Reconstitution of clathrin-coated pit budding from plasma membranes

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.     

Different HCV Genotype Distributions of HIV-Infected Individuals in Henan and Guangxi,China

Background

Due to shared transmission routes, hepatitis C virus (HCV) infection is highly prevalent among people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is associated with hepatotoxicity, leading to the negative effects on patients with HIV/HCV co-infection. In order to provide valuable information for HCV management in this particular population, we investigated the HCV genotypes in HIV-infected individuals from Henan and Guangxi, the two provinces with the most HIV-infected cases in China.

Methods

Individuals, who acquired HIV infection through various risk routes, were recruited from Henan and Guangxi. Test of antibodies against HCV (anti-HCV) was conducted, and detection of HCV RNA was performed by PCR amplification. HCV subtypes were determined by direct sequencing of amplicons, followed by phylogenetic analysis.

Results

We recruited a total of 1,112 HIV-infected people in this present study. Anti-HCV was detected from 218 (50.1%) patients from Henan and 81 (12.0%) patients from Guangxi, respectively. The highest prevalence of HIV/HCV co-infection was observed from FBDs (former blood donors) (87.2%) in Henan and IDUs (intravenous drug users) (81.8%) in Guangxi, respectively. The seroprevalence rate of HCV among people with sexual contact was significantly higher in Henan than in Guangxi (18.7% vs. 3.5%, P<0.05). The positive rate of HCV RNA in Henan and Guangxi was 30.6% (133/435) and 11.2% (76/677), respectively. Moreover, we found that 20 anti-HCV negative samples were HCV positive by PCR amplification. HCV subtype 1b (52.7%) was predominant in Henan, followed by subtype 2a (41.9%). The most frequently detected subtypes in Guangxi were 6a (35.6%) and 3b (32.9%).

Conclusion

The HCV genotype distributions were different in HIV-infected people from Henan and Guangxi. HIV/HCV co-infection was not only linked to the transmission routes, but also associated with the geographic position.     

A fine physical map of the rice chromosome 5

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1–3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for ~150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Chia-Hsiung Cheng and Mei-Chu Chung have equal contributions.     

Mitochondrial DNA Diversity in two populations of Taiwanese macaque (Macaca cyclopis)

Current genetic composition of a population is influenced by site-specific demographic history. We compared genetic diversity in two populations of Taiwanese macaques (Macaca cyclopis) with different site characteristics. Mitochondrial DNA variation in 145 individuals from one central, continuous population (Yushan) and one peripheral, isolated population (Shoushan) was ascertained by sequencing and PCR–SSCP of the 5 end hypervariable segment of the control region amplified from hair and fecal samples. All the samples from Yushan were of the same haplotype whereas those from Shoushan fell into 13 haplotypes. The mtDNA homogeneity in Yushan may indicate a local concentration of troops status of the population or the result of population bottleneck in the recent past by severe hunting. The relatively higher genetic diversity in the Shoushan population was contributed by released or escaped captive macaques. In addition, a simple method for preserving fecal sample as a DNA source is also recommended in the study.     

DNA deposition on carbon electrodes under controlled dc potentials

The native calf-thymus DNA molecule fully dispersed in solution was deposited onto highly oriented pyrolytic graphite, carbon fiber column and disk electrodes under controlled dc potentials. X-ray photoelectron spectroscopy, atomic force microscopy and electrochemical investigations indicated that network structures of DNA could be formed on various carbon electrode surfaces resulting in significant surface enlargement. The conformation, conductivity and stability of the deposited DNA layer largely depended on the concentration of the DNA deposition solution, the applied dc potential and the mode of electric field. The optimal condition for deposition of the DNA on carbon fiber disk electrode was determined as a deposition potential of 1.8 +/- 0.3 V versus 50 mM NaCl-Ag/AgCl and a deposition DNA solution of 0.1 mg ml(-1). Under this condition, the DNA was covalently bonded on the electrode surface forming a three-dimensional modified layer, generating a 500-fold enlarged effective electrode surface area and similarly enlarged current sensitivity for redox species, such as Co(phen)3(3+). A possible mechanism for the formation of DNA networks is proposed.     

Cytokine gene polymorphisms in endemic pemphigus foliaceus: a possible role for IL6 variants

Endemic pemphigus foliaceus (EPF) is a complex autoimmune disease characterized by the presence of antibodies against desmoglein 1, which lead to the loss of adhesion among keratinocytes (acantholysis). Variants of HLA class II genes have been the only genetic factors found to modulate susceptibility to EPF. This study aims at investigating the influence of cytokine genetic variants in the pathogenesis of EPF, since they may affect the expression levels of these immunomodulatory molecules. The sample included 168 patients and 189 controls and was comprised of mostly Caucasoids and Mulattos. The approach consisted of a case-control association study and the alleles were identified by mismatched PCR-RFLP. No associations were found with variants of IL1A, IL1B, IL1RN, IL4R and IL10. There was a weak negative association with the haplotype -1082G -592C (OR=0.49) of the IL10 gene in Mulattos. In regard to polymorphism -590 of the IL4 gene, a positive association with the T/T genotype (OR=2.71) and a negative association with the C variant (OR=0.37) were found. Associations with IL6 -174 variants suggest that the C/C genotype has a protective effect (OR=0.13) while carriers of the G allele are more susceptible (OR=7.66) to EPF.     

Demographic trade-offs determine species abundance and diversity

Aims Much recent theory has focused on the role of neutral processes in assembling communities, but the basic assumption that all species are demographically identical has found little empirical support. Here, we show that the framework of the current neutral theory can easily be generalized to incorporate species differences so long as fitness equivalence among individuals is maintained through trade-offs between birth and death.Methods Our theory development is based on a careful reformulation of the Moran model of metacommunity dynamics in terms of a non-linear one-step stochastic process, which is described by a master equation.Important findings We demonstrate how fitness equalization through demographic trade-offs can generate significant macroecological diversity patterns, leading to a very different interpretation of the relation between Fisher's α and Hubbell's fundamental biodiversity number. Our model shows that equal fitness (not equal demographics) significantly promotes species diversity through strong selective sieving of community membership against high-mortality species, resulting in a positive association between species abundance and per capita death rate. An important implication of demographic trade-off is that it can partly explain the excessively high speciation rates predicted by the neutral theory of the stronger symmetry. Fitness equalization through demographic trade-offs generalizes neutral theory by considering heterospecific demographic difference, thus representing a significant step toward integrating the neutral and niche paradigms of biodiversity.     

Lipids,lipid metabolism and Kaposi’s sarcoma-associated herpesvirus pathogenesis

Lipids are essential for mammalian cells to maintain many physiological functions. Emerging evidence has shown that cancer cells can develop specific alterations in lipid biosynthesis and metabolism to facilitate their survival and various malignant behaviors. To date, the precise role of cellular lipids and lipid metabolism in viral oncogenesis is still largely unclear with only a handful of literature covering this topic to implicate lipid metabolism in oncogenic virus associated pathogenesis. In this review, we focus on the role of lipid biosynthesis and metabolism in the pathogenesis of the Kaposi’s sarcoma-associated herpesvirus, a common causative factor for cancers arising in the immunocompromised settings.

Open image in new window

     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.