A novel function for proSAAS as an amyloid anti‐aggregant in Alzheimer's disease

Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti‐aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12‐month‐old APdE9 mice, and in the cortex of a human AD‐affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co‐immunoprecipitated with Aβ immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aβ_1–42 at molar ratios of 1 : 10, and this anti‐aggregation effect was dose dependent. Structure‐function studies showed that residues 97–180 were sufficient for the anti‐aggregation function against Aβ. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral‐mediated proSAAS over‐expression, blocked the neurocytotoxic effect of Aβ_1–42 in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimer's disease pathology.

     

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na⁺ transport, effects at least partially involving regulation of the epithelial Na⁺ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na⁺ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, ^V617FJAK2 or ^K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I _amil) in dual electrode voltage clamp. Moreover, I _amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I _amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I _amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I _amil. In colonic epithelium, I _amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.     

Effects of Water on Enzyme Performance with an Emphasis on the Reactions in Supercritical Fluids

ABSTRACT

Enzymes require a certain level of water in their structures in order to maintain their natural conformation, allowing them to deliver their full functionality. Furthermore, as a modifier of the solvent, up to a certain level, water can modify the solvent properties such as polarity/polarizability as well as the solubility of the reactants and the products. In addition, depending on the type of the reaction, water can be a substrate (e.g., in hydrolysis) or a product (e.g., in esterolysis) of the enzymatic reaction, influencing the enzyme turnover in different ways. It is found that regardless of the type of reaction, the functionality of enzyme itself is maximum at an optimum level of water, beyond which the enzyme performance is declined due to the loss in enzyme stability. Furthermore, mass transfer limitations caused by pathway blockage and/or by reduced solubilities of the reactants and/or products can also affect the enzyme performance at higher water levels. Controlling water content of ingoing CO₂ and substrates as well as precise management of enzyme support and salt hydrates are important strategies to adjust water level in reaction media, especially in supercritical environments.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Dietary supplementation with monosodium glutamate is safe and improves growth performance in postweaning pigs

Dietary intake of glutamate by postweaning pigs is markedly reduced due to low feed consumption. This study was conducted to determine the safety and efficacy of dietary supplementation with monosodium glutamate (MSG) in postweaning pigs. Piglets were weaned at 21 days of age to a corn and soybean meal-based diet supplemented with 0, 0.5, 1, 2, and 4 % MSG (n = 25/group). MSG was added to the basal diet at the expense of cornstarch. At 42 days of age (21 days after weaning), blood samples (10 mL) were obtained from the jugular vein of 25 pigs/group at 1 and 4 h after feeding for hematological and clinical chemistry tests; thereafter, pigs (n = 6/group) were euthanized to obtain tissues for histopathological examinations. Feed intake was not affected by dietary supplementation with 0–2 % MSG and was 15 % lower in pigs supplemented with 4 % MSG compared with the 0 % MSG group. Compared with the control, dietary supplementation with 1, 2 and 4 % MSG dose-dependently increased plasma concentrations of glutamate, glutamine, and other amino acids (including lysine, methionine, phenylalanine and leucine), daily weight gain, and feed efficiency in postweaning pigs. At day 7 postweaning, dietary supplementation with 1–4 % MSG also increased jejunal villus height, DNA content, and antioxidative capacity. The MSG supplementation dose-dependently reduced the incidence of diarrhea during the first week after weaning. All variables in standard hematology and clinical chemistry tests, as well as gross and microscopic structures, did not differ among the five groups of pigs. These results indicate that dietary supplementation with up to 4 % MSG is safe and improves growth performance in postweaning pigs.     

Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

Determination of LMF Binding Site on a HSA-PPIX Complex in the Presence of Human Holo Transferrin from the Viewpoint of Drug Loading on Proteins

Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The K_a1, K_a2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×10⁵ M⁻¹, 9.7×10⁶ M⁻¹, and 2.0×10⁴ M⁻¹, 1.8×10⁵ M⁻¹, respectively, and the n₁, n₂ values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow''s site IIA to Sudlow''s site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex.     

Generalized Baum-Welch Algorithm Based on the Similarity between Sequences

The profile hidden Markov model (PHMM) is widely used to assign the protein sequences to their respective families. A major limitation of a PHMM is the assumption that given states the observations (amino acids) are independent. To overcome this limitation, the dependency between amino acids in a multiple sequence alignment (MSA) which is the representative of a PHMM can be appended to the PHMM. Due to the fact that with a MSA, the sequences of amino acids are biologically related, the one-by-one dependency between two amino acids can be considered. In other words, based on the MSA, the dependency between an amino acid and its corresponding amino acid located above can be combined with the PHMM. For this purpose, the new emission probability matrix which considers the one-by-one dependencies between amino acids is constructed. The parameters of a PHMM are of two types; transition and emission probabilities which are usually estimated using an EM algorithm called the Baum-Welch algorithm. We have generalized the Baum-Welch algorithm using similarity emission matrix constructed by integrating the new emission probability matrix with the common emission probability matrix. Then, the performance of similarity emission is discussed by applying it to the top twenty protein families in the Pfam database. We show that using the similarity emission in the Baum-Welch algorithm significantly outperforms the common Baum-Welch algorithm in the task of assigning protein sequences to protein families.