Protein unfolding at interfaces: slow dynamics of alpha-helix to beta-sheet transition

A two-phase sequential dynamic change in the secondary structure of hen egg lysozyme (Lys) adsorbed on solid substrates was observed. The first phase involved fast conversion of alpha-helix to random/turns (within the first minute or at very low coverage or high substrate wettability) with no perceptible change in beta-sheet content. The second phase (1-1200 min), however, involved a relatively slow conversion from alpha-helix to beta-sheet without a noticeable change in random/turns. An important finding of this work is that the concentration of lysozyme in the adsorbed state has a substantial effect on the fractional content of secondary structures. Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, along with a newly-developed optimization algorithm for predicting the content of secondary structure motifs, was used to correlate the secondary structure and the amount of adsorbed lysozyme with the surface wettability of six different flat nanoporous substrates. Although three independent variables, surface wettability, solution concentration and time for adsorption, were used to follow the fractional structural changes of lysozyme, the results were all normalized onto a single plot with the amount adsorbed as the universal independent variable. Consequently, lateral interactions among proteins likely drive the transition process. Direct intermolecular force adhesion measurements between lysozyme and different functionalized self-assembled alkanethiol monolayers confirm that hydrophobic surfaces interact strongly with proteins. The lysozyme-unfolding pathway during early adsorption appears to be similar to that predicted by published molecular modeling results.     

Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a "safe" substitution.     

Cloning and characterization of DIP1, a novel protein that is related to the Id family of proteins

Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1. This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle. However, the level of this protein increases severalfold during the differentiation of HL60 cells. The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle. DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins. The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer. These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined.     

Remarkable thermal stability of doubly intramolecularly cross-linked hen lysozyme

In order to examine how a protein can be effectively stabilized, two intramolecular cross-links, Glu35-Trp108 and Lys1-His15, which have few unfavorable interactions in the folded state, were simultaneously introduced into hen lysozyme. Both of the intramolecularly cross-linked lysozymes, 35-108 CL and 1-15 CL, containing cross-links Glu35-Trp108 and Lys1-His15, respectively, showed increases in thermal stability of 13.9 and 5.2 degrees C, respectively, over that of wild type, at pH 2.7. On the other hand, a doubly cross-linked lysozyme showed an increase in thermal stability of 20.8 degrees C over that of wild type, under identical conditions. Since the sum of the differences in denaturation temperature between wild type and each of the cross-linked lysozymes was nearly equal to that between wild type and the doubly cross-linked lysozyme, we suggest that the efficient stabilization of the lysozyme molecule was the direct result of the double intramolecular cross-links.     

Identification of protein kinase C isozymes involved in the anti-proliferative and pro-apoptotic activities of 10-Methyl-aplog-1, a simplified analog of debromoaplysiatoxin,in several cancer cell lines

10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.     

Antioxidative property of T-0970, a new ureidophenol derivative

We investigated the antioxidative property of T-0970, a newly synthesized ureidophenol derivative. The inhibitory effect of T-0970 on spontaneous lipid peroxidation in rat brain was 10 times greater than those of well-known antioxidants such as butylhydroxytoluene (BHT), probucol and α-tocopherol. T-0970 also showed dose-dependent free radical scavenging activities in vitro for both superoxide anions and hydroxyl radicals. The radical-scavenging potencies of T-0970 were about 10–30 times stronger than those of BHT. We evaluated the in vivo antioxidative ability of T-0970 in the animal model of acute oxidative tissue injury in rats. Intraperitoneal injection of ferric nitrilotriacetate (Fe/NTA) caused an acute and remarkable increase in the level of thiobarbituric acid-reactive substances (TBARS) in both plasma and the liver, and also resulted in a considerable elevation of the plasma levels of GOT and GPT indicative of hepatic injury. Both oral and intravenous administration of T-0970 dose-dependently depressed these diagnostic parameters. These results indicate that T-0970 may have a therapeutic potential in various diseases associated with oxidative tissue injury.     

Risk Factors and Primary Prevention Trials for Type 1 Diabetes

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease resulting in the designated immune destruction of insulin producing β-cells, usually diagnosed in youth, and associated with important psychological, familial, and social disorders. Once diagnosed, patients need lifelong insulin treatment and will experience multiple disease-associated complications. There is no cure for T1DM currently. The last decade has witnessed great progress in elucidating the causes and treatment of the disease based on numerous researches both in rodent models of spontaneous diabetes and in humans. This article summarises our current understanding of the pathogenesis of T1DM, the roles of the immune system, genes, environment and other factors in the continuing and rapid increase in T1DM incidence at younger ages in humans. In addition, we discuss the strategies for primary and secondary prevention trials of T1DM. The purpose of this review is to provide an overview of this disorder''s pathogenesis, risk factors that cause the disease, as well as to bring forward an ideal approach to prevent and cure the disorder.     

Potential importance of lagoons as breeding grounds for bluegill Lepomis macrochirus in Lake Biwa, Japan

Exotic bluegill Lepomis macrochirus have been propagating in the littoral region of Lake Biwa and its tributary lagoons. To investigate the reproductive activity of bluegill in the prespawning season, fish samples were collected from three sites at the littoral region of the lake and their nearby lagoons. The number of females relative to the total number of specimens tended to be higher in lagoons than in the adjacent littoral sites in the lake. The females collected from lagoons had significantly larger ovaries than those from the lake. The means of gonadal somatic index of females in the lake ranged from 1.77 to 3.44, whereas those in lagoons ranged from 4.35 to 12.2. The higher temperature conditions of lagoons appeared to be responsible for the higher maturation of ovaries of the fish in lagoons. The environmental condition of lagoons is potentially suitable for the active growth of larvae, subsequently increasing their overwintering success. Therefore, earlier spawning of bluegill in lagoons might have contributed to the propagation of bluegill in Lake Biwa to some extent.     

Identification of Phthalate Ester-assimilating Bacteria

Seventeen bacterial capable of utilizing phthalate esters isolated from natural sources were identified. Based on morphological and biochemical characteristics, type of cell division, GC content in DNA, principal amino acid in the cell wall and cellular fatty acid composition, 10 isolates were identified as Nocardia erythropolis, one isolate as Pseudomonas acidovorans, another as Pseudomonas cepacia and four as members of the genus of Corynebacterium.