Labeled 3-aryl-4-indolylmaleimide derivatives and their potential as angiogenic PET biomarkers

In a continued effort to find a suitable PET tracer for visualization of angiogenic processes, we explored the 3,4-diarylmaleimide family, known to have high affinity and selectivity towards the VEGFR-TKs. One previously reported agent and three new halogen-containing 3,4-diarylmaleimide derivatives were synthesized. The four maleimide derivatives were evaluated for their affinity and selectivity towards the VEGFRs and exhibited promising results. An automated carbon-11 radiolabeling route with a total synthesis time of 50 min successfully labeled the lead compound, resulting in 1.55 ± 0.15 GBq of tracer with a radiochemical yield of 20 ± 2%, 96% radiochemical purity and a SA of 111 ± 22 GBq/μmol (EOB, n = 5). The tracer possessed high stability in in vitro blood stability tests and specific VEGFR-TK binding profiles in intact cell binding experiments. Tracer lipophilicity was evaluated in an n-octanol/phosphate buffer system giving a Log D_7.4 of 1.99 ± 0.04. For the in vivo experiments, two animal models were used. The first was a U87 glioma tumor model, frequently reported in the literature and the second, a newly developed 293/KDR tumor model. Both models were validated for VEGFR-2 expression and used in in vivo biodistribution studies. These studies revealed low accumulation and rapid washout of the tracer from tumor tissue. High accumulation of activity in the liver prompted us to examine the tracer’s in vitro stability to liver microsomes, revealing low resistance to P450 metabolism. In spite of encouraging in vitro results, the labeled lead tracer failed to accumulate in VEGFR-2 overexpressing tumors. It is possible that poor resistance to P450 metabolism reduces tracer’s circulation leading to low tumor accumulation.     

HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Background

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Methods and Findings

The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.

Conclusions

Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.     

Cisplatin effects on rhythmic functions of mice: strain and tissue dependence

The competence to preserve the optimal timing relationships between rhythmic variables enables adaptation of mammals to alternate environmental conditions. The capability to re-entrain depends on genetic factors and the nature of imposed time cues. In the present study, the authors examined in rodent models, following a cancer chronochemotherapy, cisplatin (CP), the rhythm patterns of locomotor activity and of a few biochemical variables (alkaline phosphatase and creatinine phosphokinase in kidney tissue and plasma, kidney urea nitrogen, and white blood cell count). Males of two inbred mice strains, BALB/c and c57Bl/6J, received 10 consecutive daily intraperitoneal (i.p.) injections of either saline or CP at zeitgeber time 22 (ZT22). CP administration altered the rhythms of each examined function in both strains. The type and extent of the changes varied among variables, tissues/plasma, and mouse strain. Yet, the effect of CP was not detected on all parameters, but only in ～60% of them. In addition, in the majority of the studied parameters, BALB/c and c57Bl/6J mice differed in their response to CP. The temporal parameters of period and peak time were more affected by CP than were the level ones of mesor (time series mean) and amplitude of variation. This observation may indicate the involvement of independent pathways of action upon each of the rhythm parameter sets. As a result, the rhythm phenotype of each function was modified and novel timing relationships were shaped. The results show that the circadian systems of BALB/c and c57Bl/6J mice failed to re-entrain after cessation of CP injections (tested on the first day following the 10 d course of CP administration), pointing to a direct effect of the medication on the tissues. The findings imply that optimal chemotherapeutic protocols should be tailored individually, according to the current temporal order rather than administered at a fixed predetermined circadian time. Further studies are necessary to determine which variables and rhythmic parameters could be useful to determine the optimal timing of chronochemotherapy.     

Intact cell adhesion to glycan microarrays

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.     

Circadian variation of nitric oxide synthase activity in mouse tissue

Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NO_x levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light-dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO₂+ NO₃^- (NO_x) and total protein. The NO_x concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NO_x levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.     

Tinnitus-like “hallucinations” elicited by sensory deprivation in an entropy maximization recurrent neural network

Sensory deprivation has long been known to cause hallucinations or “phantom” sensations, the most common of which is tinnitus induced by hearing loss, affecting 10–20% of the population. An observable hearing loss, causing auditory sensory deprivation over a band of frequencies, is present in over 90% of people with tinnitus. Existing plasticity-based computational models for tinnitus are usually driven by homeostatic mechanisms, modeled to fit phenomenological findings. Here, we use an objective-driven learning algorithm to model an early auditory processing neuronal network, e.g., in the dorsal cochlear nucleus. The learning algorithm maximizes the network’s output entropy by learning the feed-forward and recurrent interactions in the model. We show that the connectivity patterns and responses learned by the model display several hallmarks of early auditory neuronal networks. We further demonstrate that attenuation of peripheral inputs drives the recurrent network towards its critical point and transition into a tinnitus-like state. In this state, the network activity resembles responses to genuine inputs even in the absence of external stimulation, namely, it “hallucinates” auditory responses. These findings demonstrate how objective-driven plasticity mechanisms that normally act to optimize the network’s input representation can also elicit pathologies such as tinnitus as a result of sensory deprivation.     

Insulin rapidly stimulates binding of phosphofructokinase and aldolase to muscle cytoskeleton.

1. We report here on a novel action of insulin which shows that the hormone stimulates binding of phosphofructokinase (PFK) and aldolase to muscle cytoskeleton. 2. This effect was demonstrated both in vivo, by injection of insulin, in the tibialis anterior and gastrocnemius muscles, as well as in vitro, in the isolated rat diaphragm muscle incubated with insulin. 3. Insulin exerted this effect at physiologic range of concentrations and very rapidly (about 50% stimulation of binding occurred within 1 min). 4. The possible physiological significance of this rapid action of insulin, is to provide local ATP, generated by the accelerated cytoskeletal glycolysis, for other rapidly insulin-stimulated membrane-cytoskeleton processes.