CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.     

A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.     

The journey of a sandwich: computer-based laboratory experiments about the human digestive system in high school biology teaching

Teaching high school students about the digestive system can be a challenge for a teacher when s/he wants to overcome rote learning of facts without a deeper understanding of the physiological processes inside the alimentary tract. A series of model experiments illustrating the journey of a sandwich was introduced into teaching high school biology. Using a computer equipped with a commercially available data-acquisition system and a couple of sensors, it was possible to illustrate the basic underlying physical and chemical principles of digestion to the students. Students were able to investigate, through hands-on activities, the chewing force of the jaws, importance of the mechanical breakdown of food, enzymatic activity of pepsin and amylase, antibacterial activity of hydrochloric acid, and importance of the villi for absorption. Students found the experiments interesting and helpful for understanding the digestive process. Furthermore, the results from testing indicated that the students had a deeper understanding of the physiological processes.     

Transventricular and transpial absorption of cerebrospinal fluid into cerebral microvessels

It is generally accepted that volume of cerebrospinal fluid (CSF) is secreted in brain ventricles and flows to subarachnoid space to be absorbed into dural venous sinuses or/and into lymphatics via perineural sheats of cranial nerves. Since 99% of CSF volume is water, in experiments on cats 3H-water was slowly infused into lateral ventricle and found that it does not flow to subarachnoid space but that it is rapidly absorbed transventricularly into periventricular capillaries. When 3H-water was infused in cortical subarachnoid space, it was absorbed locally into cerebral capillaries via pia mater. On the contrary, when macromolecule 3H-inulin is applied in CSF it is very slowly eliminated in bloodstream, and, with time, is carried by systolic-diastolic pulsations and mixing of CSF bidirectionally along CSF system. Thus, CSF volume (water) is absorbed rapidly into adjacent cerebral capillaries while inulin is distributed bidirectionally due to its long residence time in CSF Previously, the macromolecules have been used to study CSF volume hydrodynamics and with this misconception of CSF physiology arose.     

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L^?1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.     

A-Kinase Anchoring Protein Lbc Coordinates a p38 Activating Signaling Complex Controlling Compensatory Cardiac Hypertrophy

In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous prohypertrophic signaling pathways in cultured cardiomyocytes. However, it remains to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3, and p38α that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. These results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.     

Enantiomerization of 3-carbethoxy-l,4-benzodiazepin-2-one: combined chiral HPLC and spectroscopic study.

Recently developed chiral HPLC columns CHIRIS AD1 and CHIRIS AD2 have been demonstrated to separate racemic, configurationally unstable ethyl-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-carboxylate (1) and its 3-methyl congener 2; fast on-column enantiomerization of configurationally unstable 1 was observed, however. Addition of 0.1% of AcOH to the eluting mixture inhibits enantiomerization, whereas the same percentage of Et(3)N completely precludes enantioseparation, suggesting base-catalysis by free beta-aminoethyl groups, present in low percentage in chiral stationary phase (CSP). When both CSPs were prepared under conditions of nonexhaustive acylation by N-DNB-alpha-aminoacids, no separation of 1 was observed. The rate of enantiomerization on CHIRIS AD2 was determined at 25 degrees C, the mechanism is discussed, and experimental results correlated with calculated relative stabilities of the tautomers la-c. Absolute (3S) configuration of (+) enantiomers of 1 and 2 was determined by comparison of their eluation profile to that of (+/-)-3 and (3S)-(+)-3, taking into account relative (psia or psie) configuration of the prevailing conformer in solution.     

Serum cholesterol concentrations in suicidal and non-suicidal male patients suffering from persistent delusional disorder

Suicidal behavior is a major health risk in psychiatric disorders, especially in affective and psychotic disorders. The neurobiology of suicidal behavior is still unclear. Suicidality has been related to a reduced cholesterol levels. The aim of this study was to evaluate serum cholesterol concentrations in suicidal and non-suicidal men suffering from persistent delusional disorder and in healthy volunteers. Results showed that serum cholesterol concentrations were significantly lower in suicidal than in non-suicidal patients and healthy controls. Also, level of psychopathology (measured by Brief Psychiatric Rating Scale) is significantly marked in the group of suicidal patients, which indicates the importance of detecting some clinical symptoms in patients with persistent delusional disorder in order to prevent suicidal behavior.     

Molecular evolution, mutation size and gene pleiotropy: a geometric reexamination

The influence of phenotypic effects of genetic mutations on molecular evolution is not well understood. Neutral and nearly neutral theories of molecular evolution predict a negative relationship between the evolutionary rate of proteins and their functional importance; nevertheless empirical studies seeking relationships between evolutionary rate and the phenotypic role of proteins have not produced conclusive results. In particular, previous studies have not found the expected negative correlation between evolutionary rate and gene pleiotropy. Here, we studied the effect of gene pleiotropy and the phenotypic size of mutations on the evolutionary rate of genes in a geometrical model, in which gene pleiotropy was characterized by n molecular phenotypes that affect organismal fitness. For a nearly neutral process, we found a negative relationship between evolutionary rate and mutation size but pleiotropy did not affect the evolutionary rate. Further, for a selection model, where most of the substitutions were fixed by natural selection in a randomly fluctuating environment, we also found a negative relationship between evolutionary rate and mutation size, but interestingly, gene pleiotropy increased the evolutionary rate as √n. These findings may explain part of the disagreement between empirical data and traditional expectations.