance. Application of the specific KAR subunit agonists SYM 2081 (10 microM), ATPA (10 microM), or iodowillardiine (1 microM) evoked currents in almost all cells tested, showing that GluR5 subunits are a component of functional KARs in LSO neurons. Electrical stimulation of ipsilateral input fibers in the presence of KAR antagonists (NS-102 and GAMS), modulators (WGA), or GYKI 52466 revealed the presence of synaptic KARs in LSO neurons.