New zeolite adsorbents for downstream processing of polyphenols from renewable resources

Commercial materials with polyvinylpolypyrrolidone and polymeric amberlites (XAD7HP, XAD16) are commonly used for the adsorptive downstream processing of polyphenols from renewable resources. In this study, beta‐zeolite‐based adsorbent systems were examined, and their properties were compared to organic resins. Batch adsorption experiments were conducted with synthetic solutions of major polyphenols. Adsorption isotherms and desorption characteristics of individual adsorbent were determined based on these results. Maximum adsorption capacities were calculated using the Langmuir model. For example, the zeolites had capacities up to 203.2 mg/g for ferulic acid. To extend these results to a complex system, additional experiments were performed on rapeseed meal and wheat seed extracts as representative renewable resources. HPLC analysis showed that with 7.5% w/v, which is regarded as the optimum amount of zeolites, zeolites A and B could bind 100% of the major polyphenols as well as release polyphenols at high yields. Additionally, regeneration experiments were performed with isopropyl alcohol at 99°C to evaluate how zeolites regenerate under mild conditions. The results showed only a negligible loss of adsorption capacity and no loss of desorption capacity. In summary, it was concluded that beta‐zeolites were promising adsorbents for developing new processes to isolate polyphenols from renewable resources.     

Determinants of Physiological and Perceived Physiological Stress Reactivity in Children and Adolescents

Aims

Abnormal physiological stress reactivity is increasingly investigated as a vulnerability marker for various physical and psychological health problems. However, studies are inconsistent in taking into account potential covariates that may influence the developing stress system. We systematically tested determinants (individual, developmental, environmental and substance use-related) of physiological and perceived physiological stress reactivity. We also examined the relation between physiological and perceived physiological stress reactivity.

Method

In a stratified sample of 363 children (7–12 years) and 344 adolescents (13–20 years) from the general population, we examined cortisol, heart rate, respiratory sinus arrhythmia and perceived physiological stress reactivity to a psychosocial stress procedure.

Results

Using multivariate linear regression models, we found that individual, developmental, environmental and substance use-related factors were related to each of the stress response indices. These determinant factors were different for each of the stress reactivity indices, and different in children versus adolescents. Perceived physiological stress reactivity predicted cortisol reactivity in adolescents only. All other relations between perceived physiological and physiological stress reactivity were not significant.

Conclusions

As physiological stress variables are often examined as vulnerability markers for the development of health problems, we maintain that it is essential that future studies take into consideration factors that may account for found relations. Our study provides an overview and indication of which variables should be considered in the investigation of the relation between physiological stress indices and illness.     

Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders

The homodimeric flavohemeprotein endothelial nitric oxide synthase (eNOS) oxidizes l-arginine to l-citrulline and nitric oxide (NO), which acutely vasodilates blood vessels and inhibits platelet aggregation. Chronically, eNOS has a major role in the regulation of blood pressure and prevention of atherosclerosis by decreasing leukocyte adhesion and smooth muscle proliferation. However, a disturbed vascular redox balance results in eNOS damage and uncoupling of oxygen activation from l-arginine conversion. Uncoupled eNOS monomerizes and generates reactive oxygen species (ROS) rather than NO. Indeed, eNOS uncoupling has been suggested as one of the main pathomechanisms in a broad range of cardiovascular and pulmonary disorders such as atherosclerosis, ventricular remodeling, and pulmonary hypertension. Therefore, modulating uncoupled eNOS, in particular eNOS-dependent ROS generation, is an attractive therapeutic approach to preventing and/or treating cardiopulmonary disorders, including protective effects during cardiothoracic surgery. This review provides a comprehensive overview of the pathogenetic role of uncoupled eNOS in both cardiovascular and pulmonary disorders. In addition, the related therapeutic possibilities such as supplementation with the eNOS substrate l-arginine, volatile NO, and direct NO donors as well as eNOS modulators such as the eNOS cofactor tetrahydrobiopterin and folic acid are discussed in detail.     

Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.     

Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus

Staphylococcal nuclease (here termed as Nuc1) is considered an important virulence factor and a unique marker widely used in the detection of Staphylococcus aureus. A second functional thermostable nuclease (here termed as Nuc2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nuc1. Recombinant Nuc2 showed nuclease activity in the zymogram test and was able to degrade various types of nucleic acids. The optimal reaction temperature and pH for Nuc2 were 50 °C and pH 10, respectively. The enzymatic activity of Nuc2 was stimulated in the presence of Ca²⁺ (0.05 mM), Mg²⁺ (0.5 mM), dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn²⁺ and Mn²⁺, and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nuc1, which was consistent with differences in the sequence pattern and structure predicted. Furthermore, a nuc1 and nuc2 double deletion mutant of S. aureus and respective complementation experiments suggest a major role for nuc1 in terms of thermonuclease activity in S. aureus.     

The population genomics of multiple tsetse fly (Glossina fuscipes fuscipes) admixture zones in Uganda

Understanding the mechanisms that enforce, maintain or reverse the process of speciation is an important challenge in evolutionary biology. This study investigates the patterns of divergence and discusses the processes that form and maintain divergent lineages of the tsetse fly Glossina fuscipes fuscipes in Uganda. We sampled 251 flies from 18 sites spanning known genetic lineages and the four admixture zones between them. We apply population genomics, hybrid zone and approximate Bayesian computation to the analysis of three types of genetic markers: 55,267 double‐digest restriction site‐associated DNA (ddRAD) SNPs to assess genome‐wide admixture, 16 microsatellites to provide continuity with published data and accurate biogeographic modelling, and a 491‐bp fragment of mitochondrial cytochrome oxidase I and II to infer maternal inheritance patterns. Admixture zones correspond with regions impacted by the reorganization of Uganda's river networks that occurred during the formation of the West African Rift system over the last several hundred thousand years. Because tsetse fly population distributions are defined by rivers, admixture zones likely represent both old and new regions of secondary contact. Our results indicate that older hybrid zones contain mostly parental types, while younger zones contain variable hybrid types resulting from multiple generations of interbreeding. These findings suggest that reproductive barriers are nearly complete in the older admixture zones, while nearly absent in the younger admixture zones. Findings are consistent with predictions of hybrid zone theory: Populations in zones of secondary contact transition rapidly from early to late stages of speciation or collapse all together.     

Retinoic acid–induced survival effects in SH-SY5Y neuroblastoma cells

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.     

Detection of mitochondrial defects by laser fluorimetry

The mitochondrial function in skeletal muscle biopsies of three patients with chronic progressive external ophthalmoplegia, having deletions of the mitochondrial DNA, was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. We detected substantially elevated steady state redox states of the mitochondrial NAD-system in the muscle fibers of these patients. Moreover, the respiratory chain-linked autofluorescence changes in the muscle fibers of these patients were larger in comparison to controls indicating substantial alterations of the mitochondrial content. These results are in line with the presence of elevated numbers of partially respiratory chain inhibited mitochondria in the skeletal muscle of chronic progressive external ophthalmoplegia patients. (Mol Cell Biochem 174: 97–100, 1997)     

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 ^−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 ^−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.     

Redox Mechanism of S-Nitrosothiol Modulation of Neuronal CaV3.2 T-Type Calcium Channels

T-type calcium channels in the dorsal root ganglia (DRG) have a central function in tuning neuronal excitability and are implicated in sensory processing including pain. Previous studies have implicated redox agents in control of T-channel activity; however, the mechanisms involved are not completely understood. Here, we recorded T-type calcium currents from acutely dissociated DRG neurons from young rats and investigated the mechanisms of Ca_V3.2 T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-penicillamine rapidly reduced T-type current amplitudes. GSNO did not affect voltage dependence of steady-state inactivation and macroscopic current kinetics of T-type channels. The effects of GSNO were abolished by pretreatment of the cells with N-ethylmaleimide, an irreversible alkylating agent, but not by pretreatment with 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor, suggesting a potential effect of GSNO on putative extracellular thiol residues on T-type channels. Expression of wild-type Ca_V3.2 channels or a quadruple Cys-Ala mutant in human embryonic kidney cells revealed that Cys residues in repeats I and II on the extracellular face of the channel were required for channel inhibition by GSNO. We propose that SNO-related molecules in vivo may lead to alterations of T-type channel-dependent neuronal excitability in sensory neurons and in the central nervous system in both physiological and pathological conditions such as neuronal ischemia/hypoxia.