Evidence for a specific microwave radiation effect on the green fluorescent protein

We have compared the effect of microwave irradiation and of conventional heating on the fluorescence of solution-based green fluorescent protein. A specialized near-field 8.5 GHz microwave applicator operating at 250 mW input microwave power was used. The solution temperature, the intensity, and the spectrum of the green fluorescent protein fluorescence 1), under microwave irradiation and 2), under conventional heating, were measured. In both cases the fluorescence intensity decreases and the spectrum becomes red-shifted. Although the microwave irradiation heats the solution, the microwave-induced changes in fluorescence cannot be explained by heating alone. Several possible scenarios are discussed.     

High mobility group proteins HMGD and HMGZ interact genetically with the Brahma chromatin remodeling complex in Drosophila

Many pleiotropic roles have been ascribed to small abundant HMG-Box (HMGB) proteins in higher eukaryotes but their precise function has remained enigmatic. To investigate their function genetically we have generated a defined deficiency uncovering the functionally redundant genes encoding HMGD and HMGZ, the Drosophila counterparts of HMGB1-3 in mammals. The resulting mutant is a strong hypomorphic allele of HmgD/Z. Surprisingly this allele is viable and exhibits only minor morphological defects even when homozygous. However, this allele interacts strongly with mutants of the Brahma chromatin remodeling complex, while no interaction was observed with mutant alleles of other remodeling complexes. We also observe genetic interactions between the HmgD/Z deficiency and some, but not all, known Brahma targets. These include the homeotic genes Sex combs reduced and Antennapedia, as well as the gene encoding the cell-signaling protein Rhomboid. In contrast to more general structural roles previously suggested for these proteins, we infer that a major function of the abundant HMGB proteins in Drosophila is to participate in Brahma-dependent chromatin remodeling at a specific subset of Brahma-dependent promoters.     

Proteasome inhibition attenuates hepatic injury in the bile duct-ligated mouse

Proteasome inhibition has recently been demonstrated to inhibit hepatic fibrogenesis in the bile duct-ligated (BDL) mouse by blocking stellate cell NF-kappaB activation. The effect of proteasome inhibition on liver injury, however, is unclear. Our aims were to assess the effect of the proteasome inhibitor bortezomib on liver injury in the BDL mouse. Liver injury was assessed in 7-day BDL mice treated with a single dose of bortezomib on day 4 after bile duct ligation. Despite NF-kappaB inhibition by bortezomib, liver injury and hepatocyte apoptosis were reduced in treated BDL mice. The antiapoptotic effect of bortezomib was likely mediated by an increase in hepatic cellular FLICE inhibitory protein (c-FLIP) levels, a potent antiapoptotic protein. Unexpectedly, numerous mitotic hepatocytes were observed in the bortezomib-treated BDL mice liver specimens. Consistent with this observation, PCNA immunoreactivity and cyclin A protein expression were also increased with bortezomib treatment. Bortezomib therapy was also associated with a decrease in numbers and activation of Kupffer cells/macrophages. In conclusion, these data suggest that the proteasome inhibitor bortezomib reduces hepatocyte injury in the BDL mouse by mechanisms associated with a reduction in hepatocyte apoptosis, a decrease in Kupffer cell/macrophage number and activation, and increased hepatocyte proliferation.     

Formation of hot spots in the plasma of a Z-pinch produced from low-density deuterated polyethylene

Results are presented from experimental studies of the plasma formation dynamics in a Z-pinch produced from a cylindrical microporous agar-agar load. The experiments were performed on the S-300 facility at a current of 2 MA and current rise time of 100 ns. To enhance the energy concentration, a deuterated polyethylene neck with a mass density of 50–75 μg/cm³ and diameter of 1–2 mm was made in the central part of the load. The spatiotemporal characteristics of the Z-pinch were studied using an optical streak camera and fast frame photography in the optical and soft X-ray spectral ranges. X-ray emission was detected using semiconductor and vacuum diodes, and neutron emission was studied by means of the time-of-flight method. It is found that, in the course of continuous plasma production, hot spots with a diameter of 100 μm form in the pinch plasma. The hot spots emit short soft X-ray pulses with a duration of 2–4 ns, as well as neutron pulses with an average neutron energy of about 2.45 MeV. The maximum neutron yield was found to be 4.5 × 10⁹ neutrons per shot. The scenario of hot spot formation is adequately described by two-dimensional MHD simulations.     

Independent origin of identical beta cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy.

