HEAT SHOCK PROTEIN MICRO-ENCAPSULATION AS A DOUBLE TOOL FOR THE IMPROVEMENT OF NEW GENERATION VACCINES

ABSTRACT

The modern vaccinology encompasses the recombinant DNA technology, protein and carbohydrate chemistry to obtain safe molecularly defined vaccines. Nevertheless most of the vaccines are poorly immunogenic because a large number of antigens are membrane proteins and consequently they are not present in their active conformation in the vaccine. Others are not as potent because they contain only B epitopes and therefore, cannot stimulate cellular memory. We have been studying the characteristics of the recombinant heat shock protein 18kDa-hsp from Mycobacterium leprae as an alternative carrier protein with a T epitope source to enhance the activity of these second generation vaccines. Here we proved that the 18kDa-hsp acted as carrier, without masking the activity of the carried antigen, with similar immune stimulatory effect when compared with ODN1668. Supramolecular aggregates of 18kDa-hsp and Mice serum albumin (MSA) were obtained using glutaraldehyde as cross linker. The Neisseria meningitides serogroup C polysaccharide (PSC, a B epitope) and the carrier protein 18kDa-hsp were co-encapsulated within Soybean phosphatidylcholine liposomes (SPC: Cho : α-Toc, 22 : 5 : 0.18 molar ratio, respectively). These liposomes were prepared in MPB buffer (20 mM phosphate, 295 mM mannitol pH 7.2) in the presence or absence of the ODN1668, TCCATGACGTTCCTGATGCT. When mice were injected with 18kDa-hsp-MSA no antibody against the MSA was observed. This means that the 18kDa-hsp acted as carrier, without masking the carried protein immune activity. Stable liposomes of 150 nm were obtained using mannitol as a cryoprotector. Genetically selected mice when injected with liposomes containing PSC and 18kDa-hsp displayed an antibody titer of 12. In contrast, in those mice injected with free PSC there was no response. The 18kDa-hsp adjuvant effect on the PSC liposomal formulation was comparable to that observed when ODN1668 was co-encapsulated with PSC. Confirming our expectations we observed that the formulation containing 18kDa-hsp conferred a memory response to the carried antigen—the Neisseria meningitidis serogroup C polysaccharide.     

蛋白质结构成对比较的新方法

介绍一种蛋白质三维结构的快速比较方法.此方法毋需初始联配,而能自动寻找和智能迭代.利用本程序对珠蛋白、丝氨酸蛋白酶、天冬氨酸蛋白酶、钙结合蛋白和溶菌酶作了系统的结构比较,取得了满意的结果.本文也讨论了衡量联配结果好坏的要素问题.     

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice: A NEW TOOL FOR STUDYING KYNURENINE PATHWAY METABOLISM IN PERIPHERY AND BRAIN*

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo^−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo^−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo^−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD⁺, did not differ between Kmo^−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo^−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo^−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.     

A NEW AND PRACTICAL ARTIFICIAL DIET FOR THE COTTON BOLL-WORM*

Abstract A new and practical artificial diet for the cotton bollworm, Helicoverpa armigera (Hübner), containing canned tomato paste has been developed. The developmental duration of larvae fed on the diet was greatly shortened because most of them only went through 5 stadia before pupation. To add certain amount of mung bean flour to partly substitute for wheat germ in the diet was beneficial to the adult reproduction. During rearing period of 12 successive generations, 87. 5%-98. 7% of the larvae survived and only less than 5 % of the pupae failed to emerge. The number of egg laid by the adults of the 11th generation averaged about 1 000/female, with a hatching rate of 86. 5%. These results suggested that the vitality of the cotton bollworm did not decline obviously after reared with the diet for two years.     

Plasmonic Nanolithography: A Review

Surface plasmon polaritons (SPPs) has attracted great attention in the last decade and recently it has been successfully applied to nanolithography due to its ability of beyond diffraction limit. This article reviews the recent development in plasmonic nanolithography, which is considered as one of the most remarkable technology for next-generation nanolithography. Nanolithography experiments were highlighted on the basis of SPPs effect. Three types of plasmonic nanolithography methods: contact nanolithography, planar lens imaging nanolithography, and direct writing nanolithography were reviewed in detail, and their advantages and shortages are analyzed and compared, respectively. Finally, the development trend of plasmonic nanolithography is suggested.     

Assembly of the Respiratory Mucin MUC5B: A NEW MODEL FOR A GEL-FORMING MUCIN*

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.     

