Isolation and characterization of cytochrome b5 from Candida tropicalis grown on alkane

Cytochrome b₅ from Candida tropicalis grown on alkane has been solubilized in three different ways (sodium cholate, trypsin, osmotic wash). After solubilization of the microsomal membrane with sodium cholate, the purification of cytochrome b₅ was achieved by DEAE-cellulose chromatography, hydroxylapatite chromatography, a second DEAE-cellulose chromatography and a Sephadex G-75 gel filtration. The purified protein had an apparent molecular weight of 16 000 ± 1 000. After solubilization by trypsin treatment or osmotic wash, the purification procedure yielded a protein with an apparent molecular weight of 12 000 ± 1 000. Though the purified proteins presented molecular weights depending on the technique of solubilization, they exhibited identical optical properties, a great stability with respect to temperature and pH, and were all autooxidable. Redox titrations revealed differences in their midpoint potential values, which were 35 ± 5 mV for the b₅ purified after cholate solubilization, —59 ± 5 mV for the b₅ purified after trypsin treatment and —65 ± 5 mV for the b₅ purified after osmotic wash.     

FOITS (fast optical in vivo topometry of human skin): new approaches to 3-D surface structures of human skin]

FOITS (Fast Optical In-vivo Topometry of human Skin) is a new technique for the three-dimensional analysis of the microstructures of the skin. Based on the surface roughness standards used in the metal-working industry, it uses computer-assisted strip analysis to process information gathered from high-speed, non-contact scanning. It is possible to visualise the mathematical data in 3D form. So far, surface relief parameters have had to be acquired indirectly by first making silicone casts of the skin and then digitalising them with image analysis, mechanical scanners or, more recent, laser profilometry and confocal microscopy. We selected the roughness parameters Ra, Rz and Rmax (DIN defined) and "positive" and "negative" volume to describe the skin surface. In 40 healthy volunteers we were able to define significant intra-individual differences in these roughness parameters, which were correlated with the location of the area of skin being scanned (p < 0.05). When healthy areas of skin were compared with areas of chronic eczema in 10 patients with neurodermatitis the differences in the above-mentioned roughness parameters were just as striking (p < 0.05). In patients with chronic atopic eczema there was a statistically significant correlation between changes in the parameters "positive" and "negative" volume, and the smoothing of the skin seen after 14 days of treatment with standard ointments (p < 0.02). Apart from its industrial uses, we see potential applications for FOITS in the investigation of quantitative and qualitative aspects of aging processes, physiological and pathological processes in the skin, and the effect of topical preparations applied to the skin.     

A novel inverse relationship between metformin-triggered AMPK-SIRT1 signaling and p53 protein abundance in high glucose-exposed HepG2 cells

AMP-activated protein kinase (AMPK) and the NAD(+)-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.     

IL-6 Indirectly Modulates the Induction of Glyceroneogenic Enzymes in Adipose Tissue during Exercise

Background

Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls.

Methodology and Principle Findings

Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6^−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6^−/− mice.

Conclusions and Significance

Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.     

ANALYSIS OF PORPHYRA RBC L DEMONSTRATES MULTIPLE MIGRATIONS OCCURRED BETWEEN THE NORTH ATLANTIC AND NORTH PACIFIC.

Ground level ultraviolet-B (UV-B; 290–320 nm) fluxes in Antarctica have been increasing due to stratospheric ozone depletion. Although mat-forming cyanobacteria are major component of freshwater algal biomass in Antarctica, little is known about their response to increasing ultraviolet radiation (UVR). The present study evaluated the sensitivity to UVR of two strains of mat-forming cyanobacteria with different cell size, Phormidium murrayi (6.0 x 3.2 μm) and Schizothrix calcicola (2.2 x 2.3 μm). Cyanobacterial photosynthesis was measured under different UV spectral quality and quantity achieved by polychromatic filters with different cutoff wavelengths and neutral density screens. The productivity and irradiance data were used to generate biological weighting functions (BWF) for the assessment of UV inhibition on photosynthesis. The kinetics of UV inhibition, as determined by PAM fluorometry, differed between the two species so that inhibition of P. murrayi and S. calcicola were modeled based on UV-irradiance and cumulative exposure, respectively. After a one hour exposure, BWF's did not differ between the two isolates of cyanobacteria despite their differences in cell size. To evaluate the negative impact of increased UV-B exposure due to ozone depletion on cyanobacteria, the BWF's were applied to two solar spectra obtained from McMurdo Station, one on a day when the ozone hole was prominent (O₃ = 170 Dobson units; DU = 10-3 cm O₃), and the other on a day with high ozone concentration (O₃ = 328 DU). The decrease in ozone level would reduce productivity by 3–8%. Seasonal variation of UVR has a bigger impact on cyanobacterial productivity than ozone depletion.     

