Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.     

Plasmodium vivax: microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections.     

Tightly bound to DNA proteins: possible universal substrates for intranuclear processes

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.     

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.     

广藿香根与根茎挥发油成分研究

应用气相色谱－质谱联用技术对广藿香根和根茎中挥发油成分进行了分析,从根油中分离出５０个色谱峰,鉴定了４７个化合物,占总量的９５．０２％,主要含广藿香酮（８１．７１％）、ｄ－苦橙油醇（２．８８％）、十六烷酸（２．２１％）、广藿香醇（１．９８％）和３,７,１１－三甲基－２,６,１０－十二烷三烯－２－醇乙酸酯（１．０９％）、广藿香醇（１．９８％）和３,７,１１－三甲基－２,６,１０－十二烷三烯－１－醇乙