New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a β(-) emitter such as (90)Y with an optical imager.     

Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins

The role of zooplankton in the control of cyanobacterial bloomsand the transfer of cyanotoxins to higher trophic levels areof great importance to the management of water resources. Manystudies have focused on the cyanobacterium Microcystis, butfew have examined the interactions between zooplankton and filamentouscyanobacteria. In this study, we provide experimental evidencefor the potential grazing of two toxic strains of filamentouscyanobacteria, Planktothrix rubescens and P. agardhii, by Daphniapulicaria, and for transfer of toxins in the planktonic foodchain. We determined clearance rates (CRs) by adult and juvenileD. pulicaria of the two Planktothrix strains, Scenedesmus acutusand a mixture of S. acutus cells with P. rubescens culture filtrate.Filament lengths were analyzed, and microcystin (MCY) presencein Daphnia was assessed using the Protein Phosphatase-2A (PP-2A)Inhibition Assay. The two Planktothrix strains were equallygrazed by D. pulicaria, but at lower CRs than S. acutus. Potentialanti-grazer toxins in P. rubescens filtrate did not inhibitDaphnia grazing. Small P. rubescens (<100 µm) filamentswere preferentially grazed by adult D. pulicaria, suggestingtheir limited ability to control a Planktothrix population duringa bloom. Large quantities of MCYs were found in unstarved Daphniapreviously exposed to Planktothrix, whereas quantities weresignificantly smaller in individuals starved for 24 h beforepreservation. This indicated a potential for transfer of toxinsin the food chain by Daphnia, especially immediately after ingestionof toxic cyanobacteria.     

Alternative antipredator responses of two coexisting Daphnia species to negative size selection by YOY perch

Our study showed for the first time in nature that two coexistingDaphnia adopted alternative life history and behavioural strategiesto cope with negative size-selection predation by gape-limitedyoung-of-the-year (YOY) perch. We evaluated the phenotypic plasticityin life history and behavioural traits of two coexisting Daphniaspecies, D. pulicaria (2 mm) and D. galeata mendotae (1.4 mm),in response to seasonal changes in predation by YOY yellow perch(Perca flavescens) in a mesotrophic lake. We expected that thelarge-sized D. pulicaria, the most likely subjected to size-selectivepredation by YOY perch, will show stronger antipredator responsesthan the small-sized D. galeata mendotae. To test this hypothesis,we examined changes in life history and behavioural traits injuveniles and adults of both species during four YOY fish predationperiods that were selected based on the presence of YOY perchin the pelagic zone and the relative abundance of Daphnia preyin their gut contents. Our study supports the scenario of negativesize-selective predation by gape-limited YOY perch on both Daphniaspecies. The electivity index indicated that no daphnids witha body length > 1.75 mm were predated by YOY yellow perch.Coexisting Daphnia exhibited phenotypic plasticity in theirantipredator defenses based on their vulnerability to seasonalchanges in size-selective predation of YOY perch. Juvenile Daphniawere the targeted prey and they responded by a decreased bodylength. Behavioural defenses were the dominant strategy usedby both adult Daphnia populations to withstand high predation.A decreased size at maturity was not employed by Daphnia, exceptat the very end of the predation period. Behavioural defensesare short-term strategy adopted to avoid predation. Both antipredatordefenses became unnecessary expenses and were no longer sustainedafter the predation period.     

Regulation of one-carbon metabolism in Arabidopsis: the N-terminal regulatory domain of cystathionine gamma-synthase is cleaved in response to folate starvation

In all organisms, control of folate homeostasis is of vital importance to sustain the demand for one-carbon (C1) units that are essential in major metabolic pathways. In this study we induced folate deficiency in Arabidopsis (Arabidopsis thaliana) cells by using two antifolate inhibitors. This treatment triggered a rapid and important decrease in the pool of folates with significant modification in the distribution of C1-substituted folate coenzymes, suggesting an adaptive response to favor a preferential shuttling of the flux of C1 units to the synthesis of nucleotides over the synthesis of methionine (Met). Metabolic profiling of folate-deficient cells indicated important perturbation of the activated methyl cycle because of the impairment of Met synthases that are deprived of their substrate 5-methyl-tetrahydrofolate. Intriguingly, S-adenosyl-Met and Met pools declined during the initial period of folate starvation but were further restored to typical levels. Reestablishment of Met and S-adenosyl-Met homeostasis was concomitant with a previously unknown posttranslational modification that consists in the removal of 92 amino acids at the N terminus of cystathionine gamma-synthase (CGS), the first specific enzyme for Met synthesis. Rescue experiments and analysis of different stresses indicated that CGS processing is specifically associated with perturbation of the folates pool. Also, CGS processing involves chloroplastic serine-type proteases that are expressed in various plant species subjected to folate starvation. We suggest that a metabolic effector, to date unidentified, can modulate CGS activity in vivo through an interaction with the N-terminal domain of the enzyme and that removal of this domain can suppress this regulation.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Cellular features of the extra-embryonic endoderm during elongation in the ovine conceptus

The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion.     

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.