Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls

The labeling of targeting peptides with (99m)Tc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac-[(99m)Tc(CO)(3)](+) core for introducing (99m)Tc in biomolecules in general and in peptides in particular. The three coordination sites available in [(99m)Tc(OH(2))(3)(CO)(3)](+) can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed-ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

A conserved role for the zinc finger polyadenosine RNA binding protein,ZC3H14, in control of poly(A) tail length

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.     

The KCNJ11 E23K polymorphism and progression of glycaemia in Southern Chinese: a long-term prospective study

Context

The KCNJ11 E23K variant is associated with type 2 diabetes mellitus (T2DM) in cross-sectional studies, but conflicting findings have been reported from prospective studies.

Objective

This study aimed to evaluate whether the E23K variant could predict glycaemic progression in a Southern Chinese population.

Methods/Principal Findings

We performed a long-term prospective study on 1912 subjects from the Hong Kong Cardiovascular Risk Factors Prevalence Study (CRISPS). The KCNJ11 E23K variant was associated with the progression to prediabetes after a median interval of 12 years on multinomial logistic regression analysis, even after adjustment for traditional risk factors (OR 1.29, P_{age, sex, BMI and fasting plasma glucose [FPG] adjusted}  = 0.02). Based on Cox proportional hazard regression analysis, the E23K variant also predicted incident prediabetes (HR 1.18, P_{age, sex, BMI and FPG adjusted}  = 0.021). However, E23K was not associated with the progression to T2DM in either multinomial or Cox regression analysis, and the association of E23K with glycaemic progression to either prediabetes or T2DM was significant only in unadjusted Cox regression analysis (P = 0.039). In a meta-analysis of eight prospective studies including our own, involving 15680 subjects, the E23K variant was associated with incident T2DM (fixed effect: OR 1.10, P = 4×10⁻³; random effect: OR 1.11, P = 0.035).

Conclusions

Our study has provided supporting evidence for the role of the E23K variant in glycaemic progression in Chinese, with its effect being more evident in the early stage of T2DM, as the subjects progressed from normal glucose tolerance to prediabetes.     

Design of Tripeptides as a Competitive Inhibitor for HMG-CoA Reductase

International Journal of Peptide Research and Therapeutics - This study presents a simple approach in design of tripeptides as a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase...     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.