Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.     

Peripheral metabolic responses to prolonged weight reduction that promote rapid, efficient regain in obesity-prone rats

Weight regain after weight loss is the most significant impediment to long-term weight reduction. We have developed a rodent paradigm that models the process of regain after weight loss, and we have employed both prospective and cross-sectional analyses to characterize the compensatory adaptations to weight reduction that may contribute to the propensity to regain lost weight. Obese rats were fed an energy-restricted (50-60% kcal) low-fat diet that reduced body weight by 14%. This reduced weight was maintained for up to 16 wk with limited provisions of the low-fat diet. Intake restriction was then removed, and the rats were followed for 56 days as they relapsed to the obese state. Prolonged weight reduction was accompanied by 1) a persistent energy gap resulting from an increased drive to eat and a reduced expenditure of energy, 2) a higher caloric efficiency of regain that may be linked with suppressed lipid utilization early in the relapse process, 3) preferential lipid accumulation in adipose tissue accompanied by adipocyte hyperplasia, and 4) humoral adiposity signals that underestimate the level of peripheral adiposity and likely influence the neural pathways controlling energy balance. Taken together, long-term weight reduction in this rodent paradigm is accompanied by a number of interrelated compensatory adjustments in the periphery that work together to promote rapid and efficient weight regain. These metabolic adjustments to weight reduction are discussed in the context of a homeostatic feedback system that controls body weight.     

Effects of chilling on tomato fruit texture

The effects of chilling on tomato ( Lycopersicon esculentum Mill cv. Caruso) texture were investigated using fruit stored at 22°C (nonchilled) or 5°C (chilled) for 28 days. or at 5°C for 15 days before transfer to 22°C to facilitate ripening during and additional 13 days (prechilled). Prechilled fruit exhibited symptoms of slight chilling injury, i.e. development of mealiness, accelerated softening relative to that of nonchilled fruit and nonuniform surface colour development. The firmness of all fruit decreased during ripening and chilled storage when measured by flat plate compression and puncture, especially during the early stages of ripening of nonchilled and prechilled fruit. The compression firmness of pericarp tissue similarly decreased during ripening of nonchilled and prechilled fruit, but was maintained during chilling. Total moisture content (ca 94%) of tissue, uronide content (32-35% w/w) and extracted β-galactosidase activity did not differ significantly ( P > 0.05) among fruit during ripening and chilled storage. The degree of uronide methyl esterification in ethanol-insoluble solids prepared from pericarp tissue (EIS) was relatively low for all fruit. i.e. <40%. EIS from which greater levels of pectinesterase were extracted (i.e. nonchilled>chilled>prechilled) exhibited decreased levels of uronide methyl esterification. Markedly elevated levels of β-glucosidase activity were extracted from prechilled EIS. Total polygalacturonase activity (mainly as PGI) and autolysis of enzyme-extracted EIS were inversely correlated ( P ≤ 0.05) only with the loss of nonchilled fruit and tissue firmness and prechilled fruit firmness. Results suggest a possible role for β-glucosidase in textural changes of prechilled fruit and tissue (e.g. loss of firmness, development of mealiness) and also implicate loss of skin strength in the softening of whole fruit during chilling.     

Repeated origin and loss of adhesive toepads in geckos

Geckos are well known for their extraordinary clinging abilities and many species easily scale vertical or even inverted surfaces. This ability is enabled by a complex digital adhesive mechanism (adhesive toepads) that employs van der Waals based adhesion, augmented by frictional forces. Numerous morphological traits and behaviors have evolved to facilitate deployment of the adhesive mechanism, maximize adhesive force and enable release from the substrate. The complex digital morphologies that result allow geckos to interact with their environment in a novel fashion quite differently from most other lizards. Details of toepad morphology suggest multiple gains and losses of the adhesive mechanism, but lack of a comprehensive phylogeny has hindered efforts to determine how frequently adhesive toepads have been gained and lost. Here we present a multigene phylogeny of geckos, including 107 of 118 recognized genera, and determine that adhesive toepads have been gained and lost multiple times, and remarkably, with approximately equal frequency. The most likely hypothesis suggests that adhesive toepads evolved 11 times and were lost nine times. The overall external morphology of the toepad is strikingly similar in many lineages in which it is independently derived, but lineage-specific differences are evident, particularly regarding internal anatomy, with unique morphological patterns defining each independent derivation.     

Effect of the estrous cycle and surgical ovariectomy on energy balance, fuel utilization, and physical activity in lean and obese female rats

This study presents an in-depth analysis of the effects of obesity on energy balance (EB) and fuel utilization in adult female rats, over the estrous cycle and immediately after surgical ovariectomy (OVX), to model pre- and postmenopausal states, respectively. Female Wistar rats were fed a high-fat (46%) diet for 16 wk to produce mature lean and obese animals. Stage of estrous was identified by daily vaginal lavage, while energy intake (EI), total energy expenditure (TEE), and fuel utilization were monitored in a multichamber indirect calorimeter and activity was monitored by infrared beam breaks. Metabolic monitoring studies were repeated during the 3-wk period of rapid OVX-induced weight gain. Component analysis of TEE was performed to determine the nonresting and resting portions of energy expenditure. Obesity was associated with a greater fluctuation in EB across the estrous cycle. Cycling obese rats were less active, expended more energy per movement, and oxidized more carbohydrate than lean rats. The changes in EB over the cycle in lean and obese rats were driven by changes in EI. Finally, OVX induced a large positive energy imbalance in obese and lean rats. This resulted primarily from an increase in EI in both groups, with little change in TEE following OVX. These observations reveal a dominant effect of obesity on EB, fuel utilization, and activity levels in cycling rats, which has implications for studies focused on obesity and EB in female rodents.     

Effects of noradrenergic neuronal activity on 3,4-dihydroxyphenylethylene glycol (DHPG) levels. Quantitation by high performance liquid chromatography

We describe a sensitive specific and simple high pressure liquid chromatographic procedure for determining 3,4-dihydroxyphenylethylene glycol (DHPG) in both plasma and tissue. DHPG is extracted from plasma or tissue extracts by adsorption onto alumina. DHPG in the alumina eluate is detected electrochemically following chromatography on a C₁₈ reverse phase column. The method is sensitive enough to detect approximately 20 pg/ml of plasma DHPG. Both clonidine (100 μg/kg) and desmethylimipramine (2.5 mg/kg) when administered to rabbits for 3 days induced significant falls in both cardiac and plasma DHPG concentrations. These experiments indicate that both tissue and plasma DHPG concentrations may be of value in assessing both the release and re-uptake of norepinephrine at peripheral sympathetic nerve endings in vivo.     

Non-cell autonomous influence of the astrocyte system xc? on hypoglycaemic neuronal cell death

Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system x_c⁻ – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system x_c⁻. Indeed, drugs known to inhibit system x_c⁻ ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system x_c⁻ (xCT). Finally, enhancement of astrocytic system x_c⁻ expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system x_c⁻ inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system x_c⁻, have a direct, non-cell autonomous effect on cortical neuron survival.