Computed Tomographic Imaging of Subchondral Fatigue Cracks in the Distal End of the Third Metacarpal Bone in the Thoroughbred Racehorse Can Predict Crack Micromotion in an Ex-Vivo Model

Articular stress fracture arising from the distal end of the third metacarpal bone (MC3) is a common serious injury in Thoroughbred racehorses. Currently, there is no method for predicting fracture risk clinically. We describe an ex-vivo biomechanical model in which we measured subchondral crack micromotion under compressive loading that modeled high speed running. Using this model, we determined the relationship between subchondral crack dimensions measured using computed tomography (CT) and crack micromotion. Thoracic limbs from 40 Thoroughbred racehorses that had sustained a catastrophic injury were studied. Limbs were radiographed and examined using CT. Parasagittal subchondral fatigue crack dimensions were measured on CT images using image analysis software. MC3 bones with fatigue cracks were tested using five cycles of compressive loading at -7,500N (38 condyles, 18 horses). Crack motion was recorded using an extensometer. Mechanical testing was validated using bones with 3 mm and 5 mm deep parasagittal subchondral slots that modeled naturally occurring fatigue cracks. After testing, subchondral crack density was determined histologically. Creation of parasagittal subchondral slots induced significant micromotion during loading (p<0.001). In our biomechanical model, we found a significant positive correlation between extensometer micromotion and parasagittal crack area derived from reconstructed CT images (S_R = 0.32, p<0.05). Correlations with transverse and frontal plane crack lengths were not significant. Histologic fatigue damage was not significantly correlated with crack dimensions determined by CT or extensometer micromotion. Bones with parasagittal crack area measurements above 30 mm² may have a high risk of crack propagation and condylar fracture in vivo because of crack micromotion. In conclusion, our results suggest that CT could be used to quantify subchondral fatigue crack dimensions in racing Thoroughbred horses in-vivo to assess risk of condylar fracture. Horses with parasagittal crack arrays that exceed 30 mm² may have a high risk for development of condylar fracture.     

Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue

We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4(+) mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific.     

Synthesis and multinuclear magnetic resonance spectroscopy of the novel ionic Pt(II) mixed-ligands complexes cis- and trans-[Pt(amine)2(pyrimidine)2](NO3)2

Novel ionic mixed-ligands complexes of the types cis- and trans-[Pt(amine)₂(pm)₂](NO₃)₂ (where pm = pyrimidine) were synthesized and studied in the solid state by IR spectroscopy and in aqueous solution by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. The results of the solution NMR characterization have shown that the isolated compounds are pure. In ¹⁹⁵Pt NMR, the cis RNH₂ complexes were observed at slightly lower fields (ave. −2441 ppm) than the equivalent trans analogues (ave. −2448 ppm). For Me₂NH, the difference between the two isomers is larger (29 ppm). The complexes are observed at lower fields (difference of 100 ppm) than the corresponding [Pt(amine)₄]²⁺ complexes, which might indicate the presence of π-backdonation in the Pt-pm bond. In ¹H NMR, the coupling constants ³J(¹⁹⁵Pt-¹H_amine) are larger in the cis compounds (38-48 Hz) than in the trans analogues (30-36 Hz). The ³J(¹⁹⁵Pt-¹H_pm) values are also larger for the cis isomers. In ¹³C NMR spectroscopy, the coupling constants ³J(¹⁹⁵Pt-¹³C_amine) are 36 Hz (ave.) for the cis complexes and 26 Hz (ave.) for the trans isomers, while the ²J(¹⁹⁵Pt-¹³C_amine) are 18 Hz (cis) and 14 Hz (trans), respectively. The ³J(¹⁹⁵Pt-¹³C_5(pm)) values are 36 Hz (cis) and 28 Hz (trans). A few ²J(¹⁹⁵Pt-¹³C_pm) couplings were observed (7-10 Hz).