A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Intracellular recording from a spider vibration receptor

The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ’s cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.     

“Hypermethylation” of anthranilic acid-labeled sugars confers the selectivity required for liquid chromatography-mass spectrometry

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of “hyper”-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.     

Molecular machinery of mitochondrial dynamics in yeast

Mitochondria are amazingly dynamic organelles. They continuously move along cytoskeletal tracks and frequently fuse and divide. These processes are important for maintenance of mitochondrial functions, for inheritance of the organelles upon cell division, for cellular differentiation and for apoptosis. As the machinery of mitochondrial behavior has been highly conserved during evolution, it can be studied in simple model organisms, such as yeast. During the past decade, several key components of mitochondrial dynamics have been identified and functionally characterized in Saccharomyces cerevisiae. These include the mitochondrial fusion and fission machineries and proteins required for maintenance of tubular shape and mitochondrial motility. Taken together, these findings reveal a comprehensive picture that shows the cellular processes and molecular components required for mitochondrial inheritance and morphogenesis in a simple eukaryotic cell.     

Pharmacological analysis of ionotropic glutamate receptor function in neuronal circuits of the zebrafish olfactory bulb

Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca(2+) imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1) interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2) interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3) AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4) ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb.     

Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.     

Coastal wetland restoration through the lens of Odum's theory of ecosystem development

Advancing ecological restoration assessments requires a more detailed consideration of species interactions and ecosystem processes. Most restoration projects rely on a few metrics not always directly linked with ecological theory. Here, we used Odum's theory of ecosystem development to assess and compare the ecosystem structure and services of created marshes (4–6 years old) with preexisting, reference marshes in a brackish water region of the Mississippi River Delta. We built ecosystem models for created and reference marshes that integrated large datasets of stomach contents, stable isotopes, and taxa abundances. Despite strong resemblance in community structure, created marshes were at an earlier succession stage compared to the reference marshes, having lower biomass (including exploited species), higher biomass turnover and production, less dependence on detritus, lower material cycling, and less energy flowing through specialist pathways. Although preserving preexisting marshes should be a priority, created marshes may still be an important tool for the restoration of coastal areas and their ecosystem services. In addition, our results show that comparisons of species biodiversity alone may fail to capture essential differences in ecosystem processes between habitats, which reinforces the importance of ecosystem modeling approaches to assess restoration projects.     

多位点DNA指纹技术在保加利亚普通田鼠中的应用探讨

DNA指纹是一种重要的现代分子遗传学标记技术（Jeffreys et al．,1985）,它所揭示的是生物体大量的、无遗传编码信息的、具有高度多态性的卫星DNA（Chen,1996）。这些DNA序列往往占据了生物体基因组总量的80％以上,由于它不编码蛋白基因,在系统发育过程中,通常不被自然选择和人工选择,使得生物变异积累形成个体基因组间的巨大差异。因此,DNA指纹受到生物学家的青睐,以用于生物个体和群体的基因组分析（Burke and Bruford,1987;Buitmap et al．,1991;Weising et al．,1995）。