Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact

Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic beta-cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of beta-cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.     

Partial characterization of a cerebral thyroid hormone-responsive protein

The thyroid hormone-responsive protein (THRP) is expressed in rat cerebral tissue and has 83% overall sequence homology with c-Abl interactor protein, Abi-2, which is a substrate for the tyrosine kinase activity of c-Abl. Within the core region of the two proteins, the sequence similarity approaches 99%. To determine whether THRP is a rat homologue of Abi-2 or is a distinct protein with unique properties, the tissue distribution of THRP and Abi-2 mRNA's was examined using a sensitive ribonuclease protection assay and probes specific for THRP and Abi-2, respectively. The THRP mRNA content of cerebral tissue (1340.0 +/- 126.5 arbitrary units) was 2.3-fold higher than Abi-2 mRNA (581.3 +/- 73.7), while the ratio of hepatic content of THRP mRNA (209.0 +/- 49.1) to hepatic Abi-2 mRNA (2923.0 +/- 378.7) was only 0.07 (P < 0.004). Very low levels of Abi-2 mRNA, but not THRP mRNA, were also found in the heart and small intestine. Experiments with PC12 cells transfected with the full-length THRP cDNA and grown in the presence or absence of a tyrosine kinase inhibitor, along with experiments where PC12 cells were cotransfected with the THRP cDNA with or without the wild-type or mutant (tyrosine kinase deficient) c-Abl cDNA, showed that THRP is tyrosine phosphorylated; however, it is not a substrate for c-Abl. These studies demonstrate that THRP and Abi-2 have distinct tissue distribution and distinct biological properties.     

Triggering of apoptosis is not sufficient to induce human immunodeficiency virus gene expression

We examined whether there is any causative link between apoptosis and HIV gene expression elicited in response to ultraviolet light (UV) and ionizing radiation (IR). We found that both UV and IR activate HIV gene expression in human T lymphoblastoid 1G5 (HIVluc) cells, but with different kinetics and magnitudes. Treatment with either type of radiation resulted in increased apoptosis, which correlated closely with HIV gene expression. The involvement of caspases in the IR response was demonstrated by using zVAD-FMK and zDEVD-FMK caspase inhibitors; both apoptosis and HIV gene expression were inhibited to similar extent. Surprisingly, treatment of 1G5 cells with FAS antibody triggered apoptosis but did not increase HIV gene expression. A correlation between increased apoptosis and gene expression was also demonstrated in human carcinoma HIVcat/A549 cells with UV whereas IR triggered apoptosis but did not activate HIV gene expression. Most significantly, UV activation of HIV gene expression, and NF-kappa-B and p38 MAP kinase, both important for efficient HIV gene expression, were not affected by treatment with the zVAD-FMK and zDEVD-FMK inhibitors. Treatment of HIVcat/A549 cells with staurosporine or scrape-loading of cells with cytochrome c resulted in apoptosis but no increase in HIV gene expression. Altogether, a direct correlation exists between apoptosis and HIV gene expression in T-cells in response to both UV and IR but this is not the case in carcinoma cells. Triggering of apoptosis per se in either cell type does not necessarily result in increased HIV gene expression. Most importantly, the apoptotic and HIV gene expression responses elicited by UV are different to some extent and can be separated.     

Hunting oxygen complexes of nitric oxide synthase at low temperature and high pressure

The reaction of nitric oxide synthase (NOS) with oxygen is fast and takes place within several steps, separated by ephemeral intermediates. The use of extreme experimental conditions, such as low temperature and high pressure, associated to rapid kinetic analysis, has proven to be a convenient tool to study this complex reaction. Stopped-flow experiments under high pressure indicated that oxygen binding occurred in more than one step. This was further corroborated by the detection of two short-lived oxy-compounds, differing in their spectral and electronic properties. Oxy-I resembles the ferrous oxygen complex known for cytochrome P450, whereas oxy-II appears to be locked in the superoxide form. Subzero temperature spectroscopy, together with an analytical separation method, revealed that the subsequent one-electron reduction of the oxygen complex is carried out by the NOS cofactor tetrahydrobiopterin (BH4). The low-temperature stabilized oxidation product of BH4 was found to be a protonated BH3 radical. Finally, work in the presence of a BH4 analog indicated that proton transfer to the activated oxygen complex is a second essential function of BH4.     

Evidence of two distinct oxygen complexes of reduced endothelial nitric oxide synthase

Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 degrees C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (k(on) approximately 2.5.10(6) m(-1).s(-1)) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at -30 degrees C. Heme-oxy II is formed more slowly (k(on) approximately equal to 3.10(5) m(-1).s(-1)) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced by N-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex.     

The use of multi-frequency EPR techniques to identify the radicals produced in irradiated beta-blockers

The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four β-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g₁=2.0086, g₂=2.0059 and g₃=2.0021 and g₁=2.0066, g₂=2.0044 and g₃=2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on β-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.     

Aggregation-resistant domain antibodies selected on phage by heat denaturation

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.     

VisRD--visual recombination detection

SUMMARY: VisRD, a program for visual recombination detection in a sequence alignment is presented. VisRD is written in Java and is designed to complement the multi-purpose phylogenetic software package SplitsTree4. AVAILABILITY: The software is freely available from http://www.lcb.uu.se/~vmoulton/software/visrd/