Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix

1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed.     

Brazilian Red Propolis Attenuates Hypertension and Renal Damage in 5/6 Renal Ablation Model

The pathogenic role of inflammation and oxidative stress in chronic kidney disease (CKD) is well known. Anti-inflammatories and antioxidant drugs has demonstrated significant renoprotection in experimental nephropathies. Moreover, the inclusion of natural antioxidants derived from food and herbal extracts (such as polyphenols, curcumin and lycopene) as an adjuvant therapy for slowing CKD progression has been largely tested. Brazilian propolis is a honeybee product, whose anti-inflammatory, antimicrobial and antioxidant effects have been widely shown in models of sepsis, cancer, skin irritation and liver fibrosis. Furthermore, previous studies demonstrated that this compound promotes vasodilation and reduces hypertension. However, potential renoprotective effects of propolis in CKD have never been investigated. The aim of this study was to evaluate the effects of a subtype of Brazilian propolis, the Red Propolis (RP), in the 5/6 renal ablation model (Nx). Adult male Wistar rats underwent Nx and were divided into untreated (Nx) and RP-treated (Nx+RP) groups, after 30 days of surgery; when rats already exhibited marked hypertension and proteinuria. Animals were observed for 90 days from the surgery day, when Nx+RP group showed significant reduction of hypertension, proteinuria, serum creatinine retention, glomerulosclerosis, renal macrophage infiltration and oxidative stress, compared to age-matched untreated Nx rats, which worsened progressively over time. In conclusion, RP treatment attenuated hypertension and structural renal damage in Nx model. Reduction of renal inflammation and oxidative stress could be a plausible mechanism to explain this renoprotection.     

A catalytic antibody produces fluorescent tracers of gap junction communication in living cells

The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with a k(cat) of 1.7 x 10(-5) s(-1) and a catalytic proficiency (k(cat)/k(uncat)K(m)) of 1.4 x 10(10) m(-1) (K(m) = 7 microm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.     

Structural and functional characterization of human NAD kinase

NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.     

Serotonin transporter: gene, genetic disorders, and pharmacogenetics

The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via modulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette's syndrome have detected signals in the chromosome 17q region where SERT is located. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs).     

Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress

Reactive oxygen species, especially hydrogen peroxide, are important in cellular signal transduction. However, excessive amounts of these species damage tissues and cells by oxidizing virtually all important biomolecules. Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. Through immunohistochemistry, we have determined that the PRDX6 protein was widely expressed in every tissue examined, most abundantly in epithelial cells. It was found in cytosol, but not in membranes, organelles, and nuclei fractions. Prdx6 mRNA was also expressed in every tissue examined. The widespread expression of Prdx6 suggested that its functions were quite important. To determine these functions, we generated Prdx6-targeted mutant (Prdx6-/-) mice, confirmed the gene disruption by Southern blots, PCR, RT-PCR, Western blots, and immunohistochemistry, and compared the effects of paraquat, hydrogen peroxide, and t-butyl hydroperoxide on Prdx6-/- and wild-type (Prdx6+/+) macrophages, and of paraquat on Prdx6-/- and Prdx6+/+ mice. Prdx6-/- macrophages had higher hydrogen peroxide levels, and lower survival rates; Prdx6-/- mice had significantly lower survival rates, more severe tissue damage, and higher protein oxidation levels. Additionally, there were no differences in the mRNA expression levels of other peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases, thioredoxins, and glutaredoxins between normal Prdx6-/- and Prdx6+/+ mice and those injected with paraquat. Our study provides in vivo evidence that PRDX6 is a unique non-redundant antioxidant that functions independently of other peroxiredoxins and antioxidant proteins.