PARTIAL CHARACTERIZATION OF A NEW MYELIN PROTEIN COMPONENT

A new protein component has been demonstrated in myelin isolated from rat whole brain and from white matter dissected from bovine, dog and rabbit brain. It is also present in rabbit optic nerve. It does not appear to be present in other subcellular fractions of rat brain. It has a molecular weight of 20,540 ± 490(S.D.), as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. A rapid procedure for the isolation of myelin is also described.     

Furanose-specific Sugar Transport: CHARACTERIZATION OF A BACTERIAL GALACTOFURANOSE-BINDING PROTEIN*

The widespread utilization of sugars by microbes is reflected in the diversity and multiplicity of cellular transporters used to acquire these compounds from the environment. The model bacterium Escherichia coli has numerous transporters that allow it to take up hexoses and pentoses, which recognize the more abundant pyranose forms of these sugars. Here we report the biochemical and structural characterization of a transporter protein YtfQ from E. coli that forms part of an uncharacterized ABC transporter system. Remarkably the crystal structure of this protein, solved to 1.2 Å using x-ray crystallography, revealed that YtfQ binds a single molecule of galactofuranose in its ligand binding pocket. Selective binding of galactofuranose over galactopyranose was also observed using NMR methods that determined the form of the sugar released from the protein. The pattern of expression of the ytfQRTyjfF operon encoding this transporter mirrors that of the high affinity galactopyranose transporter of E. coli, suggesting that this bacterium has evolved complementary transporters that enable it to use all the available galactose present during carbon limiting conditions.     

THE LASER AS A POTENTIAL TOOL FOR CELL RESEARCH

Freshly prepared hemoglobin solutions were successively irradiated up to five times with 1 MW (monochromatic wavelength) of green (530 mµ) laser power. Oxygenated hemoglobin showed no detectable change, but the spectral absorption of reduced hemoglobin showed a shift toward the characteristic curve for the oxygenated form. Intact human erythrocytes exposed to a power density of 110 MW/cm² of green laser radiation showed no appreciable change in diameter or mass, but they became transparent to a wavelength range from 400 to 600 mµ. A similar power density from a ruby laser failed to produce this bleaching effect. This response in the erythrocyte demonstrates a principle which suggests the laser as a tool for cell research: specific molecular components within a cell may be selectively altered by laser irradiation when an appropriate wavelength and a suitable power density are applied.     

一种简便的提取蚕蛹蛋白新方法

以蚕蛹为原料,采用一种简便的提取工艺提取蚕蛹蛋白质,结果表明：干蛹约占鲜蛹重的32．5％,干蛹中蛋白质含量为54．2％,成品中蚕蛹蛋白质含量为83．31％,其蚕蛹蛋白收率达干蛹的61．32％,是文献报道用减法提取收率的2．27倍,蚕蛹中蛋白质的回收率达94．33％。     

A TECHNIQUE FOR MEASURING BRAIN PROTEIN SYNTHESIS

Abstract— Mice were infused intravenously for varying periods of time with L-[U-¹⁴C]- tyrosine. The specific activity of free tyrosine in the blood and the brain, and of protein-bound tyrosine in the brain, was measured and the mean rate of protein synthesis calculated. The half-life of mixed brain proteins was found to be close to 4 days with infusions lasting 0.5–2 h.
The origin of the intracellular tyrosine pool was investigated and it was shown that 60 per cent of this was derived directly from the plasma tyrosine and 40 per cent from protein breakdown within the tissue.     

The Plastic Energy Landscape of Protein Folding: A TRIANGULAR FOLDING MECHANISM WITH AN EQUILIBRIUM INTERMEDIATE FOR A SMALL PROTEIN DOMAIN*

Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un)folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on- or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.     

CICLOGRAMA: A TOOL FOR DETECTION OF RHYTHMICITIES IN SLEEP/WAKE CYCLES

The Fourier spectral analysis of binary time series (or rectangular signals) causes methodological problems, due to the fact that it is based on sinusoidal functions. We propose a new tool for the detection of periodicities in binary time series, focusing on sleep/wake cycles. This methodology is based on a weighted histogram of cycle durations. In this paper, we compare our methodology with the Fourier spectral analysis on the basis of simulated and real binary data sets of various lengths. We also provide an approach to statistical validation of the periodicities determined with our methodology. Furthermore, we analyze the discriminating power of both methods in terms of standard deviation. Our results indicate that the Ciclograma is much more powerful than Fourier analysis when applied on this type of time series.     

Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors: A NEW MECHANISM FOR SENSING PATHOGENS*

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.