Intersubunit linker length as a modifier of protein stability: crystal structures and thermostability of mutant TRAP

The ability of proteins to self-assemble into complex, functional nanoscale structures is expected to become of significant use in the manufacture of artificial nanodevices with a wide range of novel applications. The bacterial protein TRAP has potential uses as a nanoscale component as it is ring-shaped, with a central, modifiable cavity. Furthermore, it can be engineered to make a ring of 12-fold symmetry, which is advantageous for packing into two-dimensional arrays. The 12mer form of TRAP is made by linking multiple subunits together on the same polypeptide, but the usefulness of the 12mers described to date is limited by their poor stability. Here we show that, by altering the length of the peptide linker between subunits, the thermostability can be significantly improved. Since the subunit interfaces of the different 12mers are essentially identical, stabilization arises from the reduction of strain in the linkers. Such a simple method of controlling the stability of modular proteins may have wide applications, and demonstrates the lack of absolute correlation between interactions observable by crystallography and the internal energy of a complex.     

The <Emphasis Type="Italic">COL1A1</Emphasis> gene and high myopia susceptibility in Japanese

The collagen type Ι alpha Ι (COL1A1) gene encodes the extracellular matrix component, collagen, and resides in candidate MYP5 for high myopia on the chromosome 17q22–q23.3. This locus has recently been implicated in playing an important role in the pathogenesis of experimental myopia. We investigated the association of disruptions of COL1A1 gene with high myopia by analyzing the frequency of ten SNPs in a Japanese population of 330 subjects with high myopia of −9.25 D or less and 330 randomized controls without high myopia. Two SNPs (rs2075555 and rs2269336) were significantly associated with high myopia (P < 0.05, Pc < 0.1). Two different haplotype blocks in COL1A1 were observed by the pair-wise linkage disequilibrium between the SNPs. The frequency of GGC/GGC diplotype constructed by the three SNPs (rs2075555, rs2269336, rs1107946) was significantly high (OR = 1.6) and associated with high myopia (P = 0.028, Pc< 0.084). Together our results provide the first evidence for COL1A1 as a gene associated with high myopia.     

Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-alpha secretion in Abca1-deficient macrophages

Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kappaB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kappaB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-alpha secretion.     

Peripheral tolerance and the qualitative characteristics of autoreactive T cell clones in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by autoreactive T cells specific for the mitochondrial Ag PDC-E2(163-176). We studied the ability of eight T cell clones (TCC) specific for PDC-E2(163-176) to proliferate or become anergic in the presence of costimulation signals. TCC were stimulated with either human PDC-E2(163-176), an Escherichia coli 2-oxoglutarate dehydrogenase mimic (OGDC-E2(34-47)), or analogs with amino acid substitutions using HLA-matched allogeneic PBMC or mouse L-DR53 fibroblasts as APC. Based on their differential responses to these peptides (human PDC-E2(163-176), E. coli OGDC-E2(34-47)) in the different APC systems, TCC were classified as costimulation dependent or independent. Only costimulation-dependent TCC could become anergic. TCC with costimulation-dependent responses to OGDC-E2 become anergic to PDC-E2 when preincubated with mimic, even if costimulation is independent for PDC-E2(163-176). Anergic TCC produced IL-10. One selected TCC could not become anergic after preincubation with PDC-E2(163-176)-pulsed L-DR53 but became anergic using L-DR53 pulsed with PDC-E2 peptide analogs with a substitution at a critical TCR binding site. TCC that only respond to peptide-pulsed PBMC, but not L-DR53, proliferate with peptide-pulsed CD80/CD86-transfected L-DR53; however, anergy was not induced with peptide-pulsed L-DR53 transfected with only CD80 or CD86. These data highlight that costimulation plays a dominant role in maintaining peripheral tolerance to PBC-specific Ags. They further suggest that, under specific circumstances, molecular mimicry of an autoantigen may restore rather than break peripheral tolerance.     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

Beneficial effect of GB virus C co-infection in Human Immunodeficiency Virus type 1-infected individuals

Several reports have documented a better prognosis for HIV‐1‐infected patients co‐infected with GBV‐C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV‐C co‐infection on HIV‐1‐infected patients. GBV‐C RNA was detected in 37 samples in 182 HIV‐1‐infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV‐C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV‐C viral load quantified by real‐time PCR ranged from 7.8 × 10³ to 3.3 × 10⁶ copies/ml. Weakly negative correlation was observed between GBV‐C viral load and HIV‐1 viral load in 19 HAART‐naïve patients, indicating that a higher GBV‐C viral load is associated with a greater suppression of HIV‐1 replication. A previously published in vitro study suggested that GBV‐C infection would induce up‐regulation of RANTES, leading to suppression of HIV‐1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV‐C co‐infected group than in the uninfected group (190–9,959 vs. 264–31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV‐1 replication by GBV‐C co‐infection is not mediated by up‐regulated RANTES, and thus call for another as yet unknown factor.     

Regulation of the phosphatidylinositol 3-kinase-Akt and the mitogen-activated protein kinase pathways by ursolic acid in human endometrial cancer cells

The phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the mitogen activated protein kinase (MAPK) pathway are important in the development and proliferation of various human cancers. It has been found recently that ursolic acid treatment affects growth and apoptosis in cancer cells. We sought to determine whether prominent signaling pathways, including the PI3K-Akt pathway and the MAPK (JNK, P38, and P44/42) pathway mediate these effects. Endometrial cancer cells often have high levels of phosphorylated Akt seen in conjunction with a PTEN mutation or deletion. Elevation in Akt protects the cancer cell from apoptosis. Ursolic acid treatment moderately decreased PI3K levels in SNG-II cells. Treatment also decreased phospho-Akt and phospho-P44/42 in a dose- and time-dependent fashion, dramatically in SNG-II cells and moderately in HEC108 cells. This effect was most pronounced following treatment with 50 mum ursolic acid for 72 h. Our study found inhibition of both the PI3K-Akt pathway and the MAPK pathway in two endometrial cancer cell lines, SNG-II and the poorly differentiated HEC108 cell line.     

Restoration of cone vision in a mouse model of achromatopsia

Loss of cone function in the central retina is a pivotal event in the development of severe vision impairment for many prevalent blinding diseases. Complete achromatopsia is a genetic defect resulting in cone vision loss in 1 in 30,000 individuals. Using adeno-associated virus (AAV) gene therapy, we show that it is possible to target cones and rescue both the cone-mediated electroretinogram response and visual acuity in the Gnat2 ( cpfl3 ) mouse model of achromatopsia.     

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]