The origins of the beta cardiac myosin heavy-chain (MHC) gene missense mutations that cause familial hypertrophic cardiomyopathy (FHC) in 14 families have been evaluated. Of eight different mutations, four were present in single families, while four occurred in two or more families. To investigate the origins of the four shared mutations, we defined the beta cardiac MHC haplotypes of each of the mutation-bearing chromosomes by determining the alleles present at three intragenic polymorphic loci. Two of the mutations (Arg453Cys and Val606Met) have arisen independently in each of three families, being found on different chromosomal backgrounds. A third mutation (Gly584Arg) is associated with identical haplotypes in two families with Portuguese ancestors, suggesting a founder effect. Haplotype analysis was uninformative for the fourth mutation (Arg403Gln). Thus, FHC-causing mutations have arisen independently in at least 12 of the 14 families studied, suggesting that the majority have arisen relatively recently as new mutations. This finding predicts the prevalence of disease-causing beta cardiac MHC mutations to be comparable in all population groups.     

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2.

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.     

Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE

We have developed a panel of murine monoclonal antibodies that recognize human interferon alpha. One of these mononclonal antibodies binds and neutralizes, with high affinity, all of seven tested recombinant human interferon alphas. This mononclonal antibody also neutralizes the interferon activity present in two independent pools of interferon alphas prepared following stimulation of human peripheral blood leukocytes. The complementary determining regions from this murine mononclonal antibody were transferred to a human IgG2 heavy chain and to a human kappa1 light chain. In addition, six (heavy chain) and two (light chain) amino acids were transferred from the framework regions. This generated a humanized mononclonal antibody that retained the specificity of the mouse parent. The humanized anti-interferon alpha antibody is a candidate therapeutic for those diseases, such as insulin-dependent diabetes, systemic lupus erythematosis, psoriasis and Crohn's disease, which are all characterized by pathological expression of interferon alpha.     

Light Amplification with Low-Gain Material: Harvesting Harmonic Resonance Modes of Surface Plasmon Polaritons on a Magnetic Meta-Surface

We theoretically show that it is feasible to utilize harmonic resonance modes of surface plasmon polaritons on a magnetic meta-surface for light amplification with low-gain material. The required threshold and optimal gain strength for active layer can be substantially reduced to about one-tenth as compared with those utilizing fundamental resonance modes. The findings provide a simple and practical metamaterial approach for light amplification with most of the existing active media.     

Generation of thioarsenicals is dependent on the enterohepatic circulation in rats

Three minor sulfur-containing arsenic metabolites: monomethylmonothioarsonic acid (MMMTA(V)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) were recently found in human and animal urine after exposure to inorganic arsenic. However, it remains unclear how the thioarsenicals are formed in the body and then excreted into the urine. It is hypothesized that the generation of thioarsenicals occurs during enterohepatic circulation. To address this hypothesis, male Sprague Dawley (SD) rats and Eisai hyperbilirubinuric (EHB) rats (with deficiency of multidrug resistance-associated protein 2) were orally administered a single dose of inorganic arsenite (iAs(III)) at 3.0 mg kg(-1) of body weight. Five hours after dosing, less than 1.0% of the dose was recovered in the bile of EHB rats, while more than 27% of the dose was recovered in the bile of SD rats, with the majority being monomethylarsinodiglutathione [MMA(SG)(2)] with a small amount of arsenic triglutathione [iAs(SG)(3)]. During the early time periods (3 h and 6 h) the arsenic levels in the liver, red blood cells (RBCs) and plasma of EHB rats were higher than those of SD rats, and approximately 76% and 87% of the dose was recovered in the RBCs of SD and EHB rats, respectively, at day 5 after dosing. However, there were no significant differences in arsenic concentration in urine between the two types of animal. Regarding the arsenic species in the urine of both types of rat, significant levels of thiolated arsenicals MMMTA(V) and DMMTA(V) were detected in SD rat urine, however in EHB rat urine only low levels of DMMTA(V) were detected. The present result of the metabolic balance and speciation study suggests that the formation of MMMTA(V) and DMMTA(V) in rats is dependent on enterohepatic circulation. In addition, in vitro experiments indicated that arsenicals excreted from bile may be transformed by gastrointestinal microbiota into MMMTA(V) and DMMTA(V), which are then absorbed into the bloodstream and finally excreted into the urine.     

SUSP1 antagonizes formation of highly SUMO2/3-conjugated species

Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.