Expression, purification and characterization of recombinant Z α1-Antitrypsin—The most common cause of α1-Antitrypsin deficiency

α₁-Antitrypsin (α₁AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. α₁AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe α₁AT deficiency are caused by the Z variant (E342K) of α₁AT. The presence of the Z mutation results in misfolding and polymerization of α₁AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z α₁AT production. This has created a major impediment to studying the effect of the Z mutation on α₁AT. Here we report our attempts to produce recombinant Z α₁AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z α₁AT. Cytosolic expression of the Z α₁AT gene in P. pastoris was successful. Monomeric and active recombinant Z α₁AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z α₁AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z α₁AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.     

Influence of l-rhamnosyl-d-glucosyl derivatives on properties and biological interaction of flavonoids

The anti-proliferative activity of hesperetin, hesperidin, neohesperidin and rutin was evaluated on human hepatoma cell lines (Hep G2) and correlated to their antioxidant activity. The results obtained showed strong anti-proliferative effects of hesperidin and neohesperidin, considerably higher than the other two additives. Hesperetin induced caspase-3 activation, release of LDH and endogenous accumulation of putrescine. Cell cycle distribution seems to indicate that the inhibitory effects of polyphenols on cell growth could be due to G0/G1 block, and activation of apoptotic pathway in the presence of hesperetin. Our results underline also that the glycone forms show reduced scavenging activity against DPPH, but present a remarkable inhibition of cell proliferation and low cytotoxicity.     

Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

Background

Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting.

Methodology/Principal Findings

We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called “unlicensed” NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells.

Conclusions/Significance

We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma.     

Assessment of sperm quality traits in relation to fertility in boar semen

Background  

Several studies have been published where sperm plasma membrane integrity correlated to fertility. In this study we describe a simple fluorometer-based assay where we monitored the fluorescence intensity of artificially membrane-ruptured spermatozoa with a fixed time staining with fluorescent DNA dyes.     

Adiponectin Promotes Macrophage Polarization toward an Anti-inflammatory Phenotype

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.     

Long‐term Successful Weight Loss Improves Vascular Endothelial Function in Severely Obese Individuals

Obesity is associated with increased cardiovascular risk. Although short‐term weight loss improves vascular endothelial function, longer term outcomes have not been widely investigated. We examined brachial artery endothelium‐dependent vasodilation and metabolic parameters in 29 severely obese subjects who lost ≥10% body weight (age 45 ± 13 years; BMI 48 ± 9 kg/m²) at baseline and after 12 months of dietary and/or surgical intervention. We compared these parameters to 14 obese individuals (age 49 ± 11 years; BMI 39 ± 7 kg/m²) who failed to lose weight. For the entire group, mean brachial artery flow‐mediated dilation (FMD) was impaired at 6.7 ± 4.1%. Following sustained weight loss, FMD increased significantly from 6.8 ± 4.2 to 10.0 ± 4.7%, but remained blunted in patients without weight decline from 6.5 ± 4.0 to 5.7 ± 4.1%, P = 0.013 by ANOVA. Endothelium‐independent, nitroglycerin‐mediated dilation (NMD) was unaltered. BMI fell by 13 ± 7 kg/m² following successful weight intervention and was associated with reduced total and low‐density lipoprotein cholesterol, glucose, hemoglobin A_1c, and high‐sensitivity C‐reactive protein (CRP). Vascular improvement correlated most strongly with glucose levels (r = ?0.51, P = 0.002) and was independent of weight change. In this cohort of severely obese subjects, sustained weight loss at 1 year improved vascular function and metabolic parameters. The findings suggest that reversal of endothelial dysfunction and restoration of arterial homeostasis could potentially reduce cardiovascular risk. The results also demonstrate that metabolic changes in association with weight loss are stronger determinants of vascular phenotype than degree of weight reduction.     

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.     

Caspase-dependent alteration of the ADP/ATP translocator triggers the mitochondrial permeability transition which is not required for the low-potassium-dependent apoptosis of cerebellar granule cells

We investigated ADP/ATP exchange mediated by the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore in homogenates from cerebellar granule cells en route to apoptosis induced by low potassium. We showed that, in the first 3 h of apoptosis, when maximum cytochrome c release had already occurred, adenine nucleotide translocator function was impaired owing to the action of reactive oxygen species, but no permeability transition pore opening occurred. Over 3-8 h of apoptosis, the permeability transition pore progressively opened, owing to caspase action, and further ADP/ATP translocator impairment occurred. The kinetics of transport and permeability transition pore opening were inversely correlated, both in the absence and presence of inhibitors of antioxidant and proteolytic systems. We conclude that, en route to apoptosis, alteration of the adenine nucleotide translocator occurs, resulting in permeability transition pore opening. This process depends on the action of caspase on pore component(s) other than the ADP/ATP translocator, because no change in either amount or molecular weight of the latter protein was noted during apoptosis, as measured by western blotting. Cell death occurs via apoptosis in the presence of cyclosporin A, the permeability transition pore inhibitor, thus showing that permeability transition pore opening, not needed for cytochrome c release, is also unnecessary for apoptosis to occur.     

Peripheral enkephalin hydrolysis in different animal species: A comparative study

Using column and thin layer chromatography, plasma hydrolysis of leu-enkephalin has been studied in man and several laboratory animals. The hydrolysis kinetics determined in the various species examined are considerably different. In addition, also the enzyme forms evidentiated, their molecular weight distribution and relative ratios have been found to vary greatly in the animals under test. Our data suggest that the widely different hydrolysis kinetics reported by various authors are attributable to the differences between species, rather than to differences in the analytical techniques employed.     

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies

The extent of nativeness of the stable conformation of the thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on two affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of either 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The data demonstrate that the stable structure of fragment FII as observed spectroscopically likely is one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The results support the view that the region of thermolysin composed primarily of residues 206-316 is a conformational domain of the intact protein and that isolated fragment FII retains domain-like characteristics of stable and native-like conformation.