Identifying Predictors of Activity Based Anorexia Susceptibility in Diverse Genetic Rodent Populations

Animal studies are very useful in detection of early disease indicators and in unravelling the pathophysiological processes underlying core psychiatric disorder phenotypes. Early indicators are critical for preventive and efficient treatment of progressive psychiatric disorders like anorexia nervosa. Comparable to physical hyperactivity observed in anorexia nervosa patients, in the activity-based anorexia rodent model, mice and rats express paradoxical high voluntary wheel running activity levels when food restricted. Eleven inbred mouse strains and outbred Wistar WU rats were exposed to the activity-based anorexia model in search of identifying susceptibility predictors. Body weight, food intake and wheel running activity levels of each individual mouse and rat were measured. Mouse strains and rats with high wheel running activity levels during food restriction exhibited accelerated body weight loss. Linear mixed models for repeated measures analysis showed that baseline wheel running activity levels preceding the scheduled food restriction phase strongly predicted activity-based anorexia susceptibility (mice: Beta  =  −0.0158 (±0.003 SE), P<0.0001; rats: Beta  =  −0.0242 (±0.004 SE), P<0.0001) compared to other baseline parameters. These results suggest that physical activity levels play an important role in activity-based anorexia susceptibility in different rodent species with genetically diverse background. These findings support previous retrospective studies on physical activity levels in anorexia nervosa patients and indicate that pre-morbid physical activity levels could reflect an early indicator for disease severity.     

Recombinant Adeno-Associated Virus: Efficient Transduction of the Rat VMH and Clearance from Blood

To promote the efficient and safe application of adeno-associated virus (AAV) vectors as a gene transfer tool in the central nervous system (CNS), transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH). No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×10⁹ genomic copies of AAV would be fully cleared from blood after 2 days.     

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells

The process of Ca²⁺ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with a minimal reduction of the free luminal SR [Ca²⁺] ([Ca²⁺]_FSR). Importantly, active SR Ca²⁺ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca²⁺ binding sites. A new mathematical model was developed that assumes an increasing SR Ca²⁺ buffering capacity in response to an increase of the luminal SR [Ca²⁺] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca²⁺]_FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca²⁺ binding proteins switch to standard kinetics. The importance of KonD Ca²⁺ binding properties is twofold: a more efficient Ca²⁺ release process and that [Ca²⁺]_FSR and Ca²⁺-bound to SR proteins ([Ca²⁺]_BSR) can be regulated separately allowing for Ca²⁺ release to occur (provided by Ca²⁺-bound to luminal Ca²⁺ binding proteins) without an initial reduction of the [Ca²⁺]_FSR.     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Effect of short-term testosterone treatment on leptin concentrations in boys with pubertal delay

Testosterone administration increases growth hormone (GH) secretion and decreases the plasma leptin concentration in men. We evaluated the effect of increased GH secretion due to short-term testosterone treatment on leptin concentrations. Ten boys aged 14.8 +/- 0.2 (mean +/- SE) years with transient GH deficiency caused by pubertal delay were evaluated before and after (3 months) 4 intramuscular injections of 100 mg testosterone heptylate, given at 15-day intervals. The leptin concentration decreased from 5.4 +/- 1.3 to 3. 6 +/- 1.1 microgram/l (p < 0.001), despite a weight gain of 3.4 +/- 0.5 kg. There were significant increases in body mass index (BMI), from -0.2 +/- 0.5 to 0.2 +/- 0.5 SD, p < 0.005, in GH peak after stimulation test, from 6.3 +/- 0.5 to 21.7 +/- 2.9 microgram/l, p < 0. 0003, in plasma testosterone, from 0.6 +/- 0.1 to 6.5 +/- 1.3 microgram/l, p < 0.001, in insulin-like growth factor-I (IGF-I), from 152 +/- 21 to 330 +/- 30 microgram/l, p < 0.0001, and in IGF-binding protein-3 (IGFBP-3), from 4.2 +/- 0.5 to 5.4 +/- 0.4 mg/l, p < 0.01. But there were no changes in blood glucose (4.7 +/- 0.1 and 4.8 +/- 0.1 mmol/l), or plasma fasting insulin (9.0 +/- 1.2 and 8.1 +/- 1.3 mIU/l). The leptin concentrations were positively correlated with the BMI before (p < 0.03) and after (p < 0.04) testosterone, but not with the GH peak after stimulation, or with plasma testosterone, IGF-I or IGFBP-3. The leptin and insulin concentrations after testosterone treatment were positively correlated (p < 0.04). Thus, short-term testosterone treatment of boys with pubertal delay decreases their leptin concentrations. The lack of correlation with GH secretion or with its changes, despite the dramatic increase in GH secretion, and the lack of change in insulin are additional features suggesting that testosterone increases the leptin concentration mainly by an effect on adipose tissue.     

Access to new highly potent antileukemia,antiviral and antimalarial agents via hybridization of natural products (homo)egonol,thymoquinone and artemisinin

Hybridization of natural products has high potential to further improve their activities and may produce synergistic effects between linked pharmacophores. Here we report synthesis of nine new hybrids of natural products egonol, homoegonol, thymoquinone and artemisinin and evaluation of their activities against P. falciparum 3D7 parasites, human cytomegalovirus, sensitive and multidrug-resistant human leukemia cells. Most of the new hybrids exceed their parent compounds in antimalarial, antiviral and antileukemia activities and in some cases show higher in vitro efficacy than clinically used reference drugs chloroquine, ganciclovir and doxorubicin. Combined, our findings stress the high potency of these hybrids and encourages further use of the hybridization concept in applied pharmacological research.