R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.     

The critical role of Exo84p in the organization and polarized localization of the exocyst complex

The exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. However, how the exocyst complex is assembled and targeted to sites of secretion is unclear. Here, we have investigated the role of the exocyst component Exo84p in these processes. We have generated an array of temperature-sensitive yeast exo84 mutants. Electron microscopy and cargo protein traffic analyses of these mutants indicated that Exo84p is specifically involved in the post-Golgi stage of secretion. Using various yeast mutants, we systematically studied the localization of Exo84p and other exocyst proteins by fluorescence microscopy. We found that pre-Golgi traffic and polarized actin organization are required for Exo84p localization. However, none of the exocyst proteins controls Exo84p polarization. In addition, Sec3p is not responsible for the polarization of Exo84p or any other exocyst component to the daughter cell. On the other hand, several exocyst members, including Sec10p, Sec15p, and Exo70p, clearly require Exo84p for their polarization. Biochemical analyses of the exocyst composition indicated that the assembly of Sec10p, Sec15p, and Exo70p with the rest of the complex requires Exo84p. We propose that there are at least two distinct regulatory mechanisms for exocyst polarization, one for Sec3p and one for the other members, including Exo84p. Exo84p plays a critical role in both the assembly of the exocyst and its targeting to sites of secretion.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.     

H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain

H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains.     

AtNAP1 represents an atypical SufB protein in Arabidopsis plastids

The assembly of iron-sulfur (Fe-S) clusters involves several pathways and in prokaryotes the mobilization of the sulfur (SUF) system is paramount for Fe-S biogenesis and repair during oxidative stress. The prokaryotic SUF system consists of six proteins: SufC is an ABC/ATPase that forms a complex with SufB and SufD, SufA acts as a scaffold protein, and SufE and SufS are involved in sulfur mobilization from cysteine. Despite the importance of Fe-S proteins in higher plant plastids, little is known regarding plastidic Fe-S cluster assembly. We have recently shown that Arabidopsis harbors an evolutionary conserved plastidic SufC protein (AtNAP7) capable of hydrolyzing ATP and interacting with the SufD homolog AtNAP6. Based on this and the prokaryotic SUF system we speculated that a SufB-like protein may exist in plastids. Here we demonstrate that the Arabidopsis plastid-localized SufB homolog AtNAP1 can complement SufB deficiency in Escherichia coli during oxidative stress. Furthermore, we demonstrate that AtNAP1 can interact with AtNAP7 inside living chloroplasts suggesting the presence of a plastidic AtNAP1.AtNAP6.AtNAP7 complex and remarkable evolutionary conservation of the SUF system. However, in contrast to prokaryotic SufB proteins with no associated ATPase activity we show that AtNAP1 is an iron-stimulated ATPase and that AtNAP1 is capable of forming homodimers. Our results suggest that AtNAP1 represents an atypical plastidic SufB-like protein important for Fe-S cluster assembly and for regulating iron homeostasis in Arabidopsis.     

Improved production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional cry1C gene in its chromosome

A cryIC gene, whose product is active against Spodoptera exigua, was introduced into wildtype Bacillus thuringiensis kurstaki strain YBT1520 using an integrative and thermosensitive vector, pBMB-FLCE, which was developed based on B. thuringiensis transposon Tn4430 harboring a tnpI-tnpA gene. With the mediation of TnpI-TnpA, the cry1C gene was integrated into the chromosome of the host strain. To prevent secondary integration, the integrative vector was eliminated by moving recombinant cultures to 46 degrees C for generations. Two integrative recombinant B. thuringiensis strains BMB1520-E and BMB1520-F were obtained. In recombinant BMB1520-F, the cry1C gene was expressed stably at a significant level and did not reduce the expression of endogenous crystal protein genes. Bioassay results indicated that BMB1520-E and BMB1520-F showed a higher level of activity against S. exigua third-instar larvae than did their parent strains, in addition to the high toxicity to Plutella xylostella third-instar later larvae.     

Broadening the insecticidal spectrum of Lepidoptera-specific Bacillus thuringiensis strains by chromosomal integration of cry3A

A TnpI-TnpIA-mediated and thermosensitive recombination system was developed to construct genetically modified Bacillus thuringiensis strains encoding a crystal protein particularly active against Coleopteran species. Based on B. thuringiensis transposon Tn4430, an integrative vector, pBMB-R14E, was constructed, by which the cry3A delta-endotoxin gene highly toxic to Lepidoptera was delivered into a wildtype B. thuringiensis subsp. kurstaki strain YBT1520. The cry3A gene was integrated into the chromosome of the host strain. Then the integrative vector was eliminated by moving recombinant cultures to 46 degrees C. Two recombinant B. thuringiensis strains, BMB1520-S and BMB1520-T, were obtained. In recombinant strains, the cry3A gene was stably expressed in measurable amounts and did not reduce the expression of endogenous crystal protein genes. Bioassay results showed that BMB1520-S and BMB1520-T, in addition to the activity against lepidopteran Plutella xylostella third-instar larvae present in the parental strains, exhibited a high level of activity against coleopteran Rhyllodecta vulgatissima third-instar larvae, absent from the parental strains.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

Single- and multilocus allelic variants within the GABA(B) receptor subunit 2 (GABAB2) gene are significantly associated with nicotine dependence

Twelve single-nucleotide polymorphisms (SNPs) in the human gamma-aminobutyric acid type B (GABA(B)) receptor subunit 2 gene (GABAB2) were tested for association with nicotine dependence (ND) in an extensively phenotyped cohort of 1,276 smokers and nonsmokers, representing approximately 404 nuclear families of African American (AA) or European American (EA) origin. The GABAB2 gene encodes a subunit of the GABA(B) receptor for GABA, an inhibitory neurotransmitter involved in the regulation of many physiological and psychological processes in the brain. The gene is located within a region of chromosome 9q22 that showed a "suggestive" linkage to ND. Individual SNP analysis performed using the PBAT-GEE program indicated that two SNPs in the AAs and four SNPs in the EAs were significantly associated with ND. Haplotype analysis using the Family-Based Association Test revealed that, even after Bonferroni correction, the haplotype C-C-G of rs2491397-rs2184026-rs3750344 had a significant positive association with ND in both the pooled and the AA samples. In the EAs, we identified two haplotypes, C-A-C-A and T-A-T-A, formed by SNPs rs1435252-rs378042-rs2779562-rs3750344, that showed a highly significant negative and positive association with ND, respectively. In summary, our findings provide evidence of a significant association of GABAB2 variants with ND, implying that this gene plays an important role in the etiology of this drug addiction.